PHARMACOKINETICS OF BUPRENORPHINE IN DOGS
By
V. RAVI CHANDRAN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1986
ACKNOWLEDGEMENT
I gratefully acknowledge Dr. Edward R. Garrett for his numerous and
varied contributions. I also thank him for his guidance, training,
support and facilities during my graduate career, which made this
dissertation possible.
My special thanks are extended to Dr. Jurgen Venitz for his time,
fruitful discussions and helpful suggestions, and to Dr. Larry J. Peters
and Dr. August H. Battles for animal preparations.
I wish to thank the current and past members of the "Beehive" for
their help, support, advice and friendship.
I acknowledge the members of my supervisory committee,
Dr. John H. Perrin, Dr. Hartmut C. Derendorf, Dr. James W. Simpkins,
Dr. Michael J. Katovich, Dr. C. Lindsay Devane, Dr. John A. Zoltewicz
for their contributions.
I take this opportunity to express my sincere appreciation to
Dr. Bernard Desoize, Dr. Peter Langguth, Mrs. Marjorie Rigby, Mrs. Kathy
Eberst, Mr. George Perry and Mr. Thomas Miller for their valuble help.
li
TABLE OF CONTENTS
ACKNOWLEDGEMENTS Ü
ABSTRACT iv
INTRODUCTION 1
EXPERIMENTAL 20
IV BOLUS STUDIES 30
URINARY EXCRETION OF BUPRENORPHINE 86
IV INFUSION STUDIES 131
PHARMACOKINETICS OF THE IV ADMINISTERED METABOLITE 214
SUMMARY AND CONCLUSIONS 241
APPENDIX I  PROGRAM "MULTI" 247
APPENDIX II  FITTING OF DATA TO EQUATIONS 251
APPENDIX III  KRUSKALWALLIS TEST 258
APPENDIX IV  TABLES OF RAW DATA 261
GLOSSARY OF TERMS 346
REFERENCES 348
BIOGRAPHICAL SKETCH 352
iii
Abstract of Dissertation presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PHARMACOKINETICS OF BUPRENORPHINE IN DOGS
By
V. Ravi Chandran
December 1986
Chairman: Dr. Edward R. Garrett
Cochairman: Dr. John H. Perrin
Major Department: Pharmaceutical Sciences
Specific and sensitive reversephase HPLC assays of buprenorphine
and its metabolite in biological fluids were developed with
sensitivities of 26 ng/ml using fluorimetric detection. Upon acute
bolus administration of buprenorphine in six dogs within the 0.72.6
mg/kg dose range, accurate estimation of the terminal rate constant and
the derived total body clearance were not feasible due to the lack of
sufficient quantifiable terminal plasma points at less than 5 ng/ml
sensitivity. The terminal plasma concentrations could not be increased
by increasing the bolus dose since such high doses would have
significant toxicity. This toxicity was circumvented and the terminal
plasma concentrations were increased by infusing 3.74.8 mg/kg doses of
buprenorphine over 3 h in six studies in six dogs. The terminal rate
IV
constants of the IV infusion studies averaged 34 +_ 3.7 h with an
averaged total body clearance of 212 _+ 35 ml/min. The apprent volumes of
distribution of buprenorphine referenced to the total plasma
concentration were 35 L (V , central compartment volume) and 617 L
(Vj, total body volume), indicative of a highly bound, sequestered or
lipophilic drug.
Unchanged buprenorphine is insignificantly renally (<0.5% of the
dose) and biliary (<0.5%) excreted. The major route of buprenorphine
disposition is by hepatic conjugation to glucuronide which is eliminated
into the bile (about 95%) with only small amounts appearing in urine
(<1% as metabolite). Minor metabolites excreted in the bile accounted
for about 3% of the administered dose.
Direct IV administration of the metabolite gave a terminal
halflife of 6 h. Unlike intravenously administered morphine glucuronide
which was not excreted in the bile, more than 90% of the systemically
circulating metabolite was excreted in bile and only 10% in urine.
The oral bioavailability estimated from the areas under the
buprenorphine plasma concentrationtime curve following TV and oral
administration of buprenorphine in the dogs was 36%. In a bile
cannulated dog, intraduodenally administered metabolite demonstrated 6%
enterohepatic recirculation of the conjugate.
There were no apparent correlations of the buprenorphine time
course with cardiovascular parameters such as heart rate, ECG and blood
pressure. Miotic effect was significant. Respiratory depression was
observed during the first 4 h after IV bolus injection, but not during
the infusion studies.
v
INTRODUCTION
Buprenorphine (1) is a derivative of the morphine alkaloid
thebaine. It is a strong analgesic with marked narcotic activity. Since
the midsixties, its therapeutic potential as a morphinetype analgesic
at low doses and antagonistic activity at high doses, has been well
documented.^ Buprenorphine has been claimed to have an advantage over
morphine in that the dose does not need to be increased during several
2
weeks of chronic administration.
Pharmacodynamic and Therapeutic Studies
Buprenorphine has displayed narcotic agonist and antagonist
properties in animals and man. Agonistic effects often exhibited a bell
3
shaped doseresponse curve, as occurs with pentazocine, and subjective
opiatelike effects reached a maximum at a dose of about 0.2 to 0.8 mg
4
subcutaneously in man. The onset of agonistic effects (peak effects
about 6 h after subcutaneous or IM injection) in man was slower than
with morphine but the duration of such effects was longer (about 72
5
hours) than with morphine. Also, the analgesic potency of
buprenorphine was about 25 times that of morphine (on a per unit weight
basis)
Therapeutic Trials
In a comparative study of the treatment of chronic pain of
malignant origin by intramuscularly administered buprenorphine and
morphine, 27 patients received buprenorphine (0.3 mg) and morphine (10
7
mg) in a doubleblind, singledose withinpatient study. There were no
1
2
significant differences in the intensity of analgesic effect or the time
7
to reach it. However, buprenorphine had a significantly longer
duration of action than morphine. Sedation was the most frequent side
7
effect but dizziness, nausea and vomiting were also seen. Compared to
morphine, buprenorphine showed significantly higher incidences of side
7
effects, greater severity and earlier onset, and longer duration.
Following both treatments there were small but significant decreases in
7
pulse rate, blood pressure and respiratory rate.
Antinociceptive actions (blockade of impulses at the peripheral
pain sensitive nerves) of buprenorphine and morphine given intrathecally
g
in conscious rats were compared. After intrathecal injection the peak
(30 min) antinociceptive potencies of buprenorphine or morphine were
g
similar. The analgesic profiles of buprenorphine and morphine (0.3 mg
and 10 mg respectively) were compared in a doubleblind noncrossover
9
multiple dose study (IM administration) in man. When the patient
complained of moderate to severe postoperative pain after upper
abdominal surgery, the first test dose of either drug was given. The
drugs gave an equal decrease in pain intensity, suggesting a relative
9
potency of 33:1. An average of 0.51 mg of buprenorphine or 16 mg of
morphine had to be administered for satisfactory initial analgesia. A
faster decrease in the rate of respiration was observed after
buprenorphine than after morphine, but ultimately both the drugs gave
9
the same minimum rate of respiration. These results were comparable to
7
those reported elsewhere.
An oral combination of buprenorphine and paracetamol was compared
to paracetamol in a singledose doubleblind study in man for the
initial acute treatment of postoperative pain.^ One hundred and
3
twenty patients undergoing orthopedic operations were divided into four
groups of 30 patients each. The four treatments were 1, 1.5 or 2 mg of
buprenorphine combined with paracetamol 1000 mg or paracetamol (1000 mg)
alone. There were no significant differences among the groups in
analgesia measured by the observer and by the pain intensity scoring by
the patients over the first six hour. The oral combinations of
buprenorphine and paracetamol produced a significant increase in
duration of analgesia beyond 6 hours over that of paracetamol alone at
all three dose concentrations. A significant increase in side effects
was seen only at the highest dose of buprenorphineparacetamol
combination compared with paracetamol alone.^
In a study designed to assess the development of drug dependence,
rats were chronically treated subcutaneously for 4 days with
buprenorphine.11 These rats showed only weak signs of withdrawal upon
cessation of a treatment or upon challenge with naloxone.11 More
intense withdrawal symptoms were induced when morphine was substituted
for buprenorphine. Even one injection of morphine, given 12 h after the
last buprenorphine treatment, led to withdrawal symptoms with naloxone.
Naloxone did not cause withdrawal in naive rats treated with this dose
of morphine. Thus, according to these authors,11 and contrary to a few
12 .
claims in the literature, buprenorphine induced dependence like other
opiates. The authors argue that the intensity of withdrawal is less
severe due to slow dissociation of the drug from the receptors.11
The neurochemical effects of buprenorphine were compared to those
of morphine and haloperidol in rats.^ The effect of a wide range of
doses of buprenorphine (0.001  10 mg/kg, subcutaneous administration)
was studied a) with normal concentrations of dopamine, noradrenaline,
4
5hydroxytryptamine etc. and b) with lower concentrations of dopamine
and noradrenaline in rat brain following the treatment with alphamethyl
paratyrosine (alphaMpT), which is a inhibitor of catecholamine
synthesis. Morphine and haloperidol were used as reference agents.
Buprenorphine increased the alphaMpT induced rate of dopamine depletion
but did not deplete norepinephrine. Similar results were obtained with
a higher dose (30 mg/kg) of morphine but it increased the alphaMpT
induced depletion of norepinephrine. Apparently similar effects of
buprenorphine and haloperidol on dopaminergic neurotransmission were
distinguished by pretreating the rats with naloxone (which antagonized
the effect of buprenorphine, and prevented dopamine depletion). These
neurochemical results were claimed to support the view that one site of
action of buprenorphine is on opiate receptors located on the
dopaminergic neurons.^
In a doubleblind comparison between fentanyl and buprenorphine in
supplemented nitrous oxide analgesia, buprenorphine or fentanyl (0.3 and
0.125 mg respectively administered IV) were used as supplements in 40
14
patients undergoing major abdominal surgery. Initially both narcotics
appeared to suppress tachycardia and increase arterial pressure in
response to surgery but 80% of the patients who received fentanyl
eventually reguired a further supplement of halothane (0.5%), but no
patient who received buprenorphine required halothane. Recovery from
analgesia was similar in both groups, but the duration of analgesia
after the operation was significantly greater for buprenorphine (12 h)
than fentanyl (3 h).1^
In a doubleblind randomized noncrossover trial, 47 patients
received either morphine (10 mg) or buprenorphine (0.3 mg) by regular IM
5
injection for 24 h after abdominal surgery.15 In this study, the two
drugs were equally effective at the dose ratio 1:33, buprenorphine to
7 9
morphine which is comparable to the results reported elsewhere.
One hundred twentysix patients undergoing upper and lower
abdominal surgery were studied postoperatively to compare the analgesic
effect of IM morphine, sublingual buprenorphine and self administered IV
pethidine.15 There were no significant differences among analgesic
regimens in respect to subjective pain scores or static and dynamic lung
volumes assessed at 24 h, 48 h and 5 days after operation. Sublingual
buprenorphine produced more nausea and sedation than the other regimens,
but the results were not clinically important. These authors15 report
that buprenorphine offered considerable advantages in terms of ease of
administration.
When diamorphine and buprenorphine were compared in the relief of
chest pain in man, sublingual administration appeared to be as effective
17
as the IV route, but the onset of action was slow. There were no
significant changes in the systemic or pulmonary arterial blood pressure
17
or heart rate after IV buprenorphine. A randomized doubleblind
controlled trial of equivalent doses of buprenorphine and diamorphine
showed no significant differences between the drugs in terms of pain
17 . .
relief and duration of action. The occurence of nausea, vomiting and
other side effects was similar in the two groups. The onset of action of
buprenorhine was slightly but significantly slower than that of
diamorphine.1^
Buprenorphine and pethidine were compared in a doubleblind study
of ondemand IV analgesia. Buprenorphine was about 600 times as potent
18
as pethidine. The incidence of side effects was similar with both
6
drugs. The quality of analgesia subjectively assessed was the same with
18
both drugs using this method of administration. These authors claim
that buprenorphine is a powerful analgesic agent that may be given
intravenously provided that its low potential for abuse is
substantiated.
In a smaller number of patients with chronic pain, usually due to
cancer, sublingually given buprenorphine (up to 0.8 mg 4 hourly)
provided adequate pain relief for periods up to several months, but side
effects (usually nausea and vomiting) required discontinuation of
19
treatment in about 1/3 to 1/2 of the ambulatory patients.
Following anesthesia with fentanyl in 180 patients, buprenorphine
(usually 0.4 to 0.8 mg IV) reversed some of the anesthetic effects while
producing continued analgesia that lasted about 812 h after a
20
singledose. The antagonistic activity however, was frequently
shortlived, declining rapidly after 90 to 120 min and a second dose of
buprenorphine was often required to prevent the reemergence of
20
anesthetic effects.
The efficacy of buprenorphine has been compared to lofentanil and
to saline placebo by extradural administration in the management of
21
postoperative pain in sixty patients. In a doubleblind study, these
orthopedic patients were randomly assigned to three equal groups to
determine the analgesic effects, duration of action and side effects of
the extradural administration of lofentanil (5 ug) buprenorphine (0.3
21
mg) or physiological saline. No systemic analgesics were given
before, during or after surgery, and all the patients had operations on
the lower extremities under extradural analgesia (lignocaine or
bupivacaine). Upon administration of the test drug as soon as pain
7
occured in the postoperative period, a long duration of action and a
marked analgesic effect was observed with lofentanil. A shorter duration
of action and less pain suppression occured with buprenorphine and a
rather marked placebo effect was seen with saline. The only side effect
noticed was drowsiness in 3 patients in the lofentanil group and in 2
21
patients in the buprenorphine group.
22
In a randomized doubleblind trial comparing analgesia produced
by combinations of droperidol with either buprenorphine or morphine,
buprenorphine was claimed to be as satisfactory as morphine to produce
analgesia during major surgery in 60 patients, with no difference in the
incidence of untoward side effects.
Epidural buprenorphine was investigated as a postoperative
analgesic in a randomized doubleblind study of 158 patients given
intraoperative epidural analgesia with 2% mepivacaine or 0.5%
23
bupivacaine for orthopedic surgery of the lower extremity. At the end
of the surgery, the patients were given epidurally in 15 ml saline,
either 0.15 mg of buprenorphine (n=38) or 0.3 mg (n=37). A control group
received no epidural injection (n=47). The above 3 groups received 2%
mepivacaine as intraoperative anesthetic. A fourth group (n=36) received
0.3 mg buprenorphine in 15 ml saline, after intraoperative use of 0.5%
bupivacaine. The patients rated postoperative pain. Analgesia after
0.15 mg of buprenorphine was superior to that after saline injection,
and 0.3 mg buprenorphine was superior to both saline injection and to
23
0.15 mg of buprenorphine until 12th hour. Analgesia after bupivacaine
followed by 0.3 mg of buprenorphine was not significantly different than
analgesia seen after mepivacaine followed by 0.3 mg of buprenorphine.
21
These results are comparable to those reported elsewhere.
8
Respiratory effects. The respiratory depressant activity (such as
decreased respiratory rate, increased arterial P CCL and decreased
cl Z
arterial P 0„) of single equianalgesic doses of buprenorphine and
cl Z
1 24 25
morphine appear to be similar in rats and rabbits. ' ' The extent of
buprenorphineinduced respiratory depression against dose plateaued in
26
animals, whereas such an effect was not clearly demonstrated in man,
which showed doserelated respiratory depression within the therapeutic
dose range (0.3 mg to 0.6 mg). The time to reach peak respiratory
depression in man was slower after intramuscular buprenorphine than
after morphine (3 h vs 1 h) and the duration of such an effect was
26
longer. There appears to be no completely reliable specific
antagonist for buprenorphineinduced respiratory depression since even
26
high doses of naloxone produced only partial reversal. Hcwever, the
respiratory stimulant drug doxapram has reversed respiratory depression
due to buprenorphine in a few healthy volunteers and in a few
. . . 26
patients.
Cardiovascular effects. Hemodynamic changes in healthy volunteers
after IM (0.15 to 0.6 mg), sublingual (0.4 to 0.8 mg) or oral (1 to 4
ng) doses of buprenorphine include dose related reductions in heart rate
(up to 25%) and small decreases in systolic blood pressure (about
26
10%). These results are comparable to the cardiovascular effects of
26
morphine. Similar effects occured in anesthetized patients undergoing
surgery and in a few patients with myocardial infarctions. However, in
the latter group the heart rate was found to be relatively
27
unperturbed.
Addiction potential of buprenorphine. Buprenorphine appeared to
have a lower addiction potential than the opioid agonist pentazocine in
9
animals. However the extent to which such results can be extrapolated to
1 28
man was uncertain. ' In a singledose addiction study in 5
volunteers, high (8 mg daily) intramuscular doses of buprenorphine,
administered up to 1 to 2 months produced a slowly emerging withdrawal
5 6
syndrome on abstinence from the drug. ' Though the results were
indicative of lesser addiction potential compared to morphine,
definitive statements about addiction cannot be made until it has been
more widely used in patients with chronic pain with repeated doses over
an extended period of time.^
Receptor binding studies. Receptor binding studies were undertaken
to elucidate the opioid binding characteristics of fentanyl and
29
buprenorphine, and to investigate differences between them.
Buprenorphine showed slow receptor equilibration (30 min) but with high
affinity to multiple sites. The dissociation was claimed to be slow
(halflife = 166 min) and incomplete (50% binding after 1 h). This
contrasted with the receptor binding of fentanyl, which achieved rapid
equilibrium (within 10 min) and dissociated equally rapidly (halflife =
6.8 min) and completely (100% by 1 h). Using competitive displacement
studies, it was claimed that buprenorphine displacement of fentanyl was
concentration and time dependent over the ranges (equimolar
buprenorphine and fentanyl concentrations, 2 nmol/liter) encountered in
clinical use. However, buprenorphine binding was displaced with only
. . 29
high concentrations of other opioids.
Binding of buprenorphine to the rat forebrain (telencephelon,
diencephelon and mesencephelon) was claimed to be stereospecific,
30
saturable and had high affinity. Maximum binding (Bmax) was reached
by 30 min and dissociation from the receptor was slow. The regional
10
distribution of buprenorphine binding sites in the rat brain was claimed
to be qualitatively similar to the distribution of naloxone and
dihydromorphine binding sites. The Bmax for this receptor binding of
buprenorphdne was about 2 times that for the muopiate receptor drugs
and three times the for the deltaopiate receptor ligands (such as
enkephalins). Buprenorphine was also found to be very potent in
displacing naloxone, dihydromorphine and metenkephalin. Since mu
receptors bind with exogenous opioids (such as morphine), and delta
receptors bind with endogenous opioids (such as enkephalins), the above
findings suggest that buprenorphine binds to both mu and
deltareceptors.^
Side effects.
Moderate to marked drowsiness has been reported in about 4050% of
the patients (up to 75% in some studies), but all such patients were
1 31 32
found to be easily arousable upon stimulation. ' ’ Nausea and/or
vomiting occurred in 15% of the patients. Other minor side effects (e.g.
dizziness, sweating, headache or confusion), typical of strong
analgesics, have been reported with a widely varying incidence.
Respiratory depression, as determined by laboratory measurements of
respiratory functions, does occur with buprenorphine. The extent of such
depression was similar to other opioid drugs administered in usual
clinical doses.1 However, this was not a problem in clinical studies
79
which were usually conducted in fit patients. The effect of
buprenorphine on respiration in "poor risk" patients, such as those with
respiratory diseases or congestive heart failure, has not been
determined. However, it appears that buprenorphine would have the same
potential problems as morphine in this patient group.1
11
Dosage and Administration
Buprenorphine is presently available in Europe for parenteral
use.1 The recommended dose is 0.3 to 0.6 mg by IM or slow IV injection,
repeated every 68 h as needed. Administration of buprenorphine to
patients already receiving large doses of narcotic drugs should be
undertaken with caution until the response is established, since its
antagonistic activity could conceivably cause withdrawal symptoms.1
Pharmacokinetic Studies
There is limited information available on the pharmacokinetic
properties of buprenorphine in man.1 It was stated without
documentation or citation of references that rapid absorption and peak
plasma concentrations were seen in rats on oral and IM dosings where the
oral dose was 4 times the IM dose.1 It was claimed that in primates and
in human volunteers, peak plasma concentrations were reached more slowly
after oral administration (2 h) than by IM injection (7 min).1 Drug
concentrations were stated to be detectable in blood for longer times
after oral (24 h) than IM (7 h) administration of equivalent doses. In
man buprenorphine was claimed to be excreted unchanged in the feces, and
as glucuronide and Ndealkylated buprenorphine in urine.1 References of
studies supporting these data were not given.1
In a 3 h study, peak plasma buprenorphine concentration did not yet
.33
occur in some patients after sublingual administration. In a
subsequent 10 h study with 15 postoperative patients, 5 patients
received a sublingual dose 0.4 mg of buprenorphine, five 0.8 mg and 5
received placebo at 3 h after a 0.3 mg IV dose of buprenorphine. The
plasma buprenorphine concentration was measured by a specific
34
radioimmunoassay. The plasma concentration reached a peak level in an
12
average time of about 200 min in both the 0.4 mg and 0.8 mg groups
34
(range 90360 min after the initial 3 h period). The plasma drug
concentration in the 0.8 mg group were approximately twice that in the
0.4 mg group. The absolute bioavailability was estimated to be about 55%
of the IV route for both groups by the ratio of the area under the
plasma concentration versus time (AUC) for sublingual and TV
administration. Uptake of buprenorphine from the sublingual site was
34
claimed to be complete by 5 h after the dose was given. In this
study, crossreactivity between buprenorphine and its metabolites was
not ruled out. Two modes of administration (TV followed by subcutaneous)
were carried out in each study which complicated the pharmacokinetic
analysis.
Buprenorphine kinetics were studied in surgical patients using
35
radioimmunoassay. Buprenorphine was measured in the plasma of 21
patients who received 0.3 mg IV. After 3 h, ten of these patients
received further dose of 0.3 mg TV, and 11 patients were given 0.3 mg
IM. Plasma drug concentrations were measured up to 3 h after the second
dosing. Comparison of the pharmacokinetics in the same patient, awake
and anesthetized by general anesthesia, showed that the clearance was
significantly lower (900 ml/min) in the anesthetized state compared to
the unanesthetized state (1225 ml/min). Bioavailability was claimed to
be the same for both TV and IM administered drug. The peak plasma levels
were seen at 25 min and in 10 min respectively for IV and IM dosing
35
after the second dosing. Cross reactivity among buprenorphine and its
metabolites was not ruled out in this study. The sensitivity and limits
of detection for buprenorphine were not given. Thus the terminal plasma
buprenorphine concentrations at less than 1 ng/ml are questionable.
13
Procedures for obtaining various pharmacokinetic parameters were not
given.
Plasma concentrations were correlated with clinical effects after a
single IV dose of buprenorphine (0.3 or 0.6 mg) in patients recovering
36
from surgery. Analgesia was greater at the high dose without any
apparent parallel increase in respiratory depression. Better analgesia
was reported if the first required postoperative dose of 0.3 mg has
been preceded by a similar loading dose or by the use of a larger dose
36
during surgery. This study was largely descriptive without rigorous
pharmacokinetic analysis. The plasma concentrations obtained from a
number of patients were averaged to obtain a mean concentration. This is
not a valid pharmacokinetic technique.
Metabolism and Excretion
Higher amounts of polar metabolites were seen in plasma after oral
administration than after IM in rats.'*' The premise of drug conjugation
37 38
in the gut wall was supported by studies with rat gut preparations. '
Buprenorphine has been found conjugated or Ndealkylated in bile or
tissues of animals, but unchanged in the brain. This is possibly
indicative of the fact that buprenorphine and not a derivative is
responsible for the narcotic activity.1
In a study of pharmacokinetics of buprenorphine after IM
administration to rats, dogs, rhesus monkeys and one human volunteer,
most of the dosed radioactive drug was excreted in the feces, indicating
39
biliary excretion with possible enterohepatic recirculation. After IV
administration of the tritiated buprenorphine (100 p g/kg) to the bile
duct cannulated rats, over 90% of the administered drug was excreted in
the bile within 48 h after dosing. The major metabolite in the bile was
14
buprenorphine glucuronide. Ndealkylated buprenorphine was also present.
Intraduodenal infusion of rats with bile obtained from other rats dosed
with radiolabelled drug produced a slow but extensive excretion drug
. . 39
related metabolites in the bile of the recipient animal. The plasma
concentrations were not measured in this study. The assay techniques
were not specific for the parent drug or its metabolites. Dose
dependency was not studied.
40 .
In a chronically cannulated cow, it was shown that the hepatic
extraction ratio for IV boluses of morphine, diamorphine, fentanyl,
methadone and buprenorphine increased towards a plateau value as the
portal vein drug concentration increased. The extraction ratio was
claimed to be independent of hepatic blood flow, but dependent on
concentration.
Disposition of radiolabelled buprenorphine in the rat after a
single 0.2 mg/kg IV bolus dose and continuous administration via a
41 ...
subcutaneous delivery system were carried out. After IV injection,
triexponential decay of the drug from brain was seen with halflives of
0.6, 2.3 and 7.2 h, respectively. Plasma halflives were 0.5 and 1.4 h
(the third phase was not estimated). Decay halflife of the drug from
its high affinity binding sites in brain were 1.1 and 68.7 h
respectively. Fat and lung had higher concentrations than other tissues
41
or plasma. No metbolites of the drug were detected in brain.
Unmetabolized drug excreted in the urine and feces one week after TV
injection were 1.9 and 22.4% of the dose, respectively, and 92% of the
dose was accounted for in 1 week. Urinary metabolites (%) were
conjugated buprenorphine 0.9; norbuprenorphine (free 9.4, conjugated
15
5.2); tentative 60desmethyl norbuprenorphine (free 5.4, conjugated
15.9).41
Peak plasma concentration of buprenorphine occured in 4 weeks after
s.c. implantation of a longacting radiolabelled buprenorphine (10 mg)
pellet. The apparent dissociation halflives of the drug from the lcw
and highaffinity binding sites in the brain were 4.6 and 6.8 weeks,
respectively. Fat, spleen and skeletal muscle had higher radioactivity
41
than other tissues and plasma. These authors state that highaffinity
binding of buprenorphine in brain and subsequent slow dissociation are
the factors responsible for its prolonged agonist and antagonist effects
and higher potency than other narcotic agonists.
Absorption and bioavailability. There are no published studies on
the oral absorption of this drug in man. It was claimed on the basis of
unpublished data that the peak plasma concentration of orally
administered radiolabelled buprenorphine in the rat was reached in 10
min with another peak in plasma appearing in about 58 h.1 This delayed
peak could be due to the late appearance of radiolabelled metabolites
(e.g., Ndealkylated buprenorphine and conjugates) in plasma.''' An
intramuscular dose of 20 y g/kg gave blood peak concentration similar to
that of a 100 yg/kg oral dose. In monkeys, unpublished data were cited
to support the statements that peak blood concentrations of
radiolabelled drug were reached at 2 min, 2 h and between 24 h
respectively for IM, oral and sublingual administration.'1' Also, it was
stated that in two healthy volunteers, peak blood concentrations were
reached rapidly after IM dosing (2 yg/kg) of radiolabelled
buprenorphine followed by a rapid decline. Peak concentrations were
reached slowly at 2 h after the oral administration of 15 yg/kg of the
16
drug, followed by a biexponential decline of concentration.
Concentrations as low as 0.5 to 3.5 ng/ml were claimed to be detected by
a specific radioimmunoassay technique after IV and IM administrations
1
(0.3 mg). In human volunteers a dose of 0.4 mg produced peak
concentrations of 12 ng/ml at about 2 h after oral administration.
References of studies supporting these data were not cited.1
Systemic bioavailability of buprenorphine was studied in female
rats following singledoses (200 jag/kg) administered by six different
42
routes. Relative to the 100% bioavailability from the intraarterial
route, the mean bioavailabilities were, IV 98%, intrarectal 54%,
intrahepatoporta1 49%, sublingual 13% and intraduodenal 9.7%. AUC
analysis of buprenorphine concentrations in blood showed the relative
fractions of the drug excreted (first pass) by gut, liver and lung to be
0.8, 0.5 and 0.02 respectively. In vitro absorption studies showed that
poor bioavailability of intraduodenally administered buprenorphine was
not due to slow or incomplete absorption, but due to firstpass
42
metabolism. In this study, the authors computed AUC only up to 4 h
for the plasma data. The data showed two compartment model type
disposition for the drug in plasma. AUC would be different if calculated
up to time infinity.
In the above pharmacokinetic studies, low doses and subsequent low
terminal plasma concentrations have essentially limited the estimates of
terminal rate constants and halflives of elimination. Sensitive and
selective assay techniques for buprenorphine and its metabolites in
biological fluids, and administration of large doses to a higher animal
such as dog could give acceptable pharmacokinetic parameters.
17
Protein binding. It was reported without documentation'1' that
buprenorphine was highly bound (96%) to alpha and betaglobulin
fractions of human plasma proteins in the concentration range 09 ng/ml.
The fraction of drug bound to dog plasma protein was determined by
measuring the drug concentration in plasma water after
43
ultracentrifugation. The fraction bound was estimated to be 0.945.
Binding of buprenorphine to dog plasma proteins was also determined by
partitioning the drug into red blood cells. The estimation is based upon
the presumption of established equilibria between drug in plasma water,
44
red blood cells and plasma proteins. By this technique, the fraction
43
of buprenorhine bound to plasma proteins was estimated as 0.983. This
relatively high plasma protein binding for the lipophilic buprenorphine
45
contrasts to the 2636% plasma protein binding of morphine, naloxone,
and naltrexone.46
RBC Partition. Partition studies have shown that red blood
43
cellplasma water partition coefficient of buprenorphine was 611.
45
This is in contrast to 1.11 for morphine, 1.83 for naltrexone, and
1.49 for naloxone.46
Physical properties. Fluorescence (excitation 285 nm, emission 350
nm) of buprenorphine provided excellent detection for HPLC assay in
43
biological fluids with a 5 ng/ml sensitivity. Buprenorphine
solvolysis was specificacid and specificbase catalysed. It yielded a
stoichiometric final acid degradation product (3), a fluorescent
detectable, rearranged demethoxy analogue of buprenorphine. Alkaline
43
hydrolysis produced no fluorescence products. Acid hydrolysis also
produced a fluorescentdetectable, transient dehydro intermediate (2)
that was also completely transformed into the demethoxy analogue (Scheme
18
Methyl migration
^ch2 <]
¿
' f
/CHj <]
/
¿
Scheme I
Acid Hydrolysis of Buprenorphine
19
I). Compound 2 was an excellent bioassay internal standard. Buprenorhine
was shown to be highly stable at neutral pH values, even at elevated
43
temperatures. Estimated buprenorphine pKa1 values were 8.24 and 10
for the ammonium and phenolic groups respectively. The intrinsic aqueous
43
solubility of buprenorhine was 12.7 +_ 1.2 ;ug/ml at 23C.
Assay methods. Few assay methods of buprenorhine in biological
47
fluids have been reported in the literature. A radioimmunoassay has
been used to determine plasma levels of parenterally administered
34 35
buprenorphine in dogs and humans. ' A selective ion monitoring
method (SIM) of the silylated buprenorphine in GCMS has been used to
determine the plasma levels of buprenorphine over a 203000 ng/ml
48
concentration range. A GC assay with flameionization detection of
silyl derivatives of buprenorhine was used in stability studies at 510
49
;ig/ml of aqueous solutions. An HPLC assay with fluorescence detection
43
of buprenorhine in biological fluids has been reported and its
modification and improvement is presented in this dissertation.
EXPERIMENTAL
Materials. Analytical grade solvents and reagents were used.
Buprenorphine hydrochloride, 21cyclopropy17alpha[(s)1hydroxy1, 2,
2trimethylpropyl]6,14 endo ethanotetrahydrooripavine, 1, (National
48
Institute for Drug Abuse, Rockville, MD) and the demethoxy analog, 3,
49
of buprenorphine (Addiction Research Center, Lexington, KY) were used
as received. A standard sample of 21cyclopropyl7alpha[2(3,
3dimethyllbutenyl)] 6,14 endo ethanotetrahydrooripavine, 2, was
obtained from Dr.G. Lloyd Jones of Rickett & Colman, Pharmaceutical
Division, KingstonuponHull, England.
Apparatus. An HPLC (model M6000A pump, Waters Associates, Milford,
MA), equipped with a variablewavelength fluorescence detector (model
600S Fluorescence Detector, PerkinElmer, Norwalk, CT), was used.
Injections were carried out with an auto sampler (WISP Autosampler,
Waters Associates), and the data were analysed by a microcomputer (Sigma
15, Data Station, Perkin Elmer). A separate HPLC pump (series 3B, Perkin
Elmer) equipped with a variable wavelength UV detector (model LC 75,
Perkin Elmer) was used in some studies. A laboratory centrifuge was used
in the separation of organic extract from biological fluids (Lab
Centrifuge, International Centrifuge Equipment Co., Needham Heights,
MA).50
Liquid Chromatographic Procedures. Aliquots (50100 yL) of the
solutions to be analyzed were injected into the HPLC system equipped
with a packed [packing material was C^g 5 ym Bondapakreversed phase
20
21
(ODSHypersil), Shannon Southern Products Ltd., Cheshire, U.K.] 120 mm
i.d. stainless steel column [Knauer HPLC analytical column (unpacked),
Knauer A.G. Berlin, F.R.G.] which was maintained at 40 C. The usual
mobile phase flow rate was 1.5 mL/min of a 40:60 acetonitrile:acetate
buffer (pH 3.75, 0.05M) containing 0.0004M tetrabutylammonium phosphate.
Fluorescence was effected at 285 nm excitation (slit 20 nm) and 350 nm
43
emission (slit 15 mm) and was used unless stated otherwise.
Calibration Curves in Biological Fluids
Buprenorphine. Aliquots (1 mL) of plasma, urine or bile in each of
ten 15—mL centrifuge tubes were spiked with 100 pL of 1001000 ng/mL of
buprenorphine (1). Each solution contained 50 ng/mL of the acid
degradation intermediate of buprenorphine, compound 2, as the internal
standard. The final sample contained no drug. Sodium borateboric acid
buffer (1 mL at pH 9.1, 1 M) and 4.2 mL of benzene were added to each
tube. The tubes were shaken for 20 min, centrifuged at 3000 rpm for 10
min, and 4 mL of each benzene extract was transferred to another set of
ten 15—miL centrifuge tubes. Hydrochloric acid (1 iríL, 1 M) was added to
each tube and the tubes were shaken for 10 min and then centrifuged at
3000 rpm for 10 min. After removal of benzene layer by aspiration, 1 mL
of both 1 M NaOH and pH 9.1 borate buffer (1 M) were added to each of
the remaining aqueous phases. The pH values were confirmed or adjusted
to be between 9.05 to 9.15. Benzene (4.5 mL) was added to each tube
which was shaken for 10 min and centrifuged at 3000 rpm for 10 min. The
benzene extract (4.00 mL) was transferred to a 5—miL vial (Reactivial,
Supelco, Inc. Bellefonte, Pa.) and the benzene was evaporated under a
stream of nitrogen at 55‘C. Sodium acetateacetic acid buffer (pH 3.75,
0.05 M, 100 yL) was added to each of the ReactiVials and they were
22
vortexed for 30 s, and then 75 y L of the solution was analyzed by HPLC.
Buprenorphine conjugates. Aliquots (1 mL) of plamsa, urine or bile
in each of ten 15mL centrifuge tubes were spiked with 100 y L of
1001000 ng/mL of buprenorphine. The first sample contained no drug. To
each centrifuge tube, 1 mL of 6 N HC1 was added, and autoclaved at 15
lbs/sq.in pressure for 10 min. The tubes were allowed to equilibrate to
room temperature. To each tube containing the acidtransformed demethoxy
buprenorphine, 50 y L of unconverted buprenorphine (1 y g/mL) was added
as internal standard. Excess acid was neutralized with soduim carbonate.
The pH was adjusted to 9.1 with sodium borateboric acid buffer (1 mL, 1
M) and 4.2 mL of benzene was added to each tube. The tubes were shaken
for 20 min, centrifuged at 3000 rpm for 10 min, and 4 mL of each benzene
extract was transferred to fresh 15mL centrifuge tubes. Hydrochloric
acid (1 mL, 1 M) was added to each tube and the tubes were shaken for 10
min and centrifuged at 3000 rpm for 10 min. After removal of the benzene
by aspiration, 1 rriL of both 1.00 M NaOH and pH 9.1 borate buffer (1.00
M) were added to each reamining aqueous phases. The pH values were
confirmed or adjusted to be between 9.05 to 9.15. Benzene (4.5 mL) was
added to each tube which was shaken for 10 min and centrifuged for 10
min at 3000 rpm. The benzene extract, (4.00 mL) was transferred to a 5
rriL vial (ReactiVial) and the benzene was evaporated under a stream of
nitrogen at 55'C. Sodium acetateacetic acid buffer (100 y L, pH 3.75,
0.05 M) was added to each vial (ReactiVial), vortexed for 30 s, and 75
yL of the solution was analyzed by HPLC.
Pharmacokinetic studies in dogs. Healthy mongrel male dogs (8) were
used for the pharmacokinetic investigations. Their blood analysis showed
no pathogenic abnormality or presence of microfilaria. The dogs were
23
fasted for at least 1724 h before each study and were given water ad
libitum. The animals were supported by a dog sling in a frame placed on
a laboratory table. The dogs were infused with intravenous saline (35
drops per min) for at least 3 h until drug adminstration, when the
intravenous drip was reduced to 20 drops per min. The animals were
catheterized 3 h before the study with a 30.5cm standard catheter
(Intracath, 16 GA size, Deseret Medical Inc., Sandy, Utah) in the
jugular vein after local anesthesia with mepivacaine hydrochloride
(Carbocaine hydrochloride; Winthrop Laboratories, New York, NY). Second
catheter was also implanted in a foreleg vein (vena brachialis) in most
IV infusion studies. The drug was injected directly into the jugular
catheter, followed by flushing of the catheter with 25 mL of normal
saline. The catheter was connected via a threeway stopcock (Pharmacea,
Toa Alta, PR) to the saline infusion bottle (McGaw Laboratories, Irvine,
CA). Blood samples (16 mL) were collected in heparinized Vacutainer
tubes (Becton Dickinson Vacutainers, Rutherford, NJ) after the dead
volume of the catheter was filled with 5 mL of blood by aspiration with
an extra syringe. These aspirations were carefully aseptically
reinjected into the jugular vein. The heparinized blood samples were
immediately centrifuged at 3000 rpm for 10 min. The plasmas were removed
with sterile glass pipets and were frozen until analysed.
Urine was collected from the dogs through a urinary catheter
(Polyurethane whistle tip units, 6 FR size, McGaw Laboratories) at
intervals of 1560 min for up to 24 h and at longer intervals for up to
1 week. Withdrawal times, volumes and urinary pH values were recorded
and portions of each sample were frozen until analyzed.
Infusion studies were carried out using Harvard Infusion pump
24
(Harvard Apparatus Co., Dover, MA). BuprenorphineHC1 was dissolved in
normal saline (150 mL, concentration^.7378 mg/mL), ultrasónicated for
30 min and infused into the jugular vein at the rate of 0.7026 mL/min
for 162175 min (studies 711). During infusion, blood samples were
collected from the brachialis vein. Postinfusion blood samples were
collected from both jugular and brachialis veins. In dog study 12, the
drug solution was infused into the brachialis vein (using the same drug
concentration and flow rate as above).
Dogs E, F and G underwent surgery.^ A 2% solution of thymalol
sodium was administered IV (6 mL/kg) to each dog and anesthesia was
maintained by halothane. After removal of the gallbladder, a screwcap
was placed on the opposite side of the sphincter of Oddi and the
intestine was sewn to the abdominal wall (Fig 1). At least 1 month was
allowed for recovery from the surgery before the pharmacokinetic study
of buprenorphine in the bilecannulated dogs. These dogs could be
repetitively used for bile cannulation studies by opening the screwcap
and inserting a catheter (Fast Right Heart Cardiovascular catheter, 5 F
size, C.R. Bard Inc., Billerica, MA) into the bile duct. The balloon at
the tip of the above catheter was inflated with 0.81.0 mL of air,
pulled back until the catheter was securely positioned at the inside
wall of the spincter of Oddi. Complete bile collection was effected in
such studies at intervals of 15120 min for up to 26 h.
Isolation of buprenorphine conjugate from bile. Two liquid
chromatographic glass columns (40 X 2.5 cm) were packed with nonionic
Amberlite XAD4 beads (Sigma Chemical Co., St. Louis, Mo.) by passing a
slurry of the packing material in distilled water through the column.
The perforated disk at the bottom end of the column retained the
Figure 1. Schematic of the performed surgery. After gallbladder removal, screwcap
was placed on the opposite side of the sphincter of Oddi and the intestine was
sewn to the abdominal wall. During the bile cannulation, the screwcap was replaced
by a sealed perforated rubber stopper through which a bile catheter was positioned
into the bile duct. At the end of the study, the catheter was removed and the
screwcap was replaced (See also reference 51).
gall bladder^
(removed) f
duodenum
abdominal
wall
27
Amber lite beads. Each column was washed with 500 mL of water followed by
250 mL of methanol. The columns were closed at the bottom and soaked
with distilled water overnight. Pooled bile samples (150 mL) collected
from dog studies 9 and 10 were diluted to 500 mL with distilled water.
Aliquots (250 mL) were passed through each column and washed with 300 mL
of water until the eluent was colorless. Then, 300 mL of methanol was
passed through each column. These methanolic eluates were combined and
completely evaporated to dryness under reduced pressure. The residue was
dissolved in sterile normal saline (120 mL) and the solution was
filtered through a 0.22 pm Millipore filter aided under reduced
pressure and strictly aseptic conditions. The final sterile solution was
infused into dog F (Study #13) at the rate of 14 mL/min for 8.5 min. The
pooled bile samples collected from dog study #11 were similarly except
that chromatographic separation was achieved with only one Amberlite
XAD4 column. The final sterile solution was infused into dog G (Study
#14) at the rate of 14 ml/min for 7 min.
Analysis of the conjugate by enzymatic hydrolysis. The enzyme
8glucuronidase ( 8glucuronide glucuronosohydrolase, 0.76 mg = 660,000
Fishman Units, Lot. #51F9013, Sigma Chemical Co.) was dissolved (50 mg)
in 20 mL acetate buffer (pH 3.8, 0.05 M). Aliquots (500 pL) of the
enzyme preparation and the internal standard (100 pL of compound 2 , 1
ptg/mL) were added to 500 y L of diluted bile sample (1:10,000 dilution
made with distilled water) and the total volume was adjusted to 1.6 mL
with pH 3.8 acetate buffer. The samples were incubated at 37'C for 24 h.
Diluted bile (1:10,000 dilution) samples containing no drug were spiked
with buprenorphine and treated in the same manner to establish an
appropriate calibration curve. The generated aglycone buprenorphine was
28
assayed by HPLC separation and fluorimetric detection.
Catheter binding of buprenorphine. BuprenorphineHCl was dissolved
in normal saline (150 mL, concentration = 0.7576 mg/mL of base). The
solution was passed through the plastic catheter (Intracath) without
back pressure at the rate of 0.7026 mL/min for 1 h (1 study) and 3 h (2
studies). The internal surface of the catheter was washed with 25 mL of
normal saline and dried under a stream of air. Then benzene (250 ml) was
pumped through the plastic catheter (Intracath) at the rate of 14
ml/min, evaporated in a collecting flask at 70*C under reduced pressure.
The residue was reconstituted in acetate buffer (pH 3.75, 0.05 M) and
aliquots were assayed by HPLC separation and fluorimetric detection.
BuprenorphineHCl was dissolved in normal saline (0.7567 mg/mL of
base) and passed through the plastic catheter (Intracath) at the rate of
0.7026 mL/min for 3 h. The interior surface of the catheter was washed
with 25 ni/ of normal saline. The saline was allowed to flow from an
infusion bag under the gravitational force at the rate of 45 drops/min
for 2 h and was collected (300 mL). The pH was adjusted to 9.1 and the
saline solution was extracted twice with 250 mL portions of benzene. The
combined benzene layer was evaporated under reduced pressure at 70‘C.
The residue was reconsituted in acetate buffer (pH 3.75, 0.05 M) and
aliquots were assayed by HPLC separation and fluorimetric detection.
Buprenorphine (0.7567 mg/nL) in normal saline was passed through
the plastic catheter (Intracath) for 1 h at the flow rate of 0.7026
mL/min. At the end of 1 h, the catheter was washed with 25 mL of normal
saline, and saline drip (45 drops/min) was continued. Fresh blank dog
blood (13 mL) was drawn through the catheter at 1, 2, 5, 10, 15, 20,
30, 45, 60, 120 min. The blood samples were centrifuged at 3000 rpm for
29
10 min and the supernatent plasma was analysed for buprenorphine by HPLC
separation and fluorimetric detection. The experiment was repeated
following the pumping of buprenorphine solution (using the same
concentration and flow rate as above) through the catheter for 3 h.
Invivo study. In dog study #12, buprenorphine was infused (0.5236
mg/min for 177 min) into the left brachialis vein through the indwelling
plastic catheter (Intracath). Blood samples during infusion were
collected from the jugular vein and the contralateral brachialis vein.
Upon cessation of infusion, the catheter through which the drug was
infused into the left brachialis vein was washed with 25 mL of normal
saline. Postinfusion blood samples were collected from the left
brachialis vein (site of infusion) through the plastic catheter, as well
as from the jugular and contralateral brachialis veins.
IV BOLUS STUDIES
Chromatographic assays of Buprenorphine and its conjugate. The HPLC
43
assay methods developed for buprenorphine have been published.
Chromatograms of the HPLCassayed buprenorphine are given in Fig. 2. The
acid hydrolyzable conjugate (M) assay (Fig. 3) by fluorimetric detection
(285 nm excitation, slit width 15 nm and 350 nm emission, slit width 10
nm) was equally sensitive, Table 1 shows the relevant statistics of
calibration curves. The standard errors of estimates of the
concentration about its regression on peak height ratio ranged iron +
0.5 to +_ 3 ng/ml.
Some additional linear regressions of concentrations (C, ng/ml) of
buprenorphine in plasma with their standard errors of the parameter
estimates in accordance with
C +_ SER = ( m + sm ) PHR + b + s^ Eq. 1
in the range 550 ng/ml were, C _+ 1.52 ng/ml = (61.19 _+ 1.84) PHR  1.11
+ 0.808, r = 0.9968; C + 1.52 ng/ml = (76.64 + 2.518) PHR  3.04 +
1.068, r = 0.9962; C + 1.3 ng/ml = (54.34 + 2.6) PHR  5.01 + 1.42, r =
0.9943; C + 0.38 ng/ml = (72.21 + 1.8) PHR  12.3 + 0.98, r = 0.9991. In
the buprenorphine concentration range of 50100 ng/ml, C +_ 0.97 ng/ml =
(75.49 + 1.76) PHR  13.45 + 2.1, r = 0.999; C + 2.42 ng/ml = (75.48 +
2.43) PHR  21.6 + 2.71, r = 0.9959; C + 1.78 ng/ml = (67.96 + 2.9) PHR
+ 6.02 + 3.026, r = 0.9964; C + 0.64 ng/ml = (27.94 + 0.4) PHR + 17.1 +
0.85, r = 0.9995.
30
31
Figure 2. Representative chromatograins after assay of buprenorphine (1,
60 ng/ml) with internal standard (2, 100 ng/ml) from plasma (a) and
urine (b). (The blank plasma and urine chromatograms without drug are
given underneath). Chromatogram of mixture of 25 ,yg/ml of
buprenorphine, 1, with its products 2 and 3 after acid degradation in 1
M HC1 for 3 min (c). (See also reference 43).
Figure 3. Representative chromatograms after HPLC separation followed by
fluorimetric detection of buprenorphine conjugate in plasma, urine and bile. Blank
chromatograms of biological fluids without the drug and the internal standard are
given underneath. In the chromatograms (a, b, and c) peak 3 corresponds to the
demethoxy analog of buprenorphine obtained after acid hydrolysis of buprenorphine
conjugate M. Buprenorphine conjugate produces the aglycone on acid hydrolysis
which further quantitatively rearranges into demethoxy analog (peak 3; See also
reference 43). The HPLC retention time for compound (peak) 3 is different from
buprenorphine. (See figure lc). Thus buprenorphine (peak 1) could be used as
internal standard for the conjugate assay, a) Demethoxy analog (3, 90 ng/ml) of
buprenorphine conjugate and internal standard (1, 100 ng/ml) from plasma obtained
after acid hydrolysis of plasma followed by HPLC separation, b) Demethoxy analog
(3, 80 ng/ml) of buprenorphine conjugate and internal standard (1, 100 ng/ml) from
urine obtained after acid hydrolysis, c) Demethoxy analog (3, 60 ng/ml) of
buprenorphine conjugate and internal standard (1, 100 ng/ml) from bile obtained
after acid hydrolysis and HPLC separation. In obtaining chromatograms a, b and c,
the mobile phase was 25:75 acetonitrile:acetate buffer (pH 3.75, 0.05M) plus
0.0004M tetrabutyl ammonium phosphate; flow rate 1.2 ml/min. All other
chromatographic conditions were the same as described under the subheading 'Liquid
Chromatographic procedures' in experimental section.
a
b
min
UJ
Lb
«M
il
i—•—i—i—*—•—i—i—i—r
0 2 4 6 0
min
10
Table 1  Typical statistics of plasma, and urine calibration curves for Buprenorphine (1) and
conjugate (M)
Biological
Fluid
Range
ng/ml
a
sy,x
b
m
c
s
m
bd
(D
i
f
n
r9
Plasma (1)
20100
1.38
58.96
1.19
18.18
2.69
7
0.999
2090
2.83
49.13
2.16
1.21
2.57
8
0.994
2070
0.83
74.48
1.22
5.9
0.84
5
0.999
Urine (1)
2090
1.71
53.65
1.56
4.10
1.74
7
0.998
2090
2.07
52.05
1.88
1.80
2.21
5
0.998
30100
1.84
63.51
1.19
6.21
1.92
7
0.998
Plasma (M)
550
0.65
82.07
1.78
2.94
0.5
6
0.999
50100
1.36
54.57
1.55
15.0
1.71
6
0.998
10100
2.7
58.65
1.50
1.54
1.38
13
0.996
Urine (M)
1080
1.98
117.2
3.3
2.2
1.3
9
0.997
1040
1.51
62.37
3.5
10.8
2.00
7
0.992
40100
2.73
91.29
4.73
32.61
5.42
7
0.993
a Standard error of estimate about regression of concentration (ng/ml) on peak height ratio,
k Slope. c Standard error of slope. d Intercept. e Standard error of intecept. ^ Number of
points. ^ Correlation coefficient. In buprenorphine calibration curves, demethoxy analog of
buprenorphine (compound 2, Scheme I) was used as internal standard. Buprenorphine was used as
internal standard in the assay of conjugate (M). Some additional calibration curve statistics
are given in the text.
35
In urine, examples of regression equations for buprenorphine in the
range 5100 ng/ml were, C _+ 1.59 ng/ml = (68.9 +_ 1.11) PHR  13.93 +_
1.12, r = 0.9991; C + 0.89 ng/ml = (113 + 1.89) PHR  6.8 + 0.851, r =
0.9993; C + 2.02 ng/ml = (66.5 + 1.26) PHR  7.31 + 1.17, r = 0.9977.
Examples of regression equations for buprenorphine conjugate (M) in
plasma in the range 1050 ng/ml were, C + 2 ng/ml = (57.02 _+ 2.13) PHR +
4.9 _+ 1.211, r = 0.9958; range 60100 ng/ml; C +_ 1.12 ng/ml = (39.57 _+
1.247) PHR + 22.2 +_ 1.83, r = 0.9985; range 5100 ng/ml, C +_ 1.4 ng/ml =
(73.8 j+ 0.99) PHR  2.46 +_ 0.8, r = 0.9991; range 1090 ng/ml; C +_ 1.3
ng/ml = (50.14 + 0.8) PHR  1.51 + 0.92, r = 0.9991.
Examples of regression equations for buprenorphine conjugate (M) in
urine in the range 10200 ng/ml were, C +_ 1.8 ng/ml = (82 _+ 0.621) PHR +
0.72 +_ 0.82, r = 0.9997; range 10120 ng/ml, C + 2.6 ng/ml = (92.99 +_
2.4) PHR  27 + 2.5, r = 0.998; C + 1.81 ng/ml = (162 + 2.9) PAR  13.19
+_ 1.5, r = 0.9991, where PAR = peak area ratio; range 10100 ng/ml, C +_
2 ng/ml = (66.5 _+ 1.26) PHR  7.31 +_ 1.17, r = 0.9977. An example of
regression equation for buprenorphine conjugate (M) in bile in the range
10200 ng/ml was: C +_ 6 ng/ml = (102 2.7) PHR  0.53 +_ 2.63, r =
0.9956.
Twice the standard error of estimate of buprenorphine and the
metabolite concentrations (ng/ml) on peak height ratio ranged from 15
ng/ml (Table 1), indicative of the sensitivity of the fluorimetric assay
of buprenorphine and its metabolite in biological fluids.
Plasma Pharmacokinetics and Volumes of Distribution. The plasma
concentrationtime profile of buprenorphine could be fitted to a sum of
three exponentials (Eq. 2, Figs. 49). There may not be an unique linear
sum of three exponentials Cp^ (estimated plasm concentration! that
Figure 4. Semilogarithmic plots of concentrations (as ng/ml of base) of
intravenously administered buprenorphine (1) in plasma against time (min) for the
1.4171 mg/kg dose in 22.85 kg dog A (Study #1, Table 2). In the bottom and middle
figures, the solid line represents the curve obtained by fitting the plasma data
to a sum of three exponentials in accordance with the equation (2):
Cp(ng/ml) = 986.3 e0'6823 t + 491.5 e~°'02627 t
♦ 39.8 e'0100076 1
The middle inset is the continuation of the data and fitted curve for an extended
time scale up to 2000 min. The top inset represents the data fitted to a
4compartment model in accordance with the equation:
Cp (ng/ml) = 1066 e
0.77
t . joo 0.0304
+ 488 e
t ^ .c . 0.00426
+ 45.4 e
t
L „„ 0.0003434 t
+ 22 e
Refer to the section "Validity of the terminal rate constant" for a discussion of
fitting of the data to 4comartment model.
CGNC* NG/ML
Figure 5. Samilogarithmic plots of concentrations (as ng/ml of base) of
intravenously administered buprenorphine (1) in plasma against time (min) for the
1.6369 mg/kg dose in 17.6 kg dog B (Study #2, Table 2). The solid line represents
the curve obtained by fitting the plasma data to a sum of three exponentials in
accordance with the equation (2):
Cp(ng/ml)
= 818.1 e
0.2632 t
435.7 e 0*01731 t
c. 0.000904 t
+ 54.33 e
The inset is a representation of the data and fitted curve for the initial period
of 800 min.
CGNC* NG^ni
m
C3
ro
E
c:
c:
6e
tjnnm
Figure 6. Semi1ogarithmic plots of concentrations (as ng/ml of base) of
intravenously administered buprenorphine (1) in plasma against time (min) for the
1.2023 mg/kg dose 22.5 kg dog C (Study #4, Table 2). The solid line represents the
curve obtained by fitting the plasma data to a sum of three exponentials in
accordance with the equation (2):
Cp(ng/ml)
= 2459 e
0.1173
+ 259 e °*0145 t + 31.37 e“000103
t
The inset is the continuation of data for an extended time scale up to 1500 min.
3RD 5^in 3211 9ÜD
MIN
CONO. NG/ML
— 1 J ¡ :
— □ CD C3
□ □ cd a
Tfr
lunno
Figure 7. Semilogarithmic plot of the concentrations (as ng/ml of base) of
intravenously administered buprenorphine (1) in plasma against time (min) for the
2.5632 mg/kg dose in 19.0 kg dog B (Study #3, Table 2). The solid line represents
the curve obtained by fitting the plasma data to a sum of three exponentials in
accordance with the equation (2):
Cp(ng/ml)
= 3508 e
0.4295 t
+ 1142 e
0.0217
t
+ 31.2
0.000463 t
\]ril¡Z [)\)ZZ IJU91
co
NiW
un 11 uss
^ 1 1 h
m
UUl
UUÍJI
uuun i
CCNC» NG/ML
Figure 8. Semilogarithmic plots of the concentrations (as ng/ml of base) of
intravenously administered buprenorphine (1) in plasma against time (min) for the
1.439 mg/kg dose in 24.2 kg dog C (Study #5, Table 2). The solid line represents
the curve obtained by fitting the plasma data to a sum of three exponentials in
accordance with the equation (2):
Cp(ng/ml)
0.562 t ^ ,10 0.0114 t ^ .c 0.00093 t
4075 e + 318 e + 45 e
The insets are the continuation of the data and the fitted curve for the extended
time scale up tp 3000 min.
280 *120 SGO nOÜ
CGNC» NG/ML
C3
o
g
£
s*
Figure 9. Semilogarithmic plots of the concentrations (as ng/ml of base) of
intravenously administered buprenorphine (1) in plasma ( O > Fluorescence
detection; Q , Electrochemical detection) against time (min) for the 0.7766 mg/kg
dose in dog D (Study #6, Table 2). The solid line represents the curve obtained by
fitting the plasma data to a sum of three exponentials in accordance with the
equation (2):
Cp (ng/ml) = 2519 e
0.2473
t A 0.012 t ^ OD , 0.00158 t
+ 204 e + 38.6 e
The middle inset is the representation of the data and the fitted curve for the
initial 200 min. The top inset is the plot of the weighted residuals calculated in
accordance with the equation (3) against log fitted concentrations. See fig. 10
for details on the residual plots.
.yir
'I [ID 6DD 8[][]
2ÜD
MIN
I □□□
48
best fits the actual data Cp ; instead there may be several
exp
solutions with similar minimum sum of squares. A unique solution can be
obtained only if the drug transferred into other compartments or
transformed into metabolites were analysed in their respective
52 ...
compartments. The plasma data of buprenorphine administered
intravenously to 6 dogs were fitted by nonlinear regression to a sum of
three exponentials (In study #1, Dog A, the data were also fitted to a
53
4compartment model; Fig. 4, top inset). The fitting was effected
54
with the computer program of Yamaoka et al. (See also appendix I),
where 1/Cp, the inverse of the plasma concentration was the weighting
factor (See appendix II for a discussion of different weighting
techniques). The validity of the triexponential equation,
QPcaic = P e“1T + A e~a t + B e" 31 Eq. 2
was confirmed by demonstration that regressions of the various studies
of the weighted residuals
€ ( ^exp ^calc ) / ^calc
Eq. 3
against log Cpca^c gave mean residuals £ , slopes and intercepts,
55
which were not significantly different from zero (Fig. 10, Table 2).
The residuals were randomly distributed above and below the regression
line, indicating no bias in the fitting of the chosen model. (Fig. 10)
Outliers were defined as those experimental concentrations which had
residual values, 6, greater than 2 (Eq. 3) which corresponds to a
greater than 200% deviation from the presumed best fitted value. One
such outlier in dog B at 2.5632 mg/kg (Study #3) dose of buprenorphine
was not included in the nonlinear least square curve fitting technique
Figure 10. Representative examples of the plots of the weighted residuals against
log Cpcalc values. These weighted residuals were obtained from the equation (3):
6 ( ^exp “ ^calc ] 1 ^calc
The calculated plasma values were obtained from the triexponential equation 2
fitted to the plasma data of buprenorphine: a) 1.4171 mg/kg dose study in dog A,
Study #1; b) 1.6369 mg/kg dose study in dog B, Study #2; c) the plasma metabolite
(M) residuals obtained after IV administration of buprenorphine for the 1.2023
mg/kg dose study in dog C, Study #4; d) the plasma metabolite residuals obtained
after IV administration of buprenorphine for the 1.439 mg/kg dose study in dog C,
Study #5.
The observed means, slopes and intercepts of these weighted residuals are
given in Table 2. These parameters were not statistically significantly different
from zero as confirmed by ttest. The random distribution of the residuals above
and below the regression line indicated no bias in the fitting of the chosen
model. The validity of the triexponential curve fitting to the plasma data of the
metabolite is discussed in the text.
¡JESIDUALS RESIDIA
RESIDUALS RESIDUALS
Table 2. Pharmacokinetics of intravenously administered bolus Buprenorphine (1) in dogs.
Parameter
Dog A
Dog B
Dog B
Dog C
Dog C
Dog D
Mean 1 SEM
Study No.
1
2
3
4
5
6
Dog No.
B364
B344
B344
W444
W444
W4123
Dosed , mg
32.38
28.81
48.7
27.051
34.824
20.502
Weight, Kg.
22.85
17.6
19.0
22.5
24.2
26.4
Dose mg/kg
1.4171
1.6369
2.5632
1.2023
1.4390
0.7766
Parameters from plasma data
for 1
lO^Pf b
0.3046
0.2835
0.7204
0.9089
1.1700
1.2290
0.77 1 0.17
10 Af
1.5179
1.5123
2.345
0.959
0.9132
0.9972
1.37 1 0.22
106 Bf
1.2292
1.886
0.6402
1.16
1.2922
1.8827
1.35 1 0.19
10 TT C
6.823
2.362
4.295
1.173
5.616
2.473
3.79 1 0.88
9
(1.02)
(2.93)
(1.614)
(5.91)
(1.23)
(2.8)
(2.58 1 0.74)
10¿ a
2.627
1.731
2.166
1.45
1.14
1.19
1.72 1 0.24
A
(26.4)
(40)
(32)
(47.8)
(60.8)
(58.2)
(44.2 1 5.7)
104 0
7.565
9.041
4.627
10.3
9.248
15.81
9.43 1 1.51
(916)
(767)
(1498)
(673)
(749)
(440)
(841 1 146)
Residual plots
io2 é d
1.33
1.49
3.6
6.1
2.38
2.88
Slope6 ^
0.01510.04
0.00510.04
0.01510.05
0.00410.05
0.02410.05
0.03110.036
Intercept
0.04110.08
0.00510.09
0.06610.106
0.05310.11
0.07310.1
0.02510.068
Clearances
Cl 9
tot
470
324
380
391
416
396
396 1 19
Cl h
ren
2.69(5.1)
5.24(8.2)
0.053(1.6)
0.13(1.23)
1.453(12.6)
1.91 1 0.96
Cl 1
met
467.3
318.76
379.95
390.87
415.5
394.5 1 24
2.72
2.06
1.27
3.47
2.38 1 0.47
464.6
316.7
378.7
387.4
387 1 30
ciM 1
ren
1.94(11.8)
3.6(18.5)
0.93(9.0)
4.4(30)
0.98(9.2)
2.36 1 0.7
Table 2. Continued
Parameter Dog A Dog B Dog B Dog C Dog C Dog D Mean 1 SEM
% Recoveries of buprenorphine and M in urine
Uoo'*' /dosem 0.23
0.52
0.065
UooM /dosem 0.66
0.44
0.33
Volumes of
distribution of buprenorphine
(L)
v n
c
21.2
22
10.1
558
326
637
V P
0.705
0.166
0.339
m
Parameters
iron plasma data for conjugate
(M)
104 P b
0.1071
q
105 A
1.0232
0.9295
r
106 Bf
1.043
1.172
0.888
10 tt C
1.91
o
(3.63)
HT a
2.4
1.69
— —
A
(29)
(41)
— —
104 6
2.41
3.44
6.31
(2871)
(2016)
(1098)
Residual plots.
102 é d
2.2
1.32
Slope6 ^
0.000310.07
0.01310.05
— —
Intercept^
0.02U0.123
0.03810.09
— —
0.09
0.13
0.21 1 0.08
0.79
0.13
0.47 1 0.12
9.84
7.85
7.42
13.1 1 2.73
379
450
251
434 1 59
2.054
0.816 1 0.4
0.6613
0.2087
— —
0.3257 1 0.17
1.3486
0.9620
— —
1.07 1 0.10
1.70
1.344
— —
1.23 1 0.14
6.13
(1.13)
3.66
(19)
22.75
(305)
1.06
(6.5)
0.68
(104)
4.95
(1400)
— —
3.03 1 1.57
(3.76 1 1.56)
2.1 1 0.63
(48 1 19)
4.3 1 0.9
(1846 1 391)r
3.58
0.0410.05
0.110.09
2.9
0.00710.064
0.014310.144
— —
a
Values correspond to buprenorphine base. Administered as HC1 salt, dissolved in 30 ml normal saline.
kpf' Af and values equal to P, A and B values expressed in fractions
A and B values were obtained from the nonlinear least square fitting of
of dose per ml of plasma. The P,
54
plasma data with 1/Cp weighting.
C —1
Unit for tt , a and 3 values is min . Parenthetical values correspond to
cVfean of the weighted residuals. (6 = (Q?eXp  ^Pcapc ) / Cpca ^ ). None
statistically significantly different from zero.
halflives in min.
of the mean residuals were
0 f
' These are the slopes and intercepts of plots of 6 against log fitted cPca]c The parenthetical values
correspond to standard errors. Both slope and intercept were also not significantly different from zero,
indicating that the sum of three exponentials best fits the plasma data of buprenorphine and the conjugate.
^Ratio of the dose to the total area under the plasma concentrationtime curve of buprenorphine, where the
calculated AUC = (P/^ ) + (A/a ) + (B/ 0 ) and that calculated from the trapezoidal rule (plus the quotient of
the last observed plasma level Cp(n) and 3) viere within 57% in all cases except in dog C at 1.439 mg/kg dose
level where the difference was 10%. Unit, ml/min.
^Estimates from the slopes of cumulative amounts of buprenorphine excreted ( zU ugs) renally against the area
under the plasma concentration time curve (AUC ) at that time in accordance with z U = Cl^^ AUC^
These values were calculated from the initial slopes observed (Figs. 2628), and these ratios changed as pH of
the urine changed (Fig. 26). The values in parenthesis are ^U at AUC=0 from the best linear plots of data as
shown in Figs. 2628. The plots of Au/At vs Cpt_m^ (plasma concentration at the mid point of urine
collection interval) were highly scattered in most studies (Fig. 37, see also text).
1C1 , was calculated from the difference between total clearance Cl. , and renal clearance, Cl of
met tot ren
buprenorphine.
^Cl^et ~ Clg >M was calculated using equation 35.
k 1 1—>M
Biliary clearance of M was calculated from the knowledge of Clmgt and (Cl—et  C1B ) values.
1 . M
Renal clearance of metabolite, Clrgn was estimated in accordance with equation 30 (Figs. 3234).
^Percent recoveries of buprenorphine and M in urine obtained from the quotient of the amount recovered in
urine and the total IV bolus dose of buprenorphine.
Volume of distribution of the central compartment (V ) was obtained using equation 10; P, A, and B are the
parameters from the plasma data for buprenorphine.
V, was calculated from Cl, , / r •
d tot p
^Estimated using equation 35.
^Plasma conjugate profile could not be fitted to a sum of 3 exponentials, see Fig. 17. However, the terminal
rate constant was estimated frcm the semilogarithmic plots of the terminal plasma data against time. See also
text.
rOutlier dog C at 1.2023 mg/kg dose was not included in the calculation of average and SEM.
U1
.t.
55
54
(Appendix I). The outlier was included in all other pertinent
pharmacokinetic analyses and plots, such as excretion rate, sigma minus,
and clearance plots. There were no outliers in the other studies.
Since the triexponential equation 1 adequately described the
postintravenous bolus injection data (Figs. 49), a 3compartment body
model was the simplest pharmacokinetic model for the disposition of
buprenorphine in dogs. The elimination of buprenorphine could occur from
a central compartment (C), reversibly connected with shallow (S) and
53
deep (D) peripheral compartments (scheme II):
The equation which describes the time course of the IV bolus
administered drug in the central compartment Cp as a function of time t
53
as per scheme 2 is given by
cp = (X0/Vc) [ [ (k21 —ir ) (k31 TT )/(TT  a ) (TT  B) ] e7r +
[ (k21  a ) ( a k31 ) / (rr — ot ) ( a — 3 ) ] e at +
[(k21  3) (k31 3)/(a3)(TT3)]e_et] Eq. 4
where Xq (k21 ir ) (k31  ir )/Vc(tt  a) ( tt  3 ) = P Eq. 5
X0 (k21  a ) (a k31 ) /Vc ( tt  a ) ( a 3 ) =A Eq. 6
and
XQ (k21  3 ) (k31 — 3 ) /Vc (a —3 ) ( ir — 3 ) = B Eq. 7
56
where X„ = dose, V = volume of distribution of the central
0 c
compartment.
Validity of the terminal rate constant. Proper estimation (Appendix
II) of the terminal rate constant (and halflife) depends upon a)
analytical sensitivity; b) number of terminal plasma concentration
values, the time interval between these values, and the number of
terminal halflives over which the samples were collected; c) selection
of the compartment model; and d) proper weighting of the data. Upon
acute IV bolus administration of buprenorphine in dogs at the 0.72.6
mg/kg doses used, the plasma concentrations of buprenorphine were below
20 ng/ml at 1000 min (See Figs. 49). Thus the available analytical
sensitivity of 5 ng/ml did not permit accurate estimation of the
terminal halflife. For example, at the 2.5632 mg/kg (Study #3) IV bolus
dose of buprenorphine in dog C, the estimated terminal rate constant
obtained from a semilogarithmic plot of the terminal phase plasma data
against time (n=12) was 1.7 X 10 4 (halflife = 4040 min) +_ 0.70 X
4 1
10 (SE) min Thus the range that would include the 95% confidence
limits for this rate constant would be 1.48 X 10 ^ (halflife = 47000
min) to 3.3 X 10 4 (halflife = 2111 min) min 1 (See Table 3).
The terminal halflife significantly depends upon the number of
compartments assumed. Consider dog A (Study #1). Fitting of the data
weighted by the inverse of the concentration to a 3compartment model
4 1
gave a terminal rate constant of 7.6 X 10 min (halflife = 916
min). When the same data were fitted to a 4compartment model, the
terminal rate constant estimated by using the computer program (Appendix
I) was 3.78 X 104 +0.984 X 10“4 (SE) min”1 (halflife = 1840 min,
n=3; The 95% confidence limits; + smt, where t=12.71; 433 min to time
Table 3. Statistics of the calculated terminal rate constants.
Parameter
Dog A
Dog B
Dog B
Dog C
Dog C
Dog D
Study No.
1
2
3
4
5
6
Dog No.
B364
B344
B344
W444
W444
W4123
a
n
10
10
12
13
10
10
Intercept (ng/ml)b 39.8
54.3
31.2
31.0
45.6
34.3
104 8 min 1 C
5.504
8.36
1.715
9.965
8.77
14.2
tl/2 111111
1259
829
4040
695
791
489
104 s of 8 ^
m
0.526
0.965
0.704
1.76
1.0
1.81
95% Confidence
limits for
the terminal
halflife.e
Upper limit
1615
1129
47000
1136
1074
693
Lower limit
1032
654
2111
501
623
378
95% Confidence
limits for total body clearance
(ml/min)
Lower limit
287
247
22
301
329
334
Upper limit
408
362
313
442
457
446
Number of plasma points.
K Q
' Terminal phase intercepts and rate constants were calculated from the regressions of
the logarithm of plasma concentrations against time.
d Standard errors, s values of the terminal rate constants,g , were calculated from the
m # j a
statistics of the regressions of the logarithm of the terminal plasma concentrations
against time (also see reference 59).
0 f
' 95% confidence intervals for the terminal rate constants were calculated from ttable
at a =0.05 level of significance for (n2) degrees of freedom (see also reference 59).
Cl. . was estimated using equations 8 and 9.
58
infinity; Fig. 4, top inset). This large range for the confidence limits
is attributable to the estimation of a terminal rate constant from few
(n=3) plasma values. The estimated terminal rate constants from the
semilogarithmic plots of the terminal plasma cocentrations against time,
their respective standard errors, and 95% confidence limits are given in
Table 3.
The terminal halflife estimated for studies 1,2,4,5 and 6 have
relatively less error compared to study 3 (Table 3). Yet, estimation of
the true terminal halflife depends on the number of terminal plasma
values, the time interval between these values and the number of
terminal halflives over which the samples were collected. The terminal
plasma data in studies 1,2,4,5, and 6 were not representative of the
true terminal phase since the plasma data needed to accurately estimate
the terminal halflife were below the analytical sensitivity and were
not available.
Total body clearance. The total body clearance Cl^t of a dose,
53
XQ, can be calculated from
Cl
tot
 VAUCoo
Eq. 8
where AUC^ = area under the plasma concentrationtime curve up to time
infinity. AUCt up to the last observed plasma point, Cp^ can be
calculated by the trapezoidal rule. The area from the last plasma
sampling time to infinity can be estimated from Cpn / 8 where g is the
terminal rate constant. Also, AUC^ can be explicitly calculated by
integrating equation 2 between time 0 to oo, i.e.,
AUC^ = (P/tt ) + (A/a ) + (B/0 )
Eq. 9
59
The parameters of the above equation were obtained by fitting the
buprenorphine plasma data of dogs 16 to equation 2 using the computer
54
program of Yamaoka et. al. (Appendix I). The values of the parameters
of equation 9 are given in the legends of Figs. 49 or can be calculated
from the normalized values given in Table 2. The calculated percent
contribution of the term B/6 of the terminal area to the total area in
studies 16 were 72, 68, 53, 44, 58 and 47 respectively. This
demonstrates the significance of 6 in the estimation of AUC^ and
consequently the total body clearance derived from this value (Equation
8). Thus, uncertainties in the estimates of 6 can lead to uncertainties
in the estimates of AUC and total body clearance. In the IV bolus
oo
studies (#16), the contributions of the terms P/ir and A/a were only
about half of the total area under the plasma concentration time curve.
When equation 2 was used to fit the plasma data of buprenorphine
54
(Yamaoka et. al., Appendix I), the P, ir , A, anda parameters were
estimated from the data in high range (505000 ng/ml) of plasma
concentrations. These data are held in greater confidence than the
estimates obtained from the low values of the terminal phase. Thus, if
the error in AUC estimation is primarily due to the error in the
oo
estimation of 8 , the 95% confidence limits for AUC and the derived
oo
total body clearance (Equation 8) can be estimated from the terminal
rate constant, 6 , and its standard error.
To estimate the error in AUCqq (Equation 9), the parameters P, it ,
A, anda were obtained by fitting the complete plasma data of
buprenorphine to the triexponential equation 2 (using the computer
54
program of Yamaoka et. al. , Appendix I). However, the parameters B
and 6 were obtained from the regressions of the semilogarithmic plots of
60
the terminal phase plasma data against time. The standard error value,
sm, of the terminal slope was multiplied by the tvalue (obtained from
t table for a=0.025 level of significance, two tailed for (n2) degree
of freedom; where n is the number of terminal plasma points). The
resulting g+_ t.sm permitted the estimation of the the upper and lower
95% confidence limits for AUC calculated in accordance with equation
oo
9. The upper and lower limits for the CltQt were derived from the upper
and lower limits of AUC in accordance with equation 8. These calculated
total body clearances and the respective 95% confidence limits are
reported in Table 3 for the 6 TV bolus studies in the dogs.
Volumes of distribution of buprenorphine. The plasma concentration
53
of a drug in the central compartment at time zero is given by
CpQ = P+A+B = XQ / V, Eq. 10
when an IV bolus is administered into a 3compartment body model. Vc is
the apparent volume of distribution of the central compartment. The
average Vc was 13.1 +_ 2.73 (SEM) L (Table 2). This value exceeds the
56 57
volume of blood (1.8 L) and the extracellular water (4.86.6 L) in
dogs. This indicates rapid sequestration of the drug in the
extracellular space upon bolus administration.
If the clearing organ is in the central compartment (Scheme II),
53
then the clearance from the central compartment, Clc, is given by
Cl = V k.n Eq. 11
c c 10 ^
If the drug is solely eliminated from the body through the central
compartment, the Clc is the total body clearance Cltot at any time
53
during the postdistributive phase in accordance with the equation,
61
Eq. 12
where is the overall apparent volume of distribution of the
equilibrated fluids of the body.
Thus,
Eq. 13
and
Eq. 14
If 3 or AUC have large errors, then the estimates of have large
errors. Thus the calculated distribution volumes in accordance with
equation 13 based on the best computer fit (Appendix I) of the plasma
data to triexponential equation are suspect. However, the calculated
Vj values in accordance with equation 13 (reported in Table 2) averaged
57
434 L, in excess of total body water in dogs (1115 L) and does
indicate a high degree of sequestration by body tissues.
Doseindependent pharmacokinetics of buprenorphine. The
pharmacokinetic parameters of a drug are doseindependent when all
distribution and elimination processes are first order with respect to
compartmental concentrations. The rate constants must be invariant at
highly varying administered doses and there must be no saturable first
pass metabolism. To establish whether or not there is doseindependency,
the drug is administered to the same animal at different doses. If the
plasma levels divided by the respective doses are superimposable, then
dose independency can be postulated.
62
Unfortunately dose independent pharmacokinetics of buprenorphine in
dogs could not be studied at highly varying intravenous bolus (1100
fold) dose levels. The lower limit of detecton (5 ng/ml) of
buprenorphine in plasma necessitated a certain minimal IV bolus dose to
adequately quantify terminal plasma concentrations. The fact that the
doses of buprenorphine in excess of 1.22.6 mg/kg would exhibit
significant side effects demanded an upper limit to the TV bolus dose
that could be administered.
At least two or more of the following toxic effects were observed
during a pharmacokinetic study: Defecation and muscle relaxation,
labored and forceful breathing for about 1 h after bolus dose, profuse
salivation continuing up to 4 h. The side effects observed following
rapid IV bolus injection of buprenorphine could be attributed to the
peak plasma levels (20005000 ng/ml, Figs. 49) reached immediately. All
five dogs exhibited drowsiness throughout the experiment, and the state
of general depression (characterized by lack of food intake, minimal
physical motion, lack of response to stimulus such as clapping of hands
and prolonged sleeping up to 12 h at a stretch) continued up to 15 days
depending upon the dose of buprenorphine. Higher doses produced longer
duration of these side effects.
To minimize the peak plasma concentrations of buprenorphine and the
associated side effects encountered upon TV bolus administration, and
yet to obtain adequate plasma concentration values in the terminal
phase, the higher doses of buprenorphine, 4.69, 3.85 and 3.741 mg/kg
dose in dog B, D and F (Study #7, 8 and 11), respectively, were
administered by constant rate infusion over a period of 3 h. However,
superimposition to validate dose independency is inoperative if the drug
63
is administered by two different modes (such as IV bolus and infusion).
If a relationship can be established between the plasma levels of a drug
administered by IV bolus and by IV infusion, superimposition can be
challenged by the use of the transformed IV infusion data.
Superimposition of this transformed high dose IV infusion data on low
58
dose IV bolus data was effected by the outlined procedure that
follows.
Analysis and transformation of IV infusion data. The postinfusion
data were fitted to a sum of either two (Study #7 and 8) or three (Study
53
#11) exponentials in accordance with the equation,
P' e“ Tr *tT^ + A' ea *tT* + B' e ^ *tT) Eq. 15
where T is the time at which infusion was stopped and t is the time
after initiating the infusion. The first term in the above expression is
set equal to zero when the postinfusion data are fitted to a
2compartment body model. The relationship between P and P' of equations
. 53
2 and 15 respectively is
P = P'T Tr/(le_7T T ) Eq. 16
Similarly, the relationships between A, A' and B, B' are
A = A'T a/(lea T ) Eq. 17
B = B'TS / (le~ BT ) Eq. 18
The calculated P, A, and B values were used to generate the Cp .
values of equation 2. These could be the calculated plasma
concentrations if the same dose was administered by IV bolus. These
estimated Cpca^c concentrations obtained from the infusion studies were
64
divided by the total infused dose (mg/kg) and superimposed on the
experimental values of buprenorphine (divided by the IV bolus dose in
mg/kg) obtained after low dose bolus injection in the same dog. In dog B
at three dose levels (1.64, 2.56, 4.69 mg/kg, Study #2,3 and 7), dog C
at two dose levels (1.2, 1.44 mg/kg, Study #4 and 5), dog D at two dose
levels (0.78 and 3.85 mg/kg, Study #6 and 8) and dog F at two dose
levels (0.754 and 3.741 mg/kg, Study #17 and 11) there were no apparent
dose dependencies as demonstrated by the tests of superimposition
59
(statistically confirmed by nonparametric KruskalWallis test applied
to the dosenormalised plasma concentration data. See Figs. 1114 and
the legends, also refer to Appendix III). The parameters of equations
1518 for TV infusion studies in dogs B, D and F are given in Table 5.
Plasma pharmacokinetics of the derived metabolite. The metabolite
(M) assayed in plasma was the acid hydrolyzable conjugate of
43
buprenorphine (1). This buprenophine conjugate (M) upon acid
hydrolysis presumably generated the aglycone which quantitatively
rearranged to demethoxybuprenorphine (3). Rather than assaying the
buprenorphine conjugate or the aglycone directly, this rearranged
product was assayed by HPLC separation and flúorimetric detection. Other
metabolites such as norbuprenorphine or its conjugates observed in
38 39
man ' were not detectable in dog plasma with the assay sensitivity
of 5 ng/ml. The conjugate concentration in plasma was highest at the
initial sampling time, and decreased at a rate similar to that of the
parent compound (Fig. 15). The metabolite profile in 4 IV bolus studies
could be fitted by a triexponential equation (Eq. 2). The fitting was
effected by nonlinear least square regression by using the computer
54
program (Appendix I) where the metabolite concentrations in plasma
Figure 11. Semilogarithmic plots of the concentrations of buprenorphine (1) in
plasma divided by the dose in mg/kg (cone./dose) plotted against time (min) for
the 1.6369 mg/kg (Study #2, O )> 2.5632 mg/kg (Study #3, □ ), and 4.69 mg/kg
(Study #7, , on the presumption of IV bolus administration) doses of
buprenorphine in dog B. The middle inset is the plot of the data for the first 200
min after administration, and the top inset is the continuation of the data on an
extended time scale. The data for the highest dose, 4.69 mg/kg (Study #7, (\>) was
derived from the IV infusion study in which buprenorphine was infused at the rate
of 0.5058 mg/min for 165 min. The superimposition of the infusion data on the IV
bolus data was effected by the procedure described in this chapter under the
subheading "Doseindependent pharmacokinetics of buprenorphine". The points ((\,)
for the infusion study were calculated on the premise of IV bolus administration
of 4.69 mg/kg dose of buprenorphine in accordance with the equation 2. The
parameters of equation 2 were obtained through equations 1518. The nonparametric
rank sum test (KruskalWallis test, Appendix III; see also reference 59) was used
to test the hypothesis (H') that the dosenormalised plasma concentrations at the
above three dose levels were drawn from identical distributions. The critical
value of chisquare with a =0.05 and df=2 is 5.99. The observed H' (7) is less
than 5.99. Therefore it can be concluded that there is no difference among the
three groups.
2nn 'ina 6Uti 8ua man
MIN
CONC./DOSE
ng/ml (mg/kg)~^
— □ cz
— a □ □
o □ o a
99
Figure 12. Sard logarithmic plots of the concentrations (ng/ml) of buprenorphine in
plasma plotted against time (min) for the 0.7766 mg/kg (Study #6 O) dose in dog
D. The 3.847 mg/kg dose (Study #8 Q) was administered by IV infusion over a
period of 171 min. Infusion rate of buprenorphine as base = 0.5084 mg/min. The
superimposition of infusion data on IV bolus data was effected by the procedure
described in this chapter under the subheading "Doseindependent pharmacokinetics
of buprenorphine". The points (□) represent quotient of concentrations divided by
dose that were calculated on the premise of IV bolus administration of 3.847 mg/kg
dose of buprenorphine in accordance with equation 2. The parameters of equation 2
were obtained through equations 1518. These calculated points were multiplited by
0.2019 = 0.7766/3.847 to challenge superimposition. The nonparametric rank sum
test (KruskalWallis test, Appendix III; see also reference 59) was used to test
the hypothesis (H') that the dosenormalised plasma concentrations at the above
two dose levels were drawn from identical distributions. The critical value of
chisquare with a =0.05 and df=l is 3.84. The observed H' (=0.37) is less than
3.84. Therefore it can be concluded that there is no difference among the two
groups.
I fj[jf][] Í
68
□ o
□ o
□ o
□o
□ o
□0
□o
□ o
□ o
□o
□ o
CD
CD
CD
□O
□o
CO
CD
CD
O
co
rsi
Figure 13. Semilogarithmic plots of the concentrations of buprenorphine (1) in
plasma divided by the dose (mg/kg) plotted against time (min) for the 1.2023 mg/kg
(Study #4, O) and 1.439 mg/kg (Study #5,0) dose of buprenorphine dog C. The
inset is the continuation of data on an extended time scale up to 1500 min. The
nonparametric rank sum test (KruskalWallis test, Appendix III; see also reference
59) was used to test the hypothesis (H') that the dosenormalised plasma
concentrations at the above two dose levels were drawn from identical
distributions. The critical value of chisquare with a =0.05 and df=l is 3.84. The
observed H' (=0.78) is less than 3.84. Therefore it can be concluded that there is
no difference among these two groups.
NI W
ÜD8 Ut>9 U8l> UZZ U9I
CONC./DOSE
Figure 14. Semilogarithmic plots of the concentrations (ng/ml) of buprenorphine
(1) in plasma plotted against time (min) for the 0.7542 mg/kg
(O) IV bolus dose of buprenorphine in the bile cannulated dog F (morphine
buprenorphine interaction study, #17). The 3.7408 mg/kg dose (Study #11, □ ) was
administered by IV infusion over a period of 162 min. Infusion rate of
buprenorphine as base = 0.5588 mg/min. The superimposition of infusion data on IV
bolus data was effected by the prodedure described in this chapter under the
subheading "Doseindependent pharmacokinetics of buprenorphine". The points (□)
represent quotient of the concentrations divided by dose that were calculated on
the premise of IV bolus administration of 3.7408 mg/kg dose of buprenorphine in
accordance with the equation 2. The parameters of the equation 2 were obtained
through equations 1518. These calculated points were multiplied by 0.2016 =
0.7542/3.7408 to challenge superimposition. The nonparametric rank sum test
(KruskalWallis test, Appendix III; see also reference 59) was used to test the
hypothesis (H') that the dosenormalised plasma concentrations at the above two
dose levels were drawn from identical distributions. The critical value of
chisquare with a =0.05 and df=l is 3.84. The observed H' (=0.053) is less than
3.84. Therefore it can be concluded that there is no difference among the two
groups.
n^/mi uu^/kg) J
MIN
2I6Ü
288G
3GGD
Figure 15. Semilogarithmic plots of the concentrations of buprenorphine, 1,
(O) and metabolite, M, (□) in plasma plotted against tme for a) 1.4171 mg/kg
dose of buprenorphine in dog A, Study #1; b) 1.6369 mg/kg dose of buprenorphine in
dog B, Study #2; c) 1.2023 mg/kg dose of buprenorphine in dog C, Study #4; d)
1.439 mg/kg dose of buprenorphine in dog C, Study #5. The solid lines represent
curves fitted to the plasma data of buprenorphine and M in accordance with
equation 2.
nun i
CONC. NS'ttL CONC. KG/ML
NJW
OI J6 n^L. IJl5 [J9E 1)91
8
y;
o
£
\
NIM
ODLi 095 OZt' 092 OH
75
were weighted by their inverse. The validity of triexponential equation
2 was confirmed by demonstration that the regressions in various studies
(Fig. 10) of the weighted residuals € (Eq. 3) against log Mp^^
(estimated metabolite concentations) gave mean residuals £ , slopes
and intercepts, all of which were not statistically significantly
different from zero (Table 2). The plasma concentrationtime profiles of
metabolite in the IV bolus studies (15) are given in Figs. 1620.
Maximum plasma concentration of the metabolite was observed at the
initial sampling time (about 1 min). Continued sampling gave
monotonically declining metabolite concentrations similar to the decay
of the parent compound. The parallel decays of buprenorphine and its
conjugate concentrations (Fig. 15) in plasma during the initial
distributive phase indicate that the rate determining step in the plasma
decay of the conjugate was its formation. During the terminal
elimination phase, the rate determining step in the plasma decay of
buprenorphine and its conjugate was the slow return of buprenorphine
from deep tissues to the central compartment where it could be
metabolized. This is the classical 'flipflop' pharmacokinetics for the
. 53
conjugate.
Figure 16. Semilogarithmic plots of the plasma concentrations of metabolite (M)
against time (min) for the 1.4171 mg/kg IV bolus dose of buprenorphine in 22.85 kg
dog A, Study #1. The solid line represents the curve obtained by fitting the
plasma data to a sum of two exponentials in accordance with the equation:
Cp(ng/ml) = 331 e0*024 + 33.8 e_0*000241 t
The inset is the continuation of the data for the extended time scale up to 3000
min.
MIN
Figure 17. Semilogarithmic plots of the plasma concentrations (ng/ml) of
metabolite (M) against time (min) for the 1.6369 mg/kg IV bolus dose of
buprenorphine in 17.6 kg dog B, Study #2. The solid line represents the curve
obtained by fitting the plasma data of M to a sum of three exponentials in
accordance with equation (2):
Cp (ng/ml) = 308 e 0,191 t + 267.7 e_0,0169 t
+ 33.8 e
0.000344 t
The inset is the representation of the data and the fitted curve for the initial
period of 750 min.
NI W
ÍJfJ'JC UUP? ÍJU9I !J!J? I ÍJÍJ9
1U/SN '9N03
Figure 18. Semilogarithmic plots of the plasma concentrations (ng/ml) of
metabolite (M) against time (min) for the 1.2023 mg/kg IV bolus dose of
buprenorphine (1) in 22.5 kg dog C, Study #4. The solid line represents the curve
obtained by fitting the plasma data of M to a sum of three exponentials in
accordance with the equation (2):
~ nQ1 0.602 t 0.0346 t Q 0.00191 t
Cp (ng/ml) = 1781 e + 361 e + 40.8 e
The inset is a continuation of the data and the fitted curve for an extended time
scale up tp 1500 min. The terminal halflife was estimated to be 363 min. This
value had much error due to limited analytical sensitivity, low dose and lack of
sufficient number of terminal phase plasma points.
cosía. NG'Hi
Figure 19. Semilogarithmic plots of the plasma concentrations (ng/ml) of
metabolite (M) in against time (min) for the 2.5623 mg/kg IV bolus dose of
buprenorphine (1) in the 19.0 kg dog B, Study #3. The inset represents data and
the straight line fitted to the terminal phase in accordance with the
monoexponential equation,
Cp (ng/ml) = 43.25 e
0.000631
t
CCNC, NG'ML
MIN
Figure 20. Semilogarithmic plots of the plasma concentrations (ng/ml) of
metabolite (M) against time (min) for the 1.439 mg/kg dose of buprenorphine in
24.2 kg dog C, Study #5. The solid line represents the curve obtained by fitting
the plasma data of M to a sum of three exponentials in accordance with the
equation (2):
_ , . 0.106 t A ,oc 0.0068 t Mr q 0.000495 t
Cp (ng/ml) = 727 e + 335 e + 46.8 e
The inset is a continuation of the data and the fitted curve on an extended time
scale up tp 2750 min.
1W/SN 'ONOO
MIN
URINARY EXCRETION OF BUPRENORPHINE
Sigma minus plots. If it can assumed that buprenorphine is solely
eliminated from the central compartment, the urinary excretion rate of
intact drug can be defined as
dU/dt = ku Xc Eq. 19
where is the urinary excretion rate constant, and Xc is the amount
of drug in the central compartment at time t. Integrating equation 19
. 53
between 0 to U (time; 0 to t) results in
I U E U = P"e~ 1Tt + A"eat + B"e“ Eq. 20
oo
vhere
= P k V / ir
Eq.
21
u c
= A k V / a
Eq.
22
u c
= B k V / 6
u c
Eq.
23
The values of P, A and B are same as in Eqs. 5,6 and 7, respectively if
constant renal clearance is presumed. Thus a plot of the logarithm of
the amount of unchanged drug remaining to be excreted versus time (sigma
minus plot) gives a straight line with a terminal slope equal to
 g/2.303, i.e., the same terminal slope obtained from a semilogarithmic
plot of plasma concentration (Cp) versus time.
Representative examples of the sigma minus plots for the urinary
excretion of buprenorphine are given in Fig. 21. For dog A (Study #1,
86
Figure 21. Sard logarithmic plots of the amounts of the unchanged buprenorpine (1)
remaining to be excreted in urine versus time (sigma minus plot) in accordance
with equation 20. a) Semilogarithmic fitting of the initial urine data up to 100
min for the 1.4171 mg/kg IV bolus dose of buprenorphine in dog A, Study #1. An
apparent rate constant of 0.0176/min (halflife = 39 min) was obtained, b) Fitting
of the sigma minus plot of buprenorphine in urine of dog B, Study #2 at 1.6369
mg/kg dose of buprenorphine to a sum of two exponentials. The estimated hybrid
rate constants were 0.026/min (halflife = 27 min) and 0.00176/min (halflife =
390 min), c) Sigma minus plot of urinary excretion of buprenorphine for the 1.2023
mg/kg dose of buprenorphine in dog C, Study #4. The apparent terminal phase rate
constant for the monoexponential fitting was 0.0017/min (halflife = 400 min), d)
The fitted sigma minus plot of buprenorphine in urine of dog C at 1.439 mg/kg IV
bolus dose of buprenorphine, Study #5, to a sum of two exponentials. The estimated
apparent rate constants were 0.00695/min (halflife = 100 min) and 0.000287/min
(halflife = 2412 min) respectively.
Gm* Gun i win 'inn non 121111 igihi 2111111
mih mu
88
tim
89
Fig. 21a), semi logarithmic fitting of the initial data was linear only
up to 100 min. The estimated apparent rate constant was 0.0176 min ^
(halflife = 39 min). This corresponded to the second distributional
halflife (26 min) obtained for buprenorphine from the plasma data
(Table 2). The sigma minus plot of buprenorphine in urine of dog B (Fig.
21b) at 1.6369 mg/kg (Study #2) dose showed curvature, and could be
fitted to a sum of two exponentials. The resulting hybrid rate constants
were 0.026 (halflife = 27 min) and 0.00176 (halflife = 390 min) min 1
The first halflife corresponded to the second distributional halflife
of buprenorphine (39 min, Table 2) for this dog. For dog C (Fig. 21c) at
1.2023 mg/kg dose (Study #4, Table 2), the sigma minus plot of urinary
data gave an apparent terminal phase rate constant of 0.0017 min ^
(halflife = 400 min). This corresponded with the terminal halflife of
buprenorphine (673 min) obtained from the plasma data. For the same dog
at 1.439 mg/kg dose (Study #5), sigma minus plot (Fig. 21d) showed
curvature, and could be fitted to a sum of two exponentials, and the
respective apparent rate constants were 0.00695 (halflife = 100 min)
and 0.000287 (halflife = 2412 min) min The first halflife obtained
from the urine data corresponded with the second distributional
halflife (61 min, Table 2) obtained for buprenorphine from plasma data.
The sigma minus plots for the urinary excretion of buprenorphine in
other dogs showed great scattering and reasonable estimates of the
apparent rate constants were not possible.
The halflives obtained from the various sigma minus plots shown in
Fig. 21 for the urinary excretion of buprenorphine reasonably
approximated the first and second distributional halflives of
buprenorphine in plasma. Since a minor fraction of the the dose was
90
excreted unchanged in urine (<1%) and the limit of detection of
buprenorphine was 5 ng/ml, the terminal halflife of buprenorphine in
dogs could not be readily estimated from the urinary data.
Sigma minus plots for the conjugates (M) are given in Figs. 22,23.
For dog A (Study #1), the curve could be unexpectedly and for no obvious
reason, fitted best by a simple linear equation to indicate a constant
rate of renal elimination even with decreasing plasma concentrations of
the conjugate. The excretion rate was approximately 330 ng/min,
independent of concentration of metabolite in the central compartment
(Fig. 22a). For dog B at 1.6369 mg/kg dose (Study #2) of buprenorphine
(Table 2), the sigma minus plot gave an apparent rate constant of 0.0032
min ^ (halflife = 215 min, Fig. 22b). For dog C at 1.2023 mg/kg dose
(Study #4), (Fig. 22c) an apparent rate constant of 0.0022 min 1
(halflife = 312 min) was obtained, which corresponded well with the
terminal phase halflife obtained for M from plasma data (305 min, Table
2). For dog B at 2.5632 mg/kg dose (Study #3), the sigma minus plot
(Fig. 22d) showed curvature and could be fitted to a sum of two
exponentials, and the apparent rate constants were 0.023 min ^
(halflife = 30 min) and 0.000567 min ^ (halflife = 1220 min)
respectively, where the second halflife corresponded with the terminal
halflife obtained from the plasma data of M in this dog (Table 2,
halflife = 1098 min). The sigma minus plot for M in dog C at 1.439
mg/kg dose (Study #5) showed curvature (Fig. 23) and could be fitted to
a sum of two exponentials, the apparent rate constants being 0.018 min
1 (halflife = 39 min) and 0.000812 min 1 (halflife = 853 min).
In dog C at 1.2023 mg/kg (Study #4) IV bolus dose of buprenorphine,
the terminal halflife of buprenorphine was estimated as 673 min (Table
Figure 22. Semi1ogarithmic plots of the amounts of metabolite (M) remaining to be
excreted in urine versus time (sigma minus plot) following IV bolus administration
of buprenorphine (1) in accordance with equation 20. a) The excretion data of M in
urine up to 700 min could be fitted to a simple linear equation in dog A, Study #1
at 1.4171 mg/kg dose of buprenorphine. The excretion rate was estimated to be 330
ng/min. b) Sigma minus plot of M in urine of dog B, Study #2 following 1.6369
mg/kg dose of buprenorphine, resulting in a estimated apparent rate constant
0.0032/min (halflife = 215 min), c) Sigma minus plot of M in urine for the 1.2023
mg/kg dose of buprenorphine in dog C, Study #4. The apparent rate constant was
estimated tas 0.0022/min (halflife = 312 min), d) Sigma minus plot of the urinary
excretion of M for the 2.5632 mg/kg IV bolus dose of buprenorphine in dog B, Study
#3. The datata were fitted to a sum of two exponentials in accordance with
equation 20. The estimate apparent rate constants were 0.023/min (halflige = 30
min) and 0.000567/min (halflife = 1220 min) respectively.
HIM
UII2I Il'lie \)Zr,Z 11091 1)19
cm N1W
cri niia tii9 iioi u?c 1191
¡at ,92 *92 ssrf 92 *92
o
•©
O'.
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o.
o.
o
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00I
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ssrf 92 *92 ssrt" '"nZ
Figure 23. Semi 1 ogarithmic plot of the amount of the metabolite (M) remaining to
be excreted versus time (sigma minus plot) following 1.439 mg/kg dose of
buprenorphine (1) in dog C, Study #5. The data was fitted to a sum of two
exponentials. The estimated apparent rate constants were 0.0018/min (halflife =
39 min) and 0.000812/min (halflife = 853 min) respectively.
94
SSff HI °°r\2
4l\[\ 6[IL¡ ! 2f¡fi * Gtiíl 2[¡fiti
MTN
95
2). The plasm data and sigma minus plot of M in this dog gave an
apparent terminal halflives of 305 (Fig. 18) and 312 (Fig 22c)
respectively. This metabolite halflife is therefore, an apparent
distributional halflife and not the terminal halflife.
Urinary excretion rate plots. Since xc = vc Cp in equation 19,
substituting the value of Cp from equation 2 into equation 19,
Tviii ift ^ ,iii at ± niii 8 t
dU/dt =P e + A e +B e
where P111 = P k V
u c
in , , T7
A = A k V
u c
B111 = B k V
u c
Eq. 24
Eq. 25
Eq. 26
Eq. 27
A semilogarithmic plot of excretion rate of unmetabolized drug
versus time according to equation 24 would yield a triexponential curve.
As with the semilogarithmic plasm concentrationtime plot, the terminal
exponential phase rate constant can be obtained from the terminal slope,
 3/2.303, and B111 is the extrapolated intercept of the terminal
linear phase to time zero. Similar to the treatment of the plasm data
with multicompartment characteristics, the method of residuals could be
used to obtain the parameters of the distributional phases of equation
24.
Semilogarithmic plots of A U/ At (approximations of the
instantaneous excretory rate, dU/dt), finite amounts (A U) of either 1
or M excreted during a finite time interval ( a t) against tmid
(midpoint of the collection interval) were highly scattered in most of
the studies. Thus only the data for dog A (Study #1) and B (Study #2)
are reported here. In dog A (Study #1), urine data of buprenorphine up
96
to 200 min could be fitted to log A U/ At = (—k.' /2.303) tmid +
intercept, and the apparent rate constant (k1) 0.0394 min ^ (halflife
= 18 min) corresponded with the plasma second distributional halflife
of 26 min (Table 2, Fig. 24a). In dog B at 1.6369 mg/kg dose (Study #2),
the excretion rate plot for M (Fig. 24b) gave an apparent rate constant
0.0046 min 1 (halflife = 150 min). In the same dog at this dose level,
the excretion rate plot of buprenorphine could be fitted into two
separate linear segments (Fig. 25), from which the two apparent rate
constants obtained were, 0.019 min ^ (halflife = 37 min), and 0.0011
min 1 (halflife = 622 min). Both these halflives corresponded with
the halflives obtained from the plasma data (Table 2, 39 and 654 min).
Clearances of Buprenorphine (1) and Conjugate (M)
Renal clearance of buprenorphine. Upon rearrangement of equation
19,
dU/dt = ku Vc Cp Eq. 28
Integrating between 0 to U (time; 0 to t),
t
£U = kV f Cp dt = Cl AUC. Eq. 29
u c qJ r ren t ^
where AUC^ is the area under the plasma concentrationtime curve. The
renal clearance of buprenorphine (Cl^en ) estimated from the slopes
1 1
of cumulative amounts excreted in urine EU against AUCfc averaged
1.91 _+ 0.96 (SEM) ml/min (Table 2), which indicate high protein binding
if unbound drug excreted solely by glomerular filtration.
These clearance plots (Figs. 2628) frequently did not go through
the origin and could be best characterized by one or more straight lines
conforming to the equation,
97
O
•3
Figure 24. Será logarithmic plots of the amounts (y g) of a)
buprenorphine (1) and b) metabolite (M) excreted in urine per min
( yg/min) plotted against tmid, the mid point of the urine collection
interval in accordance with the equation:
log AU/ At = (~k'/2.303) tmid + intercept
For the 1.4171 mg/kg IV bolus dose of buprenorphine in dog A (Study #1,
Table 2), the plot in accordance with the above equation for 1 gave an
apparent rate constant 0.0394/min (halflife = 18 min). For the 1.6369
mg/kg IV bolus dose of buprenorphine in dog B, Study #2, the plot in
accordance with the above equation for M gave an apparent rate constant
of 0.0046/min (halflife = 150 min).
Figure 25. Semi logarithmic plots of the amounts ( y g) of buprenorphine (1)
excreted in urine per min ( yg/min) plotted against tmid, the mid point of the
urine collection interval for the 1.6369 mg/kg IV bolus dose of buprenorphine in
dog B, Study #2, in accordance with the equation:
log AU/A t = (k'/2.303) tmid + intercept
The insets represent the data fitted into two separate linear segments as per the
above equation. The estimated rate constants were 0.019/min (a, halflife = 37
min) and 0.0011/min (b, halflife = 622 min). Note the correspondence of these
rate constants with those reported in fig. 5 for the plasma data of this dog
(Study #2, Table 2).
Dü/DT pG/MIN
lü
O
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lo o
j
,um
o
o
%
lürp
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'''O
I = :
.ül 1 ° o oO o
O O
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a
►—
o
u
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O''.
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43
H 1 1 1 1 1 h
o
I I
0(1
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T MID (MIN)
240
300
O
o
H 1 1 1 1 1 1 H 1 1
stin
! üütl lrof[]
T MID CÍ1IN)
2[lüü
2bN[]
Figure 26. Plot of the cumulative amounts ( yg) of buprenorphine (1) excreted in
urine against the area under the plasma concentrationtime curve (AUC ,
yg.min/ml) in accordance with the equation (30):
E U = Cl ( AUC.  AUCn )
ren ' t 0
where AUC^ is the area under the plasma concentrationtime curve at E U = 0. For
the 1.4171 rrq/kg IV bolus dose of buprenorphine in dog A, Study #1, the curve
shows three distinctly linear segments attributatble to the pH effect of urine.
The slope (Clren) of the initial linear segment for the pH range between 5.25.8
was estimated to be 2.69 ml/min. See also next figure.
ru juGS
i rjfi r
4
4
4l\
Gfl
IÜ
3ti
ftUCT JJG X MIN / ML
101
Figure 27. Plots of the cumulative amounts (E U, p g) of buprenorphine (1)
excreted in urine against area under the plasma concentrationtime curve at the
time of urine collection in accordance with the equation (30):
Z U = Cl [ AUC  AUCa ]
ren t 0
where AUCq is the area under the plasma concentration time curve at i U = 0. a)
For the 1.4171 mg/kg IV bolus dose of buprenorphine in dog A, Study #1, the
initial slope was estimated to be 2.69 ml/min. b) For the 1.6369 mg/kg IV bolus
dose of buprenorphine in dog B, Study #2, the initial slope ( Clren ) was
estimated as 5.24 ml/min. c) For the 1.2023 IV bolus dose of buprenorphine in dog
C, Study #4, the estimated renal clearance was 0.13 ml/min for the initial time
period up to 243 min. d) In the same dog at this dose level, estimated clearance
was 1.0 ml/min for the urine collection time between 2431270 min. In this dog,
the pH of the urine ranged between 66.5 during the initial 243 min time period;
the pH range was 5.45.7 for the time period between 243881 min.
¡rf n2 ssrr ro
35 
63.50 .
/ "
r,n
,,'Ó
52.50..
a O''
o , 
<15
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33.50
V
\
\
^ 30 
22. MI
Ib
3. MI
1 1— 1 1 f——1 1 1 1 1
5 IQ 15 211 25
DUCT JUG X MIN / ML
o r
3.MI .
x *
3.1(1 _
c °
3.10
O''
6.00 .
.''O
Kfi
^ 6/jU
^cr' °
w 6,20
5.MI 
,''ó
5.60
O
5.30
1 1 1 1 1 1 1 1 1 1
3<1 30 <12 16 50
0UC1 ¿JG X MIN / ML
ru jugs ¿igs
103
Figure 28. Plots of the cumulative amounts ( x U, p g) of buprenorphine (1)
excreted in urine against the area under the plasma concentrationtime curve (AUC
t, pg.min/ml) at the time of urine collection in accordance with the equation
(30):
X U = Cl
ren
[ AUCt  AUCq ]
where AUCq is the area under the plasma concentrationtime curve at x U = o. a)
For the 2.5632 mg/kg IV bolus dose of buprenorphine in dog B, Study #3, the
estimated renal clearance was 0.053 ml/min for the initial time period up to 271
min. b) Same as in (a), except the points up to 60 min were not included in the
regression (time; 90363 min). Estimated renal clearance was 0.03 ml/min. This low
renal clearance could be attributable to the cessation of urinary excretion of
buprenorphine during the time interval between 180240 min. c) In the same dog,
the estimated renal clearance was 0.86 ml/min for the latter time period between
12195650 min. d) For the 1.439 mg/kg IV bolus dose of buprenorphine in dog C,
Study #5, the estimated renal clearance was 0.51 ml/min for the initial time
period between 751164 min. In the same dog, regression according to the above
equation (30) gave the slope = Clren = 1.453 ml/min for the initial time period
up to 75 min. (Intercept = 12.6, r=0.997, see also Table 2).
ssrr ni ssrr m
2.20
2.14 .
2.080 .
2.020
1.96
^ 1.90
W 1.09
1.98
1.92
1.86
38
39.211
32.9(1
30.6(1
28.8(1
29
^ 28.2(1
23.9(1
21.60
19.BO
O
O'
O
O'OO
O,'
O'
H 1 1 1 1
60 68 90
ftUCT JJG X MIN / ML
98
)'Q
oo.
J3
,o
O
,'o
*
O
o
o'
9 1 h
28
H 1 1 1 h
36 49 82
ftUCT JIG X MIN / Ml.
6(1
105
106
Z u = Clren ( AUCt  AUCq ) Eq. 30
where AUCg is the area under the plasma concentration time curve at
EU=0 as estimated from the extrapolation of the best linear plots of U
versus AUC (Table 2). This nonzero intercept could be attributed to one
of several factors. When the urinary clearance (obtained from the
quotient of A U/ At against Cp^. where latter is the plasma
concentration at the mid point of successive urine collections) was
linearly related to urinary pH in dog A (Study #1, Fig. 29). Renal
clearance was low at high urine pH. If it can be hypothesized that the
43
uncharged buprenorphine in urine at high pH values (pKa' = 8.24) can
undergo renal tubular reabsorption, then lower clearances should be
observed at high urine pH values. The possible renal mechnism could be
pH dependent tubular reabsorption of neutal species and increased
excretion of ionized species at lower pH values. This is supported by
the fact that in dog B at 2.56 mg/kg TV bolus dose of buprenorphine
(Study #3), no drug was observed in the urine for a pH above 7.5 (Fig.
29) to indicate complete reabsorption of buprenorphine at these higher
pH values. However, since such a small fraction of unchanged
buprenorphine was excreted in urine, metabolic acidification of urine is
not a valid measure of counteracting narcotic toxicity due to accidental
overdosage.
45
It has been previously shown that morphine exhibits
dosedependent pharmacodynamic effects on renal processes. Decreased
renal excretory rates and reductions in urinary flow rate were observed
at high morphine doses. The urine flow was considerably less during the
Figure 29. Plots of Clren (ml/min) against urinary pH. The renal clearance was
calculated from the quotient of the urinary excretion rate ( aU/a t,
y g/min and the plasma concentration at the mid point of urine collection interval
(Cpj. . a) For the 1.4171 mg/kg TV bolus dose of buprenorphine in dog A,
Study #1, (r=0.971). b) For the 1.6369 mg/kg IV bolus dose of buprenorphine in dog
B, Study #2 (r=0.873). c) For the 2.5632 mg/kg IV bolus dose of buprenorphine in
dog B, study #3. Note the absence of renal clearance above pH 7.5. See also text.
CL KEN ML/MIN
N> IS)
801
Figure 30. Plots of urine flow (ml/min) against tmid, the mid point of
urine collection interval, a) For the 1.4171 mg/kg IV bolus dose of
buprenorphine (1) in dog A, Study #1. b) For the 1.6369 mg/kg, Stduy #2,
(O) and 2.5632 mg/kg, Study #3, (□) IV bolus doses respectively of
buprenorphine in dog B. Note the dose dependent decrease in urine flow
rate, c) For the 1.2023 mg/kg IV bolus dose of buprenorphine in dog C,
Study #4.
<*80 64Q
T MID (MIN)
URINE FLOU Ml/MIN
URINE FLCU M/MIN
2.52
Ill
first few hours after the administration of buprenorphine (Fig. 30),
which also indicated dosedependent renal elimination (Fig. 30b). This
could possibly explain the negative intercepts observed in plots in
accordance with equation 30. However, such a small fraction (<1% of the
dose) is excreted in urine, this would have no pronounced effect in the
overall doseindependent pharmacokinetics. No dependence of clearance on
urine flow rate was observed for buprenorphine (Fig. 31).
Renal clearance of the metabolite. Renal clearance of the plasma
metabolite obtained from plots (Figs. 3234) in accordance with equation
30 averaged 9.2 _+ 2.7 (SEM) ml/min (Table 2). This was higher than the
renal clearance of buprenorphine (1.72 ml/min).
Similar to buprenorhine, the plots (Figs. 3234) according to
equation 30 showed large negative intercepts. This could not be
explained as there was no pH or urine flow dependent clearance (Figs.
35,36) observed for the metabolite.
Renal clearances obtained for buprenorphine and the metabolite from
the plots of aU/a t versus cPt_mj_d were highly scattered. Some plots
(with minimum scattering) are shown in Fig. 37.
Metabolic clearance of buprenorphine. Since doseindependent
pharmacokinetics of buprenorphine in the dose range studied could not be
denied, it can be postulated that the metabolism is first order and that
the rates are dependent only on the concentrations of buprenorphine in
the central compartment. Thus the rate of formation of the total
metabolite M
dM./dt = k V Cp Eq. 31
t m c ^ ^
where dM,/dt is the rate of formation of total metabolite, k V is
t. me
Figure 31. Plots of renal clearance (Clren/ ml/min) against urine flow rate
(ml/min). The renal clearances were calculated from the quotient of the urinary
excretion rate ( aU/ At) and the plasma concentration at the mid point of urine
collection interval (Cpt_m^d). The slopes and the respective standard errors are
as follows: a) For the 1.4171 mg/kg dose of buprenorphine in dog A, Study #1,
Slope = 0.19 _+ 0.32; b) For the 1.6369 mg/kg dose of buprenorphine in dog B,
Study #2; Slope = 0.6 +_ 0.8; c) For the 1.2023 mg/kg dose of buprenorphine in dog
C, Study #4; Slope = 0.42 +_ 0.22; d) For the 1.439 mg/kg dose of buprenorphine in
dog C, Study #5; Slope = 0.24 +_ 0.3. These slopes were not statistically
significantly different from zero as confirmed by ttest.
o
*
sá
z
¡tí
.811 .
O
.6(1
d
.4(1

O
.211.
O
O
1
O
.8(1.
o °
o
.611.
o'ó'
.<111 
o
o
.2(1
o° °<9
Q— L
1 1
lili
.3(1
1 I I 1
.6n .on
URINE FI OU ML/I1IN
1 t
1.2(1
113
Figure 32. Plots of the cumulative amounts (e U, p g) of metabolite (M) excreted
in urine against area under the plasma concentrationtime curve
(AUCt, yg.min/ml) at the time of urine collection interval in accordance with
the equation (30):
EU = [ AUCt  AUCq ]
where AUCq is the area under the plasma concentration time curve at E U=0. a) For
the 1.4171 mg/kg IV bolus dose of buprenorphine in dog A, Study #1, the estimated
renal clearance for M was 11.79 ml/min for the time period between 153700 min.
The estimated renal clearance during the first 153 min was 1.94 ml/min (see table
2). b) For the 1.6369 mg/kg IV bolus dose of buprenorphine in dog B, Study #2, the
estimated initial renal clearance of M was 3.6 ml/min. c) For the 1.2023 mg/kg IV
bolus dose of buprenorphine in dog C, Study #4, the estimated renal cleamace of M
was 11 ml/min for the time period 1581270 min. However, for the initial 158 min,
the renal clearance was 4.4 ml/min (Intercept = 30.4). d) For the 1.439 mg/kg IV
bolus dose of buprenorphine in dog C, Study #5, the estimated renal clearance was
0.26 ml/min for the time period 752630 min. In the same dog, the estimated renal
clearance for the first 75 min vías 0.98 ml/min (intercept=9.2).
¿u
ru jjgs JJSS
115
Figure 33. Plot of the cumulative amount ( zU, y g) of the metabolite (M) excreted
in urine against area under the plasma concentrationtime curve
(AUCt, yg.min/ml) at the time of urine collection in accordance with the
equation (30):
l U = Cl [ AUC.  AUCn ]
ren t 0 J
where AUC^ is the area under the plasma concentrationtime curve at E U=0, for the
2.5632 mg/kg IV bolus dose of buprenorphine in dog B, Study #3. The data were
fitted to three linear segments. The regressions on these segments are given in
the legend of Fig. 34.
117
Lf>
cm
□
c:
CM
LO
□
a
¡T5
ssrf ni
AUCT jJG X MIN / ML
Figure 34. Plots of the cumulative amounts (j; U, y g) of the metabolite
(M) excreted in urine against area under the plasma concentrationtime
curve (AUCt , y g.min/ml) at the time of urine collection in accordance
with the equation (30):
ZU = Cl
ren
[ AUCfc  AUCq ]
where AUCn is the area under the plasma concentrationtime curve at
EU=0 for the 2.5632 mg/kg IV bolus dose of buprenorphine in dog B, Study
#3. a) Estimated renal clearance for the time period up to 90 min was
0.93 ml/min. b) the renal clearance for the time period 90442 min was
0.24 ml/min. c) the clearance for the time period 5415650 min was 1.8
ml/min.
sort nz sort nz  sonf ni
119
Figure 35. Plots of the renal clearance (Clren , ml/min) of the metabolite (M)
against urinary pH. The renal clearance was calculated from the quotient of the
urinary excretion rate ( AU/ At) and plasma concentration at the mid point of
urine collection interval ). The slopes with the respective standard
errors for these plots are as follows: a) For the 1.4171 mg/kg IV bolus dose of
buprenorphine (1) in dog A, Study #1; Slope = 3.42 +_ 1.59; b) For the 1.6369 mg/kg
IV bolus dose of buprenorphine in dog B, Study #2; Slope = 1.48 +_ 0.73; c) For
the 1.2023 mg/kg IV bolus dose of buprenorphine in dog C, Study #4; Slope = 3.9 +
2.51; d) For the 1.439 mg/kg IV bolus dose of buprenorphine in dog C, Study #5;
Slope = 0.067 +_ 0.18. These slopes were not statistically significantly different
from zero as confirmed by ttest.
. UVL. L
a REN ML/MIN
CL REN ML/MIN
CL REN ML/MIN
L¿ü2£2_D¿¿íE£Sj
O ,
O !
o Qd!
®>$d,
O o !
O
CL
A
o
— — rvj u) £t
• • • • • •
P m cd a an
= =2 s S O) E2 c
131
119 >
Figure 36. Plots of the renal clearance (Clren , ml/min) of the metabolite (M)
against the urine flow rate (ml/min). The renal clearance was calculated from the
quotient of the urinary excretion rate ( Au/At) and the plasma concentration at
the mid point of the urine collection interval (Cpt_m^). The slopes and the
standard errors are as follows: a) For the 1.6369 mg/kg IV bolus dose of
buprenorphine (1) in dog A, Study #2; Slope = 0.42 +_ 0.59; b) For the 1.2023
mg/kg IV bolus dose of buprenorphine in dog C, Study #4; Slope = 3.6 _+ 2.13; c)
For the 2.5632 mg/kg IV bolus dose of buprenorphine in dog B, Study #3; Slope =
1.4 +_ 1.5; d) For the 1.439 mg/kg IV bolus dose of buprenorphine in dog C, Study
#5; Slope = 0.24 +_ 0.24. These slopes were not statistically significantly
different from zero as confirmed by ttest.
2b
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211
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o
o
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o
o o
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o
o
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URINE E10U ML/MIN
O
—I
2. Ml
O
d
o
o
o
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E
+
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4 E 1 1
.6(1 .Ml
URINE FIOU ML/MIN
O
O
I
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4 I
1.2(1
H
I .Ml
123
Figure 37. Plots of the urinary excretion rate ( g/min) of
buprenorphine (1) and metabolite (M) against plasma concentration of
either 1 or H ( Cp, _ ., ng/ml ) in accordance with the equation 26. a)
For 1 at 1.6369 mg/kg1(Study #2) IV bolus dose of buprenorphine.
Estimated renal clearance was 4.7 ml/min which corresponded well with
the renal clearance (5.24 ml/min) obtained using equation 30 (See fig.
27b). b) For 1 at 1.439 mg/kg (Study #5) IV bolus dose of buprenorphine;
Estimated renal clearance was 1.38 ml/min, which corresponded well with
the renal clearance obtained using equation 30 (1.453 ml/min, see Table
2). c) For M at 1.6369 mg/kg dose of buprenorphine (Study #2). Estimated
renal clearance was 6.6 ml/min.
DLI/Df JJG/MIN OU/OT ¿JG/MIN DU/DT ¿JG/MIN
125
.40 —
.3G _
.32
.28,
.24 .
.20
. 16 
.12
.080
.040
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cb 9''
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IG
48
B TMIO HG^ML
32
G4
8D
126
the metabolic clearance (Cl^ ). The metabolic clearance of the parent
drug (Cl t) and the apparent volume of distribution (Vm) of the
metabolite that is excreted in the urine and the bile can be estimated
by integrating the above equation between 0 and t with respect to time
and considering the stoichiometry of the total metabolite
M, = k V J Cpdt=Cl ^ AUC.
t m c 0 r met t
= U + V m + B Eq. 32
m m m
where AUCfc is the area under the parent drug (1) concentrationtime
curve, U and B are the amounts of metabolite excreted into the urine
m m
and bile respectively up to that time and m is the metabolite
concentration in plasma. If it can be assumed that a constant fraction
of the hepatically formed metabolite is partitioned into the bile, i.e.,
there is a constant biliary clearance, then constant biliary clearance,
then
B = C1D AUC.
m B t
Substituting the value of B^ into equation 32,
(Cl  C1D ) AUC. = U + V m
' met B t mm
The equation can be rearranged^ into
Eq. 33
Eq. 34
U /m = V + (Cl  Cln ) AUC. /m Eq. 35
m m met B t
or U /AUC. = V m/AUC, + (Cl  C1D ) Eq. 36
m t m t ' met B ^
where ( Cl .  Cl_. ) and V can be obtained from the slopes and
met B m
intercepts of the appropriate plots of the designated quotients of AUC^
127
of the drug and cumulative amounts of M excreted in urine against the
concentration of the metabolite m, at the same time. Such plots
according to the equation 35 are given in Fig. 38. Although this method
does not require any explicit knowledge of the dose or the fraction of
the drug transformed into metabolite, it requires that all of the
metabolite be excreted in the urine, and a constant biliary clearance of
the formed metabolite prior to any possible return to the systemic
circulation. Even though an insignificant fraction of the metabolite
appears in plasma, if all of this plasma metabolite is excreted into the
urine, this method could be applied to to the urinary data to obtain the
above parameters. Regressions according to equation 35 gave the
following clearances ( Cl ^  Clg ) _+ standard error in ml/min (Table
2, Fig. 38); 2.72 + 0.5; 2.06 + 0.029; 3.47 + 0.11; 1.27 + 0.009. The
apparent volumes of distribution V of the metabolite + standard error
m —
in ml; 2054 _+ 403; 339 +_ 128; 705 _+ 498; 166 _+ 35. The latter two were
much smaller than the presumed plasma volume (1080 ml, for an hematocrit
of 0.4) in dogs.^ This result is not consistent with the assumption
that the hepatically formed metabolite appearing in the systemic
circulation is completely excreted in urine. Thus urinary excretion may
not be the only route of elimination of the systemic metabolite. In
fact, as it will be shown later, a major fraction of the systemically
circulating metabolite is excreted in bile.
Since an insignificant fraction (% recovery 0.2 _+ 0.08) of
unchanged buprenorphine is excreted in urine, it can now be postulated
that for all practical purposes, the estimated total clearance given as
396 ml/min in Table 2 (based on the unwarrented assumption of the
validities of $ and AUC) is essentially equal to the metabolic
Figure 38. Plots of the quotient of the cumulative amounts of the
metabolite and plasma concentration of the metabolite at the urine
collection time (U^/m) versus the quotient of the area under the
plasma concentrationtime curve of buprenorphine and the metabolite
level in plasma (AUC, /m) in accordance with the equation 33. a) For
the 1.2023 mg/kg IV bolus dose of buprenorhine, Study #4. The estimated
clearance, Cljj^  Cl^ was 3.47 ml/min. b) For the 1.6369 mg/kg IV
bolus dose of buprenorphine in dog B, Study #2; estimated clearance =
2.06 ml/min. c) For the 2.5632 mg/kg IV bolus dose of buprenorphine in
dog B, study #3; estimated clearance = 1.27 ml/min.
sr Be le h l
ueionu
b u?‘e ut>’2 uy i os
U9
U2M
081
dp2
E
i)9e
U2>
08>
UfS
9
6 02"L
I 1 h
o
W'iarw
Up'S D9‘E
—I 1 h
08 ‘ I
E
9
b
21
SI
E?
81
12
P2
L2
DC
621
un/n üh/m
130
clearance, Clj^^ (395 ml/min, Table 2). This integral method using
equation 35 gave the difference between the metabolic and biliary
1 M
clearances ( Cl^^.  Clg ) as 2.38 ml/min (Table 2). Thus biliary
clearance, Cl^, 387 ml/min (Table 2) value indicated elimination of
D
buprenorphine from the body virtually by metabolism.
The TV bolus doses used in the studies 16 produced terminal plasma
concentrations of buprenorphine which were below the analytical
sensitivity (5 ng/ml) and did not permit accurate estimation of the
terminal halflife. The fact that the doses of buprenorphine (1.2 to 2.6
mg/kg) used in the first 5 IV bolus studies (Table 2) exhibited
significant side effects gave a upper limit to the TV bolus dose that
could be administered. To minimize the peak plasma concentrations of
buprenorhine and the associated side effects encountered upon IV bolus
administration, yet to get a greater amount of the drug in the body to
provide adequate number of quantifiable plasma concentrations in the
terminal phase, higher doses of the drug were administered by slow IV
infusion. These studies are described in the next section.
IV INFUSION STUDIES
Plots of plasma concentrations of buprenorphine with time for the
162177 min constant rate infusion of buprenorphine (Studies 712) in 6
dogs (B,D,E,F,G and H) are shown in Figures 3944. In studies 711, the
drug was infused into the jugular vein. In these studies, during
infusion, blood samples were collected from the foreleg's brachialis
vein. In study #12, the drug was infused through the brachialis vein and
the blood samples were collected during infusion from contralateral
brachialis and jugular veins. In studies 79,11, and 12, postinfusion
blood samples were collected from both brachialis and jugular veins.
Dogs E,F and G were also bile cannulated, and the bile was collected up
to 2426 h in these dogs (Studies 9, 10 and 11).
Catheter binding of buprenorphine.
In IV infusion studies (712), buprenorphine hydrochlorie was
dissolved in normal saline (as base, concentration = 0.740.76 mg/ml, pH
= 5.255.5, infusion rate = 0.7026 ml/min) and infused into the jugular
vein (studies 711) or brachialis vein (study #12) using an intravenous
catheter placement unit (see experimental) for a period of up to 3 h.
Buprenorphine slowly partitioned into the plastic catheter during
prolonged infusion. After cessation of infusion, when blood was drawn
from the dog through the same catheter used for infusion, the drug
repartitioned into the blood and gave artifactually higher plasma
concentrations of buprenorphine. The normal saline infusion (45
drops/min) into the vein through the same catheter, which was conducted
between blood samplings, completely removed the drug from the catheter
131
Figure 39. Semilogarithmic plots of the plasma buprenorphine concentrations as
ng/ml of base (upon 0.5058 mg/min constant rate IV infusion) against time (min)
for the 4.6885 mg/kg dose in 17.8 kg dog B (Study #7, Table 5). The points (O)
and (#) represent the jugular and brachialis vein plasma concentrations
respectively of buprenorphine. The solid line represents the curve obtained by
fitting the brachialis and jugular vein plasma data to equation 39. The inset is
the data and the fitted curve for the initial 1000 min.
133
NIN
anus
□nuu
nunc
nnsj?
nnn i
m
nnn ¡
NG'ML
Figure 40. Semi logarithmic plots of the plasma concentrations as ng/ml of base
(upon 0.548 mg/min constant rate IV infusion) against time (min) for the 3.847
mg/kg dose in 22.6 kg dog D (Study #8, Table 5). The points (O) and (•)
represent the jugular and brachialis vein plasma concentrations respectively of
buprenorphine. The solid line represents the curve obtained by fitting the
experimental jugular and brachialis vein plasma data to equation 39. The inset
the data and the fitted curve for the initial 360 min.
135
Figure 41. Semilogarithmic plots of the plasma concentrations of buprenorphine as
ng/ml of base (upon 0.5101 mg/min constant rate IV infusion) against time (min)
for the 4.80 mg/kg dose in 18.6 kg bile cannulated dog E (Study #9, Table 5). The
points (O) and (#) represent the experimental jugular and brachialis vein plasma
concentrations respectively of buprenorphine. The solid line represents the curve
obtained by fitting the experimental experimental brachialis and jugular vein
plasma data of buprenorphine to equation 39. The inset is the representation of
the data and the fitted curve for the initial 1000 min.
NG'ML
137
Figure 42. Semi logarithmic plots of the plasma buprenorphine concentrations as
ng/ml of base (upon 0.5288 mg/min constant rate IV infusion) against time (min)
for the 4.0864 mg/kg dose in 22.0 kg bile cannulated dog F (Study #10, Table 5).
The points (#) and (O) represent the experimental brachialis and jugular vein
plasma concentrations respectively of buprenorphine. The solid line represents the
curve obtained by fitting the experimental brachialis and jugular vein plasma data
to equation 50. The inset is the representation of the data and the fitted curve
for the initial 1000 min.
NG'flL
139
Figure 43. Semilogarithmic plots of the plasma buprenorphine concentrations as
ng/ml of base (upon 0.5588 mg/min constant rate IV infusion) against time (min)
for the 3.7408 mg/kg dose in 24.2 kg bile cannulated dog G (Study #11, Table 5).
The points (•) and (O) represent the experimental brachialis and jugular vein
plasma concentrations respectively of buprenorphine. The solid line represents the
curve obtained by fitting the experimental brachialis and jugular vein plasma data
to equation 50. The inset is the representation of the data and the fitted curve
for the initial 1000 min.
NG/ML
141
Figure 44. Semilogarithmic plots of the plasma buprenorphine concentrations as
ng/ml of base (upon 0.5236 mg/min constant rate IV infusion) against time (min)
for the 3.83 mg/kg dose in 24.2 kg dog H (Study #12, Table 5). The drug was
infused into the brachialis vein through the indwelling catheter (Intracath, see
experimental) and plasma samples were collected from the contralateral brachialis
(#) and jugular veins (O) • The solid line represents the curve obtained by
fitting the plasma data to equation 50. The inset is the representation of the
data and the fitted curve for the initial 1200 min.
1W/5N
143
144
in about 60 min. These findings are demonstrated in the following in
vitro and in vivo studies.
In vitro studies. When 0.7562 mg/ml solution of buprenorphine in
normal saline was pumped through the catheter at the rate of 0.7026
ml/min for 1 h (31.88 mg total), the total amount of buprenorphine
recovered from the catheter by benzene extraction was 283 y g. When
buprenorphine solution (same rate and concentration as above) was passed
thorugh the catheter for 3 h (95.64 mg total), the total amounts of
buprenorphine recovered from the catheter by benzene extraction in two
studies were higher, i.e., 392 and 435 yg respectively. When normal
saline (45 drops/min) was passed through the catheter following 3 h of
the drug at the same concentration and infusion rate, 59 yg (15%) of
the catherter bound drug was recovered in the normal saline within 1
min. The total amount of burenorphine recovered in the saline in 60 min
was 394 yg, approximately the same as the amount recovered from the
catheter by benzene extraction. These results demonstrated that the
catheterbound drug redissolved in the infused saline and was removed
from the catheter completely in about 60 min.
After passing a solution (0.7562 mg/ml) of buprenorphine in normal
saline through the catheter at the rate of 0.52 mg/min for 3 h, the drug
also repartioned into the fresh blood subsequently drawn through it (See
Fig. 45).
When buprenorphine (1 mg/ml in normal saline, 30 ml) solution was
passed in 30 s through the catheter to simulate an IV bolus
admininstration, there was no binding of the drug to the catheter as
demonstrated by the fact that no drug was recovered in subsequent
benzene extraction.
145
\
CD
2
Figure 45. Semilogarithmic plots of the plasma concentrations of
buprenorphine (as ng/ml of base) against time (min). Buprenorphine
dissolved in normal saline was passed through the catheter (Intracath,
see experimental) at the rate of 0.52 mg/min for 1 h (Fig. a) and 3 h
(Fig. b). The catheter was washed with 25 ml normal saline, fresh blank
bloods were drawn through the catheter and the plasmas were analysed.
146
Metabolite solution (100 y g/ml in normal saline), passed through
the catheter at the rate of 14 ml/min for 15 min did not show any
binding to the catheter. This was demonstrated by HPLC analysis
following acid treatment of the catheter.
In vivo studies. When the drug was intravenously infused for 3 h
into the dog, the normal saline drip was subsequently maintained for 24
h. Thus the minor fraction (0.4% of the dose) of the drug that was bound
to the catheter repartitioned into saline and eventually got into the
animal. Unfortunately however, when blood was drawn through the
catheter, the drug also repartitioned into the samples of small volumes
producing artifactually higher concentrations (see Figs. 4547).
When blood was sampled from the brachialis vein upon infusion of
the drug through the plastic catheter into the jugular vein, the
postinfusion jugular vein plasma concentrations (Fig. 46, Table 4)
observed during the first 15 min of the postinfusion distributive phase
were significantly higher than the highest observed brachialis vein
plasma concentrations at the time of cessation of infusion. Similarly,
upon infusion of the drug through the plastic catheter into the left
brachialis vein, the situation was reversed, i.e., the postinfusion
left brachialis vein plasma concentrations during the first 15 min of
the postinfusion distributive phase were significantly higher than the
highest jugular and contralateral (right) brachialis vein concentrations
observed just before the cessation of infusion (Fig. 47). These results
demonstrated that the observed differences in postinfusion
buprenorphine concentrations in plasma obtained from different veins
were not due to any druginduced changes in the circulatory physiology
of the dog, but were due to the repartitioning of the catheterbound
Figure 46. Said logarithmic plots of the plasma concentrations (as ng/ml of base)
of buprenorphine plotted against time (min) following IV infusion of the drug into
the jugular vein. The points (O) and (#) represent the jugular and brachialis
vein plasma concentrations respectively of buprenorphine. a) 4.6885 mg/kg IV
infusion dose of buprenorphine in dog B, Study #7; b) 3.847 mg/kg IV infusion dose
of buprenorphine in dog D, Study #8; c) 4.8 mg/kg IV infusion dose of
buprenorphine in dog E, Study #9; d) 3.7408 mg/kg IV infusion dose of
buprenorphine in dog G, study #11. The solid lines represent the jugular vein
plasma data fitted to equation 50.
TW/9H 1U/3N
1U/3N 1U/9N
148
149
Figure 47. Semi1ogarithmic plots of the plasma concentrations of
buprenorphine (as ng/ml of base) against time (min) following constant
rate (0.5236 mg/min) infusion into the brachialis vein of dog H, Study
#12. The points (a,0) and (b, •) represent the concentrations of
buprenorphine in plasma obtained from jugular and contralateral
brachialis veins respectively. The points (□) represent the
postinfusion plasma concentrations of buprenorphine obtained from
brachialis where the blood was drawn through the indwelling catheter
used in the infusion of the drug.
150
Table 4. Postinfusion jugular and brachial
vein concentrations of buprenorphine and M in
dog D.
Buprenorphine
Jugular
vein
Time
min
ng/ml
Brachial
vein
Time
min
ng/ml
1.21
5823
2.11
771
5.67
1871
5.44
595
19.6
836
21.24
383
46.2
483
48.2
282
75.28
649
77.3
232
120.8
317
123.3
214.3
182.7
346
184.7
160.8
298.5
251.3
302
107
Postinfusion jugular and brachial vein
concentrations of buprenorphine and
metabolite in dog D (Study #8) followed by
constant rate IV infusion of buprenorphine
base into the jugular vein at the rate of
0.5084 mg/min.
151
drug into the blood to produce artifactually higher levels of
buprenorphine in the plasma samples (drawn through the catheter used in
the infusion of the drug) during the first few minutes following
cessation of infusion.
Plasma pharmacokinetics of buprenorphine.
In studies 711, the brachialis plasma concentrations of
buprenorphine during and immediately after cessation of infusion were
considered as representative of the concentrations of buprenorphine in
the systemic circulation and were used in the fitting of buprenorphine
plasma data. The post infusion plasma concentrations from brachialis and
jugular veins (except for the first 45 jugular vein plasma
concentrations immediately following cessation of infusion) were
reasonably coincident (See Fig. 46). In study 12, where the drug was
infused through the catheter into the left brachialis vein, the plasma
concentrations of the drug monitored in the jugular vein (and
contralateral right brachialis vein) were practically coincident and may
be considered as representative of the buprenorphine concentrations in
the systemic circulation (Figs. 44,47).
Two compartment model. In studies 7,8, and 9 (Table 5), the
postinfusion plamsa concentrations of buprenorphine as a function of
53
time were fitted to a sum of two exponentials in accordance with
Cp = A e at + Be
Eg. 37
54
using the computer program of Yamaoka et. al., (Appendix I). The
plasma concentrations were weighted by their inverse values in the
fittings. The validity of the biexponential equation 37 was confirmed by
demonstration that regressions of the weighted residuals (calculated in
Table 5. Pharmacokinetics of buprenorphine and M in dogs upon IV infusion.
Parameter
Dog B
Dog D
Dog E
Dog F
Dog G
Dog H
Mean ± SEM
Study #
7
8
9
10
11
12
Dog No.
B344
W4123
R5108
RB535
R5107
BW485
kg (mg/min)a
0.5058
0.5084
0.5101
0.5288
0.5588
0.5236
T (min)*3
165
171
175
170
162
177
Dose (mg)
83.46
86.94
89.274
89.9
90.53
92.68
Weight, Kg.
17.8
22.6
18.6
22.0
24.2
24.2
Dose mg/kg
4.6885
3.847
4.80
4.0864
3.7408
3.83
Parameters from plasma data for buprenorphine.
104 p ' c
0.04
0.0491
0.039
0.0427 ± 0.003
10, v
0.901
0.65
1.104
0.335
0.301
0.4115
0.62 ± 0.13
106 Bf1
1.172
2.23
0.883
0.609
0.37
0.512
1.04 ± 0.28
10 7Td
0.345
0.107
2.667
1.04 ± 0.82
o
— —
— —
— —
(20)
(64.8)
(2.6)
(29 ± 19)
10 «
2.00
4.4
0.796
0.3426
0.294
0.3523
1.36 ± 0.66
A
(35)
(15.8)
(87)
(202)
(236)
(197)
(129 ± 39)
104 3
3.583
3.433
3.8
1.334
1.319
2.965
3.45 ± 0.18e
(1934)
(2019)
(1824)
(5195)
(5254)
(2337)
(2028 ± 110)8
103 P
—
— —
— —
0.0233
0.0103
0.1837
0.072 ± 0.06
f—J
O
1 ^
>
h \
0.3086
0.49
0.2045
0.0442
0.038
0.0553
0.19 ± 0.075
105 Bf
0.1207
0.23
0.0913
0.0615
0.0371
0.0526
0.1 ± 0.03
105 AUC„ 9
1.00
1.00
1.11
0.83
0.72
1.1
10' AUCToo .
3.106
5.779
2.8605
5.08
3.95
2.7
10"5 AUCn 1
Ooo
3.912
5.72
4.19
3.66j
4.50
3.878
Residual plots
io2 e.k
1.1
15
5.5
1.32
2.4
0.72
Slope
0.015±0.055
0.03±0.09
0.14±0.13
0.019±0.083
0.025±0.1
0.01710.032
Intercept
0.018±0.11
0.09±0.17
0.22±0.26
0.048±0.16
0.025±0.2
0.024±0.06
152
Table 5. Continued. . .
Parameter
Dog B
Dog D
Dog E
Dog F
Dog G
Dog H
Mean ± SEM
Study #
7
8
9
10
11
12
Clearances
(ml/min)
CL . n
tot
213
152
213
246
201
239
211 ± 13.7
Cl— °
ren
0.40(9.5)
0.89(45)
0.13(24)
1.0(80)
—
—
0.61 ± 0.21
1>M p
met
—
—
182
261
220
—
221 ± 23
C1M ^
ren
1.6(11.5)
0.15(24)
2.3(1.04)
4.7(9.8)
—
—
3.5 ± 0.9
% Recoveries of buprenorphine and M in urine
UooVdoser
0.33
0.247
0.037
0.24
—
—
0.21 ± 0.06
UooM/doses
0.4733
0.7442
0.865
0.623
—
—
0.68 ± 0.08
Boo^/dose^
—
—
0.112
0.92
0.00
—
—
BooM/doseu
—
—
89.61
95.7
92.51
—
92.6 ± 1.76
Volumes of
distribution of buprenorphine
(L)
V V
c
31.1(31.2)
19.6(19.5)
47.1(47.1)
35.4(35.3)
69.6(69.1)
5.3(5.3)
34.7 ± 9.1
V
595
443
561
710X
580X
806
616 ± 52
a Dose in mg correspond to buprenorphine base. Administered as buprenorphine HC1 salt, dissolved in normal
saline.
Time of infusion in min.
c P'f, A'j and B' ^ are the intercepts at t=T obtained by fitting the postinf us ion plasma data to equation
54
15 by nonlinear least square curve fitting using the computer program of Yamaoka et al. where plasma
concentrations were weighted by their inverse values. The intercepts are expressed as fractions of the total
dose per ml of plasma.
ci 1
Units for ir , a and 3 values are in min . Parenthetical values correspond to halflives in min.
153
0
The mean and standard error of mean were computed for dogs B, D, E and G only. The mean ± SEM for dog F and G
were significantly different from the other four dogs. However, the terminal plasma data on these two dogs had
greater scattering (See Figs. 42,43) then the dogs B, D, E and G.
^ Af, Bf and values were calculated from the A, B and C values obtained from equations 1618 and
expressed as fractions of the total dose Xq per ml of plasma.
^ The theoretical area under the plasma concentrationtime curve of buprenorphine during the infusion phase
was calculated using equation 41 in dogs B, D and E. In dogs F, G and H, equation 52 was used.
Postinfusion area under the plasma concentrationtime curve was obtained by integrating equation 15 between
T to oo, AUC^,^ = (P'/ ir) + (A'/ “ ) + (B'/ 8 ) .
1 The total area under the plasma concentrationtime curve was calculated using the trapezoidal rule. This
area was used in the estimation of total body clearance and apparent volume of distribution of buprenorphine
(V.) in dogs.
* In study #10, the calculated area by trapezoidal rule up to 1616 min was 260508 ng.min/ml. The quotient of
the plasma concentration of buprenorphine (38.2 ng/ml) at at 1616 min and the average terminal phase rate
constant (0.000345/min) was 105234 ng.min/ml. Thus the total area was estimated as 371233 ng.min/ml.
Mean of the weighted residuals calculated in accordance with the equation 3. None of the mean residuals were
statistically significantly different from zero.
1 m
' Slopes and intercepts of the plots of C against log fitted postinfusion data. The parenthetical values
correspond to standard errors. Both slopes and intercepts were not statistically significantly different from
zero, indicative of the fact that in dogs B, D and E, the sum of two exponentials and in dogs F and G, the sum
of three exponentials best fit the postinfusion plasma data of buprenorphine.
n Ratio of the infused total dose to the total area under the plasma concentrationtime carve of
buprenorphine. The AUC^ was calculated using trapeziodal rule. The calculated total clearances in studies
712 were 203, 128, 225, 152, 194, 244 ml/min respectively which averaged 191 + 18 (SEM) ml/min.
° Estimates iron the slopes of the cumulative amounts of buprenorphine excreted ( EU, pg) renally against the
area under the plasma concentrationtime curve of buprenorphine in accordance with the equation 30. These
ratios changed as pH of the urine changed. (See Fig. 57). Values given in parenthesis correspond to EU at
AUC=0 from the best linear plots of the data shown in Figs. 57,58.
^ Cl^e^M was calculated using equation 58.
^ C1M , the renal clearance of the plasma metabolite was calculated in accordance with the equation 30,
see afso Fig. 59.
154
r,s,t,u percen1 recoveries of buprenorphine and M in urine and bile obtained iron the quotient of the amounts
recovered in urine or bile and the total infused dose of buprenorphine.
v Apparent volume of distribution of the central compartment estimated by fitting the postinfusion data to
either equation 39 (2compartment model) or 50 (3compartment model) by nonlinear least square regression
(Appendix I). The values in parenthesis were calculated from equation 10, where the values of P, A and B were
obtained through equations 1618. The mean and the standard error represents the volumes of distribution
estimated through equation 39 (or 50).
w Vj, the apparent volume of distribution of buprenorphine in the body was calculated from the ratio of
CW 6
x In dogs F, the average terminal phase rate constant (0.000345/min) rather than the estimated terminal rate
constant was used in the estimation of apparent volume of distribution.
155
156
accordance with the equation 3) against logarithm of the calculated
plasma concentrations gave mean residuals €, slopes and intercepts which
were not statistically significantly different from zero (Fig. 48, Table
5).
The parameters of the above sum of biexponential fits of the
postinfusion plasma data of buprenorphine are listed in Table 5 in
addition to the calculated parameters (equations 17,18) for an
equivalent IV bolus administration. The estimated average terminal
halflives of buprenorphine were 1934, 2019, and 1824 min in dogs B, D
and E respectively. The estimated 95% confidence limits for the terminal
rate constants are given in Table 6.
The value of , the exit rate constant from the deep
. 53
compartment was calculated from the expression
k21 = (A 6 +Ba )/(A+B) Eq. 38
where A and B were obtained from equations 17,18.
For a drug conforming to a 2compartment body model, the equation
that describes the time course of the drug in the central compartment
(k0/Vc) [ [ (k21 a) (leaT)/a(ct8)] e~at
+ [ ( 3 ~k21) (1e PT )/ 8 (a 8 )] e" 3t ] Eq. 39
where T is the duration of infusion and t is the time after initiating
infusion in min. During infusion, T=t and upon cessation of infusion, T
becomes a constant equal to the time of infusion. In the above
expression, except for Vc, all the other constants are known. Thus it
is possible to obtain V , the volume of distribution of the central
compartment by fitting the postinfusion data with the above equation
Figure 48. Representative examples of the plots of the weighted residuals against
the logarithm of calculated plasma concentations. The weighted residuals were
calculated in accordance with eguation 3:
G = (Cpexp ~ ^calc* / ^calc
a) the postinfusion plasma data of buprenorphine administered by constant rate
(0.5058 mg/min) IV infusion for the 4.6885 mg/kg dose study in 17.8 kg dog B,
Study #7; b) the postinfusion plasma data of buprenorphine administered by
constant rate (0.5084 mg/min) IV infusion for the 3.847 mg/kg dose study in 22.6
kg dog D, Study #8; c) the postinfusion plasma data of buprenorphine administered
by constant rate (0.5288 mg/min) IV infusion for the 4.0864 mg/kg dose study in
22.0 kg bile cannulated dog F, Study #10; d) the postinfusion plasma data of
buprenorphine administered by constant rate (0.5588 mg/min) TV infusion for the
3.7408 mg/kg dose study in 24.2 kg bile cannulated dog G, Study #11. The observed
means, slopes and the intercepts of these weighted residuals are given in table 4.
These parameters were not statistically significantly different from zero as
confirmed by ttest. The random distribution of the residuals above and below the
regression line indicated no bias in the fitting of the chosen model.
RESIDUALS RESIDUALS
RESIDUALS RESIDUALS
158
Table 6. Statistics of total and metabolic clearances of buprenorphine.
Parameter
Dog B
Dog D
Dog E
Dog F
Dog G
Dog H
Mean ± SEM
Study #
7
8
9
10
11
12
Dog No.
B344
W4123
R5108
RB535
R5107
BW485
Statistics for
the terminal
rate constant.
B (ng/ml) a
103.4
117.3
58.88
71.86
55.76
51.62
104 3 b
3.529
2.9078
3.177
1.76
2.0644
3.0706
104 SE of B C
0.303
0.191
0.347
0.2524
0.410
0.10
d
n
11
11
11
14
16
14
104 Upper 3 e
4.21
3.34
3.96
2.31
2.946
3.289
104 Lower 3 e
2.84
2.476
2.393
1.21
1.183
2.853
bl/2 (min)
1964
2383
2181
3938
3357
2257
Upper
2437
2800
2896
5727
5860
2429
Lower
1646
2075
1750
3000
2352
2107
105 AUC, J
tot
Upper
5.01
5.869
4.70
7.75
6.774
4.01
Lower
3.83
4.644
3.735
4.913
3.953
3.766
Cl^^ ml/min ^
203
128
225
152
194
244
191 ± 18
Upper
218
187.2
239
183
229
246
Lower
167
148
190
116
134
231
Cl h
tot
213
153
213
246
201
239
211 ± 141
1>M j
182
261
220
221 ± 23
met
a'^Terminal phase intercepts (B, ng/ml) and rate constants (3 ) were estimated iron the semilogarithmic plots
of the terminal plasma data of buprenorphine against time.
Q
Standard error of the estimated terminal rate constant ( g).
:r of terminal phase plasma points used in the estimation of terminal rate constant.
U1
vo
e95% Confidence limits were calculated from the ttable at a=0.025 level of significance for (n2) degrees
of freedom.
f In the estimation of total area under the plasma concentration of buprenorphine against time in accordance
with equation 10, the parameters P and A were obtained through equations 16 and 17. The parameters u anda
were obtained from the computer fit of the postinfusion data to equation 15. The parameters B and g were
obtained from the semilogarithmic plots of the terminal phase plasma data against time. The 95% confidence
intervals for AUC were obtained from the confidence limits estimated for the terminal rate constant assuming
no error in the P, A, ir anda values. See also discussion in the last section under the subheading "Validity
of the terminal rate constant".
^Total clearance was estimated iron the quotient of Dose/AUC where AUC was estimated from the
oo oo
plasma concentrations of buprenorphine. The 95% confidence limits for the total clearances were calculated
from the 95% confidence limits of the AUC values.
^The total clearances were calculated using trapezoidal rule.
xThe means and standard error of the means for the total clearances were calculated for the bile cannulated
dogs E, F and G only (The other dogs were not bile cannulated).
*C1q , the biliary clearances of buprenorphine as metabolite were estimated from equation 58.
B
160
161
with one unknown parameter. Curve fittings of the infusion data to the
54
above equation using the computer program of Yamaoka et al. (Appendix
I) are given in Figs. 3941 (See also Table 5). The apparent volumes of
distribution of the central compartment (V ) estimated by this
procedure were 31, 19.6, and 47.1 L respectively (Table 5). Vc was also
calculated from equation 10 where the values of the parameters A and B
(dosenormalized, shown in Table 5) were obtained through equations
17,18. The estimated apparent volumes of distribution of the central
compartment by this procedure were 31, 19.5, and 47.1 L respectively
which agreed with the above Vc values estimated by the computer fitting
of the postinfusion plasma data to equation 39.
Estimation of AUC. When the constants in equation 39 are simplified
into single constants, the following equation results during infusion:
Cp = R (e_ott 1) + S (eBt 1) Eq. 40
Integrating the above expression between 0 to T, the time infusion was
ended, gives:
AUC0_t = (R/d ) (le"at  t) + (S/e ) (1e" 3t  t) Eq. 41
where
R = (k0/VJ [ (k21a ) / a (a  B ) ] Eq. 42
and
S = (k0/Vc) [ (k21e )/ 3(3a)] Eq. 43
The postinfusion area (AUC^) under the plasma
concentrationtime curve was obtained by integrating the equation 15
between (tT)=0, the time when infusion was stopped, to time infinity
(oo),
AUC¿o = (A'/a ) + (BV 3)
Eq. 44
162
Thus, the total area is
AUC^ = AUCq_t (during infusion) + AUC^ (postinfusion) Eq. 45
These calculated areas are given in Table 5.
The total clearances, Cltot, calculated from the equation
Cltot = Dose/AUC^ were estimated as 213, 152 and 213 ml/min in dogs
B, D and E respectively (Table 5 and 6). The 95% confidence limits for
these clearances are reported in Table 6. The overall volumes of
distribution estimated from the equation 13 were 595, 443 and 561 L
respectively in dogs B, D and E, indicating high degree of sequestration
of buprenorphine into body tissues.
Three compartment model. In slow IV infusion studies 10, 11 and 12,
the postinfusion plasma concentrations of buprenorphine as a function
of time were fitted to a sum of three exponentials in accordance with
54
the equation 15 using the computer program of Yamaoka et. al.
(Appendix I, Figs. 4244). The plasma concentrations were weighted by
their inverse values. The validity of the triexponential equation 15 was
confirmed by demonstration that regressions of the weighted residuals
(calculated in accordance with the equation 3) against logarithm of the
calculated buprenorphine plasma concentrations gave mean residuals £,
slopes and intercepts which were not statistically significantly
different from zero (Fig. 48, Table 5).
The parameters of the above sum of three exponential fits of the
postinfusion plasma data of buprenorphine are listed in Table 5 in
addition to the calculated parameters (equations 1518) for an
equivalent IV bolus administration. The estimated terminal halflives of
buprenorphine in the body were 5195, 5254 and 2337 min respectively in
dogs F, G and H (studies 10, 11 and 12 respectively). The terminal
163
plasma data in studies 10 and 11 were more widely scattered (Figs. 42,
43) than in studies 7, 8, 9 and 12 (Figs. 3941,44). The terminal
halflives estimated from the semilogarithmic plots of the terminal
plasma concentrations of buprenorphine against time (and their 95%
confidence limits in parenthesis) were 3938 (30005727) and 3357
(23525860) min respectively in dogs F and G (see also Table 6). The
estimated averaged first and second distributional halflives were 29 +_
19 (SEM) min (studies 10, 11 and 12) and 129 _+ 39 (SEM) min (studies
712, see Table 5).
The values of k21 and k^, the respective exit rate constants
from the shallow and deep compartments were calculated from the
expressions'
53
k31 = 0.5 [
k21 = 0.5 [
bV(b2  4c)]
b+ V(b2  4c) ]
where
b =  ( tt B+ tt A+ 3 P+8 A+ a p+ a B) / (P+A+B)
and
Eq. 46
Eq. 47
Eq. 48
Eq. 49
c = (ctiT b+t: 8 A+a 8 P)/(P+A+B)
where P, A and B values were obtained from equations 1618.
For a drug conforming to a 3compartment body model, the equation
that describes the time course of the drug in the central compartment
. 53
is
CP,
calc
(k0/Vc) C[ (k21 “ w ) (k3l _7r ) (le* T) /" ( a  it ) ( tt  8 ) ]
[(k21 a)(k31 a)(lea T)/a(7ra)(a8)] e“at +
[(k21  8) (k31 6 ) (1e BT )/ 8 (a8 ) (8 tt )] e~6 t ] Eq. 50
where T is the duration of infusion and t is the time after initiating
tt t
164
infusion in min. During infusion, T=t and upon cessation of infusion, T
becomes a constant equal to the time of infusion. In the above
expression, except for V , all the other constants are known. Thus it
is possible to obtain Vc , the volume of distribution of the central
compartment by fitting the postinfusion data with the above equation
with one unknown parameter. Curve fittings of the infusion data to the
54
above equation using the computer program of Yamaoka et al. (Appendix
I) are given in Figs. 4143 (See also Table 5). The apparent volumes of
distribution of the central compartment (VJ estimated by this
procedure were 35.4, 69.6 and 5.3 L respectively for dogs F, G and H.
Vc was also calculated from equation 10 where the values of the
parameters P, A and B (Table 5) were obtained through equation 1618.
The estimated apparent volumes of distribution of the central
compartment by this procedure were 35.3, 69.1 and 5.3 L respectively in
dogs F, G and H, which are in agreement with the above Vc values
calculated by the computer fitting of the postinfusion data to equation
50.
Estimation of AUC for plasma buprenorphine. When the constants in
equation 41 are simplified into single constants, the following equation
results during infusion:
Cp = Q (e“ 7Tt 1) + R (e_a 1 1) + S (e“3 1 1) Eq. 51
Integrating the above expression between 0 to T, the time infusion was
ended, gives:
AUCq_t = (Q / 7T ) (1e" " 1  t) + (R / a ) (le"a  t) +
 Bt
(S / 6) (1e
t)
Eq. 52
165
where
Q = (k0/VJ [ (k21TT ) (k31 TT ) / IT ( a TT )(*(? )]
R = (k0/vc) [(k21a ) (k31a ) / a (ir a) (a  3)]
Eq. 53
Eq. 54
and
S = (k0/Vc)[(k2l"3)(k3re ) / 3 («3 ) ( 3tt ) ]
Eq. 55
The postinfusion area under the plasma concentrationtime curve
was obtained by integrating the equation 15 between (tT)=0, the time
when infusion was stopped, to time infinity (oo),
AUCóo = (P'/17 ) + (A'/a ) + (B'/P )
Eq. 56
Thus, AUC = AUCfl^ + AUC' , and the calculated areas are given
in Table 5.
The total clearances, Cltot, calculated from the equation Cl^^
= Dose/AUC^ were 246, 201 and 239 ml/min (see also Table 5) for dogs
F, G and H respectively. The average total body clearance estimated for
all 6 dogs was 211 +_ 13.7 (SEM) ml/min (Table 5 and 6). The 95%
confidence limits for these clearances are reported in Table 6. This
value is lower than the total clearance obtained from IV bolus studies
(396 _+ 19 (SEM) ml/min, Table 2). Thus over estimation of Clt t value
in IV bolus studies could be attributed to the lack of sufficient number
of quantifiable terminal plasma points to obtain reasonable estimates of
terminal rate constant, and the derived total body clearance. The
overall volume of distribution V^ estimated from the equation 13 was
616 +_ 52 (SEM, n=6) L, indicating high degree of sequestration into body
tissues, consistent with the observations from IV bolus studies (434 L,
Table 2).
Plasma pharmacokinetics of the derived metabolite (M). The
brachialis and jugular vein plasma concentrations of buprenorphine
166
conjugate were apparently same in the postinfusion phase (Figs. 4952).
Similar to the case of IV bolus administration of buprenorphine, where
the highest plasma concentration of the metabolite occured immediately
following the bolus dose, the highest plasma concentration of the
metabolite occured immediately after the cessation of infusion of
buprenorphine (Figs. 4952). This is possible only when the hepatically
derived metabolite is so rapidly formed as well as eliminated from the
systemic circulation to mimic the buprenorphine concentrations in
plasma.
In all three bile cannulation studies (911), no detectable plasma
concentration of the metabolite was observed after 12 h following
initiation of infusion (duration of infusion = 165175 min; see Figs.
51,52). However, when the bile catheter was removed at 26 h and the
screwcap (Fig. 1) was replaced, the bile to flowed normally into the
duodenum, and metabolite reappeared in the plasma (studies 9 and 11,
Figs. 51,52). This strongly indicates that the enterohepatic
recirculation of the metabolite resumed when the bile was no longer
collected completely.
In dog G, buprenorphine conjugate (9.08 mg) was administered
intraduodenally and it appeared in plasma (Fig. 53). This provided
additional evidence for the enterohepatic recirculation of the
metabolite.
A minor fraction (6.2%) of the intraduodenally administered
metabolite was recovered in bile. Neither drug nor metabolite were
detectable in urine. These results confirmed the fact that the terminal
halflife of buprenorphine in a given dog would not be affected by bile
cannulation and complete bile collection.
Figure 49. Sard logarithmic plots of the experimental jugular vein (O) and
brachialis vein (#) plasma concentrations (ng/ml) of the hepatically derived
acidhydrolyzable metabolite (M) against time (min) for the 4.6885 mg/kg IV
infusion dose of buprenorphine in dog B, Study #7. The inset is the representation
of the plasma concentrations of the metabolite up to 500 min.
IW'SN
O
o
o
o
o
2nnn
o
o
, °
f
3Í1ÜG
O
luna
MIN
I
H
‘inuu
]—gh
bnnü
168
Figure 50. Semi logarithmic plots of the experimental jugular vein (O) and
brachialis vein (•) plasma concentrations (ng/ml) of the hepatically derived
acidhydrolyzable metabolite (M) against time (min) for the 3.847 mg/kg IV
infusion dose of buprenorphine in dog D, Study #8. The inset is the representation
of the plasma concentrations of the metabolite up tp 700 min.
170
HI W
[](]fJ9 UU8l> ÜÜ9C DIM DUG!
Figure 51. Semilogarithmic plots of the experimental jugular (O) and brachialis
(#) vein plasma concentrations (ng/ml) of the hepatically formed
acidhydrolyzable metabolite (M) against time (min) for the 4.80 mg/kg IV infusion
dose of buprenorphine in dog E, Study #9. The solid line represents the curve
obtained by fitting the postinfusion data to the equation: C = Co exp(kt) where
Co = concentration at time when infusion was stopped and k is the apparent first
order elimination rate constant. The middle inset represents the fitted
postinfusion plasma data up to 600 min, and the top inset is the brachealis (#)
plasma concentration of metabolite during infusion and postinfusion jugular vein
(O) plasma concentrations of metabolite. The experimental plasma points (0)
around 1200 min correspond to blood that were sampled but no metabolite was
detectable in plasma at the analytical sensitivity of 5 ng/ml.
IW'SN
MIN
172
Figure 52. Semi logarithmic plots of the experimental jugular vein (O) and
brachialis vein (0) plasma concentrations (ng/ml) of the metabolite (M) against
time (min) for the 3.7408 mg/kg IV infusion dose of buprenorphine in dog G, Study
#11. The experimental plasma points around 1200 min (0) correspond to blood that
were sampled, but no metabolite was detectable at the analytical sensitivity of 5
ng/ml.
174
MIN
□num nuns auns UDUt» muz
NG/tll
175
o
o
i
240 360
MIN
480 GOO
Figure 53. Semilogarithmic plot of the plasma concentrations of
buprenorphine glucuronide (ng/ml) plotted against time (min) following
intraduodenal administration of 9.08 mg of the metabolite to dog G.
176
Urinary excretion of buprenorphine and metabolite. Some examples of
sigma minus plots (equations 2024) for buprenorpine and metabolite are
given in Figs. 54,55. Urinary excretion rate plots (logAU/ At versus
tmid) were scattered, and only the examples which followed the equation
24 are presented in Fig. 56. The rate constants obtained from the slopes
of these plots are given in legends of figures 5456. They corresponded
to the first and second distributional halflives obtained from the
plasma data (Table 5).
Renal clearance of buprenorphine and metabolite. The renal
clearance of buprenorphine in dog E at 4.8 mg/kg dose showed a dramatic
pH dependency (Study #9, Fig. 57). The values of renal clearance for
buprenorphine and metabolite calculated from the slopes in accordance
with the equation 30 are given in Table 5 (See also Figs. 58,59). The
urine pH ranges were narrower in other studies (Appendix IV, Tables
ATV2b,4b,5b,7b,8b,10b). Thus any apparent pH effects on renal
clearance of buprenorphine in many could not be detected due to lack of
enough pH variability. The metabolite showed neither pH nor urine flow
dependent renal clearance (Fig. 60a,b). Buprenorphine clearance was not
urine flow dependent (Fig. 60c). These results were consistent with
those obtained from the IV bolus studies.
Biliary excretion of buprenorphine and metabolite. Analysis of
buprenphine conjugate excreted in bile by HPLC following acid hydrolysis
and gglucuronidase hydrolysis (see experimental) gave identical
results (Fig. 61). In study #11, cumulative amounts of the conjugate
excreted in bile analysed by HPLC following acid and 3 glucuronidase
hydrolysis respectively were 83.75 and 83.1 mg.
177
Figure 54. Semilogarithniic plots of the amounts of the unchanged
bupenorphine (1) remaining to be excreted in urine versus time (sigma
minus plot) in accordance with the equation 20. a) Semilogarithmic
fitting of the postinfusion urine data to a sum of two exponentials for
the 3.847 mg/kg IV infusion dose of buprenorphine to dog D (Study #8,
Table 5). The estimated hybrid rate constants were 0.0072/min (half life
= 96 min) and 0.000276/min (half life = 2507 min), b) Sigma minus plot
of the postinfusion urinary excretion of buprenorphine for the 4.8
mg/kg IV infusion dose of buprenorphine in dog E (Study #9, Table 5).
The apparent rate constant for this monoexponential fitting was
0.00036/min (half life = 1931 min ). These estimated rate constants
correspond with the rate constants obtained from the plasma data (Table
5) in these studies.
Figure 55. Semilogarithmic plots of the amounts of the metabolite (M) remaining to
be excreted in urine versus time (sigma minus plot) following IV infusion of
buprenorphine in accordance with equation 20. a) Sigma minus plot of metabolite in
urine of dog B (Study #7) following 4.6885 mg/kg IV infusion of buprenorphine,
resulting in an estimated apparent rate constant 0.0013/min (halflife = 553 min),
b) Sigma minus plot of metabolite in urine of dog D (Study #8) following 3.847
mg/kg IV infusion dose of buprenorphine. The estimated apparent rate constant was
0.00037/min (halflife = 1877 min), c) Sigma minus plot of the urinary excretion
of metabolite for the 4.8 mg/kg IV infusion dose of buprenorphine in dog E (Study
#9). The estimated apparent rate constant was 0.0034/min (halflife = 203 min), d)
Sigma minus plot of the urinary excretion of metabolite for the 4.0864 mg/kg IV
infusion dose of buprenorphine in dog F (Study #10). The data were fitted to a sum
of two exponentials in accordance with equation 20. The estimated apparent rate
constants were 0.00567/min (halflife = 122 min) and 0.00009/min (halflife = 7700
min) respectively.
$sr1 nx*ni ssrf ni
ibib jjss tu¿tu ues
IODO
H 1 1 1 1 1 1 1 *—I 1
6tm I2QQ I8ÜQ 2*1(10 3000
MIN
179
Figure 56. Semilogarithmic plots of the amounts ( y g) buprenorphine
excreted in urine (a, b) and bile (c) per minute plotted against tmid,
the mid point of the biological fluid collection interval, in accordance
with the reduced form of the equation (22):
Log aU/ At = (k'/2.303) tmid + intercept
for a) 4.8 mg/kg IV infusion dose of buprenorphine in dog E (Study #9)
resulting in an estimated apparent rate constant of 0.0031/min
(halflife = 225 min); b) 4.6885 mg/kg IV infusion dose of buprenorphine
in dog B (Study #7) with an estimated apparent rate constant of
0.0157/min (halflife = 44.3 min); and c) dog E at the same above 4.8
mg/kg IV infusion dose of buprenorphine (Study #9), the biliary
excretion rate (ug/min) of buprenorphine could be fitted to a sum of two
exponentials resulting in apparent rate constants of 0.031/min
(halflife = 22.2 min) and 0.0028/min (halflife = 251 min)
respectively.
DlvDT JjG'MN OU/DT JJG'MIN DU/OT pG'MIN
181
Figure 57. Plots of cumulative amounts ( E U, y g) of buprenorphine excreted in
urine against the area under the plasma concentration time curve (AUCt,
y g.min/ml) in accordance with the equation (28):
Z U = Cl (AUC AUCn )
ren ' t O'
where AUCg is the area under the plasma concentrationtime curve at £ U=0. For the
4.8 mg/kg IV infusion dose of buprenorphine in dog E (Study #9), the plot showed
four distinctly linear segments attributable to the pH effect on renal clearance
of buprenorphine. The renal clearance of buprenorphine estimated from the slope of
the linear segment for the pH range between 6.146.39 (inset) was 0.134 ml/min.
I6Ü 2‘IN
^UCT JJG X MIN / ML
inn
8U
32n
183
Figure 58. Plots of the cumulative amounts ( IU, yg) of buprenorphine
excreted in urine against area under the plasma concentrationtime curve
at the time of urine collection in accordance with the equation (28):
ZÜ = Clren (AUCt  AUC0 )
where AUCp is the area under the plasma concentrationtime curve at E U
= 0. a) For the 4.6885 mg/kg IV infusion dose of buprenorphine in dog B
(Study #7), the clearance estimated from the initial slope was 0.37
ml/min; b) For the 3.847 mg/kg IV infusion dose of buprenorphine in dog
D (Study #8), the estimated renal clearance was 0.9 ml/min for the
initial period up to 600 min; c) For the 4.0864 mg/kg IV infusion dose
of 1 in dog F (Study #10), the estimated clearance was 1.0 ml/min for
the urine collection time between 200800 min.
sgrfm . ssrí rn sarfnx
185
Figure 59. Plots of the cumulative amounts ( eU, yg) of metabolite (M) excreted in
urine against area under the plasma concentrationtime curve at the time of urine
collection in accordance with the equation (28):
EU = Clren (AUCt  AUC0 )
where AUCq is the area under the plasma concentrationtime curve atEU = 0. a)
For the 4.6885 mg/kg IV infusion dose of buprenorphine in dog B (Study #7), the
clearance estimated for the time period 1001800 min was 5.36 ml/min; b) for the
3.847 mg/kg IV infusion dose of buprenorphine in dog D (Study #8), the estimated
renal clearance for the initial period up to 12000 min was 1.714 ml/min; c) For
the 4.8 mg/kg IV infusion dose of 1 in dog E (Study #9), the estimated renal
clearance from the initial slope was 2.33 ml/min; d) For the 4.0864 mg/kg IV
infusion dose of buprenorphine in dog F (Study #10), for the time period between
200500 min the renal clearance of metabolite estimated as 21.2 ml/min, and
between 6001400 min the clearance was 1.5 ml/min.
ssrf ra ssrt nz
sarf nz ssrf n3
187
Figure 60. Plots of renal clearance (ml/min) of buprenorphine and metabolite (M)
against urine flow (ml/min) and pH. The renal clearances were calculated from the
quotient of the urinary excretion rate ( aU/ At, p g/min) and plasma concentration
of buprenorphine or metabolite (ng/ml) at the midpoint of urine collection
interval. The slopes and the respective standard errors are: a) For the 4.6885
mg/kg TV infusion dose of buprenorphine in dog B (Study #7), the estimated slope
of the plot of renal clearance of metabolite versus urine flow (ml/min) was 0.064
+_ 0.3. b) For 3.847 mg/kg IV infusion dose of buprenorphine in dog D (Study #8),
the slope of the plot of renal clearance of metabolite versus pH was 0.25 +_ 1.2.
c) In dog E (Study #9), at 4.8 mg/kg IV infusion dose of buprenorphine, the slope
of the plot of the renal clearance of buprenorphine versus urine flow was 0.1 _+
0.1. These slopes were not statistically significantly different from zero as
confirmed by ttest.
ClREN «.✓MIN
O
O
z
Ckr
O
o.5n T
6.35
fi..
5.25
4.50 ..
3.35..
3 
2.25
1.5(1  
.35  
8,
o o
■4 1 h
o
o
o
.. D.
O O
o °
O o
o o
o
6.U8Q
6.36
H 1 1 h
PH
6.64
6.32
O.
O
1
3.20
189
Figure 61. Plots of the cumulative amounts (IBM, mg) of buprenorphine conjugate
excreted in bile of dog G (Study #11) at 3.7408 mg/kg IV infusion dose of
buprenorpine. The conjugate amounts were estimated by HPLC separation and
fluorimetric detection of demethoxybuprenorphine (3) obtained after acid
hydrolysis (O) of the buprenorphine conjugate and by HPLC separation and
fluorimetric detection of buprenorphine obtained after g glucuronidase hydrolysis
(□) of the buprenorphine conjugate. The cumulative amounts of the buprenorphine
conjugate obtained by these two different hydrolysis techniques were 83.75 (O)
and 83.05 (□) mgs respectively.
12H IÜ8LI M4Í1 I Bill]
MIN
ZBM MG
c:
I6T
192
Sigma minus plots of the biliary excretion of the unchanged
buprenorphine (Studies 10,11, Fig. 62) and hepatically formed metabolite
(Studies 9,10,11, Fig. 63) are from the data obtained during the entire
bile collection period (26 h). These plots were apparently linear
(except study #11, Fig. 63c). The estimated apparent halflives are
reported in the figure legends. These halflives corresponded to the 2nd
distributional halflives of buprenorphine in plasma (Table 5).
Biliary clearance of buprenorphine and metabolite. In bile
cannulated dogs E, F and G, the percentages of the total doses excreted
in bile as unchanged buprenorphine were 0.112, 0.92 and 0.00
respectively (Table 5). The biliary clearances of buprenorphine were
estimated from the plots of the cumulative amounts of the unchanged
buprenorphine excreted in bile against the areas under the plasma
concentrationtime profile of buprenorphine in plasma in accordance with
the equation
IB = Cl^ [AUCt  AUCq] Eq. 57
where AUCq is the area under the plasma concentrationtime curve of
buprenorphine when IB=0. In the bile cannulated dogs E and F (Fig. 64,
Table 5), the estimated biliary clearances for unchanged buprenorphine
were 0.16 and 3.2 ml/min respectively.
In the bile cannulated dogs E, F and G, the average of the total
dose excreted in bile, assayed as acid hydrolyzable conjugate (M) of
buprenorphine, was 92.6 +_ 1.8 % (SEM) (See Table 5). This is in contrast
to morphine, where 20% of the dose was excreted in the bile of dogs as
45
morphine glucuromde.
Since a minor fraction of the hepatically formed metabolite was
excreted in urine, the term in equation 32 can be neglected. Thus
193
IDO:
CT
=1
CCT
UJ
Figure 62. Semilogarithmic plots of the amounts of buprenorphine
remaining to be excreted in bile versus time (Sigma minus plot)
following IV infusion of buprenorphine in accordace with equation 20. a)
Sigma minus plot of buprenorphine in bile of dog E (Study #9) following
4.8 mg/kg IV infusion dose of buprenorphine. The estimated apparent
elimination rate constant was 0.0032/min (halflife = 220 min), b) Sigma
minus plot of buprenorphine in bile of dog F (Study #10) following
4.0864 mg/kg IV infusion dose of buprenorphine. The estimated apparent
rate constant was 0.002/min (halflife = 340 min). See also Table 5.
Figure 63. Semi logarithmic plots of the amounts of the hepatically
formed metabolite (M) remaining to be excreted in bile versus time
(Sigma minus plot) following IV infusion of buprenorphine in accordace
with equation 20. a) Sigma minus plot of metabolite in bile of dog E
(Study #9) following 4.8 mg/kg IV infusion of buprenorphine, resulting
in an estimated apparent elimination rate constant of 0.0038/min
(halflife = 181 min). b) Sigma minus plot for the biliary excretion of
metabolite in dog F (Study #10) following 4.0864 mg/kg IV infusion of
buprenorphine. The estimated apparent elimination rate constant was
0.0029/min (halflife = 241 min), c) Sigma minus plot of metabolite in
bile of dog G (Study #11) upon IV infusion of 3.7408 mg/kg dose of
buprenorphine. Estimated apparent excretion rate constant was 0.0026/min
(halflife = 268 min).
195
196
Figure 64. Plots of the cumulative amounts (E B, y g) of buprenorphine
excreted in bile against the area under the plasma concentrationtime
curve (AUCt , yg.min/ml) at the time of urine collection interval in
accordance with the equation:
E B = Clg [AUCt  AUCq ]
where AUCq is the area under the plasma concentrationtime curve of
buprenorphine at E B=0. a) For the 4.8 mg/kg IV infusion dose of
buprenorphine in dog E (Study #9), the estimated biliary clearance of
buprenorphine was 0.16 ml/min. b) For the 4.0864 mg/kg IV infusion dose
of buprenorphine in dog F (Study #10), estimated biliary clearance was
3.24 ml/min.
197
equation 32 can be written as
B
m
1>M
= Cl ' AUC  V m
met t m p
Eq. 58
where B is the cumulative amount of metabolite (M) collected in bile,
Cl^e^M = apparent metabolic clearance of buprenorphine as M, AUCfc
= area under the plasma concentrationtime curve of buprenorphine at
time t, Vm is apparent volume of distribution of the metabolite and m^
is the metabolite concentration in plasma. Thus the apparent metabolic
clearance, C1^^M of buprenorphine as M can be estimated by
multiple linear regression of the cumulative amounts of the hepatically
formed metabolite (M) excreted in bile against the area under the plasma
concentrationtime curve of buprenorphine and the corresponding
metabolite concentration in plasma. For dogs E, F and G, these apparent
metabolic clearances obtained from the equation 58 were 182, 261 and 220
ml/min (Fig. 65, Table 5 where areas were calculated using trapezoidal
rule) and the apparent volumes of distribution of the metabolite were
13.6, 281 and 67.6 L respectively. Plots of A B/ At against •
the plasma concentrations of either buprenorphine or the metabolite were
usually scattered. Some examples are given in Fig. 66.
Minor metabolites in bile. Three additional peaks that could be
assigned to minor metabolites in the bile were observed in the
chromatograms of the bile samples from dogs E, F and G (studies 911).
Typical chromatograms obtained after acid and 3glucuronidase enzyme
hydrolysis of bile are presented in Fig. 67.
Norbuprenorphine rearranges in acid to form
demethoxynorbuprenorphine. When bile was analysed by HPLC following acid
hydrolysis, the peak with the lowest retention time had the same
Figure 65. Plots of the cumulative amounts ( IBM, yg) of the
hepatically formed metabolite M excreted in bile against time (min). The
solid lines represent the cumulative amounts of the metabolite excreted
in bile calculated in accordance with equation 58 where the metabolic
clearance of the parent compound and the apparent volume of the
distribution of the metabolite were estimated from the multiple linear
regression of the cumulative amounts of the conjugate excreted in bile
against area under the plasma concentrationtime curve of the parent
compound and the meatbolite concentration in plasma, a) Dog E at 4.0864
mg/kg IV infusion dose of buprenorphine, Study #9; b) Dog F at 4.0864
mg/kg IV infusion dose of buprenorphine, Study #10; c) Dog G at 3.7408
mg/kg IV infusion dose of buprenorphine, Study #11. The estimated
metabolic clearances wre 182, 261 and 220 ml/min and the apparent
volumes of distribution of the metabolite were 13.6, 281 and 67.6 L
respectively.
XBfljJGS IBM UGS
199
Figure 66. Plots of excretion rate ( Au/ At, p g/min) of either buprenorphine or
metabolite (M) excreted in bile or urine against the plasma concentration of
buprenorphine or M (ng/ml) in accordance with equation 26. a) For the 3.847 mg/kg
IV infusion dose of buprenorphine in dog D (Study #8), the estimated renal
clearance of buprenorphine was 0.8 ml/min. b) For the 3.7408 mg/kg IV infusion
dose of buprenorphine in dog G (Study #11), biliary clearance of metabolite was
estimated as 1146 ml/min. c) For the 4.8 mg/kg IV infusion dose of buprenorphine
in dog E (Study #9), apparent biliary clearance of buprenorphine as hepatically
eliminated metbolite was estimated as 184 ml/min. d) In the same study #9, biliary
clearance of buprenorphine as unchanged drug was estimated as 0.22 ml/min.
DB/DT (JG/P1IN OBM/OT jJG'MN
400
360 _.
32tl .
280
2*10..
2110  .
160  
120
80
40
40
£
h 1 1 1 1 y
60 120 180
M TMID CNG/MLJ
'O
201
Figure 67. The chromatographic peaks corresponding to 1, 4, 5 and 6 are
the aglycones generated from the conjugated metabolites following
3glucuronidase hydrolysis (l=buprenorphine; 4=norbuprenorphine;
5,6=unknown metabolites). The chromatographic peaks 3, 7, 8, 9 and 10
correspond to rearranged aglycones generated after acid hydrolysis of
the conjugated metabolites (3=demethoxybuprenorphine;
7=demethoxynorbuprenorphine; 8=rearranged aglycone of conjugated
metabolite 5; and 9,10 are the two rearrangement products presumably
generated from the aglycone (6) upon acid hydrolysis, a) Chromatograms
obtained after 8glucuronidase hydrolysis of buprenorphine and other
minor metabolite conjugates. The peaks 1 and 4 correspond to the
aglycones buprenorphine and norbuprenorphine respectively. The peaks 5
and 6 correspond to aglycones of unknown minor metabolites, b) The
standard chromatogram of bile spiked with norbuprenorphine (4, 60 ng/ml)
and buprenorphine (1, 60 ng/ml). c) Chromatograms obtained after acid
hydrolysis of conjugates of buprenorphine and other minor metabolites
(3=demethoxybuprenorphine; 7=demethoxynorbuprenorphine; 8,9 =acid
rearrangement products of the aglycones (5 and 6) generated from the
conjugates of these minor metabolites). d) The peaks 4 and 5 from the
chromatogram (a) were collected by HPLC separation and subjected to acid
hydrolysis. The resulting hydrolysed products were chromatographed by
HPLC separation and flúorimetric detection (7=demethoxynorbuprenorphine;
8=acid rearranged product of the minor metabolite 5 generated from the
respective conjugate). e) Standard chromatogram of
demethxoynorbuprenorphine (7) obtained after acid hydrolysis of bile
spiked with 50 ng/ml of norbuprenorphine (4). f) The peak 6 from
chromatogram (a) was collected by HPLC separation and subjected to acid
hydrolysis. The resulting solution was chromatographed by HPLC
separation and flúorimetric detection. This gave two peaks 9 and 10.
203
204
retention time as a reference standard of demethoxynorbuprenorphine (see
Fig. 67 and the legend). Norbuprenorphine was not observed in bile
before acid or enzymatic hydrolysis. These results indicate that the
aglycone norbuprenorphine generated following enzymatic hydrolysis or
the rearranged demethoxynorbuprenorphine generated after acid hydrolysis
were presumably derived from the conjugate of norbuprenorphine. These
findings were confirmed (see Fig. 67) by acid and 3glucuronidase
hydrolysis using a standard sample of norbuprenorphine (obtained front
Reckitt & Colman Co., KingstonUponHull, England). The other two
unidentified minor metabolites were hypothesized to be conjugates as
they were extractable from the bile only after acid hydrolysis. None of
the minor metabolites were detectable in plasma or urine
(norbuprenorphine detection limit, 5 ng/ml).
Plots of the cumulative amounts of the minor metabolites collected
in bile are shown in Figs. 6870. In these plots, the cumulative amount
"til
of the 1 1 metabolite was estimated from the expression:
N
hZ
j=i
(PHRi X DF X V)
Eg. 59
where Pffit is the peak height ratio of the i^ metabolite
(buprenorphine was the internal standard), DF is the dilution factor, V
is the volume of bile collected at each interval, and N is the number of
bile samples. The total amounts of the norbuprenorphine collected in
these studies (911) were 0.936, 0.744 and 1.07 mg respectively. Thus
the percentage of the total dose excreted in bile as norbuprenorphine
conjugate were 1, 0.83 and 1.2 respectively.
Figure 68. Plots of the cumulative amounts of the minor acid hydrolyzable
conjugates of buprenorphine, norbuprenorphine conjugate, 4, (O), conjugates of
unknown metabolites, 5, (□) and 6 (\>) excreted in bile against time for the 4.8
mg/kg IV infusion dose of buprenorphine (Study #9) in dog E. Ai's of the left
ordinate represent the summation of PHR X DF X V where PHR = peak height ratios
obtained by acid hydrolysis, HPLC separation followed by flúorimetric detection of
these minor metabolites using buprenorphine as internal standard; DF= dilution
factor; V = volume of bile excreted during a bile collection interval. (See also
Fig. 67 for these chromatograms). Calibration curve using norbuprenorphine as
internal standard gave the actual amounts of the acid hydrolyzable conjugate of
norbuprenorphine (4) excreted in bile. The right ordinate represents these
cumulative amounts of the norbuprenorphine conjugate, 4, (O) excreted in bile in
mg.
8GGD_
1.00
O O
e b
H
960
O O O O
.. 0.75
g B B B
0.50
I 1 h
0.25
I28G I6GQ
206
Figure 69. Plots of the cumulative amounts of the minor acid hydrolyzable
conjugates of buprenorphine, norbuprenorphine conjugate, 4, (O)/ conjugates of
unknown metabolites, 5, (□) and 6 ((\>) excreted in bile against time for the
4.0864 mg/kg IV infusion dose of buprenorphine (Study #10) in dog F. Ai's of the
left ordinate represent the summation of PHR X DF X V where PHR = peak height
ratios obtained by acid hydrolysis, HPLC separation followed by fluorimetric
detection of these minor metabolites using buprenorphine as internal standard; DF
dilution factor; V = volume of bile excreted during a bile collection interval.
(See also Fig. 67 for these chromatograms). Calibration curve using
norbuprenorphine as internal standard gave the actual amounts of the acid
hydrolyzable conjugate of norbuprenorphine (4) excreted in bile. The right
ordinate represents these cumulative amounts of the norbuprenorphine conjugate, 4
(O) excreted in bile in mg.
208
Figure 70. Plots of the cumulative amounts of the minor acid hydrolyzable
conjugates of buprenorphine, norbuprenorphine conjugate, 4, (O)t conjugates of
unknown metabolites, 5, (□) and 6 (t\) excreted in bile against time for the
3.7408 mg/kg IV infusion dose of buprenorphine (Study #11) in dog G. Ai's of the
left ordinate represent the summation of PHR X DF X V where PHR = peak height
ratios obtained by acid hydrolysis, HPLC separation followed by fluorimetric
detection of these minor metabolites using buprenorphine as internal standard; DF
dilution factor; V = volume of bile excreted during a bile collection interval.
(See also Fig. 67 for these chromatograms). Calibration curve using
norbuprenorphine as internal standard gave the actual amounts of the acid
hydrolyzable conjugate of norbuprenorphine (4) excreted in bile. The right
ordinate represents these cumulative amounts of the norbuprenorphine conjugate, 4
(O) excreted in bile in mg.
210
211
The sigma minus plots for these minor metabolites in terms of the
amounts are shown in Fig. 71. These plots were apparently linear
during the bile collection period. The apparent rate constants estimated
from these plots are given in figure legends. These rate constants were
similar among the minor metabolites and also corresponded with the rate
constant obtained from the sigma minus plot of the major metabolite (see
Fig. 63 and the legend). These results indicate parallel conjugation of
all the metabolites in the liver.
Figure 71. Sard logarithmic plots of the theoretical amounts of the minor
metabolites (norbuprenorphine, O; unknown metabolites, □ and tb )
against time, a) The estimated apparent rate apparent rate constants in
accordance with equation 20 were 0.0027/min (O)/ 0.0034/min (□), and
0.0036/min (11s) in dog E at 4.8 mg/kg IV infusion dose of buprenorphine
(Study #9). b) In dog F at 4.0864 mg/kg IV infusin dose of
buprenorphine, the estimated apparent rate constants were 0.0023/min
(O) f 0.0025/min (□) and 0.002/min ((\). c) In dog G at 3.7408 mg/kg IV
infusion dose of buprenorphine, the estimated apparent rate constants
were 0.0021/min (O)f 0.0019/min (□), and 0.0021/min (bj . Note the
correspondence of these rate constants with the rate constants obtained
for the major metabolite M in respective studies (see legend for Fig.
63).
213
's.~
O,
XD
ti.
□
—I
I6QQ
1440
PHARMACOKINETICS OF THE IV ADMINISTERED METABOLITE
Semilogarithmic plots of plasma concentrations of the metabolite
against time after 7 and 8.5 min constant rate infusion of the
metabolite in studies 13 and 14 in dogs F and G respectively are shown
in Figures 72,73 (Table 7). Dog G was bile cannulated and the bile was
collected up to 22 h in this dog (Study #14).
The postinfusion plasma concentrations of the metabolite were
fitted to a sum of three exponentials in accordance with equation 15
54
using the computer program of Yamaoka et. al. (Appendix I). The
plasma concentrations were weighted by their inverse values. The
validity of the triexponential equation 15 was confirmed by
demonstration that regressions of the weighted residuals (calculated in
accordance with the equation 3) against the logarithm of the estimated
concentrations of the metabolite gave mean residuals C, slopes and
intercepts which were not statistically significantly different from
zero (Fig. 74, Table 7).
The parameters of the sum of three exponentials that best fit the
postinfusion plasma data of buprenorphine are listed in Table 7. In
addition, the calculated parameters (equations 1518) for an equivalent
IV bolus administration are given. The terminal halflives of the IV
administered metabolite were 392 and 319 min respectively in dogs F and
G (Studies 13,14). The estimated average terminal halflife of the
metabolite in plasma following IV administration of the parent drug was
1846 min (Table 2). The fact that terminal halflife of the IV
214
Figure 72. Semilogarithmic plots of the concentrations (ng/ml) of intravenously
administered buprenorphine conjugate (M, metabolite) in plasma against time (min)
for the 1.889 mg/kg dose in the 20.2 kg nonbile cannulated dog F (Study #13,
Table 7). The solid line represents the curve obtained by fitting the experimental
plasma data to equation 50. The inset is the representation of the data and the
fitted curve for the initial 650 min.
1W/SN
216
Figure 73. Semilogarithmic plots of the concentrations (ng/ml) of the
intravenously administered buprenorphine conjugate (M, metabolite) in plasma upon
constant rate (1.7738 mg/min) IV infusion of the conjugate plotted against time
(min) for the 0.5153 mg/kg dose in 24.2 kg bilecannulated dog G (Study #14, Table
7). The solid line represents the curve obtained by fitting the experimental
jugular vein plasma data to equation 50. The inset is the representation of the
data and the fitted curve for the initial 500 min.
1W/SN
MIN
28Ü
I I2Ü
M[1U
218
219
Table 7. Pharmacokinetics of TV administered Metabolite.
Parameter
Dog F
Dog G
Study #
13
14
Dog No.
RB535
R5107
kQ (mg/min)a
T (min)b
4.4944
1.7738
8.49
7.03
Dose (mg)
38.16
12.47
Weight, Kg.
20.2
24.2
Dose mg/kg
1.889
0.5153
Parameters from plasma data for the metabolite.
104 P'£C
4.32
1.80
105 A'
106 B'f
8.12
3.00
4.80
5.00
10 2 ^
5.78
17.0
9
(4)
(12)
10z a
1.19
2.8
(25)
(58)
10° 6
1.77
2.2
(319)
(392)
104 P, e
105 A
106 B
5.47
8.53
3.01
3.06
5.27
5.04
10' AUC. 1
105 AUC° 9
8.22
1.17
6.1
0.632
Too
Residual plots
102 0. h
2.2
0.9
Slope1
0.00640.02
0.00150.03
Intercept1
0.040.07
0.00630.06
Clearances (ml/min)
Cl ^
11 tot
55(5455)
166(162176)
ciM 1
ren
23(93)
31(18)
P1M m
U1B
—
151n
% Recoveries of the metabolite in urine and bile.
UooM/dose°
38 9.5
BooM/dose^
— 91.3
Volumes of
distribution of the metabolite (L).
V *
c
1.75(1.6) 2.75(2.75)
V
31 76.5
220
Table 7. Continued . . . .
a Dose in mg corrospond to buprenorphine conjugate assayed by acid
hydrolysis followed by HPLC separation and fluorimetric detection.
Time of infusion in min.
Q
P'f, A'f, and B'^ are the intercepts obtained by fitting the
postinfusion plasma data to equation 15 by nonlinear least square curve
fitting using the computer program of Yamaoka et al. (Appendix I) where
plasma concentrations were weighted by their inverse values. The
intercepts are expressed as fractions of the total dose per ml of
plasma.
^ Unit for it , a and 3 values is min . Parenthetical values corrospond
to halflives in min.
Pft Aand values were calculated using equations 1618 and
expressed as fractions of the total dose per ml of plasma.
The theoretical area under the plasma concentrationtime curve of
buprenorphine during the infusion phase was calculated lasing equation
52.
^ Postinfusion area under the plasma concentrationtime curve was
obtained by integrating equation 15 between T to oo, AUC =
(P'/tt ) + (A1 / a ) + (B'/ 3). 100
Mean of the weighted residuals calculated in accordance with the
equation 3. None of the mean residuals were statistically significantly
different from zero.
1,) Slopes and intercepts of the plots of 6 against log fitted
postinfusion data. The parenthetical values corrospond to standard
errors. Both slopes and intercepts were not statistically significantly
different from zero, indicating that the sum of three exponentials best
fits the postinfusion plasma data of buprenorphine conjugate.
Ratio of the infused total dose to the total area under the plasma
concentrationtime curve of buprenorphine conjugate. The AUC was
calculated from equations 52 and 56. The values in parenthesis
correspond to 95% confidence limits.
1 Estimates from the initial slopes of the cumulative amounts of
buprenorphine conjugate excreted ( £U, yg) renally against the area
under the plasma concentrationtime curve of the conjugate in accordance
with the equation 30. Values in parenthesis corrospond to EU at AUC=0
from the best linear plots of the data shown in Figs. 77,78.
m Clg was calculated from a plot of the cumulative amount of the
conjugate excreted in bile against the area under the plasma
concentration time profile of the conjugate.
221
Table 7. Continued . . . .
This biliary clearance was calculated from the expression, f.Cl ,
where f is the fraction of the dose excreted in bile. The biliary
clearance, estimated from a plot of the cumulative amounts of the
systemic metabolite excreted in bile was 300 ml/min, see Fig. 77d. This
clearance estimted from the initial slope was bile flow dependent (Fig.
79a).
°'p Percent recoveries of the conjugate in urine and bile obtained from
the quotient of the amounts recovered in urine or bile and the total
infused dose of the conjugate.
^ Apparent volume of distribution of the central compartment estimated
by fitting the postinfusion data to equation 50 by nonlinear least
square regression (Appendix I). The values in parenthesis were
calculated from equation 10, where the values of P, A and B were
obtained through equations 1618.
r V. was calculated from the ratio of Cl. , / r
d tot v
222
Figure 74. Plots of the weighted residuals against the logarithm of the
calculated plasma concentrations. The weighted residuals were calculated
in accordance with equation 3:
6 = ( ^exp “ ^calc5 / ^calc
a) The residuals calculated for the postinfusion jugular vein plasma
data of buprenorphine conjugate (M, metabolite) administered by constant
rate (4.4944 mg/min) IV infusion for the 1.889 mg/kg dose study in 20.2
kg nonbilecannulated dog F (Study #13, Table 7); b) The residuals
calculated for the postinfusion jugular vein plasma data of
buprenorphine conjugate (M, metabolite) administered by constant rate
(1.7738 mg/min) IV infusion for the 0.5153 mg/kg dose in 24.2 kg
bilecannulated dog G (Study #14, Table 7). The observed residual means,
slopes and intercepts were not statistically significantly different
from zero as confirmed by ttest. The random distribution of the
residuals above and below the regression line indicated no bias in the
fittina of the chosen model.
223
administered metabolite was shorter than the hepatically derived
metabolite supports the hypothesis that the ratedetermining step for
metabolite elimination on buprenorphine administration is not the
elimination of the metabolite. The slowest process, the rate of return
of the drug from the deepest tissues, must be the rate determining step
in overall disposition of the drug and the hepatically derived
metabolite.
The volumes of distribution of the central compartment V , were
obtained by fitting the postinfusion data to equation 50 using the
54
computer program of Yamaoka et.al. (Appendix I, see also Figs.
72,73). The values of Vc estimated by this procedure were 1.72 and 2.75
L in dog F and G (Studies 13, 14) respectively. The V was also
estimated from equation 10, where the values of the parameters P, A and
B were obtained through equations 1618. The estimated apparent volumes
of distribution of the central compartment by this procedure were 1.6
and 2.75 L respectively in dogs F and G (Studies 13 and 14).
The estimated total clearances (Cltot =Dose/AUCoo, see equations
5156 for AUC estimations) for the metabolite were 55 and 166 ml/min
respectively in dogs F and G (Studies 13 and 14). The 95% confidence
limits for these clearances are reported in Table 7. The overall
apparent volumes of distribution of the metabolite in the body estimated
in accordance with equation 13 were 31 and 76.5 L respectively in dogs F
and G (Studies 13 and 14).
Urinary and biliary excretion of the metabolite. The sigma minus
plots (equations 2024) for the metabolite excretion in bile and urine
are given in Fig. 75 and the estimated apparent rate constants are
reported in the figure's legend. Metabolite excretion in bile had an
Figure 75. Semilogarithmic plots of the buprenorphine conjugate (M,
metabolite) remaining to be excreted in bile/urine versus time (sigma
minus plot) in accordance with equation 20. a) Semilogarithmic fitting
of the postinfusion urine data to a sum of two exponentials for the
1.889 mg/kg IV infusion dose of the metabolite in the
nonbilecannulated dog F (Study #13, Table 7). The estimated hybrid
rate constants were 0.0229/min (halflife = 30 min) and 0.0012/min
(halflife = 578 min). b) Sigma minus plot of the postinfusion biliary
excretion of the metabolite for the 0.5153 mg/kg IV infusion dose of the
metabolite in the bilecannulated dog G (Study #14, Table 7). The
apparent rate constant for this monoexponential fit was 0.0051/min
(halflife = 139 min). In the same dog G, the sigma minus plot of the
postinfusion urinary excretion of the metabolite could be fitted to a
sum of 2 exponentials. The estimated hybrid rate constants were
0.0216/min (halflife = 32 min) and 0.005/min (halflife = 141 min).
sari sari' wai^ua 3 sari um"Vinz
225
226
estimated apparent rate constant of 0.05 min 1 (halflife = 140 min),
an intermediate value between second distributional and terminal
halflives obtained from the plasma data (See Table 7). Urinary and
biliary excretion rate plots were scattered; the two examples which
followed equation 24 are given in Fig. 76. The rate constants obtained
from these plots are reported in the legends of figures 75 and 76. They
corresponded to the second distributional and terminal halflives
obtained from plasma data (See also Table 7).
Renal and biliary clearances of the metabolite. The percentages of
the total intravenously administered conjugate dose excreted in urine
were 38 and 9.5% respectively in dog F (Study #13, nonbile cannulated)
and dog G (Study #14, bile cannulated). In dog G, the percentage of the
metabolite excreted in bile was 91.3. About 80% of the administered
metabolite was recovered in the bile of dog G (Study #14) within the
first 2 h. This is in constrast to the pharmacokinetics of the
intravenously administered morphine glucuronide which was solely
. . 45
eliminated in the urine.
Biliary and renal clearance plots for the intravenously
administered conjugate in bile and urine are shown in Figs. 77 and 78
for dogs F and G (Studies 13 and 14) respectively. The plots for dog G
showed two distinctly different linear segments (Fig. 77 c,d). The
biliary and urinary clearances of the metabolite estimated from the
initial slopes were 300 and 31 ml/min respectively. However the
clearances estimated from the expression (f.Cltot) where f is the
fraction of the dose excreted through a given elimination route, the
biliary and urinary clearances in dog G were 151 and 16 ml/min
respectively. The higher biliary and urinary clearances of the
227
Figure 76. Semilogarithmic plots of the excretion rate (A BM/A t or
AU/ At) of the buprenorphine conjugate (M, metabolite) plotted against
tmid, the mid point of the biological fluid collection interval in
accordance with equation 24. a) For 0.5153 mg/kg IV infusion dose of the
metabolite in dog G (Study #14, Table 7), the biliary excretion data was
fitted to a sum of three exponentials. The estimated hybrid rate
constants were 0.0213/min (halflife = 33 min), 0.0079/min (halflife =
88 min) and 0.0021/min (halflife = 330 min). See also Table 7 for the
correspondence of these estimated rate constants to those obtained from
plasma data, b) In the same dog G (Study #14), the urinary excretion
data for the metabolite was fitted to a sum of two exponentials. The
estimated hybrid rate constants were 0.0215/min (halflife = 32 min) and
0.004/min (halflife = 173 min).
Figure 77. Plots of the cumulative amounts (z BM, z U, y g) of the intravenously
infused buprenorphine conjugate (M, metabolite) in accordance with equation 30. a)
For 0.5153 mg/kg IV infusion dose of the metabolite in the bilecannulated dog G
(Study #14, Table 7) the apprent urinary clearance of the conjugate estimated from
the initial slope was 31 ml/min. b) In the same dog G (Study #14), the biliary
clearance estimated from the initial slope was 300 ml/min. c, d) Complete profiles
of the clearance plots for the metabolite in urine and bile respectively in dog G
(Study #14).
sari uai sari uaz
229
230
Figure 78. Plot of the cumulative amount of the intravenously infused
buprenorphine conjugate (M, metabolite) excreted in urine against the
area under the plasma concentrationtime curve in accordance with
equation 30. For the 1.889 mg/kg IV infusion dose of the conjugate in
the nonbilecannulated dog F (Study #13, Table 7), the estimated
urinary clearance of the metabolite was 23 ml/min.
231
metabolite during the initial period (upto 4 h) was attributable to the
bile and urine flow dependent biliary and urinary clearances of the
metabolite (see Fig. 79a,b). However, urinary clearance was not pH
dependent (Fig. 79c).
The clearances estimated from the plots of the urinary or biliary
excretion rates against the plasma concentration of the metabolite at
the midpoint of the biological fluid collection interval (Fig. 80)
corresponded with the clearanaces estimated from the sigma minus plots
(See figure legends 77,78 and 80).
Oral Bioavailability of Buprenorphine in Dogs.
Buprenorphine is almost completely hepatically metabolized in dogs.
First pass metabolism is ancitipated upon oral administration. The
amount, A, of the orally administered dose Xg, that eventually reaches
the systemic circulation unchanged is:
A = (1f. )
ípm
f X
a 0
= f.X„
Eg. 60
where f. is fraction of the dose eliminated by firstpass metabolism
ípm
before reaching the systemic circulation, f is fraction of the dose
administered that is absorbed unchanged and f is the fraction of the
dose that eventually reaches the systemic circulation intact. The
equation 60 is valid if there is no saturable firstpass liver
metabolism and no gut wall metabolism. Since buprenorphine showed
doseindependent pharmacokinetics in the dose range studied, the total
body clearance of the drug administered IV is
Cl
IV
tot
= XÍ7 / AUCIV
0 oo
Eq. 61
For the same drug administered orally, assuming that there is not gut
Figure 79. Plots of the apparent biliary/urinary clearances of the
buprenorphine conjugate (M, metabolite) against bile/urine flow and
urine pH. The apparent urinary/biliary clearances were calculated from
the quotient of the excretion rate and the plasma concentration of the
metabolite at the mid point of the biological fluid collection interval.
The respective slopes and intercepts are: a) For the 0.5153 mg/kg IV
infusion dose of the conjugate in the bilecannulated dog G (Study #14,
Table 7), the estimated slope of the plot of the biliary clearance
against bile flow was 5276 +_ 212. This was statistically significant
slope as confirmed by ttest. b) In the same dog G (Study #14), the
estimated slope of the plot of the urinary clearance against the urine
flow was 5.2 +_ 1.3. This slope was also statistically significant as
confirmed by ttest. c) In the same dog G (Study #14) the slope of the
plot of urinary clearance against pH was 15 +_ 5.5 which was not
statistically significant.
6.24 6.68 1.12 1.56
CL REN ML/MIN
CL REN ML/MIN
CL 811 E ML/MIN
— — bjLOLjtkXkincn
oiNjcDHdcnMaoAO
I 1 1 1 k 1 1 1 1 1
/ O
o
o
o
O '
o o
o
3
0
6
\
4?
o
o
—  MuicjAAincn
tnpoco.azacnr'jcnxiO
— — N N) N) U U ^
a a>KjmcaAa>£jjr>:n
oaoociaocicia
CD
C*
U
ci¬
en
CD
co
r i
m
o
a. _i
r~ ro
\
u
CT.
CD
m
4 1 1 1 1 1 1 1 1 1
\
o
A
Óv
U» '
\o
0)
O V
'P
ro
uj
oj
83.88
Figure 80. Clearance plots of the amounts of the buprenorphine conjugate
(M, metabolite) excreted in urine or bile per unit time against the
plasma concentration of the metabolite at the mid point of the
collection interval, a) For the 1.889 mg/kg IV infusion dose of the
metabolite in the nonbile cannulated dog F (Study #13, Table 7), the
estimated urinary clearance was 14.3 ml/min. b) The urinary clearance of
the metabolite in dog G (Study #14) was estimated as 36 ml/min. c) In
the same dog the biliary clearance plot showed curvature. The values
outside the parenthesis correspond to the instantaneous biliary
clearance and values within the parenthesis correspond to the mid point
of the bile collection interval in min.
Niw/3fí iQ/na n!u/3(1 iQ/na Niu/ort^Wraa
235
2D
16
18
M
12
ID
8
6
O /
/
/ O
/
/
/
“"(3
«£
34 D
b
H 1 1
68Q ID2D
M THID NG^ML
—I—
13RD
O
—I
HDD
2CD T
16C  _
I6D..
140..
120..
IQ0
80
60
40  
O 322.59(46.6)
O 396.8(83.9)
C
20 0 258.7(114.7)
<*£
+
340
S8G
1020
M 7M!0 NG'HL
105.8(12) O
—I 1 1
1360 1000
236
wall metabolism and no saturable firstpass metabolism
Eq. 62
Since total body clearance is the same irrespective of the route of
administration, equations 61 and 62 can be equated and on rearrangement,
the fraction of the dose that eventually reaches the systemic
circulation is
.oral
X™ AUC°r*
0 oo
f = (1f. ) f = ——T
lpm a Yoral
0
Eq. 63
.IV
oo
Plots of the plasma concentrationtime profile of buprenorphine and
conjugate are presented in Fig. 81 for dog B and D following 84.7 and
87.11 mg oral doses of buprenorphine respectively. In dogs B and D
(Study #15,16), the apparent bioavailability f of buprenorphine
estimated in accordance with equation 63 (Table 8) were 6 and 3.7%
respectively.
If the following assumptions are valid, i.e., a) the drug is
eliminated from the body virtually completely by hepatic metabolism, b)
there is no gut wall metabolism, c) there is no saturable firstpass
metabolism, d) when the drug is delivered to the liver through either
portal vein or hepatic artery, the extent of firstpass metabolism is
the same, and e) a constant fraction of the hepatically derived
metabolite reaches the systemic circulation, then the areas under the
plasma concentrationtime curves of the metabolite obtained after oral,
AUC^(©ral)' 311(1 IV administration, AUC^, of the parent
compound can be compared to estimate the fraction of the dose absrobed
unchanged:
Figure 81. Semilogarithmic plots of the plasma concentrations of buprenorphine (a,
c) and metabolite (b, d) against time following oral administration of
buprenorphine. a) Buprenorphine in plasma following 84.7 mg oral dose of
buprenorphine in in dog B, study #15. b) Metabolite in plasma in the dog B in the
same study (#15). c) Buprenorphine in plasma following 87.11 mg oral dose
buprenorphine in dog D, study #16. d) Metabolite in plasma in dog D in the same
study (#16).
238
TU/9N
1U/3N
1U/3N
1U/9H
239
Table 8. Oral bioavailability of buprenorphine.
Parameter
Dog B
Dog D
Study #
15
16
Dog No.
B344
W4123
Weight, Kg.
17.8
22.6
TV Dose (mg)
83.46
86.94
Oral Dose (mg)
84.7
87.11
Areas under the plasma
concentration
time curve.
IV a
AUC
oo
391290
572107
AUC0ral
OO
23706
21207
AUC^
1(IV)
163454
370001
AUCi(oral)
16549
74289
f b
0.06
0.037
f C
a
0.1
0.2
f. d
ípm
0.40
0.82
The areas under the plasma concentrationtime curve up to the last
observed plasma point were measured by the trapezoidal rule. The areas
from the last observed plasma sampling time to infinity were estimated
from the ratio of the last plasma concetration and g , where g was
estimated from the semilogarithmic plot of terminal plasma
concentrations against time following IV administration. Same g values
were used for the AUC estimations of the metabolite concentrations in
plasma. Unit for area: ng.min/ml.
Fraction of the absorbed dose that eventually reaches the systemic
circulation unchanged,
c
Fraction of the orally administered dose absorbed unchanged (that is
delivered to the liver intact). The values were estimated in accordance
with equation 64.
Fraction of the absorbed drug that undergoes first pass metabolism
(Eg. 65).
240
f
a
= Aüdf, ,,
1(oral)
xf /
AUC^
C1(IV)
.oral
‘O
Eq. 64
Thus the calculated percentages of the dose absorbed were 10 and 20
respectively in dogs B and D (Study #15,16, Table 8), and the fractions
of the doses cleared by firstpass metabolism before reaching the
systemic circulation, estimated in accordance with the equation
f. = 1  (f/f ) Eq. 65
lpm v a' ^
were 40 and 82% respectively in dog B and D (Study #15,16). The extent
of first pass metabolism estimated from the ratios of the total body
clearance and hepatic blood flow (360400 ml/min in a 20kg dog^)
were 0.6 and 0.43 in dogs B and D. This contradiction may be explained
by one or more of the above assumptions not being valid. Studies with
37 38
rat gut preparations ' have demonstrated gut wall metabolism of
buprenorphine. Thus any significant gut wall metabolism would
overestimate the firstpass metabolism calculated in accordance with
equation 65.
SUMMARY AND CONCLUSIONS
The pharmacokinetic dispositon of buprenorphine in dogs can be
adequately described by a 3compartment body model (Figs. 49).
Buprenorphine readily distributes into shallow and deep compartments
following rapid IV bolus injection. The proper estimations of the
terminal rate constants and the total body clearances were not feasible
following acute IV bolus (0.72.6 mg/kg, studies 16) doses of
buprenorphine. This was attributed to the analytical sensitivity which
did not permit estimations of buprenorphine plasma concentrations below
5 ng/ml. The fact that doses (1.22.6 mg/kg) of buprenorphine
administered in the first 5 IV bolus studies exhibited significant side
effects gave an upper limit to the maximal IV bolus dose that could be
administered. To minimize initial peak plasma concentrations of
buprenorphine and the associated side effects, buprenorphine was
administered to six dogs (studies 712) by slow IV infusion which
delivered larger amounts of the drug to the animal and provided adequate
number of quantifiable terminal plasma concentrations.
Buprenorphine was shown to bind to the plastic material of the
indwelling catheter used in the slow infusion of buprenorphine into the
veins of dogs. This resulted in artifactually higher levels of
buprenorphine in blood samples drawn through the same catheter
immediately following cessation of infusion. This necessitated drawings
of blood from a different vein (vena brachialis or jugularis) for the
time intervals duringinfusion as well as postinfusion.
241
242
The estimated terminal halflives and the derived total body
clearances from these IV infusion studies averaged 2080 _+ 110 min (SEM,
n=4) and 212 +_ 10 ml/min (SEM, n=6) respectively.
Only a minor fraction (<0.5%) of the intravenously administered
buprenorphine was eliminated unchanged in the urine. There was no renal
clearance when urine pH was above 7.2 (Fig. 57). Assuming that only the
unionized buprenorphine (pKa' = 8.24) can undergo renal tubular
reabsorption, then lower renal clearance can be anticipated at higher pH
values. This is supported by the fact that in dog B (study #3) and dog E
(study #9), no drug was observed in the urine at pH values above 7.3,
indicating complete tubular reabsorption of buprenorphine at these
higher pH values. However, since such small fractions of buprenorphine
are excreted in urine, therapeutic acidification of urine is not a valid
measure of counteracting narcotic toxicity due to accidental overdosage.
Also, changes in the renal clearance due to pH variability would not
have significant effects on the doseindependent pharmacokinetics of
buprenorphine. The renal clearance of buprenorphine was not urine flow
dependent.
The dosenormalized plasma concentrations were superimposable
(0.74.8 mg/kg IV bolus and infusion doses, Figs. 1114), demonstrating
doseindependency in the dose range studied.
Buprenorphine conjugate was the only metabolite detectable in
plasma. The plasma concentrationtime data of this metabolite paralleled
the the plasma concentrationtime data of buprenorphine (Fig. 15).
Following IV bolus administration of buprenorphine, the highest plasma
concentrations of the metabolite occured within 2 min (Fig. 15). In TV
infusion studies, the highest concentrations of the metabolite occurred
243
immediately after the cessation of infusion of buprenorphine (Figs.
4952) and were coincident with the maximal buprenorphine concentrations
in plasma. The parallel decays of buprenorphine and the conjugate in
plasma (Fig. 15) during the distributive phase indicated that the rate
determining step in the plasma decay of the conjugate is the rate of
return of the drug from the shallow compartment. Similarly during the
terminal elimination phase, the rate determining step in the plasma
decay of both buprenorphine and its metabolite is the slow return of
buprenorphine from the deep compartment to the central compartment where
it could be metabolized.
In bile cannulated dogs (studies 911), no detectable plasma
concentrations of the metabolite were observed after 16 h following
initiation of infusion. However, when the bile catheter was removed at
26 h and the screwcap (Fig. 1) was replaced, the bile flowed normally
into the duodenum and the metabolite reappeared in plasma. This strongly
indicated enterohepatic recirculation. Upon intraduodena1
administration of the buprenorphine conjugate, it appeared in plasma,
providing further evidence for the enterohepatic recirculation of the
metabolite. In the same study, a minor fraction (6.2%) of the
intraduodenally administered metabolite was recovered in bile as
conjugate. Free buprenorphine was not detected in plasma, but observed
in small amounts in urine. These results confirmed that the
enterohepatic recirculation of buprenorphine conjugate would have
negligible effects on the terminal halflife of buprenorphine.
The fraction of the IV administered buprenorphine that was
recovered as conjugate in the bile of dogs (E, F and G) averaged 92.6 +_
1.8 % (SEM). About 0.7% of the IV administered buprenorphine was
244
recovered in urine as conjugate. A minor fraction (<0.5%) of the
administered buprenorphine was recovered unchanged in the bile of dogs E
and F (studies 9, 10). This may be due to the enzymatic hydrolysis of
the conjugate in bile. No free buprenorphine was detected in the bile of
the third dog (study #11).
Three additional HPLC peaks that could be assigned to minor
metabolites were observed in the chromatograms of the bile samples.
These were hypothesised to be conjugates since they were extractable
from the bile only after acid or enzymatic hydrolysis. When the bile was
analysed by HPLC following acid hydrolysis, the peak with the lowest
retention time (Fig. 67) had the same retention time as a reference
standard of demethoxynorbuprenorphine. Norbuprenorphine rearranges in
acid to form demethxoynorbuprenorphine. Norbuprenorphine was not
observed in bile before acid or enzymatic hydrolysis. These results
indicated that the aglycone generated following enzymatic hydrolysis, or
in fact, the rearranged demethoxybuprenorphine generated after acid
hydrolysis were presumably derived from the conjugate of
norbuprenorphine.
Pharmacokinetics of intravenously administered buprenorphine
conjugate conformed to a 3compartment body model (Scheme II). The
terminal plasma halflives estimated in two studies were 5.25 and 6.5 h
respectively (in dogs F and G, studies 13 and 14). The estimated average
terminal halflife of the conjugate in plasma following TV bolus
administration of buprenorphine was 31 h (Table 2). The fact that the
terminal halflife of the IV administered metabolite was shorter than
the hepátically formed metabolite (following IV administration of
buprenorphine) confirmed the hypothesis that the slowest process, the
245
rate of return of buprenorphine from the deep compartment, must be the
rate determining step in the overall disposition of the hepatically
formed metabolite.
In one study (dog G, study #14), the percentage of the IV
administered metabolite excreted in the bile was 91.3%. About 80% of the
IV administered metabolite was recovered in bile within the first two
hours. These results are in contrast to the pharmacokinetics of
intravenously administered morphine glucuronide which was solely
. . 45
eliminated in the urine.
The total body clearances estimated from the ratios of the doses of
the drug to the total areas under the plasma concentrationtime curve of
buprenorphine averaged 220 ml/min in the bile cannulated dogs E, F and
G. The metabolic clearances of buprenorphine estimated in accordance
with equation 58 averaged 220 ml/min in these three dogs (Table 5). The
estimated biliary clearances of the intravenously administered
metabolite in dogs F and G were 32 and 151 ml/min respectively (Table
7). In all studies, the plasma metabolite data following the IV
administration of the parent compound paralleled the buprenorphine
plasma data (Figs. 3944, 4952). These results led to the hypothesis
that when buprenorphine was delivered to the liver hepatocytes through
the hepatic artery or portal vein, the conjugate formed at the initial
stages of buprenorphine transit through the liver presumably reaches
rapid equilibration with the systemically circulating metabolite. As the
drug is transported deeper into the liver tissues, the formed metabolite
is presumably "trapped" in these hepatocytes. Thus the metabolite can no
longer partition into the systemic circulation and can be conceived as
being susceptible to direct biliary excretion. The fraction of the
246
hepatically formed metabolite that reaches the systemic circulation was
estimated from the ratios of the areas under the plasma metabolite
concentrationtime curve following IV administration of the metabolite
and the parent compound. The estimated percentages of the hepatically
formed metabolite reaching the systemic circulation were 6 and 12%
respectively in dogs F and G. This is in contrast to morphine
45 ...
pharmacokinetics where 3070% of the hepatically derived metabolite
reaches the systemic circulation.
Buprenorphine is almost completely hepatically metabolized in dogs.
First pass metabolism is anticipated upon oral administration. The
extent of the firstpass metabolism estimated from the ratios of total
body clearance and the hepatic plasma flow (360400 ml/min in a 20kg
dog~^) averaged 0.59 +_ 0.09 (SEM) in studies 712. Thus about 60% of
the drug was cleared during one pass through the liver. The absolute
bioavailability of buprenorphine estimated from the areas under the
plasma concentrationtime curve following oral and IV administrations
were 6 and 3.7% respectively in dogs B and D (studies 15 and 16). This
low bioavailability was attributed to poor absorption rather than
firstpass metabolism.
APPENDIX I
PROGRAM "MULTI"
1 DIM TU(50),CU(50),PH(50),VOL(50)
10 PRINT"* MULTILINES FITTINGS *"
20 PRINT:PRINT"DEFINE EQUATIONS AT 1000,1100,1200,1300,1400":PRINT
30 PRINT "CP AND T ARE DEPENDENT AND INDEPENDENT VARIABLES."
35 PRINT:PRINT"P(1), P(2),.... ARE PARAMETERS TO FIT."
36 PRINT:PRINT TAB(8);"GCTO 840 IF DIVERGED.":FP=0
37 DIMME$(3):ME$(0)="GAUSSNEWTON":ME$(1)="DAMPING GAUSSNEWTON"
38 ME$ (2) ="MARQUARDT" :ME$ (3) ="SIMPLEX"
40 PRINT:FORI=0 TO 3:PRINT"(";I;") ";ME$(I);" METHOD":NEXT
45 PRINT:INPUT"WHICH ALGORITHM DO YOU SELECT";AL
49 PRINT:PRINT"* I BELIEVE YOU HAVE DEFINED EQUATIONS *"
50 INPUT"SUBJECT NAME";N$
51 PRINT"INPUT FILE NAME";: INPUT F$
52 OPEN"I",#l,F$
54 INPUT"NUMBER OF LINES" ;IN:DIMNL (IN)
55 INPUT"WEIGHT OF DATA (0,1,2)";IW:INPUT"NUMBER OF PARAMETERS";M
56 PORI=lTOLN: PRINT "NUMBER OF POINTS ("; I; ") ";: INPUT#1, NL (I) : NEXT
57 INPUT#1,N2
60 N=0:FORI=lTOLN:N=N+NL(I) :NEXT:DIMTX(N) ,CV(N) ,A(M,M+1) ,P(M) ,X(M,M)
65 NL(0)=0:BS=0:FORJ=lTOIN:BS=BS+NL(Jl):PRINT
70 POR I=lTONL(J):PRINT"T";J;"(";I;"), CP";J;"(";I;")";:INPUT#1, TX(BS+I), CV(
BS+I)
75 NEXT I,J:IF AL=3 THEN 3000
76 FORI=lTCN2:INPUT#l,TU(I),CU(I),PH(I),VOL(I):NEXTI
77 PC=.0001:CF=100:IF FP=0 THEN DIMCS(N,M):FP=1
78 PRINT:INPUT"DT FOR JACOBIAN (0.10.0001)";DT:PRINT:FOR I=lTOM
80 PRINT"INITIAL P(";I;")=";:INPUT#1, A(I,0):P(I)=A(I,0):NEXT:CLOSE#1:GOSUB 40
00:S1=SS
140 PRINT "INITIAL SS=";SS:PORK=lTOlOO:GOSUB 7000:GOSUB 7400:GOSUB 6000:JJ=0
490 JJ=JJ+1:IF JJ>25 THEN 730
500 FOR 1=1 TO M:P(I)=A(I,0)+A(I,M+1):NEXT:GOSUB 4000
510 DS=ABS(SlSS):IF AL<>2 OR SS=0 THEN 590
520 FW=0:FOR I=1TOM:PW=PW4X(I,0) *A(I,M+1) 4CF*A(I,NB1) *A(I,M+1) :NEXT
530 IF DS/PW>.75 THEN CF=CF/2
540 IF DS/PW<.25 THEN CF=5*CF
590 IF DS<=PC*Sl THEN 730
600 IF AL=1 AND SS>Sl THEN FOR 1=1 TO M:A(I,M+1)=.5*A(I,M+1):NEXT:GCTO 490
630 FOR 1=1 TO M:A(I,0)=P(I):NEXT:Sl=SS:PRINT:PRINT"LOOP=";K
640 IF AL=1 THEN PRINT "DAMP=";JJ
660 FOR 1=1 TO M:PRINT"P(";I;")=";P(I):NEXT:PRINT"SS=";SS:NEXT
730 REM CHANGE TO PRINTER
731 PRINT#2:PRINT#2,"*";N$;"* BY ";ME$(AL);" METHOD":PRINT#2,"WEIGHT=1/CP«(
";IW;")"
733 IF AL<>3 AND N>M THEN GOSUB 8000
734 IFSS=0 THEN PRINT"AIC=INFINITE":GOTO 740
735 PRINT"AIC=";N*LOG(SS)+2*M
740 IF AL=3 THEN PRINT"ALPHA=";AA;" BETA=";BB;" GAMMA=";CC:PRINT
742 IF AL<>3 THEN PRINT"DT=";DT
247
248
745 IF AL=2 THEN PRINT"FACTOR=";CF
750 FORI=lTOM:PRINT#2,"FINAL P";I;"=";P(I);
760 IF AL<>3 AND X(I,0)>0 AND N>M THEN PRINT#2," S.D.=";SQR(X(I,0)*SS/(NM)
);
810 PRINT#2:NEXT:PRINT#2,"FINAL SS=";SS:BS=0:PORJ=lTO LN:BS=BS+NL(Jl)
820 PRINT#2:F0RI=1T0NL(J):T=TX(BS+I):ON J GOSUB 1000,1100,1200,1300,1400
830 PRINT#2,"T";J;"=";T;" CP";J;"=";CP;" (";CV(BS+I);")":NEXTI,J
840 PRINT:PRINT"WHICH ALGORITHM DO YOU SELECT?"
850 INPUT"(0,1,2,3 OR 1)";AL:IF AL<0 THEN END
860 IF AL=3 THEN 3000
870 GOTO 77
1000 CP=P(1)*EXP(P(2)*T)+P(3)*EXP(P(4)*T)+P(5)*EXP(P(6)*T):RETURN
1100 CP=(P(1)*P(2)/(P(2)P(3)))*(EXP(P(3)*T)EXP(P(2)*T)):RETURN
2000 FORJS=lTOM:PT=P(JS) :P(JS)=FT+DT:ON J GOSUB 1000,1100,1200,1300,1400
2020 DD=CP:P(JS)=PTDT:ON J GOSUB 1000,1100,1200,1300,1400
2030 CS(BS+I,JS)=(DDCP)/(2*DT):P(JS)=PT:NEXT:RETURN
3000 AA=1:BB=.5:CC=2:SG=1E10:PC=.00001
3025 PRINT:FORI=lTOM:PRINT"INITIAL P (";I; ") ";:INPUT A(I,1) :NEXT
3030 FOR J=2 TO MPl:POR 1=1 TO M:A(I,J)=2*RND(1)*A(I,1)+.01*(RND(l).5):NEXT I
,J
3040 FORK=l TO M+l:FORI=lTOM:P(I)=A(I,K):NEXT:GOSUB 4000:A(0,K)=SS:NEXT
3070 PRINT:FOR 1=1 TO M+1:PRINT"SS";I; "=";A(0,I) :NEXT:GCTO 5000
3080 SR=0:SL=1E10:FORJ=lTOM+l:IF SRA(0,J) THEN JL=J:SL=A(0,J)
3100 NEXT:SR=0:FOR J=1 TO M+1:IF JOJH AND SRPC*SG THEN SG=SR:GOTO 3080
5040 FOR 1=1 TO M:P(I)=A(I,JL):NEXT:SS=A(0,JL):GCTO 730
6000 IF NP=1 THEN A(1,2)=A(1,2)/A(1,1):RETURN
6020 RM=ABS(A(1,1)):FOR IS=1 TO NP:FOR JS=1 TO NP
6050 IF RMM
B
Biliary clearance of drug as metabolite (ml/min).
Cl—
U1B
Biliary clearance of the unchanged drug (ml/min).
Cl
ren
Renal clearance of the unchanged drug (ml/min).
M
Cl
ren
Renal clearance of the metabolite (ml/min).
Cl
met
Metabolic clearance of the drug (ml/min).
Cp
Drug concentration in plasma at time t (ng/ml).
%
Drug concentration in plasma immediately following IV bolus
injection (ng/ml).
f
Fraction of the orally administered dose that eventually
reaches the sytemic circuation unchanged.
f
a
Fraction of the orally administered dose that reaches the
liver unchanged.
f.
lpm
Fraction of the absorbed dose that undergoes firstpass
metabolism.
346
Apparent first order intercompartmenta1 transfer rate
constants, where i=l,2, . . . j=l,2, . . . i^j,
(l/min).
Apparent first order rate constant for the metabolite
formation in the central compartment for a drug that
distributes in the body in accordance with a multicompartment
body model (l/min).
Zero order infusion rate constant (mg/min).
Apprent first order elimination rate constant for the drug
from the cental compartment (l/min).
Rate of metabolite formation in the central compartment.
Time (min).
Time at which zero order infusin is terminated (min).
Amount of the metabolite excreted in urine to time t ( g).
Apparent volume of distribution of the drug in the central
compartment (L).
A proportinality constant relating the amount of the drug in
the body to the drug concentration in plasma at pseudosteady
state (L).
A proportionality constant relating the amount of the
metabolite in the body to the metabolite concentrations in
plasma on the presumption that plasma metabolite
concentrations are representative of the metabolite
concentrations in the equilibrated fluids of the body (L).
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BIOGRAPHICAL SKETCH
V. Ravi Chandran was born May 3, 1955 in India. He received B.Pharm
(Honors) degree in 1977 and M.Pharm in 1979 from Jadavpur University,
Calcutta. He was then employed as a research pharmacist in Lister
Laboratories, Bombay. He came to University of Florida in 1981 and
obtained a masters degree in pharmaceutical sciences in 1983. After
completion of the Ph.D. he will be employed as Senior Research
Pharmacist at SterlingWinthrop Research Institute, Albany, New York.
352
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Edward R.
ett, Chairman
Graduate Research Professor
’harmacy
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
ames W. Simpkins
Professor of Pharmacodynamics
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
John A. Zoltewicz
Professor of Chemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
C. Lindsay (Devane
Associate Professor of
Pharmacy Practice
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
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Pharmacodynamics
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
J/ Ch ^ g _/■
Hartmut C. Derendorf 7~~
Assistant Professor of
Pharmaceutics
This dissertation was submitted to the Graduate Faculty of the College
of Pharmacy and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosphy.
December 1986
Dean, College of Pharmacy
Dean, Graduate School
UNIVERSITY OF FLORIDA
3 1262 08554 3998