INTERACTION OF GRISEOFULVIN WITH METOCLOPRAMIDE, PROPANTHELINE AND PHENOBARBITAL IN THE RAT by FAKHREDIN JAMALI Pharm.D., University of Tehran, 1969 M.Sc, University of British Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1976 FakhreSih Jamali, 1976 DOCTOR OF PHILOSOPHY in the Faculty of Pharmaceutical Sciences Division of Pharmaceutics In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of th i s thesis for sc h o l a r l y purposes may be granted by the Head of my Department or by his representatives. I t i s understood that copying or pu b l i c a t i o n of th i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Faculty of Pharmaceutical Sciences The University of B r i t i s h Columbia Vancouver, B.C. V6T 1W5 Date: October £L / fqj4 i ABSTRACT Phenobarbital pretreatment, in man and rat, has been observed to decrease the plasma levels attained after administration of solid dosage forms of griseofulvin. Various mechanisms including induced metabolism, increased gastrointestinal motility, diminished dissolution rate or a complex mechanism involving enzyme induction, first-pass metabolism and distribution rate-limited elimination of griseofulvin have been suggested. This present investigation was initiated in an attempt to cl a r i f y the mechanism of this interaction. Intravenous administration of griseofulvin in control and phenobarbital dosed rats confirmed that no obvious differences existed in griseofulvin metabolism under the experimental conditions. The influence of gut motility on the absorption of griseofulvin administered as single oral doses of either 100 mg/kg in 0.5% Tween 80 (suspension) or 50 mg/kg in 100% polyethylene glycol (PEG) 600 (solution) was evaluated by pretreating rats with single intraperitoneal doses of either 10 mg/kg metoclopramide hydrochloride or 5 mg/kg propantheline bromide two hours prior to griseofulvin administration. Metoclopramide pretreatment accelerated the absorption rate of griseofulvin suspension (the time of peak-plasma level, T was shortened from 6 to 3 hours) but max reduced i t s total absorption by 50%. Conversely, this treatment regimen increased the total absorption of griseofulvin by 230% when administered in solution form. Propantheline preadministration, however, retarded the absorption rate ( T m a x prolonged from 6 to 19 hours) but increased the total absorption of a suspension of griseofulvin by 50% The propantheline treatment regimen elicited a sharply contrasting effect by decreasing the total absorption (23%) of griseofulvin when given in solution form. i i I t was therefore, concluded that a) the i n t e r a c t i o n of g r i s e o f u l v i n with metoclopramide and propantheline i s formulation-dependent and b) the gut m o t i l i t y has a s i g n i f i c a n t influence on the absorption of g r i s e o f u l v i n . The i n t e r a c t i o n between g r i s e o f u l v i n and phenobarbital was evaluated by administering s i n g l e o r a l doses of a) suspension of 100 mg/kg g r i s e o f u l v i n i n either 0.5% or 2% Tween 80, b) suspensions of 20 or 100 mg/kg g r i s e o f u l v i n i n 70% PEG 300, or c) solutions of 50 mg/kg g r i s e o f u l v i n i n 100% PEG 600. Test r a t s received si n g l e o r a l doses of 15 mg/kg sodium phenobarbital 24 hours p r i o r to g r i s e o f u l v i n administration. The t r e a t -ment reduced the t o t a l absorption of g r i s e o f u l v i n administered i n 0.5% Tween 80 by 50%; The extent of the i n t e r a c t i o n was reduced to an i n -s i g n i f i c a n t l e v e l on increasing the concentration of Tween 80 from 0.5% to 2%. Phenobarbital did not a l t e r the apparent rate of g r i s e o f u l v i n absorption since the peak plasma concentrations were coincident between control and t e s t animals. Phenobarbital treatment did not a f f e c t the a v a i l a b i l i t y of g r i s e o f u l v i n when administered as suspensions or solutions i n PEG as r e f l e c t e d by equivalent areas under the plasma curves at the same dose l e v e l i n test and control animals. It can therefore be concluded that the i n t e r a c t i o n between g r i s e o f u l v i n and phenobarbital i n the r a t i s a) a formulation-dependent phenomenon and b) not caused by either enzyme induction or an increased gut m o t i l i t y . I t i s concluded that when g r i s e o f u l v i n i s administered as a suspension i n 0.5% Tween 80 the phenobarbital-griseofulvin i n t e r a c t i o n i n the rat i s caused by a complex mechanism r e s u l t i n g i n a diminished d i s s o l u t i o n rate and subsequent decreased absorption. Supervisor, i i i TABLE OF CONTENTS Page 1. INTRODUCTION 1 2. LITERATURE SURVEY 4 Pharmacokinetics of Griseofulvin 8 Pharmacokinetics of Griseofulvin Following Intravenous Administration 8 Pharmacokinetics of Griseofulvin Following Oral Administration ; . . . . 11 Distribution of Griseofulvin 19 Elimination of Griseofulvin ; 21 Griseofulvin-Phenobarbital Interaction 24 3. EXPERIMENTAL • 30 Assay Procedure 30 Calibration Curves 30 a. Standard Solutions after Dissolving Griseofulvin in Benzene 30 b. Standard Solutions after Extraction , 31 Gas-Liquid Chromatography of Griseofulvin 35 Internal Standard Solution 40 Extraction of Griseofulvin from Plasma . 40 Dosage Forms 42 Identification of the Materials 43 Equilibrium Solubility of Griseofulvin in the Presence of Polyethylene Glycol 300 and Tween 80 44 Animals and Treatments 44 Plasma Level Studies . . . . 47 Gastric Emptying Experiments 48 Determination of 4-Demethyl Griseofulvin 49 a. Silanization of the Column 49 b. Utilization of Different Columns 51 c. Derivatization of 4-Demethyl griseofulvin j 55 Treatment of Data 57 Continued... iv TABLE OF CONTENTS (Continued) Page 4. RESULTS AND DISCUSSION 60 Determination of Griseofulvin 60 Gas-Liquid Chromatography of Griseofulvin 60 Extraction Procedure 61 Equilibrium Solubility of Griseofulvin in the Presence of PEG- 300 and Tween 80 . . . . 61 Absorption of Griseofulvin Following Oral Administration of Different Dosage Forms 64 Effect of Motility Modifiers on Gastric Emptying 69 Effect of Motility Modifiers on Griseofulvin Plasma Levels . . . . 75 Single Oral Dose of Griseofulvin Suspended in 0.5% Tween 80 . . 75 Single Oral Dose of Griseofulvin Dissolved in 100% PEG 600 . . . 83 Multiple Dose and Long Term Treatment with Propantheline . . . . 90 Intravenous Studies 99: Griseofulvin-Phenobarbital Interaction . . . . . . 109 Griseofulvin Suspensions in Tween 80 110 Griseofulvin in PEG . . . . . . . . 120 5. SUMMARY AND CONCLUSION 137 6. REFERENCES . 140 7. APPENDICES 143 Appendix I, Identification of the Materials Appendix II, Computer Programme V LIST OF TABLES Table Page 3-1 Griseofulvin (Gris) Concentration, and the corresponding Peak Height Ratios (Gris/D) of the Standard Solutions 34 3-II Griseofulvin Recovery Following its Extraction from Aqueous Solutions Containing Rat Plasma 36 3- III Administered Dosage Forms and Different Treatments Applied to the Rat 46 4- 1 Percent Griseofulvin Remaining in Stomach and the Corresponding Plasma Levels in the Rat following Oral Administration of Single Doses of 100 mg/kg Griseofulvin in 0.5% Tween 80 71 4-II Percent Griseofulvin remaining in Stomach following Oral Administration of Single Doses of 50 mg/kg Griseofulvin in Polyethylene Glycol 600 to the Control, Metoclopramide Hydrochloride Pretreated Rats (10 mg/kg) and Propantheline Bromide i Pretreated Rats (5 mg/kg) 73 4-III Plasma Levels of Griseofulvin (Micrograms per Milliliter) following Oral Administration of 100 mg/kg in 0.5% Tween 80 to Control and Test Animals , 77 4-IV Plasma Levels of Griseofulvin (Micrograms per Milliliter) following Oral Administration of 100 mg/kg in 0.5% Tween 80 to Rats Receiving Propantheline (5 mg/kg, i.p.), two hours prior to Griseofulvin Administration . 78 4-V Peak Plasma Levels (Cmax) and its Time of Occurrence (Tmax) Following Administration of 100 mg/kg Griseofulvin in 0.5% Tween 80 to the Control, Metoclopramide Hydrochloride (10 mg/kg) and Propantheline Bromide (5 mg/kg) Pretreated Rats 80 4-VI Area Under Plasma Concentration-Time (y.;g.hr.ml •"?") of Griseofulvin Following Oral Administration of Single Doses of 100 mg/kg of the Drug in 0.5% Tween 80 to the Control, Metoclopramide Hydrochloride Pretreated and Propantheline Bromide Pretreated Rats . . . 81 Continued... vx Table 4-VII 4-VIII 4-IX 4-X 4-XI 4-XII LIST OF TABLES (Continued) Plasma Levels of Griseofulvin (Microgram per Milliliter) following its Oral Administration in a Single 50 mg/kg in PEG 600 Dose in Control and Test Rats Plasma Levels of Griseofulvin (Micrograms per Milliliter) after a Single Oral Dose (50 mg/kg) in PEG 600 in Control and Test Rats . . . . . . . . Area Under Plasma Concentration-Time Curves, AUC (yg.hr.ml--*-) and the Peak Plasma Concentrations, Cmax (yg/ml) Following Oral Administration of Single Doses of Griseofulvin (50 mg/kg) in Polyethylene Glycol 600 (solution) to the Control, Metoclopramide Hydrochloride Pretreated (10 mg/kg) and Propantheline Bromide Pretreated (5 mg/kg) Rats Plasma Levels of Griseofulvin (y g/ml) following its Oral Administration (100 mg/kgiin 0.5% Tween 80) to Animals receiving either Single or Multiple Doses of Propantheline Plasma Levels of Griseofulvin (y g/ml) following its Oral Administration (100 mg/kg in 0.5% Tween 80) to the Control and Test Rats. Control Animals were treated Intraperitbneally with 1 ml of Distilled Water while Test Rats received 5 mg/kg Propantheline Bromide Intraperitoneally Twice Daily 7 days Prior to and During the Plasma Studies Biological Half-Life of the Post-Distribution Phases (0.693/g) and Area Under the Griseofulvin-Plasma-Time Curves (AUC) in the Rat after Intravenous Administration of Single Doses of the Drug in Poly-ethylene Glycol 600. Test Rats received Single Doses of Metoclopramide Hydrochloride (10 mg/kg), Propantheline Bromide (5 mg/kg) or Sodium Pheno-barbital (15 mg/kg) prior to the Griseofulvin Administration Page 85 86 88 93 97 105 4-XIII Plasma Levels of Griseofulvin (Micrograms per Milliliter) following Oral Administration of 100 mg/kg in 0.5% Tween 80 in the Control and Test Rats. Phenobarbital (15 mg/mg) was given Orally to the Test Rats, 24 hours prior to Griseofulvin Administration 112 Continued... v i i LIST OF TABLES (Continued) Table Page 4-XIV Area Under Plasma Concentration-Time Curves, AUC (yg.hr.ml - 1) of Griseofulvin Following Oral Administration of Single Doses of 100 mg/kg of the Drug in 0.5% Tween 80 to the Control and Phenobarbital Pretreated Rats 113 4-XV Peak Plasma Levels, Cmax and their Time of Occurrence, Tmax Following Oral Administration of 100 mg/kg of the Drug in 0.5% Tween 80 to the Control and Phenobarbital Pretreated Rats . . . . . 114 4rrXVI Griseofulvin-Plasma Levels .h(3.0ifcmg)/l'kg±daliIy (f-olr n^/daysj^iincgejase'd cggiQ)l±gg£. blood flow by, 3!7.-ab7*5%-2inv the 3r7at.,75%. Although i n a system of blood flow-rate l i m i t e d metabolism, enzyme induction would not a f f e c t the rate of metabolism, i t may very well increase the extent of metabolism. However, t h i s seems u n l i k e l y , because g r i s e o f u l v i n i s extensively metabolized following i t s administration (,25-29). Only trace q u a n t i t i e s of i n t a c t g r i s e o f u l v i n have been recovered from the urine of r a t s (25),-mice (26) and man (27). The recovered quantity of the i n t a c t g r i s e o f u l v i n i n rabbits (28) and dogs (,29) has been n e g l i g i b l e . This suggests that the extent of g r i s e o f u l v i n metabolism cannot be increased.cany f£ur.ther by pre-administration of phenobarbital. A f t e r c a r e f u l study of the reported i n t e r -a ction between g r i s e o f u l v i n and phenobarbital ( 16.-20^), i t became apparent that the evidence accumulated was i n s u f f i c i e n t to point to the p r i n c i p a l 7 mechanism involved in this interaction. Further examination of the griseofulvin-phenobarbital interaction was, therefore, necessary. 8 Pharmacokinetics of Griseofulvin The kinetics of absorption, distribution and elimination of griseofulvin have been studied in man ( 3 2 ) , rabbit ( 3 3 ) , dog ( 3 4 ) and to a limited extent in the rat ( 35 . ) . Pharmacokinetics of Griseofulvin Following Intravenous Admin- istration The absorption kinetics of any drug are best studied when a complete understanding of its distribution and elimination is obtained after intravenous (iv) administration. Before initiation of an absorption study, as in this case to look for a possible interaction, i t is necessary to evaluate the pharmacokinetics of a drug following iv dosing. This rules out any unusual gut metabolism or" first-pass metabolism. These phenomena may likely be overlooked after oral administration of the drug unless suitable comparisons are made to the iv route. A known amount of drug is introduced into the body intravenously and the basic pharmacokinetic parameters describing distribution and elimination are estimated by following the time-course of the drug. The pharmacokinetic parameters for distribution and elimination are then estimated in the absence of incomplete absorption, gut metabolism or first-pass metabolism. In the case of poorly soluble drugs, such as griseofulvin, iv administration presents certain problems, e.g., a dose of 200 mg of griseo-fulvin would require approximately 20 1 of saline to dissolve the drug. Different solvents have been used to overcome the problem. Bedford et al (:35) dissolved griseofulvin in 75% N,N-dimethylformamide in water while Rowland et al ( 3 2 ) used polyethylene glycol (PEG) 300 to prepare iv preparations of griseofulvin. 9 Nevertheless, after iv administration i t has been reported that the decline of griseofulvin plasma levels in man, dog and rabbit can be described by a two-compartmental open model (page 107). The following equation defines such a^ .model: . . -at . „ - pt s Cp = A e + Be H (1) r o o where Cp represents the plasma concentration at any time, t, A and B are the ordinate concentration intercepts of the distribution and post-distribution phases at t = 0. The rate constants of the distribution and post-distribution phases are a and 3. The half-life (tJg) of the distribution phase (^ **^ "*) has a been reported to be approximately 4, 6, and 60 min in the dog ('3.4), rabbit (.33) and man ( 3 2 ) respectively. The post-distribution phase has a longer th (°' 6 9 3) ; 0;83^hrminedpgC >1328„..hr;in rabbit andbfromQ9.;5ihrrrto 21.0 "hr i n man. .... The amount of the drug in the central compartment at time t, Xg, can be calculated by multiplying the right hand stifdelqof lEqty ifcb.yytKe volume of distribution, Vd: X,, = Vd(A e" a t + B e" e t) (2) B o o The volume of distribution is an indication of the extent of distribution of the drug in the b o d y . In humans ( 3 2 ) and dogs (3.4) Vd has been reported to be approximately 56 and 10 1 respectively. In the rabbit (3. 3 ) , however, i t has been observed that the Vd for this drug is not constant. This was attributed to inconsistent plasma levels and consequently the area under the plasma concentration vs_ time curves (AUC) even at constant dosage. 'A ... twofold increase in the administered iv dose had no effect on the area under the plasma-level vs time curve (AUC). The mean AUC ± 95% confidence level after a single iv dose of 5 mg/kg of griseofulvin in 5 rabbits was observed to 10 be 350 ± 134 yg/min/ml, while i t is 306 ± 81 yg/min/ml following a single dose of 10 mg/kg in 10 rabbits. No adequate explanation for this unusual observation has been offered. The volume of distribution of the central compartment, Vd, is calculated using Eq. 3: Vd = dose/i».(A + +3B ) (3) C O 0 o From Equations 1 and 3 i t becomes obvious that inconsistent plasma levels (Cp) will give rise to inconsistent Vd, since A q and B q are proportional to Cp, and Vd is indirectly proportional to the former parameters. The area under the concentration v& time curve is usually accepted as being directly proportional to the amount of drug in the body. However, after iv administration to rabbits, i t does not seem to be a valid assumption. • / In the rat, Bedford et al ( 3 5 ) anticipated another difficulty. They suggested that after iv administration of griseofulvin more than 90% of.the drug dwasppe-lim-inated t'from...the! canimaltne-.c during the. first few minutes. No explanation was given for this phenomenon. Nevertheless, the rapid elimination of the drug in the first few minutes post-dosing introduces difficulties in estimating pharmacokinetic parameters of griseofulvin in the rat. Likewise and contrary to expectation, Chiou and Riegelman (through reference 3 6 •, Figs; 3 and 4), obtained an AUC following a single 50 mg iv dose of griseofulvin to a dog which seemed to be substantially lower than those of equal doses of orally administered solid dosage forms. This is inconsistent with the fact that cumulative urinary excretion of the major metabolite of griseofulvin following iv administration was higher than those of an orally administered dose ( 3 6 ) . 11 Pharmacokinetics of Griseofulvin Following Oral Administration -., Due to the low solubility properties (15 mg/1 at 37°C)t griseofulvin absorption from the gastrointestinal tract is erratic and incomplete. Katchen and Symchowicz (6) found a good correlation between dissolution rate of griseo-fulvin in simulated intestinal fluid and its absorption in man. They explained that, i f the gastrointestinal fluid is saturated with respect to griseofulvin when a dose of 500 mg is administered, a minimum of 33 1 of fluid is required to transfer the drug from the gastrointestinal tract into the systemic circulation, as shown in the following calculation: Dose _ 500 mg _ _„ Solubility 15 * mg/1 " Therefore, i t appears reasonable to suggest that the absorption of griseofulvin is dissolution rate limited. Twenty four hours after administration of griseofulvin Davis et_ al (2 8 ) recovered 55% of an oral dose of griseofulvin from the alimentary tract of the rat, of which 37.5% was in the faeces. Rowland et al ( 3 2 ) reported that, in man, from 27 to 72% of oral doses of micronized griseofulvin was absorbed, giving an indication of the extreme variability of the absorption process. In cats, Bedford e_t al ( 3 5 ) demonstrated that the major griseofulvin absorption site is the upper part of the small intestine. They ( 3 5 ) admin-istered griseofulvin directly into ligated segments of the gastrointestinal tract and studied the blood levels of the drug. The highest blood level was obtained when the drug was introduced into the duodenum, with the jejunum and ileum next. The absorption from the stomach was considerably lower andj- . blood 'levels '-dec-lined tbezerolSahourssafter.vtehewmaximumiplasma level was . attained. 12 Alternatively, Chiou and Riegelman (37) hypothesized that . there might be a limited residence time through a particular region of the gut where absorption is optimal. These reports suggest'.that: the absorption, of .griseofulvin:,: : from the gastrointestinal tract is limited by two major factors, viz., the dissolution rate and the gastrointestinal motility. Any factor influencing these two will consequently alter the absorption of an orally administered dosage form of griseofulvin. Several investigators have introduced different means to improve the absorption of griseofulvin through accelerating the dissolution rate. Kraml et al (7) suggested the use of micronized powder of griseofulvin in the preparations. They 0 ) noticed a substantial increase in the griseofulvin plasma level in the rat when the micronized powder was administered in an aqueous suspension as compared to the regular powder. The improved absorption is due to the fact that micronization will give rise to a higher surface area. This increases the rate of dissolution and consequently the absorption rate of the drug. Kraml e_t al (7) observed that,, addition of surfactant to aqueous and corn o i l suspensions failed to significantly affect griseofulvin-serum levels. "rr.These-authors used 0.5% Perminal BXN and 0.5% Nekal BX78 in aqueous suspensions and 0.077% Aerosol OT and 0.077% Tween 80 in corn o i l suspensions. In contrast to the observations of Kraml et al Duncan et. al (8) noticed. .•/.-/ that, addition of 0.5% lecithin Glidden R.G. or Perminal BXN into the suspension Of griseofulvin gave rise to higher blood levels in rats. They showed that the method of incorporation of the surfactant into the suspension influences 13 the absorption of the drug. When g r i s e o f u l v i n and the surface-active agents were mixed p r i o r to the addition of water, s i g n i f i c a n t l y higher g r i s e o f u l v i n blood l e v e l s were observed as compared to when the surfactant i s f i r s t dissolved into the water and the g r i s e o f u l v i n added afterwards. Chiou and Riegelman (36,37) administered g r i s e o f u l v i n dispersed i n a s o l i d matrix of polyethylene g l y c o l (PEG) 6000 to man and to dogs and noticed higher (approximately threefold) and more constant absorption as compared to that obtained a f t e r administration of tablets of micronized drug. I t was concluded that a f t e r the exposure of the griseofulvin-PEG preparation i n the g a s t r o i n t e s t i n a l f l u i d , the drug might p r e c i p i t a t e i n the c o l l o i d a l or submicron form which could Be. dissolved rapi'd-l'yVv .-'rr': Recently, a simple method of preparation was introduced (38) which has yielded g r i s e o f u l v i n dosage forms with higher r e l a t i v e b i o a v a i l a b i l i t y . In t h i s method g r i s e o f u l v i n formulations are prepared d i r e c t l y from the fermentation substance. The therapeutic e f f e c t of these preparations has been tested on patients with scalp i n f e c t i o n s and has been reported to be s i g n i f i c a n t l y higher than that achieved with g r i s e o f u l v i n t a b l e t s . These authors a t t r i b u t e d the improved effectiveness to the fa c t that,, i n these preparations, g r i s e o f u l v i n i s i n i t s "natural v e h i c l e " , and therefore i t can be absorbed more r e a d i l y and e l i c i t s i t s e f f e c t more completely. In man, the serum g r i s e o f u l v i n concentration has been observed to be almost doubled when the drug i s administered with a high f a t meal ( 9 ) . This observation was a t t r i b u t e d to an increase i n absorption due to e i t h e r a change i n m o t i l i t y or an increased e m u l s i f i c a t i o n caused by an increased b i l e s ecretion. Kraml et^ a_l (7) also observed two- to t h r e e f o l d higher serum l e v e l s following administration of g r i s e o f u l v i n suspended i n corn o i l . However, 14 Bloedow and Hayton (3.9,) have recently administered griseofulvin to the rat in a series of l i p i d and l i p i d - l i k e vehicles and noticed that only digestable • polar vehicles (e.g., Tween 80) caused an increase in griseofulvin plasma levels. These vehicles, they explained/,^rfo'rme^raBfstable-im'o'nolayer on" water ?and iwater,. da'ssbiv.edcan ifchem'.siThei bioavailability of. *ver-' i , griseofulvin in other classes of lipids was either lower or equal to 'that observed following administration of the drug in an aqueous suspension. Recently, Bates and co-workers studied the.absorption of griseofulvin from different liquid preparations in the rat (12) and man (.40). These authors (12) administered griseofulvin suspensions in corn o i l and griseofulvin emulsions in corn oil/water to the rat and compared the results with those of aqueous suspensions. The highest percent absorption with the smallest coefficient of variation was observed after the oil/water emulsion with the o i l suspension next. The aqueous suspension was reported to have the lowest bioavailability and exhibited the highest coefficient of variation. However, the authors (12) observed that the time to attain maximum plasma level was significantly longer when the drug was given in an o i l suspension or an oil/water suspension as compared to the aqueous suspension. In humans, also, Bates and Sequeira (40) have recently shown that - : griseofulvin was more rapidly, uniformly and completely absorbed when the drug was administered in an oil/water emulsion than when i t was administered in either tablet form or in an aqueous, suspension. These authors (40.) suggested that, the inhibitory effect of emulsified corn o i l on the gastric emptying process could be responsible for the increased absorption. The slower rate of gastric emptying coupled with the inhibitory effect of l i p i d on the motility 15 of the upper part of the small intestine causes a smaller amount of un-dissolved griseofulvin to be released at any particular time and become exposed to the absorptive s i t e . In other words, a smaller amount of micron-tzed griseofulvin with a longer residence time in the absorptive region has a better chance of being absorbed. As w i l l be discussed later, this confirms the observations of Jamali and Axelson (4.1,42) regarding the significance of gut motility on the absorption of griseofulvin in the rat. To further support their hypothesis, Bates and Sequeira (140) pretreated -a human subject with propantheline, an agent capable of retarding the gastrointestinal motility, prior to the administration of a single oral dose of micronized griseofulvin in an aqueous suspension. As a result, an increase in the maximum urinary excretion rate along with an increase in total recovery of the major metabolite in the urine was noticed. Since upon administration griseofulvin i s metabolized almost completely, an increase in the recovery of the metabolite indicates an increase in the absorption of the. drug. The stomach has a limited capacity for absorption of many drugs, while the proximal segment of the small intestine has been noticed to be the major site of absorption for many drugs, such as griseofulvin ( 3 5 ). This fact is attributed to the large surface area of this segment of the gut relative to that of the stomach. Consequently, a change in the residence time of a drug in the gut may influence absorption. Manyc drugs have been observed to influence the gut motility and consequently the residence time of a co-administered drug. Metoclopramide ( 4 3 - 4 , 5 ) and propantheline (46) are two commonly used drugs to respectively increase and decrease motility of the gastrointestinal t(GI>)ttr?iet. It has been observed that an increase in the 16 g a s t r i c emptying rate caused by metoclopramide increases the rate of absorption of many drugs such as paracetamol (47), t e t r a c y c l i n e (48), p i v a m p i c i l l i n (48) and ethanol (4=)). On the other hand, a reduced m o t i l i t y caused by propantheline or atropine reduced the rate of absorption of digoxin (13), paracetamol (47), t e t r a c y c l i n e (4.8), p i v a m p i c i l l i n (4.8), ethanol (4.9), sulfamethoxazole (5,0), r i b o f l a v i n (14) and phenolsulfonphthalein (15). With regard to the t o t a l absorption, however, m o t i l i t y modifiers a f f e c t only the absorption of drugs with inherently poor absorption c h a r a c t e r i s t i c s such as digoxin (13), r i b o f l a v i n (14) and phenolsulfonphthalein (15). In these cases, metoclopramide generally decreased the t o t a l absorption of the drugs while a n t i c h o l i n e r g i c agents •'. -increased: c-the •. ^ d s r e l a t i v e a v a i l a b i l i t y . I t i s i n t e r e s t i n g to note t h a t A although propantheline treatment increased the blood l e v e l s of digoxin when the drug was given i n tablet form, i t f a i l e d to influence t h i s parameter following administration of a s o l u t i o n of the glycoside (13). It i s also worth mentioning that, among the above mentioned drugs r i b o f l a v i n possesses an exceptionally i n t e r e s t i n g absorption c h a r a c t e r i s t i c . I t i s absorbed by a s p e c i a l i z e d absorption process i n the human small i n t e s t i n e (50.yjjl).Therefore i t s absorption i s dependent upon the residence time i n the small i n t e s t i n e . Propantheline treatment prolonged the residence time, thereby increasing the t o t a l absorption of r i b o f l a v i n (14). Grounse (9) and Bates and Sequeira (j30)- pointed to another mechanism that might be involved i n the absorption of g r i s e o f u l v i n when administered with f a t t y meals or i n l i p i d v e h i c l e s . Administration of l i p i d s may stimulate the secretion of b i l e . B i l e contains p h y s i o l o g i c a l surface active agents which are known to have important r o l e s i n the absorption" process of Hours Figure '2-1/' . B i l i a r y flow, b i l i a r y b i l e acid concentration and b i l i a r y acid excretion in contrail rats and rats treated with "phenobarbital (75 mg/kg) once da i l y for four days. The soiid ; l i n e s represent the results from the control group, and the dashed l i n e s represent the results from the phenobarbital treated r a t s . From C.D. Klaassen, J. Pharmacol 176, 743, 1971. Exp. Ther. 18 many drugs (,52-59 ). Kimura elt a_l ('59) studied the e f f e c t of sodium taurocholate (STC), a major b i l e s a l t , on the absorption of a series of drugs i n the r a t . They noticed s i g n i f i c a n t but v a r i a b l e changes i n the absorption of the drugs, depending upon t h e i r physicochemic.al nature of absorption c h a r a c t e r i s t i c s . As a r e s u l t of simultaneous perfusion of 20 mM STC (the c r i t i c a l m i c e l l e concentration, CMC, 2.9 mM f o r STC) d i r e c t l y to d i f f e r e n t cannulated segments of the small i n t e s t i n e the percent absorption of sulfaguanidine increased while the opposite e f f e c t was noticed with imipramine and quinine (.59). These authors a t t r i b u t e d the increased absorption of sulfaguanidine to the e f f e c t of b i l e s a l t on the "structure of the absorption surface" and the decreased absorption of imipramine and quinine to the " l o s s of thermodynamic a c t i v i t y of the drugs due to the formation of the m i c e l l a r complexes with the b i l e s a l t " . Thus, an increase i n the concentration of the physiologic surface-active agents caused by stimulation of b i l e secretion may a l t e r the d i s s o l u t i o n rate a i v i r1and phenobarbitone-dosed males (n = 12). Standard errors are between 0.3 and 0.1. (Reference 17 .) 26 follow-up of the time course of the drug i n the same rat and provides only an estimation of the curve of the blood-level vs_ time. Furthermore, i n th e i r repeated phenobarbital treatment study the griseofulvin-blood l e v e l was measured only at one point (4 hours post-administration). To explain the p o t e n t i a l mechanism involved i n g r i s e o f u l v i n -phenobarbital i n t e r a c t i o n , B u s f i e l d and co-workers suggested that pheno-b a r b i t a l reduces the griseofulvin-blood l e v e l due, perhaps, to i t s a b i l i t y to induce the metabolism of g r i s e o f u l v i n . Further substantiation f o r t h i s was t h e i r observation that, i n v i t r o , the barbiturate accelerated the metabolism of g r i s e o f u l v i n by the rat l i v e r s l i c e s . This i s consistent with the observation of L i n et_ a l (66.) which was discussed e a r l i e r . However, l a t e r , Riegelman et a l (2'0) disputed t h i s hypothesis. They administered g r i s e o f u l v i n intravenously as w e l l as o r a l l y to man pretreated with repeated o r a l doses of 30 mg/kg (three times d a i l y f o r four days) and studied the griseofulvin-plasma l e v e l s i n the subjects. Riegelman et a l observed that phenobarbital treatment reduced the griseofulvin-plasma l e v e l s when griseo-f u l v i n was administered o r a l l y . However, the treatment f a i l e d to a l t e r the plasma l e v e l s of an intravenously administered dose of g r i s e o f u l v i n (Fig. 2-5). Furthermore, phenobarbital pretreatment did not influence the k i n e t i c s of the elimi n a t i o n of g r i s e o f u l v i n regardless of the route of administration; the slopes of the p o s t - d i s t r i b u t i o n phase of the griseofulvin-plasma level-time curves f e l l into the same range i n both control and test subjects (Fig. 2-5). This i s of p a r t i c u l a r importance. The slope of the p o s t - d i s t r i b u t i o n phase fo r a two-compartment open model i s described by Equation 4: ^ e l ^ l ' ? l o p e ="a(2.303) ( 4 ) 27 Figure 2-5. Griseofulvin plasma levels as a function of time of one human subject in presence and absence of phenobarbital treat-ment. -In; control experiment ? the'subjectr received^' either a single intravenous dose (100 mg in PEG 300) .or a single tablet of 500 mg: orally; in the test experiment the subject received 30 mg phenobarbital orally three times .daily for four days prior to the griseofulvin administration. (Reference 20 .) 28 Thus i t is directly proportional to the overall elimination rate constant, k^^. As mentioned before, the elimination of griseofulvin is mainly associated with its metabolism. Therefore any changes in the rate constant for metabolism of the drug would be expected to influence the slope of the post-distribution phase of the plasma level vs time curve. Stimulation and inhibition of the metabolism of a drug would result in a greater or smaller slope of the post-distribution phase respectively. The observed parallel slopes in the control and test subjects coupled with the phenobarbital's failure to reduce the griseofulvin-plasma levels following administration of an iv dose.suggested that the observed interaction was unlikely to occur at the level of metabolism (20). Since the interaction has been observed only following oral administration, Riegelman et al suggested that phenobarbital decreased the absorption of griseofulvin rather than inducing its metabolism. They proposed two potential mechanisms to explain this reduced absorption: phenobarbital either reduced the degree of dispersion of the tablet granules or increased the motility of the proximal segment of the small intestine where griseofulvin absorption appears to be maximal. The first mechanism causes a reduction of the surface area, therefore, dissolution of griseofulvin would be retarded. The second mechanism results in a reduced residence time of the poorly absorbed drug in the absorptive site, thereby decreasing its total absorption. Both mechanisms 6f-fered:jw.er-el based on the ability of phenobarbital to enhance the flow of bile. An increase in the volume of the bile may accelerate the motility of the gut and force the drug past the absorptive site before i t can be absorbed. Alternatively, considering the effect of bile salts on the dispersion and dissolution of poorly soluble drugs, Riegelman et al suggested that the change in the bile flow may be responsible for the reduced absorption of griseofulvin following the administration of phenobarbital. Other mechanisms have also been proposed in an attempt to explain this interaction. Axelson (24) hypothesized that the reduced griseofulvin-plasma level might be due to a complex mechanism involving enzyme induction, first pass metabolism and distribution rate-limited elimination of the drug. For example, i f the elimination of griseofulvin was rate-limited by its access to the liver rather than by its metabolism per se, enzyme induction would have no effect on the half-life of the drug but may significantly increase the fraction of the dose which is metabolized on the first pass through the liver upon oral administration. In such a case the slope of the post-distribution of the plasma level-time curve would not be an indication of the rate of metabolism but would rather be an indication of the rate of blood flow to the metabolizing organ (blood flow-rate limited elimination). However, as mentioned before, since in control rats as well as in other examined species griseofulvin is extensively metabolized (more than 9 9 % of ah 260°, and 280°). The GLC conditions were as follows: column, 1.83 m x 0.6A cm (6 f t x 0.25 in) glass tube packed with 10% on 80-100 mesh Chromosorb W; column temperature, as mentioned above; injection port temperature, 250°; detector temperature, 350°; carrier gas (5% methane, 95% argon) flow, 40 ml/min. As can be seen in Fig. 3-7, the result obtained using a column packed with OV-101 also cannot be considered satisfactory. In fact, no peak due to 4-DMG was observed even after an injection of 20 ng of the metabolite. This is contrary to expectations. Due to the lesser polarity ascribed to OV-101, i t was expected that reduced absorption would be observed and as a consequence improved peak resolution would result after using this stationary phase. The loss of the peak, however, may have resulted from a firm binding between the stationary phase and the metabolite. c. Derivatization of 4-Demethylgriseofulvin - The griseofulvin metabolite, 4-DMG, differs from i t s parent compound by lacking a methyl group in the 4 position. To improve the assay, attempts were made to convert 4-DMG to griseofulvin. The metabolite was darivatized to i t s methylated form, R 21 griseofulvin, using MethElute , an effective methylating agent. The procedure uti l i z e d in the derivatization was as follows: to each of 0.5 ml solutions containing 200, 400 or 600 pcg/5 y 1 4-DMG in benzene, in 15 ml teflon-stoppered centrifuge tubes 25 y1 of MethElute (0.2 M in methanol) was added. The contents of the tubes were shaken for 30 sec and Trimethyl anilinum hydroxide, Pierce Chemical Co., Rockford, 111. 10 _• H 1 M 1 1 -1 >"4 8 12 16 20 24 28 Minutes Fig u r e 3-8.Gasz-chromatpgrams^ obtained f o l l o w i n g i n j e c t i o n of 200 and 400 peg •methylated 4-DMG ( i n 5« yl) i n t o a column packed with 3% OV 25 on"Chromosorb:W; attenuation 8X. 5 yl of each of the solutions were separately injected into the GLC using the same gas chromatograph parameters as described for the griseofulvin determination. Figure 3-8 depicts chromatograms obtained from injections of 5 yl of solutions of derivatized 4-DMG containing 200 and 400 peg of the metabolite. Although derivatization occurred, the resulting peak heights were extremely variable and Inconsistent after injection of the different solutions. The unfortunate extreme variability of the derivatization plus the lack of availability of pure 4-DMG metabolite resulted in the discontinuance of this portion of the project. In some instances, addition of the reagent resulted in the total disappearance of the peak. This might have resulted from destruction of the compound unde'rathenconditions'.of'the experiment. Treatment of Data - Mean plasma levels of griseofulvin and the deviation from the mean were calculated for each experiment. The plasma levels were plotted as a function of time and the area under the curves (AUC) were measured using a planimeter. The Student's t-test was used to statistically measure the significance of the differences at a = 0.05. 22 A digital computer programme was developed to best-fit the experimental data points and also evaluate pharmacokinetic parameters following a single intravenous injection of a given drug. The programme is based on the Equations ('!)-:"-. ""-I.B.M. 370/168. 58 Cp = Ae" a t + Be" e t ( 1 ) which describes a two compartment open model. The computer f i r s t f i t s - (& the terminal part of the curve based on Cp = Be and then using the method — c t t residualsal i t f i t s the curve describing the distribution phase (a = Ae ). Addition of these two exponential curves, i.e., the distribution and post-distribution curves, results in a single bi-exponential curve.which defines the time-course of the disappearance of the drug from the central compart-ment. The computer computes the calculated slopes of both exponential processes with their correlation of coefficient and also the intercepts of each curve with Y-axis (A and B respectively). In the next step the following parameters are computed: biological half-lives of the distribution Tj (ALPHA) and post-distribution, Tt (BETA) phases; the plasma concentration at zero time, Cpo; the volume of distribution of the central compartment, vc; the f i r s t order rate constant for the distribution to the second compartment, k^; the f i r s t order rate constant for the distribution back to the central compartment, k^^; the f i r s t order rate constant for the overall elimination of the drug from the central compartment, kel; the area A B under the plasma concentration vs time curve, AUC = — + -j-; and, clearance CL (Appendix I"i) . 59 4. RESULTS AND DISCUSSION Determination of Griseofulvin Gas-Liquid Chromatography of Griseofulvin - The pulsed-mode linear electron-capture detector used for the griseofulvin assay provided the necessary sensitivity and specificity desired for plasmaSlevel studies. Excellent correlation (r = 0.999, Table 3-1) was found between peak height ratio and the amount of the drug i n the range from 16 peg to 48 ng of griseofulvin , (Fig. 3-1). This analytical procedure for the detection of griseofulvin in plasma samples represents a substantial improvement over the procedure described by Shah et a l (67). Under their experimental.. . tffondift-iQhsjjrit Shahse eiuttejLrs found linearity from 100 peg to 7 ng. Further, the technique described herein detects only those compounds with highly electron-iwA^hxtfc^^ i s , therefore, selective. The resulting chromatograph therefore contains only peaks due to griseofulvin, diazepam and occasionally some minor non-interfering compounds. The resolution of the peak is excellent, allowing precise quantitation using the peak-height ratio method (Fig. 3-2); Under, such conditions, the retention time was 2.08 and 6.24 minutes for diazepam and griseofulvin respectively. Diazepam was found to be a suitable internal standard. The diazepam peak appeared 2.08 minutes after the injection which i s 4.16 minutes apart from the griseofulvin peak. The resultant peak was sharp and repro-ducible. However, the electron-capture response to diazepam has been observed to be linear only in a limited concentration range (68 ). Since the internal standard technique requires the diazepam to be present in a constant concentration, this factor was of no consequence. Extraction Procedure - The applied method of extraction was simple and efficient. The mean percent griseofulvin recovery after extraction was 9 9 . 2 ± 2 . 8 (Table 3-II). This level of efficiency permits determination of trace quantities of griseofulvin in the biological samples. This is of particular importance when the time course of the drug is being studied. The griseofulvin plasma concentration in some rats declined to very low levels 48 hours after administration of griseofulvin. This concentration ranged from 0 . 0 0 1 to 0 . 9 6 ug/ml of griseofulvin in plasma (Tables 4-HI, 4-IV, 4-VII, 4-VIII, 4-X, 4-XIII, 4-XV, 4-XVI, 4-XVIII, 4-XX and 4-XXII) and for this reason, large volumes of blood were subject to analysis. In these cases animals were sacrificed and 0 . 5 to 1 ml of blood wase collected via a cardiac puncture. Nevertheless, attempts were made to maintain the lower limit of the assay at an approximate concentration of 40 pcg/5 ,ji 1 (.vl"5'•,ng/ml ' plasma), because lower griseofulvin levels would result -in large variations in the determinations of peak height. Addition of 0 . 1 ml of PEG 3 0 0 , PEG 6 0 0 , 0 . 5 % and 2% Tween 8 0 , or 1 ml of 50% ethanol to the aqueous phase prior to the extraction had no effect on the percent griseofulvin recovery. Equilibrium Solubility of Griseofulvin in the Presence of PEG 3 0 0 and Tween 80 Since the griseofulvin-phenobarbital interact ion has i3es?r. fmay;ula:}.o:cturj oca.tir at the level of dissolution, several formulations (suspension and solution) were prepared and administered to control and phenobarbital treated rats. Prior to tt'hese;pexp.erfimentaji, i t was necessary to determine the solubility of griseofulvin in the suspensions. Therefore the 61 equilibrium solubility of the drug in various solutions containing different concentrations of PEG 300 and Tween 80 was measured. Fig. 4-1 depicts the concentration of griseofulvin in the supernatant vs_ percent PEG in d i s t i l l e d water at room temperature and 37°. Each data point represents the mean of two determinations. Preliminary determinations showed that the supernatant was saturated with griseofulvin prior to 48 hours. As can be seen the solubility of griseofulvin increases with an increase in percent PEG 300. This is particularly profound when PEG content exceeds 60%. At room temperature, 3.20 mg/ml griseofulvin i s dissolved in an aqueous solution containing 70% PEG 300. After administration of 1 ml of this suspension, because of the l i p i d soluble nature of griseofulvin, a high plasma peak level should be observed in a short period of time. Further absorption, however, as w i l l be discussed later, may depend on the dissolution rate of griseofulvin in the surrounding f l u i d . Griseofulvin equilibrium solubility was determined also in the supernatant of preparations containing 0.5, 2 and 5% Tween 80 at room temperature. The griseofulvin concentration in the supernatant of a l l three preparations was 0.015 mg/ml. This is the same as griseofulvin solubility in water. Two explanations could be given for this observation: 1) In the range of 0.5 to 5%, Tween 80 might act only as a wetting agent without any solubilizing effect; or 2) Griseofulvin and Tween 80 might yield a complex phase which in turn sediments along with the solid phase in the process of centrifugation. Since the c r i t i c a l micelle concentration of Tween 80 is approximately 0.05%, i t i s unlikely that in the range of 0.5 to 5% that solubilization or some other interaction does not occur. Visual examinations revealed that an emulsion-like phase existed in the suspensions containing Tween 80. However, after centrifugation of the suspensions, a clear supernatant was obtained. It is possible for griseofulyin to be incorporated into this phase and to sediment 20 40 60 80 100 % PEG 300 Figure 4-1 . Equilibrium solubility of griseofulvin in aqueous solution's containing different concentrations of polyethelene glycol 300 at room temperature '( A ) and 37° (, # )• Each point represents average of 2 measure-ments . 63 during the process of centrifugation. Lack of a complete griseofulvin-Tween 80 phase diagram does not permit further interpretation of this observation. 64 Absorption of Griseofulvin Following Oral Administration of Different Dosage Forms The griseofulvin-phenobarbital interaction in the rat was previously reported (17) after a single dose of 100 mg/kg griseofulvin suspension (in 0.5% Tween 80) was administered orally to the rat. To i n i t i a t e this study on the mechanism of the interaction and also to ascertain the reproducibility of the previous observation the same dosage form was adopted in this work. Furthermore, since there have been questions as to whether the phenobarbital effect on the griseofulvin-plasma levels In the rat^.is due to a reduced absorption or induced metabolism, different dosage forms with improved dissolution properties were administered in an attempt to obviate the problem of dissolution rate. Fig. 4-2 depicts the griseofulvin-plasma concentrations vs time plots following the oral administration of single doses of griseofulvin in 0.5% Tween 80 (100 mg/kg), 2% Tween 80 (100 mg/kg), 70% PEG 300 (20 and 100 mg/kg) and 100% PEG 600 (50 mg/kg) to the rat. Each curve represents data from one rat with closest griseofulvin-plasma levels to the mean (n = 4 to 8). The absorption properties of griseofulvin were found to be highly dependent upon the dosage form. With regard to the attainment of the maximum griseofulvin-plasma level (Tmax) which i s an indication of the rate of absorption, the administered preparations f e l l into two distinctly different categories: a) suspensions and solutions containing PEG with very rapid absorption rates (Tmax, 15 to 30 min), and b) suspensions containing Tween 80 with slow absorption rates (Tmax, approximately 6 hr). The very short onset of absorption observed following administration of the drug in PEG can be explained in terms of the high avai l a b i l i t y of dissolved griseofulvin. Griseofulvin is a highly l i p i d soluble 65 drug. Once dissolved in PEG, griseofulvin readily crosses the gut membrane and appears in the plasma. The observation that the administration of 1 ml of suspensions containing either 5 or 25 mg drug resulted in the same Cmax (4.2 and 4.8^ig/ml respectively, Tables 4-XXI and 4-XXIII) supports this auggestion. Both suspensions contained 3.2 mg griseofulvin in the dissolved form (Fig. 4-1). This relatively small quantity of the available griseofulvin appears in the plasma as a sharp peak shortly after the administration (Fig. 4-2). Further absorption, however, requires a longer time, because the solid griseofulvin must f i r s t dissolve and then be absorbed. Dissolution of griseofulvin even in PEG is very slow, e.g., to dissolve a sample of 5 mg griseofulvin in pure PEG 300 or 600 required approximately 1 hour stirring with a magnetic s t i r r e r . A second griseofulvin-plasma peak also appeared at 6 to 8 hours post-dosing. This peak was of small magnitude (Fig. 4-2). An increase of fivefold in the administered dose (from 5 to 25 mg) resulted in only a small increase in AUC (from 11.2 to 18.5 j^g/ml/hr, Tables 4-XXI and 4-XXIII). This observation suggests that the absorption of solid griseofulvin is possibly dissolution rate limited. It is interesting to note that when griseofulvin i s administered in the PEG vehicle, absorption commences from the time immediately following administration (the f i r s t blood samples usually contained the highest Cmax, i.e., Tmax ( 15-20 min). This observation seems consistent with a significant absorption of the readily available dissolved griseofulvin in the stomach. The gastric emptying studies (Fig. 4-4) revealed that after one hour, approximately 84% of the griseofulvin-PEG 600*osolution could be recovered from the stomach, i.e., following administration of griseofulvin-8. Hours Figure 4-2. Griseofulvin-plasma level vs time following oral administration of single doses in 0.5%,;Tween 80(100 mg/kg), Q , 2% Tween 80 (100 mg/kg), • , 70% polyethylene glycol 300 (20 mg/kg), • , 70% polyethylene glycol 300 (100 mg/kg), O ,and 100% polyethylene glycol 600 (50 mg/kg), e • Each curve represents data from one rat closest to the mean. 6 7 PEG preparation, Tmax occurs when the drug is mainly in the stomach. Alternatively, i t appears more likely that a portion of the dissolved griseofulvin may be emptied from the stomach and absorbed quite rapidly even though the majority of the dose may s t i l l be found in the stomach at one hour. Among the administered dosage forms, griseofulvin-PEG 600 (solution) showed the highest Cmax (10.98^.g/ml, Table 4-IX) and, considering the dose (50 mg/kg), the highest AUC (37.85 i^g/ml/hr, Table 4-IX). This observation is consistent with that of Chiou and Riegelman ( 3 6 ) who admini-stered different preparations of griseofulvin to dogs and found that the absorption from a griseofulvin-PEG 400 solution was much faster and more complete. However, the absorption of griseofulvin even when administered in solution form, is by no means complete, because, as will be discussed later, the extent of absorption following administration of griseofulvin in solution to metoclopramide pretreated rats is threefold higher than in control rats. The relative bioavailability (AUC) of the preparations containing 70% PEG 300 (11.2 and 18.5^g/ml/hr for 20 and 100 mg/kg doses respectively) was significantly lower than that of pure PEG 600. This is likely due to the fact that in griseofulvin-PEG 600 preparations a l l of the drug (12.5 mg) was in solution while in 70% PEG 300 preparations only 3.20 mg of the administered doses (5 or 25 mg) was in the dissolved form. Absorption of griseofulvin from preparations containing Tween 80 was also erratic and incomplete. The mean Cmax and mean AUC following administration of 100 mg/kg griseofulvin in 0.5% Tween 80 was 6.69M g/ml and 68 43.28 ug/ml/hr respectively. Addition of 2% Tween 80, however, reduced the Cmax to 3.64 yg/ml and AUC to 30.00 yg/ml/hr, i.e., increase in Tween 80 resulted in a significant decrease in absorption. Recently Bloedow and Hayton (39;) administered 50 mg/kg griseofulvin in pure Tween 80 (solution) to the rat and noticed a Cmax of 1.53 yg/ml, Tmax of 8 hr and AUC of 15.8 yg.hr ml \ i.e., a solution of griseofulvin in pure Tween 80 has lower relative availability than aqueous suspensions containing 0.5 or 2% Tween 80. The critical micelle concentration (CMC) of Tween 80 is approximately 0.05%. Concentrations used in this research were well above the CMC (0.5 and 2%). In this concentration range Tween 80 is expected to act as a solubilizing agent as well as a wetting agent. Griseofulvin, in a suspension containing Tween 80, seems to have been incorporated into a complex phase with a very low apparent availability. The significantly lower AUC observed following administration of 100 mg/kg griseofulvin in 2% Tween 80 as compared to the same dose in 0.5% Tween 80 supports this hypothesis. A slow release would give rise to a long Tmax and, due to the removal of the drug from the absorptive sites before complete absorption, a lower AUC would be obtained. As can be seen in Tables 4-III and 4-VII the griseofulvin concen-tration in the terminal part of the plasma-level vs time curves fluctuates considerably and makes an accurate evaluation of the biological half-life of the drug virtually impossible. This is perhaps because of the continuous absorption of griseofulvin throughout the gastrointestinal tract. This observation was consistent in almost a l l of the curves regardless of the received treatments or administered dosage forms. 69 Effect of Motility Modifiers on Gastric Emptying The principal griseofulvin absorption site has been suggested to be the proximal segment of the small intestine (35). Absorption of the drug from the stomach when administered in solid dosage forms is very limited. Thus, i t appears reasonable to suggest that the gastric emptying rate affects the rate of absorption of griseofulvin. The longer the drug remains in the stomach the longer w i l l be the time required for attainment of Cmax. Meto-clopramide and propantheline have been used in this study to increase and decrease respectively the gastric emptying rate and gastrointestinal motility. In order to ascertain the influence of these agents on the gastric emptying, their effects were studied by determining the percent of'amr^s' ; • administered dose of griseofulvin remaining in the stomach as a function of time. The results of gastric emptying experiments are detailed in Tables 4-1 and 4-II and their mean values are shown in Figs. 4-3 and 4^ -4 follow-ing the administration of suspension (0.5% Tween 80) and solution forms (PEG 600) of griseofulvin respectively. Since the gastric absorption of griseofulvin (particularly following the administration of suspension forms) is limited,, the disappearance of the drug from the stomach can be considered to be mainly due to the gastric emptying process rather than the absorption process. The stimulating effect of metoclopramide on the gastric emptying rate is clearly demonstrated by the fact that, one hour after administration, only 24% of griseofulvin in suspension and 62% of griseofulvin in solution remained in the stomach as compared to 76% and 83% of suspension and solution respectively in the control rats (Figs. 4-3 and 4-4). rc o rd fi o -H CO c •H W) C •H •H fi QJ > rH 4H O 0) CO •H CD -H C 0) O

r- AUG, Cmax, AUC, Cmax, 37.6- 15.32 126.6 20.34 3784 5.83 38.8 13.10 138.6 25.42 27.5 .3.87 55'. 5- 12.01 84.6 22.03 16.4 3.51 30.1 6.09 142.4 34.81 :-2«:2 :3.40 37.9 11. 63 115.0 33.03 17.8 3.40 41.3 12.33 126.4 25.25 22.6 28.9 8.12 119.4 17.60 13.5 3 2.6 9.23 158*2 37.20 6.1 Mean, 37.8 10.98 126.4 26.96 21.2 4.00 Standard error, 2.96 1.051 7.73 2.551 3.46 0.48 Mean AUC/dose, hr.ml~ 1.10 3 ' 3?03 10.11 1.69 Coefficient of 22.1 27.1 17.3 26.8fe 46.3 27.0. variation, %, Statistical difference, s s s , s 1. Determined by the Student's t-test; a=0.05 (2 tailed); s, significant. 89 Consequently, the undissolved drug travels through the gastrointestinal tract very slowly, permitting more drug dissolution and subsequent absorption. The oral solution studies support the preceding explanation. With an oral solution of griseofulvin, the drug i s readily available and a dissolution process i s less significant; the absorption is more rapid and begins from the moment that the dosage form is in contact with the absorptive site. Some precipitation of the dissolved fraction may occur but i t s consequence on the absorption w i l l be less significant than that of a suspension form as i s evidenced by the observed AUC. Metoclopramide pretreat-ment markedly increased the bioavailability of the solution. This is because the transit time in the gastrointestinal tract has been shortened, and there is less chance for precipitation of griseofulvin in the stomach. Therefore there i s no dissolution process to delay absorption, and the drug is readily absorbed. However, propantheline pretreatment induces a longer residence time of the drug in the stomach where the absorption of griseofulvin is limited. This delayed release of the drug to the absorption site may result in either precipitation of the drug or in degradation of griseofulvin. Davis et a l (28) noticed a continuous disappearance of griseofulvin from the alimentary canal of the rat corresponding with the f a l l in blood level. They attributed this observation to griseofulvin destruction in the gut. However, in this work addition of 1 ml d i s t i l l e d water to 1 ml of griseo-fulvin in PEG 600 solution in a test tube caused the drug to gradually pre-cipitate into fine crystals. This observation, coupled with the fact that a large percent of unchanged griseofulvin was recovered from the stomach of the propantheline pretreated rats 11 hours after griseofulvin administration, 90 suggests that precipitation rather than destruction of the drug may be responsible for the poor relative bioavailability. In contrast' to the observed effect of propantheline on griseofulvin solution, Manninen e_t a l ( . 1 3 ) noticed that the absorption properties of digoxin, when given in solution form, were unaffected by the treatment with the anticholinergic drug. This difference can perhaps be explained by the different solubility properties of these two drugs, i.e., following admin-istration, upon longer residence time in the stomach griseofulvin may precipitate and yield a lower relative availability while digoxin may remain in so'Sufei6*nita"ndi s.ub'setq.uently. b.e ;abIs,oj-Aed>., Multiple Dose and Long Term Treatment with Propantheline - The results of multiple dose treatment with propantheline are detailed in Table 4-X. Figure 4-7 a,b,c depicts plasma levels of griseofulvin following administration of a single dose of 100 mg/kg griseofulvin in 0.5% Tween 80 to rats treated intraperitoneally with 5 mg/kg propantheline bromide at 2 hours prior to and at 6, 14 and 26 hours post-griseofulvin dosing. Table 4-X and Figs. 4-7 d and e show the results of an oral administration of a single dose of 100 mg/kg griseofulvin to rats pretreated with a single intraperitoneal dose of 5 mg/kg propantheline bromide. The latter group of rats was used to simultaneously study the effect of single and multiple treatment of propantheline on the plasma levels of griseofulvin suspension. The results, D f long-term treatment with propantheline on,vgriseofulvin-ipl?asmaofle,vel!S:ofuiyin are detailed in Table 4-XI and are plotted as griseo-•-' fulvin plasma concentration vs_ time in Fig'i( 4-8. Test animals received doses of 5 mg/kg propantheline bromide twice a day for seven days prior to and during the experiment. 9 1 -Figure 4-7, ,(a,b,and c).. . For details see next page -* i g u * 4- • f.;Ca'D% ana •£) . .tor a e u u see neict 6 t page 9 2 4 12 20 28 36 44 52 Hours Fi g u r e 4 - 7 : . ^ G r i s e o f u l v i n - p l a s m a c o n c e n t r a t i o n s f f bilowinsgr-oral a d m i n i s t r a t i o n of s"±tt|iie^ iii^ o6e'i3j;.••. of 100 mg/kg g r i s e o f u l v i n i n 0 i 5% Tween 80 to the r a t s p r e t r e a t e d i n t r a p e r i t o n e a l l y with either, a .single, dose/•".of ^ propantheline (2 hours prior to jgriseofulyin;' "rats d and ~e) :or multi^^ at' 6,.. 14..and 24 hour's. after' gri-seofulvin administration;' rats, a, ;b and -c. in.- preceding" page) -~\ Arrows'indicate the" time of propanthelineadministration. Table 4-XJ Plasma Levels of Griseofulvin (jjgY ml).M-olt^liig-it s lOMl'i A4minlsEratiofiO(jbgQlqgg/kgOiri%QT5%-TwSen 80) to Animals Receiving either a.SingleiposeopjfwJ>.r.opantKeiine',26H6iirs'nP.r$ort'to, Griseofulvin Administration .or. Multiple. Doses at. 2^-Hours -Prioretc ka'h(ia*aty6, 14 and 24 Hours After Griseofulvin Administration. latRat Hours 10 14 17 20 26 30 48 72 Propantheline a 0.398 1.102 1.878 1.180 1.149 3.129 1.800 0.622 0.361 — — Pretreated (5 mg/kg, i.p., b — 0.461 8.91 1.295 3.525 3.260 1.726 0.548 0.584 1.020 0.000 at -2, 6, 14 and 26 hours). c 0.844 1.777 3.882 5.245 2.910 4.669 1.082 0.375 0.237 — — Propantheline d — 0.334 0.726 4.235 2.609 1.694 0.854 0.241 0.282 Pretreated (5 mg/kg, i.p. e 0.565 0.440 2.534 1.920 1.955 5.232 1.604 0.945 — at -2 hours) 94 12 20 28 36 44 52 Hours Fig u r e 4 -8" ( a, b, and c ). iFor. i e t a i l s s!ee3p§fel^96. ho G r i s e o f u l v i n - P l a s m a L e v e l s , ug/m-L -i i r o c l-i CO K3 N3 O N3 oo Lo 4> N3 H i 96 Figure 4-8 . Griseofulvin-plasma concentrations following oral administration of single doses of 100 mg/kg g r i s e o f u l v i n in 0.5% Tween 80 to the rats treated i n t r a p e r i t o n e a l l y with 5 mg/kg propantheline bromide twice d a i l y , 7 days prior to and during the plasma: studies, • :.'(ffats ^ $hrQHgh:hj):-X*see last, two preceding pages), (h=l). ' . Table 4-XIT. Plasma Levels of Griseofulvin (ug.^l)1' f o l a o w i n ^ Tween;.80) to the Control and Test Rats. Control Animals were treated Intraperitoneally with 1 ml of Di s t i l l e d Water while Tests Rats received 5 mg/kg Propantheline Bromide Intraperitoneally Twice Daily 7 Days Prior to and During the Plasma Studies. Rat Hours ConfrcIs 2 4 6 11 14 17 20 24 28 48 Controls 1 1.188 5.982 3.843 0.517 0.523 0.291 0.235 0.030 0.036 0.010 2 1.637 3.565 5.143 0.547 0.450 0.432 0.588 0.501 0.082 0.000 Propantheline a 1.015 2.481 1.156 4.093 3.139 5.082 1.375 Long-term Pretreated b 0.647 1.021 1.200 1.892 3.715 — 1.874 1.458 0.479 0.019 c 2.190 1.383 0.729 1.907 2.280 1.208 2.799 2.156 — — d 1.785 0.568 1.041 1.973 1.819 0.928 1.507 2.264 0.000 0.015 e 2.595 1.468 1.730 1.078 0.641 — 0.429 0.328 0.146 ' — f 0.875 0.795 0.819 3.401 — 2.954 2.960 0.340 0.453 — g 1.136 8.588 4.855 3.290 2.035 1.837 0.787 0.000 0.378 0.000 h 1.681 3.544 4.505 2.445 4.149 5.063 2.858 0.902 0.282 0.000 98 Following repetitive treatment with propantheline, a l l of the griseofulvin-plasma levels-time curves share the unusual characteristic of multiple peaks" and-.4, demonstrated f tendency to fluctuate. This observation was also noticed to a lesser degree in some^of the single treatment studies which were described previously (Table 4-V). Attention should be paid to the fact that Fig. 4-4, which, depicts the results obtained after treatment with single doses of propantheline, is expressed in terms of mean plasma levels vs time. As a result of averaging the data points, the fluctuations are obscured and the resulting curve appears like a smooth curve. No correlation between the time of propantheline treatment and frequency of occurrence of the peak plasma levels was found. Further, no reasonable explanation for inconsistency of the plasma levels can be given except the possible inter animal variations in response to the pharmacologic effect of propantheline. Nevertheless, the ge'lafeiveilbioavaiMhMity of griseo-fulvin'y] as a result of multiple treatments with propantheline is higher than that of controls. The explanation outlined in single propantheline treatment studies.is also valid in repetitive treatment studies, namely that propantheline causes retardation of gastrointestinal motility. As a result of>delayed' emptying ct^e^ratWo~P^ f&dmvit-heatstomalchr.mays .x*.- :. be reduced; the drug w i l l travel slowly through the small intestine and w i l l be exposed to the absorptive sites. These effects may respectively give rise to: a) reduced rate of absorption, reflected in a longer time for the peak plasma level to be reached; and b) an increase in the extent of absorption, indicated by a larger AUC. Nevertheless, these two experiments, which are conducted in a manner 99 analogous to c l i n i c a l use ( i . e . , therapeutic administration of propantheline), c l e a r l y demonstrate the unexpected g r i s e o f u l v i n absorption p r o f i l e one may observe i f g r i s e o f u l v i n and propantheline are administered concomitantly. These observations also point to the need f or follow-up experimentation i n humans to examine t h i s p o t e n t i a l i n t e r a c t i o n . G r i s e o f u l v i n represents a unique model drug due to i t s exceptionally e r r a t i c d i s s o l u t i o n and absorption c h a r a c t e r i s t i c s . Thus the observed findings with g r i s e o f u l v i n may lead to further experimentation^;' with other poorly absorbed drugs such as dicoumarol. Intravenous Studies - Although there i s no evidence i n the l i t e r a t u r e to suggest that e i t h e r metoclopramide or propantheline influence the metabolism of any drug, i t appeared necessary to investigate whether they have any e f f e c t on the k i n e t i c s of g r i s e o f u l v i n metabolism. Therefore g r i s e o f u l v i n was administered intravenously to the cont r o l r a t s and to those pretreated i n t r a -p e r i t o n e a l l y with s i n g l e doses of eit h e r metoclopramide (10 mg/kg) or propan-th e l i n e (5 mg/kg) 2 hours p r i o r to the g r i s e o f u l v i n administration. This experiment was also conductedoon rats pretreated with si n g l e o r a l doses of 15 mg/kg sodium phenobarbital 24 hours p r i o r to the griseofuLvin. administration. Phenobarbital i s a known enzyme inducer. B u s f i e l d et a l ( 1 7 ) , who reported the griseofulvin-phenobarbital i n t e r a c t i o n i n the r a t , suggested that the i n t e r -action occurs at the l e v e l of metabolism. This hypothesis was questioned by Riegelman et a i ( 2 0 ) . In man, they (20 ) suggested that the g r i s e o f u l v i n -phenobarbital i n t e r a c t i o n r.eBul-.tejd' ea from a reduced absorption rather than induced metabolism. In t h i s study, the findings of the i v experiments,' coupled with those of o r a l studies, provide;.!, a better understanding of the mechanism of the i n t e r a c t i o n . 100 Figures 4-9 and 4-10 depict plots of plasma concentration and logarithm of plasma concentration of griseofulvin vs time following a single iv dose of 26 mg/kg of the drug on a control rat. The curves are fitt e d by computer, based on the assumption that the pharmacokinetics of griseofulvin in the rat follows a biexponential equation: C P = A.e- a t + B . e - S t £1, The calculated parameters for each set of data are given in: Appendix II.. The biological half-lives of the post-distribution and the AUCs of griseofulvin are shown in Table 4-XII. The biological h a l f - l i f e of the post-distribution phase was observed to be reproducible and ranged from 1.9 to 2.4 hours in both control and test animals, i.e., pretreatment with phenobarbital, meto-clopramide and propantheline had no significant effect on the slope of the post-distribution phase of griseofulvin. This is of particular interest; the slope of the elimination (post-distribution) phase after iv administration is defined by the following equation: . Hi k21 s l o p e = = ~ a(2.303) where aais the rate constant of the distribution phase and the rest of the terms are defined on pager1072.v In the case of griseofulvin, since"less than •U%.tof. an iv dose is excreted unchanged '(iii arine.a(66.);? t-he^rate constant of elimination, k is almost exclusively associated with the metabolism of the drug. Therefore, since there is a direct relation between the slope of 101 ( 3 D C0NT.02 I 15.0 TIME(HR) - 1 — 27.0 0.0 18.0 21.0 24.0 30.0 F i g u r e 4-9. Computer ^fi«ed-.^ c o n c e n t r a t i o n s vs time f o l l o w i n g intravenous • a d m i n i s t r a t i o n of a s i n g l e dose of 26 mg/kg g r i s e o f u l v i n d i s s o l v e d i n p o l y e t h y l e n e g l y c o l 600 to the rat,(h=l). 1 0 2 o CD 0.0 . C0NT.02 3.0 6.0 15.0 TIME(HR) 30.0 4-1:0- Computer C • f3!ttfcdV l o g a r i t h m of plasma c o n c e n t r a t i o n s " I S/5» a d m i n i s t r a t i o n of a s i n g l e F i g u r e vs time curve f o l l o w i n g intravenous., ifse of 26 mg/kg g r i s e o f u l v i n d i s s o l v e d "in p o l y e t h y l e n e g l y c o l 600 to the rat„.( n=l ) . 103 the terminal part of the curve and k ^ , any changes i n the g r i s e o f u l v i n metabolism rate constant must be r e f l e c t e d i n the slope of the post-d i s t r i b u t i o n phase and consequently, the h a l f - l i f e of the drug. The fa c t that, i n both control and test animals, the terminal part of the curves are p a r a l l e l ( F ig. 4-11) leads to the conclusion that pretreatment with pheno-b a r b i t a l , metoclopramide and propantheline has not s i g n i f i c a n t l y influenced the metabolism of g r i s e o f u l v i n i n the r a t . Despite the reproducible b i o l o g i c a l h a l f - l i v e s , large deviations were observed among other calculated pharmacokinetic parameters. These v a r i a t i o n s were p a r t i c u l a r l y noticed i n the AUC values (Table 4-XII). Further-more, the calculated AUCs following i v administration of g r i s e o f u l v i n were found to be generally smaller than expected, because the mean AUC/dose obtained following o r a l administration of g r i s e o f u l v i n i n PEG 600 to the metoclopramide -1 3 pretreated rats was 10 hr.ml .10 (Table 4-IX), while t h i s value following i v -1 3 i n j e c t i o n of g r i s e o f u l v i n was approximately 6 hr.ml .10 (Table 4-XII). These r e s u l t s cannot be r e a d i l y interpreted due to the l i m i t e d number of blood samples taken during the d i s t r i b u t i o n phase. In j e c t i o n of the g r i s e o f u l v i n -PEG 600 preparation into the t a i l vein caused noticeable paling of the t a i l during the f i r s t hour, making the blood sampling extremely d i f f i c u l t . Never-theless, the t t of the p o s t - d i s t r i b u t i o n phase was found to be consistent throughout the experimentation. The d i s t r i b u t i o n phase plays an important r o l e i n the evaluation of pharmacokinetic parameters. Bedford e_t a l (35) suggested that, i n the r a t , following a single i v dose of 20 mg/kg g r i s e o f u l v i n more than 90% of the drug disappeared from the blood wi t h i n f i v e minutes. Due to the experimental design, the frequency of blood sampling during the 104 3 9 15 21 27 3 9 15 21 27 Hour s Hours F i g u r e 4-.ifi. Computer .fitted* " l curves of l o g a r i t h m of plasma c o n c e n t r a t i o n s vs time and r e s i d u a l c o n c e n t r a t i o n s vs time f o l l o w i n g i n t r a v e n <3us.o a d m i n i s t r a t i o n of s i n g l e doses of g r i s e o f u l v i n / p o l y e t h y l e h e g l y c o l 600 to a c o n t r o l r a t X a ), and r a t s p r e t r e a t e d with s i n g l e doses of meto-clopramide ( b ) , p r o p a n t h e l i n e ( c ), or p h e n o b a r b i t a l ( d ),.,( n = 1) . 1 0 5 Table 4-XII Biological Half-Life of the Post-Distribution Phases(0.693/- B) and Area^. Under the Griseofulvin-Plasma-Time Curves(AUC) in the Rat after Intravenous Administration of Single Doses of the Drug in Polyethylene Glycol 600. Test Rats received Single Doses of Metoclopramide Hydro-chloride(10 mg/kg), Propantheline Bromide(5 mg/kg) or Sodium Pheno-barbital(15 mg/kg) prior to.the Griseofulvin Administration. Dose, 0.693/B, AUC,_1 AUC/Dose. mg/kg. hr. u g.hr.ml . hr.ml--'-. lO-^ t Controls, 20 20 T w Phenobarbital 40, j pretreated, 20 , 16 T t Metoclopramide 20 ^ pretreated, 20"; t Propantheline 20 ^ pretreated, 20 2.4 27.0 5.40 2.0 22.2 2.4 29.6 5.92 2.3 42.5 4". 25 2.4 33.1 6.62 2.1 25.2 6.30 2.1 25.2 5.04 2.1 25.8 5.16 2.1 61.2 12,24 2.1 "<25..2 '5.04 Y) Solution of 10 mg/kg griseofulvin in 100% PEG 600. t) Solution of 5 mg/kg griseofulvin in 100% PEG 600. 106 f i r s t few minutes of experimentation was limited. Similar results have been observed by Chiou and Riegelman ( 3 6 ) . In Fig. 3 of their paper, the plasma concentrations of griseofulvin obtained after administration of various dosage forms ( a l l corrected to a 50 mg dose) to a dog are presented with another curve obtained following iv administration of a single dose of 50 mg griseofulvin. The AUCs of a l l of the oral administrations seem to be substantially larger than those of the intravenous. No explanation appears to have been given for this observation by Chiou and Riegelman. Two possible mechanisms may be responsible for the observed low AUC after iv administration: (a) the drug may precipitate in the systemic circulation immediately after the injection due to i t s very low water solubility; or (b) i t may be eliminated at a significantly higher rate during the f i r s t few minutes. The f i r s t hypothesis seems unlikely; reproducible half-lives of the post-distribution phase would seem to indicate that no re-distribution of the drug due to dissolution of the precipitated griseofulvin has taken place. If the drug were precipitated in the systemic circulation one would expect to observe a gradual dissolution of the drug in that region which in turn would give rise to irreproducible post-distribution half-lives. The second mechanism, i.e., higher rate of elimination in the 1 0 7 f i r s t few minutes, could easily be responsible for the observation. The disappearance of a given drug from the central compartment takes place according to the following rate equation: dc 'dt k e l C - ~ k12 C + k21 T (-9) dc where - is the rate of disappearance of the drug from the central compartment, C is the drug ccajnQiiat-jitxor.,. k ^ is the rate constant for the elimination of the drug from the central compartment, k ^ and k2^ are the ratecconstants for the distribution of the drug into the other compart-ment (s)and for the redistribution of the drug back into the central compartment respectively, and T is the drug con aniount ci O I 1 i n other compart-ment (s) as shown in the following diagram: iv 12 k21 k ^ and k^^ can be considered as hybrid rate constants/where more than two compartments are involved. 108 As shown in Eq. 9 the rate of disappearance ( - -^ ~) is directly proportional to the drug amount in the central compartment. If k^, k^ a n <^ k.^ remain constant (Eq. 9), only the amount terms will be variable and any increase in the amount will give rise to an increase in the rate of disappearance. After injection of a dose of 5 mg griseofulvin into a 250 g rat the amount of the drug in the systemic circulation will rise sharply. Therefore, either the rate of disappearance increases and a large portion of the drug crosses the central compartment and distributes into other compartments or i t will be eliminated in the very first few minutes after administration. The main objective of the iv experiments was to show that the pre-treatments with phenobarbital, metoclopramide and propantheline did not influence the biological half-life and hence metabolism of griseofulvin. The results of absorption studies also support the hypothesis that these treatments did not influence the rate of griseofulvin metabolism. Since alternative experimentation served to clarify the interaction between griseofulvin and phenobarbital, continued study of the pharmacokinetics of griseofulvin in the rat was not performed. An induced metabolic system for a drug would be expected to result in reduced plasma levels. Conversely, a metabolic inhibition would give rise to an increase in the plasma levels. These alterations in the metabolism of a drug should be observed irrespective of the dosage form or the route of administration. Formulation-dependent griseofulvin-plasma levels observed in the rats pretreated with metoclopramide and propantheline (as discussed earlier) imply that these motility modifiers do not affect the rate of griseofulvin metabolism in the rat. As will be discussed later, the effect of phenobarbital on the griseofulvin-plasma levels was also formulation-dependent . 1 09 Griseofulvin-Phenobarbital Interaction The significance of gut motility on the absorption of griseo-fulvin has been ishpwn>earli-e.ri and sthis part vof." thejsthesis i s devoted to clarifying the mechanism of the observed interaction between griseofulvin and phenobarbital. The griseofulvin-phenobarbital interaction has been observed when griseofulvin i s given orally (;16,1 7,1.8>,203). It is also interesting to note that, in previous reports (ll'6 s J 17,«18» 20") regarding this observation, griseo-fulvin had been administered in solid dosage forms, i.e., tablets in man and suspension (in 0.5% Tween 80) in the rat. Therefore, i t appeared necessary to investigate the griseofulvin-phenobarbital interaction after administration of solutions and more rapidly dissolving suspensions. Unless precipitation occurs following administration, the effect of the dissolution process should be minimal, and more information regarding the mechanism of the interaction may be provided. Thus, in this work, the griseofulvin-phenobarbital interaction was studied following administration of single doses of the following preparations: 1) 100 mg/kg griseofulvin in 0.5% Tween 80 (Fig. 4-12) 2) 100 mg/kg griseofulvin in 2% Tween 80 (Fig. 4-13) 3) 50 mg/kg griseofulvin in 100% PEG 600 (Fig. 4-14) 4) 20 mg/kg griseofulvin in .70% PEG 300 (Fig. 4-15) 5) 100 mg/kg griseofulvin in 70% PEG 300 (Eig. 4-16). A l l preparations except griseofulvin-PEG 600 are in suspensions. Griseo-fulvin concentrations in the supernatants of the formulations were measured 110 and reported in the equilibrium solubility measurement section. Griseofulvin-PEG 600 formulation was a clear solution of the drug. A suspension of griseofulvin in 0.5% Tween 80 was administered to examine the reproducibility of the reported phenobarbital interaction ( 1 6 ) . The suspension of griseo-fulvin in 2% Tween 80 was used to study the effect of an increase in the surfactant concentration and hence to simulate the influence of bile salts on the observed interaction. Because of the relatively high solubility of griseofulvin in PEG, this vehicle was used in an attempt to prepare more rapidly dissolving preparations. However, as mentioned earlier, administration of griseofulvin in pure PEG 600 caused noticeable change in the appearance of the gut along with an apparent tendency to retain water. Since such changes might influence the absorption of griseofulvin, suspensions of the drug in 70% PEG 300 were used to further study the interaction. This solvent produced no noticeable effect on the gut appearance. The observed changes in Cmax, Tmax and AUC caused by phenobarbital pretreatments following administration of different griseofulvin preparations are summarized and compared with those of motility modifiers in Table 5-T... -Griseofulvin Suspensions in Tween 80 - Plasma levels following oral administration of a single dose containing 100 mg/kg griseofulvin in 0>5% Tween 80 to the control rats and those pretreated orally with single doses of sodium phenobarbital are shown in Table 4-XIII. The mean plasma-griseofulvin levels vs time plots obtained from control and test animals are shown in Fig. 4-12. It has been hypothesized (20) that the griseofulvin-phenobarbital interaction may be due to the a b i l i t y of the barbiturate to increase gut motility. In this work, phenobarbital pretreatment, like Hours Figure 4-12. Mean griseofulvin -plasma levels vs time following oral administration of single doses of 100 mg/kg in 0.5% Tween 80 to the control, A (n=6) and phenobarbital pretreated,O (n=6) rats. Error bars represent standard error of the mean. Table 4-XIII Plasma Levels of Griseofulvin (Micrograms per M i l l i l i t e r ) following Oral Administration of 100 mg/kg i n 0.5% Tween 80 i n the Control and Test Rats . Phenobarbital (15 mg/kg) was given Orally to the Test Rats, 24 hours prior to Griseofulvin Administration. Hours Rat 12 24 48 Griseofulvin 1 suspension 2 control 3 4 5 6 Mean (SE) a 1.881 2.670 1.656 6.009 1.482 2.533 0.633 5.557 1.378 5.680 3.841 5.224 1.812 4.592 (0.441) (0.664) 5.962 7.236 7.679 7.061 6.013 6.920 6.613 7.018 6.020 6.048' 6.567 6.717 (0.399) (0.22Q) 2.251 . 0.520 4.528 1.508 3.743 0.214 3.249 0.036 1.234 0.432 3.817 2.014 3.137 0.787 (0.489) (0.322) 0.238 0.058 0.280 0.120 0.120 0.147 0.813 0.161 0.174 0.293 0.117 (0.107) (0.024) Phenobarbital 1 1.251 4.236 4.822 3.372 1.177 0.326 0.087 0.003 sodium 2 2.162 2.835 3.926 4.073 0.740 0.389 0.062 0.012 (15 mg/kg, 3 1.256 1.745 2.391 3.640 0.836 0.219 0.008 0.001 Orally) 4 1.620 2.984 3.301 1.815 0.673 0.136. 0.012 0.000 predosing 5 2.483 2.720 2.035 1.362 0.564 0.093 0.030 0.003 6 1.420 2.033 2.811 3.203 0.815 0.535 0.071 0.010 Mean 1.699 2.759 3.214 2.911 0.801 0.283 0.045 0.005 (SE) a (0.209) (0.355) (0.421) (0.439) (0.085) (0.068) (0.013) (0.002) S t a t i s t i c a l significance ns . s s s s ns s s a) Standard error of the mean in parentheses b) Determined by Student's t-Test (2 t a i l e d ) ; P<0.05 1 1 3 TableT4-XIV Area Under Plasma Concentration-Time Curves, AUC (ugrhr.ml ^) of Griseofulvin Following Oral Administration of Single Doses of 100 mg/kg of the Drug in 0.5% Tween 80 to the Control and Phenobarbital Pretreated Rats. Control, Phenobarbital pretreated, 37^ .5 26.98 53.0 25.8 37.3 19.3 37.8 19.6 40.4 19.2 52.96 22.2 Mean, 43.2 22.1 Standard error, 3.13 1. 39 Mean AUC/dose, hr.ml 1.73 0.87 103 Coefficient of 17.8 15.5 variation, %, ^ St a t i s t i c a l difference » s 1. Determined by the Student's t-test; a=0.05 (2 tailed); s, significant. 114 Table 4-XV Peak Plasma Levels, Cmax and their Time of Occurence, Tmax Following Oral Administration of 100 mg/kg of the Drug in 0.5% Tween 80 to the Control and Phenobarbital Pretreated Rats. .Phenob arb i t al 1 Control > pretreated, Cmax, u g/ml. Tmax, hr. Cmax, ru g/ml. Tmax, hr. 7c. 35 6 4.82 5 7.68 5 4.10 6 6.92 6 3.64 6 7.02 6 3.30 5 6.02 6 2.72 5 6.05 6 3.20 6 i Mean, 6.84 5.8 3.63 5.5 Standard error, 0.28 0.17 0.30 • 0.22 ;Coefficient of 9.9 7.0 20.4 10.0 variation, %, i Statistical difference . s ns 1. Determined by the Student's t-test; a=0.05 (2 tailed) s,significant. 1 1 5 metoclopramide pretreatment, reduced both griseofulvin plasma levels and the area under the griseofulvin-plasma concentration-time curve (AUC). The observed effects of phenobarbital are in agreement with the report of Busfield et a l (16). Although both drugs decreased the AUC to the same extent, i.e., by about.50% (Tables 4-VI and 4-XIV), the effect of metoclopramide preadministration on the mean peak plasma level (Cmax) was more profound than that of phenobarbital pretreatment (Tables 4-V and 4-XV). The former drug reduced the mean Cmax by 62% while the latter lowered this parameter by 47%. A very significant difference between these observed interactions was that phenobarbital, unlike metoclopramide, did not shorten Tmax (Table 4-XV). This might be an indication that the barbiturate did not influence gastric emptying. Metoclopramide has two distinct effects on the Gl tract. It shortens the residence time in the stomach, where absorption is limited, and i t increases the motility of the proximal part of the intestine, i.e., the principal absorption site for griseofulvin. The former effect results in a more rapid absorption and is mostcobviouswwhentthe drug is given in suspension form. The latter effect of metoclopramide, i.e., increased motility of the proximal part of the intestine, however, causes the drug to be forced past the absorption site and gives rise to a decreased absorption. This effect is clearly shown in the suspension studies, where, as a result of shorter residence time at the absorption site, the AUC is reduced significantly (Table 4-VI). Therefore, the observed effect of phenobarbital on the plasma levels of griseofulvin (in 0 .5% Tween 80) seems to suggest that the barbiturate mimics the second effect of metoclopramide, i.e., decreases the transit time in the region of the absorption site. But at this stage one Hours Figure 4-13. Mean plasma levels vs_ time following oral administration of .1.00 mg/kg griseofulvin in 2% Tween 80 to the control, O (n=4) and phenobarbital pretreated, # (n=5) rats. Error bars represent standard error of the mean. • ' Table 4-XVT; Griseofulvin-Plasma Levels (au g/ml) following Oral Administration, of Single Doses of 100 mg/kg Griseofulvin in 2% Tween 80 to the Control Rats and Rats Pretreated orally with Single Doses of 15 mg/kg Phenobarbital Sodium 24 Hours Prior to the Griseofulvin Administration. Hours Rats 1.5 4 6 8 12 24 Controls, 1 1.097 1.208 2.723 1.960 0.436 0.314 2 1.119 3.884 4.597 2.652 1.095 0.353 3 1.123 3,501 3.332 1.781 0.911 -4 3.068 4.222 3.930 2.852 0.880 -Mean, 1.602 3.204 3.645 2.311 0.830 0.333 Standard error, 0.488 0.681 0.401 0.260 0.140 0.019 Phenobarb i t a l pretreated, 1 2 0.434 1.034 0.967 0.812 2.620 2.416 1.063 0.700 0.721 0.952 0.457 0.295 3 3.480 2.447 3.447 2.599 1.010 -4 1.340 2.321 1.961 1.765 - 0.369 5 - . 0.873 3.151 2.186 ,„„2.394 _ _ J ) . 921 . 0.192 _ .. • ^ ~ "- • -• Mean, 1.432 1.940 2.526 1.707 0.901 0.328 Standard error, 0.532 0.452 0.255 0.322- 0.125 0.056 Stati s t i c a l significance,! ns2 ns S3 ns ns ns 1. Determined by Student's t-test ; a=0.05 . 2. Not significant. 3. Significant. Table 4-XVII Area Under Plasma Concentrations-Time Curves, AUC (uu g.hr.ml j) ,Peak Plasma Concentrations, Cmax ( y g/ml) and their Time of Occurence, Tmax (hr) of Griseofulvin Following Oral Administration of Single Doses of 100 mg/kg of the Drug in 2% Tween 80 to the Control and Phenobarbital Pretreated Rats. Control, Phenobarbital pretreated, AUC Cmax Tmax AUC Cmax Tmax 19.6 2.72 6 17.1 2.62 6 35.2 4.60 6 15.7 2.42 6 27.4 3.50 4 32.6 3.45 6 37.6 4.22 4 22.1 2.32 4 21.8 3.15 4 Mean, Standard error, 30.0 4.09 Mean AUC/dose, hr.ml - 1.10 3 1.20 Coefficient of variation, %, Stati s t i c a l difference 27.3 3.76 0.41 22.0 5.0 0.58 23.1 21.9 2.79 5.5 1.64 0.22 0.39 0.88 16.8 17.5 15.7 ns p<0.1 ns 1. Determined by the Student's t-test;;(2 tailed); ns, not significant. 11 9 cannot rule out the influence of other possible mechanisms (e.g., reduced dissolution due to a reduction in bi l e salt concentration or blood flow rate limited elimination). To c l a r i f y the problem, a suspension containing 2% Tween 80 was prepared and administered to the rat in the presence and absence of pheno-barbital. It was expected that a fourfold increase in the concentration of the surfactant should mask the surfactant effects of the bile salts. Fig. 4-13 and Table 4-XVI depict the griseofulvin-plasma levels vs time curves following administration of single doses of the suspension of griseofulvin in 2% Tween 80 in the presence and absence of phenobarbital. Although pheno-barbital pretreatment reduced the plasma levels of griseofulvin i t s effect was significant only at Cmax (a = 0.1). The treatment also failed to significantly influence AUC and Tmax (Table 4-XVII). These observations indicate that a fourfold increase in the concentration of Tween 80 has significantly reduced the extent of the interaction. Phenobarbital pretreat-ment followed by the administration of a suspension of griseofulvin in 0.5% Tween 80 reduced the mean Cmax by $0%, while under identical conditions but using 2% Tween 80 the barbiturate lowered the mean Cmax by only 31%. Therefore, i t i s reasonable to suggest that a decreased dissolution rate may be mainly responsible for the griseofulvin-phenobarbital interaction. TablesA^XIV and 4-XVII show that an increase in the Tween 80 concentration caused a decrease in the relative bioavailability of griseo-fulvin. Therefore a shorter residence time in the region of the absorptive sites would be expected to decrease the availability of the preparations with higher Tween 80 concentrations s t i l l further.- Thus, the griseofulvin-1 20 phenobarbital interaction is unlikely to result from an increase in motility. • Addition of 2% Tween 80 to the griseofulvin suspension has perhaps reduced the relative bioavailability of the drug to such an extent that phenobarbital treatment cannot reduce i t further. Phenobarbital treatment and an increased concentration of Tween 80 may influence the absorption of griseofulvin by the same mechanism. It is feasible to hypothesize that the pretreatment with phenobarbital followed by the administration of griseofulvin in 0.5% Tween 80 might have retarded the dissolution of griseofulvin by reducing the concentration of the b i l e salts. The mechanism of such an effect is not clear . because the change in the bile constituent as a result of pheno-barbital treatment has not yet been f u l l y studied. Furthermore, since the addition of extra surfactant to the preparation did not completely eliminate the effect of phenobarbital on the plasma levels of griseofulvin other mechanisms could also be involved in the interaction. However, preparations containing PEG provide more information regarding the mechanism of the interaction. Griseofulvin in PEG - The plasma levels following a single oral dose of 50 mg/kg griseofulvin in PEG 600 (solution) in the control and phenobarbital pretreated animals are shown in Table 4-XVIII and their means are plotted as functions of time in Fig. 4-14. Phenobarbital predosing failed to significantly affect the plasma levels of griseofulvin when the drug was administered in the solution form (Fig. 4-14, Tables 4-XVIII and 4-XX). This further suggests that, despite the similarity in the result from 0.5% Tween 80 suspension studies, the effects of phenobarbital and metoclopramide on the plasma levels of griseofulvin may not be due to the same mechanism. 10 12 24 Hours Figure 4-14. Mean griseofulvin-plasma level-vs time- following oral administration..of single doses of 50 mg/kg in 100% polyethylene glycol^.600 to the control, a (n=8) and phenobarbital pretreated, • (n=6) rats. Error bars represent standard error of the mean. Table" 4-XVI-II Plasma L e v e l s o f G r i s e o f u l v i n (Microgram p e r M i l l i l i t e r ) f o l l o w i n g i t s O r a l A d m i n i s t r a t i o n o f a S i n g l e 50 mg/kg i n PEG 600 Dose i n C o n t r o l and T e s t R a t s . P h e n o b a r b i t a l Sodium ( 1 5 mg/kg) was g i v e n O r a l l y t o t h e T e s t A n i m a l s , 24 h o u r s p r i o r t o G r i s e o f u l v i n A d m i n i s t r a t i o n : HOURS R a t s 0.33 0.5 1 2 3 4 6 8 12 24 48 G r i s e o f u l v i n 1 15.321 8.120 5.440 3.280 1.613 0.856 0.451 0.854 0.500 0.030 c o n t r o l 2 — 13.098 7.946 4.403 2.554 1.304 0.4'87 1.344 0.751 0.032 0.024 3 — 12.008 8.704 5.512 4.531 5.227 5.781 2.607 0.308 0.148 0.005 4 — 6.093 3.671 1.921 2.416 1.526 2.099 1.523 0.473 0.233 0.018 5 — 11.677 6.896 3.000 1.337 1.021 2.251 2.486 0.508 0.290 0.036 6 12.331 — 8.120 4.915 — 1.280 0.675 0.471 0.816 • — — 7 8.124 — 3.466 — 1.191 0.450 0.623 0.983 0.310 0.001 8 — 9.233 — 4.204 — 1.380 0.724 0.591 0.814 0.204 0.028 Mean 10.2275 11.238 7.243 4.108 2.824 1.818 1.665 1.262 0.688 0.208 0.020 ( S E ) a (2.100) (1.309) (0.754) (0.4415) (0.528) (0.491) (0.639) (0.314 (0.082) (0.062) (0.005) P h e n o b a r b i - 1 t a i sodium 2 (15 mg/kg, 3 o r a l ) 4 p r e d o s i n g 5 6 Mean ( S E ) a S t a t i s t i c a l b s i g n i f i c a n c e 15.301 8, .739 5.461 4.318 3.900 1.125 1.356 0.840 0.391 0.057 8.530 4 .779 1.685 0.871 0.737 0.183 0.233 0.736 0.314 0.014 18.246 6. .821 3.893 2.393 1.304 1.000 1.140 0.411 0.556 0.026 7.677 — 4.008 — • 1.411 2.010 0.528 0.724 0.380 0.018 14.464 — 8.263 — 2.560 0.594 0.560 0.803 0.318 — 9.360 — 5.440 — 2.850 0.487 0.911 0.798 0.210 0.021 12.283 6. .779 4.792 2.527 2.127 0.900 0.788 0.719 0.362 0.027 (1.763) (1. 143) (0.894) (0.997) (0.482) (0.263) (0.172) (0.064) (0.047) (0.008) ns ns ns ns ns ns ns ns ns ns a) S t a n d a r d e r r o r o f the mean i n p a r e n t h e s e s b) Determined by S t u d e n t ' s t - t e s t , (2 T a i l e d ) ; P ^ 0 . 0 5 Table 4-XIX Area Under Plasma Concentration-Time Curves, AUC (y g.hr.ml ) and the Peak Plasma Concentrations, Cmax (yg/ml) Following Oral Administration of Griseofulvin(50;mg/kg) in Polyethylene Glycol 600' ( solution ) to the Control and Phenobarbital Pretreated Rats. Phenobarbital Control, pretreated, AUC, Cmax, AUC, Cmax, 37.6 15.32 43.8 15.30 55.5 12.01 24.4 8.53 30.1 6.09 35.7 18.25 41.3 12.33 35.3 7.68 28.9. 8.12 41.4 14.46 32.6" 9.23 35.2 9.36 Mean, 37.7 10.52 36.0 12.26 Standard error, 4.05 1.36 2.75 1.76 -1 3 , - - • Mean AUC/dose, hr.ml .10 3.01 — 2.88 — Coefficient of variation, %, 26.3 . 31.7 18.7 35.2 Stati s t i c a l difference!, • ns ns 1. Determined by the Student's t-test; a=0.05 (2 tailed); ns, not significant. 1 24 Riegelman ejt a_l ( 2 0 ) hypothesized that phenobarbital might increase the motility of the proximal part of the intestine through i t s effect on bile .flow -since the stimulating effect of phenobarbital on bil e flow has been observed by several investigators ( 21 '-t2 3 ). Nevertheless, the present evidence implies that the influence of phenobarbital on the absorption of griseofulvin is unlikely to result from an increase^ in motility. If the observed effect of phenobarbital on the plasma levels of griseo-fulvin were due to an enhanced motility, a change in the AUC would be expected after preadministration of phenobarbital regardless of the form in which griseofulvin is administered. A shorter residence time in the intestine resulting from increased motility, without a simultaneously enhanced gastric emptying, would be expected to cause a decreased total absorption of griseofulvin irrespective of the form in which i t is administered. In fact the same AUCs were observed with or without pheno-barbital pretreatment when griseofulvin was administered i n solution (Table 4-XIX). AUC for the controls and phenobarbital pretreated rats were significantly lox^er than those pretreated with metoclopramide (Tables 4-IX and 4-XIX). This i s perhaps due to precipitation of the drug i n the stomach as a result of a longer residence time in this region where absorption is limited. Following precipitation, the limiting effect of dissolution rate w i l l be introduced into the absorption process. Therefore, although some of the readily available drug is absorbed prior to precipitation, a major fraction of the administered griseofulvin may be in the form of undissolved crystals (precipitated) which w i l l be absorbed only i f sufficient time at the absorptive site i s allowed for dissolution. This is additional support for the suggestion 1 2 5 that an increase in peristalsis ( i f any) caused by phenobarbital has no significant role in the griseofulvin-phenobarbital interaction. If the transit time in the upper part of the small intestine were to be significantly decreased by phenobarbital, the AUC in phenobarbital treated rats following administration of the solution form should have been decreased. t However, since griseofulvin in PEG 600 preparation caused a noticeable change in the gut appearance, PEG 600 i t s e l f might have an effect on peristalsis. It is possible that the absorption properties of griseo-fulvin might have been altered by the vehicle in both control and test rats. In an attempt to c l a r i f y this point, two different griseofulvin dosage forms containing 100 mg/kg drug in either 70% PEG 600 or 70% PEG 300 in d i s t i l l e d water were prepared and their effects on the gut were tested. Single doses of 100 mg/kg griseofulvin in either 70% PEG 300 or 70% PEG 600 (1 ml) were administered to two separate groups of 5 rats. One rat from each group was sacrificed at either 1, 3, 5, 8 or 12 hours post-dosing and after an abdominal incision the gut was examined for any abnormalities. While the 70% PEG 600 preparation showed a noticeable increase in the volume of gut f l u i d , the 70% PEG 300 preparation exerted no effect on the gross appearance of the gut. Therefore, 70% PEG 300 was chosen as a vehicle to administer griseofulvin orally at two different concentrations (20 and 100 mg/kg). In addition the effect of pretreatment with phenobarbital on the griseofulvin-plasma levels was studied. The griseofulvin-plasma levels following administrat ion of 20 and 100 mg/kg griseofulvin in 70% PEG 300 are shown in Tables 4-XX and 4-XXII and their means are plotted as a function of Hours Figure 4-15. Mean griseofulvin plasma levels vs time following oral administration of 20 mg/kg of the drug in 70% polyethylene glycol to the control, A and phenobarbital pretreated, A rats (n=4). Error bars represent standard error of the mean. Table 45XX Griseofulvin-Plasma Levels C'U g/mL) following Oral Administration of Single Doses of 2© mg/kg Griseofulvin in 70% Polyethylene Glycol 300 to the Control Rats and Rats Pretreated Orally r with Single Doses of 15 mg/kg Phenobarbital Sodium 24 Hours Prior to the Grd:sedr;ulv;inMdm'in'istration. Hours . Rats 0.25 1 3 5.16 6.5 8 13 24 Controls, 1 3. 768 3.044 0.420 0.250 0.191 0.111 0.430 0.002 2, 4. 466 2.037 0.585 0.578 1.542 0.626 0.362 0.041 3 5. 318 3,763 0.624 0.468 0.354 0.292 0.255 0.030 4 3. 303 1.180 0.411 0.321 1.871 0.470 - -Mean, ' 4. 214 2.506 0.510 0.404 0.989 0.375 0.349 0.024 Standard 0. 439 0.566 0.055 0.073 0.421 0.111 0.051 0.012 error, Phenobarbital 1 9-955 6.938 1.320 0.287 0.467 0.132 0.079 0.016 pretreated, 2 6. 760 3.080 0.287 0.063 0.095 0.123 0.161 0.028 3 4. 631 2.619 0.188 1.085 0.485 0.399 0.252 0.038 4. 3. 718 1.317 0.718 0.550 1.417 0.425 - 0.003 Mean, 6. 266 3.488 0.628 0.496 0.616 0.270 0.164 0.021 Standard 1. 385 1.209 0.258 0.220 0.282 0.082 0.050 0.007 error, Statistical''' .2 ns ns ns ns ns ns ns ns significance, 1. Determined by Student's t-test; a=0.05. 2. Not significant. Table 4-XXI Area Under Plasma Concentrations-Time Curves, AUC (u g.hr.ml ) and the Peak Plasma Concentrations, Cmax ( u g/ml) Following Oral Administration of 20 mg/kg Griseofulvin in 70% Polyethylene Glycol 300 to the Control and Phenobarbital Pretreated Rats. Control, AUC, . 9.3 .13.1 12.3 .10.-3 Cmax, 3.77 4.47 5.32 3.30 Phenobarbital pretreated, AUC, 16.8 8.6. 11.4 10.4 Cmax, 9.95 6.76 4.63 3.72 Mean, Standard error, Mean AUC/dose, hr.ml-1.103 Coefficient of variation, %, 11.2 0.88 2.24 15.7 4.21 0.44 20.8 11.8 6.27 1.76 1.38 2.36 29.8 44.2 Statistical difference,,, ns ns 1. Determined by the Student's t-test (2 tailed); a=0.05; ns, not significant. ho CO Figure 4-16, Plasma levels of griseofulvin vs r time following administration of 100 mg/kg of the drug in 70% polyethylene glycol 300 to the control and phenobarbital pretreated rats. Key, Q t control° (n=4) ; #2,stest (n=4) ; error bars represent standard error of the mean. Table 4-XXII Griseofulvin-Plasma Levels ( yg/ml) following Oral Administration of Single Doses of 100 mg/kg Griseofulvin in 70% Polyethylene Glycol 300 to the Control Rats and Rats Pretreated Orally with Single Doses of 15 mg/kg Phenobarbital Sodium 24 Hours Prior to the Griseofulvin Adminis-tration . Hours Rats 0.25 1 2.5 5.25 6.5 8 13 24 Controls, 1 4.988 1.415 0.264 0.765 0. 230 1.016 1.008 0.011 2 3.735 1.013 2.097 0.825 0. 932 1.294 0.339 0.082 3 : 7.536 - 2.089 0.512 0. 425 1.256 1.372 0.130 4 3.115 .0.903 0.703 0.611 1. 938 . 0.832 0.711 — Mean, 4.843 1.110 1.288 0.678 0. 881 1.099 0.857 0.074 Standard 0.978 0.155 0.473 0.143 0. 382 0.108 0.219 0.034 error, Phenobarbital 1. 3.436 1.630 0.400 0.155 0. 279 1.148 0.668 0.033 pretreated, 2,: 4.372 0.905 1.660 1.447 3. 164 1.992 1.008 0,020 3 3.144 1.047 0.457 0.474 0. 486 0.500 0.764 0.004 4 5.325 2.130 1.681 0.813 2. 371 0.911 0.373 0.011 Mean, 4.069 1.428 1.049 0.722 1. 575 1.138 0.837 0.017 Standard 0.494 0.282 0.359 0.276 0. 708 0.315 0.076 0.006 error, Statistical significace,! ns2 iis ns ' ns ris ns ns ns" 1. Determined by Student's t-test; ct=0.05 . 2. Not significant Table 4-XXIII Area Under Plasma Concentrations-Time Curves, AUC (pg.hr.ml ) and the Peak Plasma Concentrations, Cmax ( u g/ml) Following Oral Administration of 100 mg/kg Griseofulvin in. 70% Polyethylene Glycol 300 to the Control and Phenobarbital Pretreated Rats. Control, AUC Cmax Phenobarbital pretreated, AUC Cmax 15.8 4.99 12.2 3.44 17.8 3.73 25.6 4.37 26.4 7.54 11.7 3.14 14.2 3.11 18.3 5.32 Mean, 18.5 4.84 16.9 4.07 Standard error 2.73 0.98 3.26 0.49 -1 3 Mean AUC/dose, hr.ml .10 0.72 - 0.61 -Coefficient of variation, %, 29.5 40.4 38.5 24.3 Statistical difference^, ns ns 1. Determined by the Student's t-test (2 tailed); a=0.05; ns, not significant. 132 time in Figs. 4-15 and 4-16 respectively. Pretreatment with phenobarbital did not reduce the griseofulvin-plasma concentrations of either of the 70% PEG 300 preparations. This is due, perhaps, to the wetting and solvent properties of PEG. Administration of 20 and 100 mg/kg griseofulvin in 70% PEG 300, like griseofulvin in PEG 600, yielded a rapid rise in the plasma concentration. It i s suggested that this i s due to the absorption of the dissolved fraction of drug in the dosage form. Attainment of approximately the same Cmax following administration of both preparations (4.2 to 6.3 and 3.1 to 5.3 jj'gV'ml for 20 and 100 mg/kg doses respectively) supports this suggestion (Tables 4-XXI and 4-XXIII). The remaining fraction of the drug (undissolved fraction), however, was absorbed slowly and incompletely. Both preparations had the same concentration of the drug in the supernatant (3.2 ug/ml) but were different in their total concentration of griseofulvin (5 or 25 mg/ml). Therefore, the increase in AUC from 11 to 17-18 ug/ml/hr following administration of the higher dose, although small, may be related to the absorption of the undissolved fraction in the two dosage forms. The appearance of a second plasma-concentration peak after 6 to 8 hours (Tables 4-XX and 4-XXII) may also result from absorption of drug from the previously undissolved fraction of griseofulvin in the dosage form. Phenobarbital seemed to f a i l to reduce the absorption of either the dissolved or the undissolved fraction of griseofulvin when given in 70% PEG 3Q0. These observations further suggest that the observed griseofulvin-phenobarbital interaction may result from a reduced dissolution rather than an increased gut motility. If the gut motility had increased following pretreatment with phenobarbital, i t should have affected the absorption of the undissolved fraction of the dose. This fraction of the dose, due to very slow dissolution rate, requires a longer residence time in the absorptive sites to be dissolved 133 and subsequently absorbed. Therefore, a reduced residence time i s li k e l y to reduce the total absorption of griseofulvin (i.e., as reflected by AUC). Absorption studies in this investigation have revealed that the availability of griseofulvin in PEG, unlike that of griseofulvin in Tween 80, is high. Once the drug i s dissolved in PEG i t w i l l be absorbed very rapidly (Tmax 20 min), while the griseofulvin absorption from Tween 80 is relatively slow. Bloedow and Hayton (39) reported a Tmax of eight hours following administration of griseofulvin in pure Tween 80 (solution). In this investigation values of Tmax obtained after administration of a suspension of griseofulvin in 0.5 or 2% Tween 80 were about 6 hours. A delayed Tmax coupled with the observed reduced AUC on increasing the concentration of Tween 80 imply that the absorption rate of griseofulvin decreases with increasing concentration of Tween 80. Phenobarbital pretreatment reduces the plasma levels of griseofulvin when the drug is administered either in tablet (in man) or in suspension forms containing Tween 80. These dosage forms, unlike PEG preparations, share the property of being very slowly absorbed. It is possible that bile salts accelerate the griseo-fulvin release rate from these preparations. Preparations containing PEG, however, may not, due to the solvent effect of the 1 3 4 vehicle, be affected by this change. Therefore, the griseofulvin-plasma levels remain unchanged after pretreatment with phenobarbital. The mechanism of this effect, however, is not completely clear. An in vitro dissolution study of griseofulvin in different dosage forms and in the presence of b i l e from control and phenobarbital pretreated rats may c l a r i f y the problem. Originally i t was hypothesized ('2.4,) that the reduced plasma levels might be related to a complex mechanism involving enzyme induction, first-pass metabolism and distribution rate-limited elimination of griseofulvin. The fact that phenobarbital pretreatment had no effect on the plasma levels of griseofulvin when the drug was administered in PEG ruled out these p o s s i b i l i t i e s , because such effects are expected to be independent of the dosage forms, i.e., the interaction would have taken place regardless of the; availability of the 1 3 5 dosage form. Furthermore, phenobarbital's failure to reduce the griseofulvin plasma levels following administration of the drug in PEG coupled with the observed identical biological half-lives in control and test rats (Table 4-1) support the suggestion ( 2 0 ) that the enzyme induction had no significant role in the griseofulvin-phenobarbital interaction. Therefore i t i s suggested that the griseof ulvin-phenobarbital interaction results r from reduced availability and/or reduced dissolution rather than induced metabolism or decreased transit time in the region of absorption. Phenobarbital has been observed to reduce plasma, concentration of" many other drugs, such as dilantin, dicumarol ( 6 9 ) and digoxin ( 7 0 ). A l l these drugs, lik e griseofulvin, show low solubility in aqueous media, but unlike the latter lack h,hlgh>ali%©.ph4jiic .-pr.o.p^ rMes."' rT-he'tshoBtefJ^-bi'ological half-l i f e observed in phenobarbital pretreated subjects suggests that induced metabolism; is responsible for the reduced dicumarol-plasma levels.'' However, the reducedidicumarolrplasma levelpmaynalsodbeupartlyi'duehtc5 reduced-' absorpV 5, 191 (1966). 11. W.L. Chiou and S. Riegelman, ibid., 59, 937 (1970). 12. P.J. Carrigan and T.R. Bates, ibid., 62, 1467 (1973). 13. V. Manninen, A. Apajalahti, J. Melin and M. Karesoja, Lancet, 1_, 398 (1973). 14. G. Levy, M. Gibaldi and J.A. Procknal, J. Pharm. Sci., 61_, 798 (1972). 15. J.J. Ashley and G. Levy, ibid., 6>2, 688 (1973). 16. D. Busfield, K.J. Child, R.M. Atkinson and E.G. Tomich, Lancet, J2_, 1042 (1963). 17. D. Busfield, K.J. Child and E.G. Tomich, Brit. J. Pharmacol., 22, 137 (1964). 18. E. Lorenc, Missouri Med., 64, 32 (1967). 19. A.H. Conney, C. Davison, R. Gastel amd J.J. Burns, J:. Pharmaol. Exp. Ther., 130, 1 (1960). 20. S. Riegelman, M. Rowland and W.L. Epstein, J.A.M.A., 213, 426 (1970). 21. CD. Klaassen, J. Pharmacol. Exp. Ther., 168, 218 (1969). 22. CD. Klaassen, J. Pharmacol. Exp. Ther., 176, 743 (1971). 23. G. Paumgartner, W. Horak, P. Probst and G. Grabner, Arch. Pharmak, 270, 98 (1971). . 24. J.E. Axelson, Grant, No. MA 5358, Medical Research Council, Ottawa, Canada (1973). 25. S. Symchowicz and K.K. Wong, Biochem. Pharmacol., JL5, 1595 (1966). 26. C. Lin, R. Chang, J. Magat and S. Symchowicz, J. Pharm. Pharmacol., 24, 911 (1972). 141 27. C. Lin, J. Magat, R. Chang, J. McGlotten and S. Symchowicz, J. Pharmacol. Exp. Ther., 187, 415 (1973). 28. B. Davis, K.J. Child and E.G. Tomich, J. Pharm. Pharmacol., 13, 166 (1961). 29. P.A. Harris and S. Riegelman, J. Pharm. Sci., 5_8, 93 (1969). 30. R.A. Branch, D.G. Shand, R.W. Wilkinson and A.S. Nies, J. Pharmacol. Exp. Ther., 184, 515 (1973). 31. E.E. Ohnhaus. S.S. Thorgeirsson, D.S. Davies and A. Breckenridge, Biochem. Pharmacol., 20, 2561 (1971). 32. M. Rowland, S. Riegelman and W.L. Epstein, J. Pharm. Sci., 5_7, 984 (1968). 33. L.J. Fischer and S. Riegelman, ibid., 54, 1571 (1965). 34. W.L. Chiou and S. Riegelman, ibid., 58, 1500 (1969). 35. C. Bedford, D. Busfield, K.J. Child, I. MacGregor, P. Sutherland and E.G. Tomich, A.M.A. Arch. Dermatol., jU, 137 (1960). 36. W.L. Chiou and S. Riegelman, J. Pharm. Sci., 59_, 937 (1970). 37. W.L. Chiou and S. Riegelman, ibid., 60, 1376 (1971). 38. Antitinea Capitis Team, Institute of Dermatology, Kiangsu, Chin. Med. J., (9) 163 (1973). 39. D.C. Bloedow and W.L. Hayton, J. Pharm. Sci., _65, 328 (1976). 40. T.R. Bates and J.A. Sequeira, ibid., 64, 793 (1975). 41. J.E. Axelson and F. Jamali, presented to the Research Symposium and Research Conference of the Association of Faculties of Pharmacy of Canada, Ottawa, Canada, May 20, 1974. 42. F. Jamali and J.E. Axelson, presented to the Academy of Pharmaceutical Sciences, Pharmaceutics Section, San Francisco meeting, April, 1975. 43. A.G. Johnson, Gut, _12, 421 (1971). 44. T.H. Howells, T. Khanam, L. Kreel, C. Seymour, B. Oliver and J.A. Davies, Brit. Med. J., 2, 558 (1971). 45. H.I. Jacoby and D.A. Brodie, Fed. Proc, 25_, 200 (1966). 46. L.S. Goodman and A. Gilman, "The Pharmacological Basis of Therapeutics", MacMillan Publishing Co., New York, f i f t h edition, 1975, p.523. 47. J. Nimmo, R.C. Heading, P. T o t h i l l and L.F. Pres'cott, Brit. Med. J., 1_, 587 (1973). 48. G. Gothoni, P. Pentikainen, H.L. Vapaatalo, R. Hackman and K?A. Bjorksten, Annals of Clin. Res., 4.,' 228 (1972). 49. D.O. Gibbons and A.F. Lant, Clin. Pharmacol. Ther., 17, 578 (1975). 50. G. Levy and W.J. Jusko, J. Pharm. Sci., 55, 285 (1966). 142 51. W.J. Jusko and G., Levy, ib i d . , 56, 58 (1967);. 52. T.R. Bates, M. Gibaldi and J.L. Kanig, ibid., 55, 901 (1966). 53. M. Gibaldi and CH. Nightingale, ibid., 57, 1354 (1968). 54. S. Feldman and M. Gibaldi, ibid., 58, 425 (1969). 55. S. Feldmanaand M. Gibaldi, ibid., 58, 967 (1969). 56. CH. Nightingale, R.J. Wynn and M. Gibaldi, ibid., 58, 1005 (1969). 57. S. Feldman, M. Salvino and M. Gibaldi, i b i d . , 59, 705 (1970). 58. CH. Nightingale, J.E. Axelson and M. Gibaldi, ibid., 60, 145 (1971). 59. T. Kimura, H. Sezaki and K. Kakemi, Chem. Pharm. Bull., 20, 1656 (1972). 60. V.P. Shah, W.L. Epstein and S. Riegelman, J. Clin. Invest., 53, 1673 (1974). 61. M.J. Barnes9and B. Boothroyd, Biochem. J., 7<3, 41 (1961). 62. S. Symchowicz, M.S. Staub and K.K. Wong, Biochem. Pharmacol., 16, 2405 (1967). 63. C. Lin, R. Chang, J. Magat and S. Symchowicz, J. Pharm. Pharmacol., 24, 911 (1972). 64. S. Symchowicz and K.K. Wong, Biochem. Pharmacol., 1_5, 1601 (1966). 65. S.A. Kaplan, S. Riegelman and K.H. Lee, J. Pharm. Sci. ^ 55^ , 14 (1966). 66. C Lin and S. Symchowicz, Drug Metab. Rev., j4, 75 (1975). 67. V.P. Shah, S. Riegelman and W.L. Epstein, J. Pharm. Sci., 61, 634 (1972). 68. J.E. Axelson, unpublished work. 69. S.A. Cucinell, A.H. Conney, M. Sansur and J.J. Burns, Pharmacol. Ther., 6, 420 (1965). 70. A. Kaldor, G. Somogyi, L.A. Debreczeni and B. Gachalyi, Int. J. Clin. Pharmacol., 1_2, 403 (1975). 71. P. Aggeler and R. O'Reilly, J. Lab. Clin. Med., 74, 229 (1969). 1 4 3 7. APPENDICES 144 Appendix I' Identification of the Materials Identity of griseofulvin>.and propantheline bromide was verified by determining their melting points'!". Sodium phenobarbital was identified 2 mass spectrometrically . The-mass speetras of griseofulvin, 4- demethyl-griseofulvin, metoclopramide ihyjir.plch^^ propantheline bromide were also obtained.-The results"are as followes: Griseofulvin, Molecular weight, 352.:3 . '3"' • ° Melting point , ,(220) 219.6-' Mass spectra, Mass/charge, 138 69 214 310 215 140 321 123 352 139 Relative 100 89 64 63 61 44 34 32 31 30 intensity, Sodium Phenobarbital, Molecular weight,of 232.2-the acid, 3 .Massf spectra* , Mass/charge, 204 l i 7 146 ;17.4 91 143- 232. Relative 100 43 20 12 10 8 7 intensity, ^Determined 'using a Mettler FP2-FP11 Capillary' Melting Point 'Apparatus:. 21.Determined using a Finnigan 3200 with 6100 Data System; direct solid probe; electron energy of 30 electron volts. 3.-.Verified, Atlas of Spectral Data and Physical Constants for Organic Compounds, J.Gv.Grasselli,. C.R.C. Press, Cleveland,,Ohio, 1975, (reported value- in parentheses). 145 Metoclopramide - hydrochloride.monohydrate Molecular weight of the base, 299..8 ' =Melting point, 141° . with decompositidn. Mass spectra, Mass/charge, 86 99 58 87 184 10 16 299 Relative intensity, 100 75 38 28 22 20 16 4 Propantheline Bromide, Molecular weight of the base, 353.0 Melting range, 4 (156- 159.0° 3 162) Mass spectra, - . -» 'J ; Jo2 / ' ' Mass/charge,. 181 114 86 310 23 338 21 182 7 24 253 311 353 Relative intensity, 100 89 67 53 52 42 31 16 15:14 13 11 4 4-Demethyl griseofulvin, Molecular weight, 337.8 Mass spectra, Mass/charge, 152 138 69 153 296 201 338 123 200 323 Relative intensity, 100 91 61 57 50 47 43 37 36 31 Verified, U.S.P. XIX, 1975; reported value in parentheses. 1 4 6 Appendix II A Computer Programme The following programme was developed to evaluate pharmacokinetic parameters following intravenous administration of a single dose of a given drug based on the Eq. Cp = Ae~ a t + Be" ^ Z; M I C H I G A N TERMINAL SYSTEM FORTRAN G l 4 1 3 3 6 ) 00 0 1 C0C2 0C0 3 CCC5 0 0 0 6 _coo7_ O J oa 0 0 0 9 r-i' 0 0 1 1 0 0 1 2 -0.013-0 0 1 4 C J 1 5 3 0 1 7 C02G 00 2 1 0 0 2 3 0 0 2 4 1 :',•> ? KCRRE 1 0 - 2 5 - 7 5 1 5 : 2 8 : 55 10 100 SL'CRCUTINE K 0 R R E I T , Z , N , A, B, R, » ) DIMENSION T i l ) ,111) SUMSCT=0. Si;VT = r SUMTZ-0. SUMZ=0. _£LNSCZ=C : DC 100 I = l , . \ SUMr=Sl,'MT + T I I I •SI f'S<,'T..= S m S . QTrT l l ! » n i | TZ = S I :MTZ + T ! I I - - 2 I I ] S I . M Z = S U M Z + Z ( i) ~SLK.SC2=.SLM_S C i * . l U . U U . U J Crr-.T INUt SL'M2=SU>'TtSo,''T -IXEJ-i= ii.«S CZ.'..Si=Sl_KX2 I F { C f N . L E . l . c - 1 5 ) R E T U R N AA = .\wSUMTZ-SUM.T*SUMZ i / 0 E > i _ K= ( S U M S C T * S U M Z - S U M T Z » S U M T ) / D E N tip. = 0 E N- ( N « S U M SC Z-S UM Z » S U M Z ! F = ftf /;o3 TLSHX-1000 10 .»<= i;. V.R I TE ( 6 , 1 ) N ,A,:',B,R " 6 . 5 X , ' R = < , 6 6 . 3 ) RE TURN .ii£-i.^4Jl.<.LJJiT.£&CE£X=i.^£J.a. »j?Tir,\s vQPT ICNS — i S J . A J . i S X NO ERR'JRS E F F E C T * ID,EBCDIC,SOURCE,NOL 1ST , NiJDFCK . L O A C N O M A P EFFECT'.' NAME = KCRRfc , L I N E C N T = 57 SXU8££_SiAI£>l£;,.tS_= 2J.^PR PGR A'.- , A T I S T I C S * NC O I A S N C S T I C S GENERATED I N IN _1C0.4_ 1. 0 0 0 2 . 0 0 0 3 . 0 0 0 4. OilO 5. 0 0 0 6 . 0 0 0 -7_ooa_ 3 . 0 0 0 9 . 0 0 0 l l . G C O 1 2 . 0 0 0 - a s . O G O _ 1 4 . 0 0 0 1 5 . 0G0 _ i 6 . . 0 i i a _ 1 7 . 0 0 0 1 8 . 0 0 0 — 1 9 . 0 0 0 — 2 0 . 0 0 0 2 1 . 0C0 _ 2 2 - O 0 a _ 2 3 . 0 0 0 2 4 . 0 0 0 - 2 5 . 0 0 0 — 2 6 . 000 2 7 . 0 0 0 _ 2 & - a Q G _ IN KCR.PE *) IBM 370/168 MICHIGAN TERMINAL SYSTEM FORTRAN G( 41326) CAIN 10-25-75 15:28: 55 C001 0002 0003 0004 CCC5 0006 0003 0009 0010 0011 0012 0014 0015 .001 6 0017 0013 ___13_ 0020 0021 J023 0024 -.002 5-0026 0027 no ? ? 0029 0030 0032 0033 0034 0035 _fl.0_3.6_ 00 37 0038 0040 0041 nn4-> 0043 0044 on 4 5 0046 0 04 7 0049 0050 0051 0052 CIMENS ION 2(100 ),T (100),ZL!100! 10GICAL*1 CASEN0.8) EQUIVALENCE { C PM AX , CP SCL ) ,ITMAX,TSCL) I r . G t f Al El FT I — 1234 N=L _L3_ READ15,13,END=400)CASENO __QRMA_(-8.A.U_ READ r5,12,EN0 = 400) TMAX , CPMI N ,CPMAX 12 FORMAT ( 3 F 1 0 . 2 ) PI p T j ^ T ^ r i i g n PR r o s r . i . I F . n . 10 READl5,1,END=100)T(N),2(N) N = N + 1 c-.__io_i.c_ F0PMAT(2F10.3) 100 N = N-1 TF(M . r-o n i r.r TO 40n KRITE(6,14)CASENG 14 FORMAT!1H1,/20X,8A1//I 11 FORMAT (5X,- TIME- ,10X,'CP' / 5X,4( ' + ' ) ,9X,4(' +' )/ 1/3X,F8.3,5X, F8 . 3 i CO 110 1=1, N 71 1 M - ? i T 1 — _ 110 CONTINUE REAC(5,2)NBETA _2 EJ3£i_AT_U--__ FORMAT(1 HI ) !..DEX=N-NBETA + 1 nn ? P 0 I=TM,-.FY,M 200 2 ( I ) - A LOG10(Z(I)) CALL K.ORREI Tl I NO EX) ,Z( INDEX) ,N6ETA,A,B,R1 ,£3 70) _F_LX£J_-,-4..J - — : — 4 F0F.MATI/1X,20.•*•>//) • 6=10.**B _ r P C T A T<- M f ^ i i q P F T A : -BETA=ALOG(10.)*A INf.EX=INDEX—1 K_J DO 300 1 = 1 , INDEX BT=BETA*T< I ) 250 15 I F ( Z ( K ) .LE. 0.) GO TO 250 Z ( K) = Al CG10( 21 K) ) K = Kt 1 — • — GO TO 300 WRITE16,15 )T( I ) CPgt . , . .T . / / * Y , I T H F W M I ' F GIVEN F f " T=' F8.2, DELETFI.')-300 CONTINUE INDEX=K-1 CAU ' T l f l T i M NT'" • A P , R P , P ? . E 3 7 0 I A=10 .**BP C ALPHA IS fINUS ALPHA .M or.i i n . i c A P _ 29. 000 3C. 000 31. 000 3 2. 0OQ. 33.000 34.000 _35.-00.0_ 36.000 37.000 _3_-0_0-V.R IT E (6 , 5 ) FORMAT!///, r o 7 - A + R 5X, 'FINAL RESULTS:•// 1 39.000 4 0. 000 _4.1-000_ 42.000 43.000 __4—OO0-45.000 46.000 _A.7.-000-48.000 4y. 000 51.000 52. 000 _53~000_ 54. 000 55.000 56. 000-57. 000 58.000 -5.9-0.0 0-60. 000 61.000 63.000 64.000 __.5-0_i.a_ 66.000 67. 000 ___,a—aao-69.000 7 0. 000 __J_l-.-0.OC--72.000 73.000 —IA-aao_ 75.000 76. 000 ..77. OOQ-78.000 79. 000 o n . n i . n PAGE P00 1 81. 000 82. 000 63.000 _-MICHIGAN TERMINAL SYSTEM FORTRAN G(41336) MAIN 10-25-75 15: 28 : 55 PAGE P002 0054 G055 0056 on s 7 RFAD15, 1 )XBZ VC=XPZ/CPZ AK21 = A*VC*(ALPHA-F3ETA)/XBZ— ALPHA AKFI = AI PHA*8FTA/AK -, 1 84.000 85. 000 8 6 . 0 0 0 R 7 AO fl 005S 0059 CiOM A K12=-[ALPFA + BETA + AK21 + AKEL) *AUC=—A/ALPHA-3/BETA C1.=XI,2/AUC ... 88. 000 8 9 . 0 0 0 9 0 000 0061 0062 6 TAL=—AL0GI2. )/ALPHA VJR IT E ( 6 , 6 ) A .ALPHA , R.2, TAL EliaaAIi//5X, ' A=' . F l 1 , f i t . 5 X . ' -A l PHA= • . F l 1 . A. ? Y . I I • RF*. 3 , > I t , s y , 9 1 . 000 9 2 . 0 0 0 q -3 f; f) 0 0064 C C 6 5 ** T 1/2 (ALPHAi =• ,F11.6> T6E=-AL0G(2. l/BETA klP-LLEi 6-. J . JJ3.. Jj r J A. R! .TRF 9 4 . 0 0 0 9 5 . 0 0 0 0066 • nr-A7 7 FORMAT C P 0 = > , F 1 1 . 6 , 5 X , • X B 0 = ' , F l 1 . 6 , 5 X , • V C = • , F l 1 . 6 ) WP IT E 16 , 9 I AK 1 2, AK 2 1, AK EL , AUC ,C L EJJZtiAXI./y_5X J , ;02=' .F_Ll_i>.5X. 'K?l-t f F] l fr, sx , .K Fl = ' ,F1 1 , A / / q x , i j - 3 * - U j _ l U 100.000 101. 000 A 11 r — i n ? A n n C071 nr. 7 5 * ' , F U . 6 , 1 0 X , ' C L = ' , F 1 1 . 6 ) I FI PLOT I) GO TO 1234 nx = T<:ri / i n . — i i i — i i ; / . n u n 103.000 104 .000 0073 CC74 r 7 CALL A X I S I O . , 0 . , "TIME! HR)',-6,10., C . , C . , D X ) DY=CPSCL/10. — C A LI—A.X I_S-(XU.^JD—.UiiiCfA'' K f M f. G / MI | , t p , i n ,QH , , .I , , PY ) L-Q 5 *. Q.Q-.Q 106 .000 107. 000 1 r\ p n A A 0076 00 77 "07M 00 120 I=1,N XX = T ( I l /DX YY = 7I i n / n y . I U-C-* -U U U 109 .000 11C. 000 i l l Ann 0079 0C30 0,18 1 i ?n 2L( I ) = ALOG10(ZL ( I ) ) CALL SYMBOL (XX ,YY , . 1 , 3 , 0 . ,-1) I » u U Li 112 .000 113. 000 • 1 1 L. A A A 0CS2' 0083 n n a 4 X=T(1) XX=X/DX ... Y = A*PXPf Al P H l * X l + 3 « F » B r S F T » « » ) i~i , it».uU.U 115. 000 116. ocd CC35 C C 86 •ir,a YY=Y/DY CALL PL0T (XX ,YY ,3 ) ..f.AII P! OT( y y , Y Y r ?> 1-LX.-0 0 0 118. 000 119 .000 1 A A A C083 CC39 r-r.or, 320 XX=XX+.01 X=XX*0X i = M E J i P l l l P H A * V U R * F V O l n F T A i Y ] X £\J »-U U J 121 .000 122. 000 CC91 C092 YY=Y/DY CALL P L0T !XX ,YY ,1 ) —LFJ.X.. . I F . n m i m m 1.2-3- QG Q 124. 000 125. 000 0094 0095 CALL S Y M B O L ( 6 . , 5 . , . 1 , C A S END ,0 . , 8) CALL A X I S ( 0 . , 1 4 . ,< TIME(HR)> , - 8 , 1 0 . ,0.,0.,DX) CAM P i n T i n . , 1 4 . , 1 1 ji.26-.-G-G 0 127. 000 128. 000 17C A A A CC97 0098 nnQQ LL L O T ( 0 . , 2 4 . , 2 )CPMAX = AL0G10(CPMAX) —CP'-'Ui-uu r.r-. l n (r P u i M J L-*Cx . . .U .LJJJ 13 0. 000 131.000 0100 0101 23 C 0Y=10./ICPMAX-CPMI N) YY=14. . VY=YV+nv 1-32-.-000 133. 000 134.000 0103 0 104 n i .-,<; CALL SYNBOHO. ,YY , . 1, 1 5 , 0 . ,-1 ) IF (YY . L T . 24 . ) GO TO 330 —CAJJ—_0 149. 000 15 0.000 —15.1..000 152.000 153.000 —15-4 ..000 155.000 156. 000 -1.57...000 158.000 159.000 -16-0.-0-00 161. 000 162.000 -163.000 164. 000 16 5.000 _16£_X«H)_ 400 FORMAT!//5X,'***# ERROR RETURN FRCK KCRRE'//5X ,•**** PROCESS * I NAT ED CUE TO PAD CATA 1) —GI1_TX 1?34 WR!TF(6,3) CALL PLOTND STOP : 167. 000 168.000 _16.9..0U__ 17C. 000 171.000 _L72._0.Qa TERM 173.000 174.000 -113 ..000 176.000 177.000 • 173. 003-179.000 0145 'OPTIONS * P P T_I.L'1-1S_ Ef-i D IN EFFECT* IN—f_E£_LCJj__ *STAT1ST ICS* *STATI STI CS* Nn =apoj<: IM M.. jfi ID,EECD1C,SOURCE,NOLI ST,NODECK.LOAD,NOMAP -NA.MF = M-AiN r I T M F r f . ' T = SOURCE STATEMENTS NO DIAGNOSTICS GENERATED 145,PROGRAM SIZE 5696 NO STATEMENTS FLAGGED IN THE F X F f t l T T P . N T F R M I M g T F I - 1B0VE COMPILATIONS. $R IV.2 EXECUTION BEGINS 1 5 0 In order to run the programme the following information should be punched in the computer cards: Card number Column Input 1 1-8 Lable,ee,g., CONT. 02 2 1—7 Scale orl the time-axis, e.g., 30. 8-15 Lower limit on* the plasma level-axis, e.g., 0.001 16-23 Upper limit on the plasma level-axis, e.g., 20. 3-n 1 Time of sampling, e.g., 1. 11 Plasma level, e.g., 6.309 Note, one card for each plasma level and the corresponding time of sampling; n, number of data cards following card number 3 n+_ 1 $END n+2 4 Number of data points in the $-phase, e.g., 4 n+3 1 Administered dose, e.g., 6.5 n+4 1 $END n+5 1 $R PL0T:Q PAR= , commanding plotting n+6 1 $SIG 151 The print-out containes the data (time of sampling and plasma levels), calculated parameters and f i t t e d plasma level vs time and logarithm of plasma level vjs time curves. Each data set i s labeled as follows: CONT,control rats PHEN, phenobarbital pretreated rats MET, metoclopramide pretreated rats PROP, propantheline pretreated rats A number following the label indicates the rat number i n the category, e.g., CONT 02 indicates rat number 2 of the controls. The resulted plasma level-time and the logarithem of plasma l e v e l -time curves and the calculated parameters are presented i n following pages. 152 CONT.Ol TIME CP 4-.+-. 0.860 8.235 "2 ."860 2.85V' 4.280 1.158 11.660 0.162 22.620 0.006 3 POINTS: SLOPE* -0.123473 INTEPCEPT= 4.106844 P=-1.000 2 POINTS: SLOPE* -0.343314 INTERCEPT* 9.905205 R*-1.000 FINAL RESULTS: A= 9.905205 -ALPHA- -0.790510 (-1.000) T 1/2 (ALPHA) = 0.876836 B= 4.106844 -BETA * -0.284307 (-1.000) T 1/2 (BETA) = 2.438022 CP0= 14.012049 XH0= 5.CC0000 VC = 0.356836 K12 = 0.122704 AUC* 26.975235 K21* 0.432672 KEL= 0.519441 CL- 0.185355 154 155 CONT.02 TIME CP >. — 1.000 6.3C9 3.000 2.100 /. n.m 1 . 1 1 S 9.000 0.253 12.000 C.09C 23.000 0.002 <, POINTS: SLOPE* -0.151248 INTERCEPT* 5.214C19 R=-0.999 2 P0IN1S: .m.f_ - f . M 7 « 7 INTERCEPT* 8.26381 2 R=-1.000 FINAL RESULTS: A* 8.263E12 -AlPhA* -1 . 145529 l - l . C O & l T 1/2 (ALPHA) * 0.605089 B" 5.214C19 -BETA = -0.348262 (-0.999) T 1/2 (BETA) * 1.990301 CPO- 1 3 - 4 7 7 8 3 1 X80= 6.5C000C VC* 0.482273 r...5'.« K?l = C.656692 KEL* 0.607506 AUC* 22.185486 1 CL* 0.292984 : _= : — : • — — 156 CONT,03 TIKE CP 1.170 t.119 2.500 2.450 4.000 1.213 5.17C 0.752 12.000 C.1CC 23.000 0.004 3 POINTS: SLCPE= -0.127503 INTERCEPT* 3.414751 R*-1.000 3 POINTS: SI OPE* -0. 548774 INTERCEPT* 22.763e70 R*-0.996 FINAL RESUL IS: A* 22.71.3870 ALPHA* -1.2635S7 l - C . 5 . 1 T 1/2 I ALPHA ) = 0.548551 B* 3.41H751 BETA = -C. 293587 l-l.COO) T 1/2 (BETA) = 2.360963 CPO* 26.17E619 XBO* 5.CC0OOC VC= 0.190996 K12* 0.254035 K21= 0.420116 K E l * 0.883031 AUC* 29.646271 CL* 0.168655 138 159 PHEN.Ol TIME CP + »f + H H 1.000 11.376 <:.-.0 4.984 6.000 1.097 12.000 C.267 24.000 0.006 — — 3 POINTS: ~ — SLOPE* -0.128666 INTERCEPT* 7.507994 R* -0.997 2 POINTS: SLOPE* -0 .628743 INTERCEPT* 25.214798 R*-1.000 FINAL RESLLTS: — *->.21WStl -ALPHA* -1 .47076C ( -1.C00) T 1/2 (ALPHA) = 0.471285 B- 7.5C7S94 -BETA = -0 .296265 ( -0.997) T 1/2 (BETA ) = 2.339622 CP0= 32.72.778 XBO* 10.000000 VC* 0.305597 K12- 0.431083 K21= 0.565743 KEL* 0.770197 AUC* H2.486221 CL= 0. 2353 7C • ~ : t 160 161 162 PHEN ,02 TIKE CP **** • + + + v _ ____ l . ooo 9.37C 3.170 3.021 4. 000 1.4 19 5.500 0.711 9. 170 C.263 12.000 0.103 23.000 C. C05 4 POINTS: SLOPE = -C.123299 INTERCEPT* 3.364924 R*-1.000 3 POINTS: SLCPE* -0.404743 INTERCEPT* 19.783463 R=-0.960 F1NAL RESULTS: A* 19.763463 -ALPHA* -0.931956 (-0.960) T 1/2 (ALPHA) = 0.743756 B= 3.364924 - B E T A * -0.283906 (-1 .000) T 1/2 (BETA) * 2.441465 CP0= 23.148376 X&0= 5.C00OC0 VC* 0.215998 K12* 0.137986 K21* C.378109 KEL* 0.699767 TuC = 33.0CC124 CL* 0.151148" 163 1 64 165 PHEN.03 TIME CP 1.170 t.COl 3.000 2.440 4.000 1.591 5. SOC C.75C 9.000 0.373 12.200 0. 155 23.000 0.003 4 POINTS: SLOPE* -0.14CG49 INTERCEPT* 6.006315 R*-0.994 THE VALUE GIVEN FOR T * 4.00 OELETEO 2 POINTS: SLCFE* -0.572000 INTERCEPT* 8.830572 R=-1.000 FINAL RESULTS: A = 8.83C572 -ALPHA* -1.317078 ( -1 . c o o t T 1/2 (ALPHA) = 0.526277 B = 6.C06315 —BETA = -C.324317 ( -C.994I T 1/2 (BETA) * 2.137249 CP0= 14.836887 XBO* 4.000000 VC* 0.269598 K12* 0.326992 K21* 0.726210 KEL = 0.588192 AUC* 25.224533 CL* 0.158576 166 O o PHEN.03 167 168 / TIME + f f f METfl. Cl CP t+ »<• > 1 .OOC 7. 103 3.200 2.233 4.330 1.245 5.5CC 0.932 9.COO 0.299 12.170 C.C85 .3.000 0.003 4 POINTS: SLOPE= -0.142361 INTERCEPT^ 5.3B169C R--0.999 THE VALUE GIVEN FCR T = 4.33 DELETED 2 POINTS: SLOPE- -0.439533 INTERCEPT- 8.874506 R--1.0C0 F I N AL RESULTS: A = 6.874506 ALPHA = -1.012061 (-1.C00) T 1/2 (ALPHA) = 0.684887 8 = 5.3-165C BE TA = -C.327643 (-0.999) T 1/2 I BETA) = 2.114263 CPO- 14.25-19- >B0= 5.00000C VC= 0 .350725 K12 = C.167692 K21 = C.566135- KEL = 0.566077 AuC = 25.1-4158 CL= 0.LS8637 169 170 171 c ' TIME •. fr f MET0.02 CP > 0.660 11.006 -.000 3.60C 4.170 1.600 5. 5CC C.682 12.000 0.C96 23.OCC C.002 3 POINTS: SLOPE- -C.144298 INTERCEPT* 4.57SC74 R=-0.999 3 P O I N T S : SLCPE* -C.331641 INTERCEPT* 9.169435 R = -0.957 F I N A L RESULTS: A* 9.169435 -ALPHA * -0.763631 ( -0.9571 T 1/2 ( ALPHA) * 0.907699 6 = 4.579C74 - e E T A * -0.332258 1 -0.999) T 1/2 (BETA) = 2.086173 CPU* 13.7465C6 X80* 5.CC000C VC = 0.3o3b76 K12 = 0.08665C K21* C.475931 KEL* 0.533107 AUC* 25.789368 CL* 0.19387U 172 173 174 PROP,01 TIME CP 0.167 16.711 0.670 14.085 4.067 4 .4 76 5.720 2.760 a.830 1.215 23.OCO C.013 3 POINTS: SLOPE- -0.142024 INTERCEPT- 23.2C1E74 R- -1 .000 THE VALUE GIVEN FOR T= 0.17 DELE1 ED THE VALUE GIVEN FOR T= 0.67 DEL ET EC THE VALUE GIVEN FCP T - 4 .07 'OEL ET ED * * * * ERROR RETURN FROM K.ORRF * * * * PROCESS TERMINATED CUE TC BAD DATA 175 r V T I ME • t+ + PROP,C3 CP *i + + /• 0. 3 70 12.589 2.450 3.B2C 5.78'. 3.858 8.700 1.34 1 23.200 29.200 0.012 0.003 3 POINTS: SLCPE= -0 .129626 INIEBCEPT= 16.634018 R=-0.997 THE VALUE GIVEN FOR T= 0.37 OELETED THE V'-LUE GIVEN FOR T= 2.45 DELETED THE VALUE GIVEN FOR T= 3.82 OELETED . * * * * ERROR RETURN FROM KORRE *<•*« PROCESS TERMINATED OLE TC BAD LATA