Br. J. clin. Pharmac.

(1979), 7, 101-105

BIOAVAILABILITY OF CHLORPROPAMIDE
M. EVANS1, R.C. GLASS', M. MITCHARD2 * B.M. MUNDAY2 & R. YATES2**
2

Research Department, The Boots Company Limited, Nottingham and Department of Clinical Pharmacology, The Medical School, The University of Birmingham, Birmingham B 1 5 2TJ

'

1 The serum profiles of chlorpropamide obtained following single doses of two tablet preparations and from a suspension formulation have been compared in healthy volunteers. The amounts of chlorpropamide absorbed from the three formulations were similar but the drug was absorbed more rapidly from the suspension than from either tablet formulation. There was a small but therapeutically insignificant difference in the rate of absorption of the drug from the two tablets. 2 Changes in blood glucose concentrations were found to be related to the drug serum profile characteristics of the formulations.

Introduction

Methods

Chlorpropamide is a sulphonylurea used effectively in the treatment of maturity onset diabetes. It is a potent, long acting hypoglycaemic agent which acts by stimulating insulin secretion. Many workers (Beaser, 1959; Carlozzi, lezzoni & Silver, 1959; Handelsman, Levitt & Calabretta, 1959; Knauff, Fajans, Ramirez & Conn, 1959; West & Johnson, 1960) investigated aspects of the pharmacokinetics of chlorpropamide in the late 1950's and recently Taylor (1972) reported a more detailed study. Although the American Food and Drug Administration authority have listed chlorpropamide tablets as a formulation with a potential or known bioequivalence problem (Federal Register 1975), only two bioavailability studies appear to have been reported in the literature (Monro & Welling, 1974 and Taylor, Assinder, Chasseaud, Bradford & Burton, 1977). In both of those studies, Diabineseb, 250 mg, was compared with other tablet formulations of chlorpropamide but no attempt was made to compare availability characteristics from tablets with the availability of the drug from suspension nor to correlate availability characteristics with hypoglycaemic effects. It was therefore of interest to compare the bioavailabilities of two generic tablet 250 mg each containing preparations, chlorpropamide, with the availability of chlorpropamide from a 250 mg dose of a laboratory prepared suspension and to determine whether differences in bioavailability were reflected in different degrees of hypoglycaemia. Present addresses: *LERS, 31 Avenue PB Couterier, 92 Bagneux, Paris and **I.C.I. Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire SK10 4TG.

Chlorpropamide tablets BP, 250 mg, (A) (The Boots Co. Ltd), Batch no. FER 10075 and Diabinese, tablets, 250 mg, (B), (Pfizer Ltd), batch no. 403-17058 were used. Tablets A were manufactured from the same batch of chlorpropamide powder from which the suspension was prepared. The powder had a weight mean particle size of 16 gm with 99% (by weight) < 36 gim and 1% < 5 gm as measured by a Coulter Counter fitted with a 140 gm tube. The suspension (C) was freshly prepared on each occasion in the following manner. Tween 80 solution (0.05%, 10 ml) recently (within 48 h) prepared in distilled water was added to 250 mg of chlorpropamide BP in a 50 ml glass bottle. This was shaken for 15 s and the suspension immediately drunk from the bottle. The bottle was subsequently washed with four 25 ml portions of water, each washing being drunk by the volunteer; a similar volume of water (110 ml) was taken with each of the tablet pre-

parations. The study was approved by the Queen Elizabeth Hospital Medical Research Ethical Committee. Details of the study were subsequently explained to each volunteer and their informed consent obtained. Subjects selected were nine male volunteers, aged 18-35 years, in whom no abnormality was found on clinical examination or in haematological or biochemical profiles. Subjects were excluded if there was a history or family history of diabetes, of renal, hepatic or thyroid disease or if there was any history of drug allergy, fainting, unexplained loss of consciousness or of epilepsy. None had taken any drug for at least 2 weeks before the study.

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M. EVANS, R.C. GLASS, M. MITCHARD, B.M. MUNDAY & R. YATES

Subjects were required to fast from 22.00 h on the night before each part of the study but were allowed one unsweetened cup of tea or coffee on rising, and a sandwich lunch at 13.00 h. Consecutive parts of the study were carried out in a hospital ward at 2 week intervals commencing at 09.00-09.30 h. The study was a randomized, three-way cross-over, following a latin square design. A plastic intravenous cannula was inserted into a forearm vein to facilitate blood sampling. Heparinized normal saline was used to prevent blood clotting in the cannula and care was taken to ensure that blood samples were not contaminated by the heparinized saline. Blood samples (10 ml), collected from a threeway tap immediately before and at 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 8.0 h after the chlorpropamide dose, were allowed to clot and the serum immediately separated in order to minimize haemolysis. These sera were stored at -20C until analysed. Extra blood samples were collected on each occasion except at 8 h for blood sugar estimation. Further blood samples (10 ml) were then obtained by venepuncture at 24, 48, 72, 120, 144 and 168 h and the serum chlorpropamide concentrations determined. Chlorpropamide was assayed as its methyl derivative by a gas chromatography method using tolbutamide as an internal standard. To serum (1 ml) was added internal standard solution (1 ml of 0.01 N NaOH containing 20 jg tolbutamide) and 1 N HCI (250 jil). After gentle mixing by hand, the mixture was percolated through a small column of XAD-2 resin (< 30 mesh, 70 mm x 4 mm) followed by distilled water (2 x 2 ml) and the eluate discarded. Methanol (4 ml) was then percolated through the column and the eluate collected in a pointed centrifuge tube. Methanol was removed by heating (400C) in a strong current of air and the remaining aqueous solution was extracted thoroughly with dichloromethane (5 ml) by repeated aspiration with a Pasteur pipette. After centrifugation, the organic layer was transferred to a pointed test tube and evaporated to dryness by heating (400C) in a current of air. The residue was then methylated by rinsing down the tube with a solution of diazomethane in hexane (500 jl) followed by heating (600C) for 10 min. Hexane was removed t#a current of air and the bottom of the tube was thoroughly rinsed with methyl acetate (12 jlI). An aliquot (2 j1) of this solution was then injected onto the gas chromatography column. Blank serum spiked with varying amounts of chlorpropamide, 0-40 jg, gave a straight line for the calibration curve. At 10 jg mlP' the coefficient of variation was 0.9% (n= 10). The XAD-2 columns were regenerated by washing with methanol (10 ml) followed by water (10 ml). Gas chromatography conditions were: 6 ft x J in silanized stainless steel column packed with 5% Carbowax 20M on 100/120 mesh Gas Chrom Q; He carrier gas 50 ml min-', column temperature 2350C, injector 2500C,

detector (FID) 295C, attenuation 4 x 10-'0A f.s.d. The retention times of the methyl derivatives of tolbutamide and chlorpropamide were 6 and 8 min respectively. Blood glucose concentrations were determined on a Technicon Automatic Analyser in the Clinical Chemistry Department of the Queen Elizabeth Hospital, Birmingham, using the glucose oxidase method of Trinder (1969). Parameters of the three formulations were analysed using analysis of variance and where a significant difference (P < 0.05) occurred among the formulations, Duncan's Multiple Range Test (1955) was applied to determine which pairs of formulations were significantly different (P < 0.05). Areas under the serum concentration versus time curves were calculated using the trapezoidal rule. Results

Chlorpropamide serum concentrations

The means of the serum concentrations, areas under serum concentration versus time curves, peak serum

concentrations, times of peak serum concentrations and also their statistical analyses are shown in Table 1. No significant difference was found in the amount of drug absorbed from the three formulations as reflected in the areas under their serum concentration versus time curves. This was also true of the means of individual peak serum concentrations. Other than the 24 h samples there were no significant differences among the three formulations in the mean serum concentrations at sampling times greater than 3 h. Significant differences were found in the rates of absorption of chlorpropamide from the suspension compared with that of either tablet formulation. At 0.5 h and 1 h after ingestion of the dose the mean + s.e. mean serum concentrations of drug were 1.3 + 0.4 and 10.5 + 2.5 gg mln[ for tablet A, 4.8 2.7 and 12.4 + 4.5 jg ml[1 for tablet B, 23.4 + 4.9 and 29.6 2.0 jg ml-' for the suspension C. The drug was absorbed more rapidly from the suspension than from either tablet formulation but there was no significant difference in absorption rate between the tablets. The means of the times after dosing of individual peak serum concentrations were 6.6 + 0.6 h for tablet A, 4.2 + 0.8 h for tablet B and 1.8 + 0.6 h for suspension C. These differences were significant.
Blood glucose levels
The mean blood glucose levels obtained from the three chlorpropamide preparations and a statistical analysis of the data are shown in Table 2. The mean + s.e. mean maximum decrements in blood glucose concentrations of 0.67 0.08, 0.78 + 0.12 and

BIOAVAILABILITY OF CHLORPROPAMIDE

103

0.91 + 0.14 mmol 1-I for tablet A, tablet B and suspension C respectively were not significantly different. However, the means of the times of maximum decrease of both 3.44 + 0.60 h for tablet A and 2.56 + 0.44 h for tablet B were significantly different from that 1.06 0.13 h of suspension C, but there was no significant difference between the two tablets. There was also a significant difference among the mean blood glucose concentrations at 1 h after administration of the drug. The mean glucose concentration for suspension C was significantly lower than that of tablet A, but there were no significant differences between tablet B and the suspension nor between the two tablets.

Discussion

The chlorpropamide serum profile of the Diabinese@ tablet (B) used in this study showed a slower initial

rate of absorption of drug than was found by Monro & Welling (1974) but from 2 h onwards the profile was very similar to that in the earlier study with a prolonged absorption/distribution phase of at least 8 h followed by a slow elimination phase. The profile for the suspension indicates that the prolonged absorption/distribution phase is not due to an excessively long time of disintegration of the tablet in vivo. The serum profile of the suspension showed that the drug was absorbed more rapidly from this preparation than from either tablet as indicated by the significantly higher serum concentrations of chlorpropamide during the initial absorption phase and also by the earlier peak serum concentrations. Similar amounts of drug, as reflected in the areas under the serum concentration versus time curves, were absorbed from each of the three formulations. The present study demonstrates that the two tablet preparations of chlorpropamide have similar bioavailability characteristics. The areas under the serum

Table 1 Mean + s.e. mean chlorpropamide serum profiles of nine fasting volunteers after single 250 mg oral doses of three different preparations in a cross-over study
Parameter

Preparation

TabletA

Tablet B

Statistics ANOVA Duncan's multiple Suspension C (Among treatments) range test A A B and and and B C C

Til,me (h)
Mean serum concentration at:

(jg ml-,)

0 0.5 1 2 3 4 5 6 8 24 48 72 120 144 168

0 1.3 +0.4 10.5 +2.5 17.8 +3.0 25.3 +3.0 26.1 +2.8 29.2 1.4 30.6 + 1.7 30.2 +2.2 24.1 +1.4 16.9 + 1.7 11.3 +1.2

0 4.8 2.7

12.4+4.5 23.0 3.4 29.1 +1.1 29.1 +1.6 32.7 +2.5 28.8 +1.0
32.5 1.9 25.0 1.4 15.7 1.0 11.3 0.9 5.2 +0.7

5.40.9

3.40.7 2.4+0.6

4.2.+0.7 3.0+0.6

0 23.4+4.9 29.6 +2.0 30.3-+ 2.2 26.8 +1.7 27.3 +2.0 28.5 + 1.6 25.9 1.5 25.7 1.7 19.6 1.7 14.9+ 1.8 9.4+ 1.0 4.5+0.6 3.3 +0.4 1.9 + 0.5

P<0.001
0.001 0.05 NS NS NS NS NS < 0.05 NS NS NS NS NS

Mean area under serum 2028.1 + 173.9 2068.6 + 126.8 1768.7 143.9 NS concentration versus time curve (jg h ml-1) Mean of peak serum 35.3 +2.2 NS 32.5 1.7 35.8 2.0 concentrations (jig ml-1) Mean of times of peak 4.2 0.8 1.80.6 <0.001 6.6 +0.6 serum concentrations (h) ANOVA = Analysis of variance fot complete cross-over design. Duncan's multiple range test: + = difference between formulations is significant at P=0.05; = difference between formulations is not significant at P=0.05.
-

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BIOAVAILABILITY OF CHLORPROPAMIDE

concentration versus time curves were almost identical and at no time were there statistically significant differences between serum chlorpropamide concentrations obtained from the tablet formulations. The mean peak serum concentrations of drug were peak B yielded tablet but similar concentrations at an earlier time. It has been shown (Carlozzi et al., 1959) that mean predose, steady state serum chlorpropamide concentrations of 80 to 100 gg ml-' were maintained in volunteers by a single daily dose of 250 mg. These levels are four to five times higher than the 24 h values reported in this study and indicate the extent to which drugs with a long half-life, such as chlorpropamide, accumulate when taken regularly. In general, mean steady state serum drug concentrations are determined by the amount of drug absorbed and are independent of the rate of absorption provided that this is much faster than the rate of apparent elimination. The small formulation effect on rate of absorption observed in this study will therefore not produce different mean steady state serum chlorpropamide levels. This is important when considering the oral sulphonylurea hypoglycaemic agents, since it has been demonstrated

that the degree of hypoglycaemia produced is related to the plasma concentration of the drug, which is in turn proportional to the dose (West & Johnson, 1960). These authors reported decreases in blood sugar of 12 to 21 mg 100 ml-' (equivalent to 0.66 to 1.16 m mol 1-l) 30 min after 250 mg of chlorpropamide was given intravenously to 'normal subjects'. These values are similar to those obtained in this study after oral administration of the same dose. Comparison of the results shows that there was a very good correlation between bioavailability characteristics of the three formulations as measured by chlorpropamide serum profiles and the pharmacological effect of the drug. It is of interest to observe that a greater fall in blood sugar was obtained more quickly after the suspension than after the tablets; in other words there was a correlation between hypoglycaemic response and serum chlorpropamide concentrations attained after acute administration of the different preparations. However, as argued above, this observation is clinically irrelevant as the differences will become insignificant when the drug is taken in a normal multidose regime.

Table 2 Mean s.e. mean blood glucose profiles of nine fasting volunteers after single 250 mg oral doses of three chlorpropamide preparations in a cross-over study
Parameter Preparation

TabletA

Tablet B

Statistics ANOVA Duncan's multiple Suspension C (Among treatments) range test A A B and and and B C C

Time (h) NS 4.77 0.14 4.700.16 0 4.75 0.10 NS 4.44 0.10 4.21 +0.14 0.5 4.58 + 0.07 (mmol 1-1) 1 4.380.10 4.240.16 3.88 +0.13 P< 0.05 NS 4.08 +0.09 2 4.250.09 4.14+0.11 NS 4.43 + 0.08 4.22 0.07 3 4.350.09 NS 4 4.290.08 4.21 0.05 4.42 0.08 NS 4.26 0.03 4.33 + 0.07 5 4.22 0.09 NS 6 4.31 +0.07 4.26 0.07 4.32 0.07 0.67 + 0.08 Mean of individual maximum 0.78+0.12 0.91 +0.14 NS decrements (mmol 1-1) * Mean of individual times 3.440.60 1.06+0.13 P<0.01 2.560.44 to reach maximum decrements (h) * +0 ANOVA = Analysis of variance for complete cross-over design. Duncan's multiple range test: + = difference between formulations is significant at P=0.05; - = difference between formulations is not significant at P=0.05.
Mean blood concentration at

-00

M. EVANS, R.C. GLASS, M. MITCHARD, B.M. MUNDAY & R. YATES

105

References
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MONRO, A.M. & WELLING, P.G. (1974). The bioavailability in man of marketed brands of chlorpropamide. Eur. J. clin. Pharmac., 7,47-49. TAYLOR, J.A. (1972). Pharmacokinetics and biotransformation of chlorpropamide in man. Clin. Pharmac. Ther., 13, 710-718.
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(1959). A laboratory and clinical study of chlorpropamide and tolbutamide. Ann. N.Y. Acad. Sci., 74, 632-642.
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BRADFORD, P.M. & BURTON, J.S. (1977). Plasma concentrations, bioavailability and dissolution of chlorpropamide. Eur. J. clin. Pharmac., 11, 207-212. TRINDER, P. (1969). Determination of glucose in blood using glucose oxidase with an alternate oxygen acceptor. Ann. Clin. Biochem., 6, 24-27. WEST, K.M. & JOHNSON, P.C. (1960). Metabolism and relative hypoglycaemic potencies of four sulfonylureas in man. Diabetes, 9,454-458.

(1959). Metabolic studies of chlorpropamide in normal men and diabetic subjects. Ann. N.Y. Acad. Sci., 74, 603-617.

(Received February 28, 1978)