Plasma concentrations of pyrimethamine and sulfadoxine and evaluation of pharmaco-kinetic data by computerized curve fitting

Bulletin of the World Health Organization, 60 (1): 115- 122 (1982) Plasma concentrations of pyrimethamine and sulfadoxine and evaluation of pharmacokinetic data by computerized curve fitting E. WEIDEKAMM,1 H. PLOZZA-NOTTEBROCK,2 I. FORGO,3 & U. C. DUBACH4 For the determination of the plasma concentration profiles of pyrimethamine and sulfadoxine after the administration of 1 tablet of Fansidar, highly specific analytical methods are needed as thepyrimethamine concentration is low (0.2 - 0.02 mg/litre) and the concentration ratio ofthe two components in theplasma is high (>1: 500). The microbio- logical method described in this paper fulfils these requirements for high specificity and sensitivity (the sensitivity limit for sulfadoxine is I mg/litre and for pyrimethamine is 0.013 mg/litre). Pharmacokinetic data were evaluatedfor 14 volunteers after administration of) tablet of Fansidar, and a computer simulation of multiple dosing (I tablet per week) was performed. The properties of the antimalarial Fansidar are based on the synergistic effect of its two components, which act as reversible inhibitors of two enzymes: sulfadoxine inhibits the plasmodial dihydropteroate synthetase (EC 2.5.1.15) and pyrimethamine blocks the tetrahydrofolate dehydrogenase (dihydrofolate reductase, EC 1.5.1.4). Since these two enzymes are located in sequence in the folate biosynthesis pathway of the plasmodia, the selective chemotherapeutic effi- cacy of the individual components is potentiated. As a consequence, the dose of both components can be reduced and accordingly the risk of side-effects mini- mized. In addition, the development of plasmodial resistance. against this antimalarial will be retarded because of the simultaneous presence of pyri- methamine and sulfadoxine. In the present paper, we describe a specific and sensitive microbiological assay to determine the concentrations of pyrimethamine and sulfadoxine in plasma. Plasma concentration profiles of 14 volun- teers who received 1 tablet containing 25 mg of pyrimethamine plus 500 mg of sulfadoxine were analysed, and the pharmacokinetic data obtained were used for a computer simulation of the plasma concentrations of the two components during weekly administration. ' Biochemist, Pharma Research Division, F. Hoffmann- La Roche, Basel, Switzerland. 2 Microbiologist, Pharma Research Division, F. Hoffmann-La Roche, Basel, Switzerland. 3 Resident, University Medical Outpatient Department, Kantons- spital, Basel, Switzerland. 4Director, University Medical Outpatient Department, Kantons- spital, Basel, Switzerland. MATERIALS AND METHODS Subjects Twelve male Caucasian volunteers (aged 21 -42 years and weighing 60- 85 kg) and two female Cau- casian volunteers (aged 20 and 21 years and weighing 57 and 63 kg) were selected for this study, which was performed at the University Medical Outpatient Department in Basel. The investigations were carried out in accordance with the principles laid down in the Declaration of Helsinki. All volunteers were healthy without renal, hepatic, or gastrointestinal disorders. Free and informed consent for the investigation was given by all subjects. No other drugs were taken during the trial, or for two weeks beforehand. Medication One tablet containing 25 mg of pyrimethamine and 500 mg of sulfadoxine was administered to each volunteer with 200 ml of tap water following over- night fast. One hour later a normal breakfast was served. Blood sampling Blood samples of 10 ml were collected in Vacu- tainers immediately before drug administration and 0.5, 1.5, 3, 6, 8, 24, 32, 48, 75, 120, 168, 216, 240, and 264 hours after the medication. Potassium ammonium oxalate was used as anticoagulant. The blood samples were centrifuged at 1000 g for 20 min, 4152 -115 116 E. WEIDEKAMM ET AL. and the plasma supernatant was stored in glass tubes at - 20 °C until analysed. Microbiological assay Pyrimethamine. Aliquots of 2 ml of plasma were adjusted to pH 11 with 2 mol/litre NaOH and extracted twice under vigorous agitation with 18 ml of chloroform using a partition funnel. The combined chloroform extracts, which contained all of the pyrimethamine, were evaporated to dryness with nitrogen and then dissolved in 2 ml of 0.067 mol/litre sodium/potassium phosphate buffer solution, pH 6.6. Pyrimethamine standard solutions were prepared by dissolving 27 mg of pyrimethamine in 5 ml of dimethylformamide using a sonification bath., Sodium/potassium phosphate buffer (0.067 mol/ litre, pH 6.6) was added to give a total volume of 100 ml; appropriate standard concentrations were prepared from this solution by dilution with the above-mentioned phosphate buffer. Metal cylinders (inner diameter 6 mm, height 10 mm) were placed on large agar plates (24.3 cm x 24.3 cm) and 200-Al samples and standard solutions (concentration range 13 - 370 Ag/litre) were applied in quadruplicate to the plates. The medium used for the agar plates was Bacto Folic Acid Assay Medium (FAAM)0 with the addition of 1 jig of folic acid, 27 g of Bacto Difco Agar, and 50 mg of 4-aminobenzoic acid per litre. Streptococcusfaecalis ATCC 8043 b was used as the test organism for the dermination of pyrimethamine. 100 ml of FAAM broth (75 g of FAAM and 1 ng of folic acid per litre) were sterilized at 120 °C for 5 min and inoculated with a loop of S.faecalis grown overnight on blood agar dishes at 37 'C. The inoculated broth was incubated for 16 h at 37 'C and centrifuged at 1500 g for 15 min. The bacterial sediments were resuspended and diluted 1: 400 in 0.15 mol/litre NaCl, and 1 ml of this suspension was added to 110 ml of FAAM medium, gently mixed, and poured onto the plates. After incubation of the agar plates at 37 'C for about 18 h, the inhibition zones were measured with a slide gauge and recorded automatically on teletype chart paper. Sulfadoxine. The plasma supernatant remaining after the chloroform extraction was acidified with 0.5 ml of 2 mol/litre HCI and mixed thoroughly; 18 ml of chloroform was added, shaken mechanically for 15 min, and centrifuged at 1500 g for 10 min. The lower chloroform phase containing the sulfonamide was evaporated to dryness with nitrogen. The residues were resuspended with 2 ml of 0.067 mol/litre phos- a Difco Laboratories, MI, USA. American Type Culture Collection, MD, USA. phate buffer, pH 6.6. Samples of 200 yd were assayed on large agar plates, using a "special agar", free from 4-aminobenzoic acid, and containing 30 g of vitamin- free casamino acids,' 10 g of glucose, 3 g of NaCl, and 30 g of Bacto Difco agar per litre of distilled water. Sterilization and storage were carried out as described for pyrimethamine. Sulfadoxine standard solutions were prepared by adding 27 mg of sulfadoxine to 2 ml of water and 0.15 ml of 2 mol/litre NaOH, mixing thoroughly, and making up to 100 ml with water. From this stock solution, standard concentrations between 1.1 and 90 mg/litre were prepared using 0.067 mol/litre sodium/potassium phosphate buffer, pH 6.6. The test organism used for the determination of sulfadoxine was Bacillus subtilis ATCC 6633, in the form of a 1: 100 dilution of a commercial spore sus- pension. Aliquots of 0.4 ml of this dilgtion were mixed with 110 ml of agar medium and incubated at 37 °C for about 9 h. Samples of 1 ml of this suspen- sion were then mixed with 110 ml of the "special agar" and poured into the agar plates. Aliquots of 200 Al of the standard solutions or plasma extracts were placed in the cylinders on the plates, incubated for 10 h at 37 IC, and assayed as described for pyrimethamine. Complete separation of pyrimethamine from sulf- adoxine in the plasma was verified by means of a com- bined thin-layer chromatography-bioautographie test (1). Linearity and sensitivity Pyrimethamine. A plot of log pyrimethamine con- centrations against corresponding inhibition zone diameters for the "spiked" plasma samples showed a linear correlation in the concentration range 13.3 - 370 Ag/litre (coefficient of correlation, 0.9974). The average recovery rate in the concentra- tion range 0.04T4.5 mg/litre was 103.1 ± 11.6%. Sulfadoxine. A similar regression line for sulf- adoxine showed a linear correlation in the con- centration range 1.1 - 90 mg/litre (coefficient of correlation, 0.9941). The average recovery in the con- centration range 3.3 - 90 mg/litre was 98 ± 4%o. Data analysis Both pyrimethamine and sulfadoxine exhibit two-compartment pharmacokinetics, and plasma level-time curves are best fitted by non-linear regression analysis according to Cp = A e - c' + B*e-' + C.e-kat. Computer programs, as de- scribed elsewhere (2), were used for the evaluation of pharmacokinetic data. ' Difco Laboratories, MI, USA. PLASMA CONCENTRATIONS OF PYRIMETHAMINE AND SULFADOXINE 117 The pharmacokinetic parameters obtained from these single-dose experiments were used to simulate the plasma concentration profiles of pyrimethamine and sulfadoxine during weekly dosing of 1 tablet of Fansidar (i.e., the recommended dosage schedule for malaria prophylaxis in adults). RESULTS AND DISCUSSION In Tables 1 and 2, a pharmacokinetic characteriz- ation of all tested subjects is given in terms of Cm. (maximum plasma concentration measured), tmax (time at which the maximum concentration of drug in the plasma occurs), t1/2 (terminal elimination half- life), AUCo-.O (area under the plasma level-time curve extrapolated from zero to infinity), K4 (apparent volume of distribution of the central compartment), and Cl, (systemic plasma clearance of the drug). The last two parameters are calculated as: D.f D-fVc= andCls =Cl Yo AUCo -00 where D = dose; f = fraction of administered dose that is absorbed (taken as 1 for both components as no intravenous injec- tion is possible in man); and YO = extrapolated intercept of zero-time plasma concentration. Pyrimethamine (Table 1) After administration of 1 tablet of Fansidar, con- taining 25 mg of pyrimethamine, maximum plasma concentrations between 0.127 mg/litre (D.E.) and 0.398 mg/litre (J.F.) were measured (mean value, 0.214 ± 0.065 mg/litre). These peak concentrations were reached after 4.2 ± 2.7 h. The average elim- ination half-life for all 14 volunteers was 95.5 ± 30.6 h. Individual half-lives varied between 46.1 h (E.H.) and 150 h (D.E.). Our results are in agreement with the data pub- lished by Cavallito et al. (3), who found an average elimination half-life of 84.3 h (range 79 - 93 h), a tmax of 4 h, and a Cmax of 0.5 - 1 mg/litre after admin- istration of 100 mg of pyrimethamine to 4 volunteers. Jones & Ovenell (4) determined the elimination half- life in 11 volunteers who received 12.5 mg of pyri- methamine, and found a mean value of 77.4 h (range 35 - 174 h) with a corresponding tmax of 4 - 8 h, and a Cmax of 0.080 - 0.150 mg/litre. Stickney et al. (5) analysed the pyrimethamine plasma concentrations of 10 volunteers and obtained an average elimination half-life of 92 h. Sulfadoxine (Table 2) The mean value of the sulfadoxine half-life for all 14 volunteers was 184.1 ± 32.8 h (range 113 - 226 h). A maximum concentration of 63.2 + 10.8 mg/litre was observed in the plasma 3.7 (+ 1.7) h after administration of 1 tablet of Fansidar (sulfadoxine content, 500 mg). Similar elimination half-lives for sulfadoxine have been published elsewhere: by Peck et al. (6) (194 ± 44 h), Bohni (7) (179 h), and Hall (8) (200 h). Single-dose curves One of the volunteers (R.F.), whose pyrimeth- amine and sulfadoxine plasma profiles were represen- tative of the average response seen in this study, has been selected for the demonstration of the concen- tration-time curves (Fig. 1 and 2). Both curves show clearly the distributive phase, where the drug concen- tration in the plasma decreases more rapidly than in the postdistributive phase. Multiple dosing The recommended dosage for adults during malaria prophylaxis is 1 tablet of Fansidar per week. Using the microconstantsd derived from the plasma profile curves in Fig. 1 and 2, a dosing interval of 7 days, and an apparent volume of the central compart- ment of 4 litres for sulfadoxine and 43 litres for pyri- methamine, a computer simulation was performed of the plasma concentrations during multiple dosing (Fig. 3 and 4). The "average" plasma concentration of a drug in steady-state, Css, can be calculated from: Css= (oCdt) IT = (oF Cdt) /r where r = dosing interval and ol Cdt = AUCo-OO. This equation assumes that the volume of distribution and the elimination rate are constant over the entire dosing period. Using this equation, it was found that: Cs, = 0.155 mg/litre for pyrimethamine, after about 4 weekly administrations (6.64 half-lives are required to reach 99%1o of the steady-state plasma level of a drug); and Cs, = 98.4 mg/litre for sulfadoxine, after about 7 weekly administrations. d Apparent first-order elimination and intercompartmental distribution rate constants. E. WEIDEKAMM ET AL. Table 1. Plasma parameters and pharmacokinetics of pyrimethamine in 14 human volunteers after administration of 1 tablet of Fansidar, containing 25 mg of pyrimethamine Elimination half-life C. t., AUCo, VI C/. Subject tl/2(h) (mg/litre) (h) (mg.h.litre ') (litres)(ml/h) E. H. 46.1 0.197 1.5 9.4 68.6 2659.6 R. M. 76 0.179 6 22.7 138.6 1101.3 K. K. 115.5 0.200 6 28.8 64.1 868.1 R. F. 105 0.273 3 26.0 42.7 961.5 W. A. 118.8 0.222 1.5 24.6 60.5 1016.3 N. G. 92 0.173 8 22.5 32.3 1111.1 K. L. (female) 148 0.185 2 20.8 61.1 1201.9 D. E. (female) 150 0.127 8 16.2 104.6 1543.2 A. F. 54 0.195 1.5 11.2 78.0 2232.1 B. W. 74 0.256 8 20.3 81.4 1231.5 M. P. 91 0.229 1.5 18.2 59.3 1373.6 J. F. 89 0.398 6 15.0 63.4 1666.7 E. W. 78 0.156 3 15.6 95.6 1602.6 K. S. 100 0.210 3 15.7 112.9 1592.4 Mean ± SD 95.5 ± 30.6 0.214 ±0.065 4.2 ± 2.7 19.1 ± 5.6 75.9 ± 28.6 1440.1 ± 503 Table 2. Plasma parameters and pharmacokinetics of sulfadoxine in 14 volunteers after administration of 1 tablet of Fansidar, containing 500 mg of sulfadoxine Elimination half-life C., tW AUCo, C/, Subject tl,2(h) (mg/litre) (h) (mg.h.litre ') (litres) (ml/h) E. H. 173.3 66.0 3 14 308 3.9 34.9 R. M. 187 56.1 1.5 13 201 8.4 37.9 K. K. 210 64.7 6 17 814 3.9 28.1 R. F." 203.4 67.3 3 16 533 4.0 30.2 W. A. 210 53.8 6 16 947 8.9 29.5 N. G. 216.6 67.7 3 18 591 6.3 26.9 K. L. (female) 226 76.0 6 20 899 6.2 23.9 D. E. (female) 185 54.2 4 12 824 4.9 38.9 A. F." 151 51.0 4 4 784 4.8 104.5 B. W. 113 72.1 4 9 080 3.9 55.1 M. P." 146 41.0 6 6 971 7.7 71.7 J. F. 165 75.0 2.5 14 542 6.8 34.4 E. W. 219 61.9 '1.5 16 532 4.8 30.2 K. S. 172 78.1 1.5 15 329 5.1 32.6 Mean ± SD 184.1 ± 32.8 63.2 ±10.8 3.7 ± 1.7 14 168 ± 4532 5.7 ± 1.72 41.3 ± 22.1 ' Plasma concentrations were determined microbiologically; all other samples were analysed with the Autoanalyzer (Bratton- Marshall reaction). 118 PLASMA CONCENTRATIONS OF PYRIMETHAMINE AND SULFADOXINE mg/ I Plasma 0.4 - 0.2- 0.1 - 0.08_ 0.06- 0.04- t C 0.5 0.052 1.5 0.228 3.0 0.273 6.0 0.190 8.0 0.237 24.0 0.139 32.0 0.135 48.0 0.125 75.0 0.117 120.0 0.077 168.0 0.047 216.0 0.041 264.0 0.027 0 0.0211 0.01- II I I I I I I I I Il l II I I I I 0 28 56 84 112 140 168 196 224 252 280 Time (hours) Fig. 1. Plasma concentration profile of pyrimethamine after administration of 1 tablet of Fansidar (25 mg of pyri- methamine and 500 mg of sulfadoxine) to volunteer R.F. mg/ I Plasma 100- 80- t C 0.5 19.4 1.5 63.5 3.0 67.3 6.0 58.7 Ar '7O.V. J,u.i 60- 24.0 54.832.0 53.1 _ . 48.0 48.1 75.0 44.2 40- 120.0 36.9 168.0 32.2 20- 216.0 27.1 240.0 24.0 264.0 23.6 10 1 I I , I I I I I I I I I I I I I 0 28 56 84 112 140 168 196 224 252 280 Time (hours) Fig. 2. Plasma concentration profile of sulfadoxine after administration of 1 tablet of Fansidar (25 mg of pyrimeth- amine and 500 mg of sulfadoxine) to volunteer R.F. 119 E. WEIDEKAMM ET AL. mg/ I Plasma 0.4- 0.36- 0.32 0.28 0.24- 0.2- 0.16- 0.12- 0.08- 0.04 0 0 10 20 30 40 50 60 70 80 90 100 Time (days ) Fig. 3. Computer simulation of pyrimethamine plasma concentration produced by weekly administration of 1 tablet of Fansidar. Pharmacokinetic parameters are taken from the single dose response shown in Fig. 1. (Volunteer R.F.). mg/I 150 135 120 105 90 75 60 45 30 15 0 Plasma Fig. 4. Computer simulated sulfadoxine plasma levels produced by weekly administration of 1 tablet of Fansidar. Pharmacokinetic data are taken from the single dose response in Fig. 2. (Volunteer R.F.). I I I I I I I I I I I I I I I I I I I I 1 0 10 20 30 40 50 60 70 80 90 100 Time (days) 120 PLASMA CONCENTRATIONS OF PYRIMETHAMINE AND SULFADOXINE 121 Since each drug dose is administered in the post- distributive phase of the preceding dose, the extent to which both components accumulate in the plasma can be calculated from: R = I/(I-e-r) (where ,B = apparent first-order disposition rate constant; ,B = 0.693/tl/2). This accumulation factor R is 1.4 for pyrimeth- amine and 2.2 for sulfadoxine. In spite of the different elimination half-lives of the two Fansidar components, a harmful drug accumu- lation or a decrease of the drug concentrations below the therapeutic range does not occur with the recom- mended prophylactic dosage of 1 tablet per week. The microbiological assay method described for pyrimethamine and sulfadoxine benefits from the fact that no microbiologically active metabolites of the two components are present in the plasma (7). The test organisms used are specific for the individual substance classes, but the large difference in concentration of pyrimethamine and sulfadoxine necessitated separation of the compounds before analysis, to ensure that the results were reliable. With a sensitivity of 13 Ag of pyrimethamine/litre and 1 mg of sulfadoxine/litre the microbiological assay is comparable with recently reported chemical methods. Thus, Jones & Ovenell (4) reported a high- pressure liquid chromatography method for pyri- methamine with a sensitivity limit of 10 jtg/litre. Simmons & de Angelis (9) described a thin-layer chromatography method that allowed the detection of 5 ng of pyrimethamine by fluorescence scanning. A new chemical method of determination for sulfadoxine using the Autoanalyzere has a higher sensitivity (0.1 mg/litre), but the standard deviation for the recovery rate in the concentration range below 10 mg/litre is ±25Vo. Internal Roche Report RCR B-86'631. ACKNOWLEDGEMENTS We wish to thank Mrs R. Portmann and Mr A. Witschi for their invaluable technical assistance. RESUME CONCENTRATIONS PLASMATIQUES DE LA PYRIMETHAMINE ET DE LA SULFADOXINE ET EVALUATION DE DONNEES PHARMACOCINETIQUES PAR ETABLISSEMENT D'UNE COURBE A L'ORDINATEUR Description d'une nouvelle methode microbiologique de dosage de la pyrimethamine et de la sulfadoxine dans le plasma humain. La methode analytique comprend l'extraction fractionn6e des deux composants et leur deter- mination quantitative par la methode d'immunodiffusion en gelose. Pour cette experience, les germes choisis ont ete: pour la sulfadoxine, Bacillus subtilis ATCC 6633, et pour la pyrimethamine Streptococcus faecalis ATCC 8043. La limite de sensibilite inferieure pour la pyrimethamine est de 13 ng/ml et l'extraction aboutit A un rendement de 103,1 ± 11,6% dans la fourchette de concentration allant de 0,04 a 4,5 tg/ml. La limite de sensibilite pour la sulfadoxine est de I gg/ml et la quantite obtenue par extraction s'eleve A 98 ±4% dans la gamme de concentration de 3,3 A 90,0 zg/ml. Les concentrations plasmatiques de 14 volontaires en bonne sante ont ete determinees apres administration d'un comprime de 25 mg de pyrimethamine et de 500 mg de sulfadoxine. Les profils de concentration obtenus ont ete le mieux decrits par une cinetique A deux compartiments non lin6aires. La periode d'elimination finale moyenne calcul&e a partir de ces donn&es etait de 95,5 ± 30,6 heures pour la pyrimethamine et de 184,1 ± 32,8 heures pour la sulfa- doxine. Les concentrations plasmatiques maximales ont &6 atteintes apres 4 heures environ et s'e1evaient a 0,214 ± 0,065 itg/ml dans le cas de la pyrimethamine et a 63,2 ± 10,8 ug/ml dans le cas de la sulfadoxine. En utilisant les donnees fournies par des etudes executees A I'aide d'une dose unique, on a realise une simulation par ordinateur d'administration a doses multiples (un com- prime/semaine) afin d'obtenir des informations sur les courbes de concentration plasmatique. En outre, les concen- trations (<moyennes>) de m6dicaments a l'etat d'equilibre et les facteurs d'accumulation des deux composants ont e evalues dans le plasma. 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