EurJ Clin Pharmacol (1992) 42:171-174

Crl a ll @ Springer-Verlag 1992

Disposition of quinine in plasma, red blood cells and saliva after oral and intravenous administration to healthy adult Africans L. A. Salako and A. Sowunmi Department of PharmacoIogy and Therapeutics, University of Ibadan, Ibadan, Nigeria

Summary. T h e p h a r m a c o k i n e t i c s of quinine has b e e n studied in ten healthy adult Africans after intravenous infusion and oral ingestion of a 500 m g dose. B l o o d and saliva samples were collected over 48 h and quinine in plasma, red cells and saliva was d e t e r m i n e d by H P L C . Quinine was rapidly and almost completely a b s o r b e d after an oral dose, with absorption half-life of 0.53 h, a tmax of 1--3 h and a bioavailability of 88 %. Analysis of the i.v. data gave an a p p a r e n t v o l u m e of distribution of 3.6 1. kg - 1 and a plasma clearance of 0.19 1-kg- ~. h ~. T h e concentration-time curves for plasma, red cells and saliva had declining phases were approximately parallel, giving a similar half-life that in all three media. T h e half-lives after the i. v. infusion also did not different f r o m those after oral administration. T h e dose was well tolerated by b o t h methods of administration.

Key words: Quinine, Malaria; pharmacokinetics, red blood cells, plasma, saliva, adverse effects

Malaria is a m a j o r public health p r o b l e m in most of the developing countries of tropical Africa. With the developm e n t and spread of Plasrnodium falciparum resistant to chloroquine, the first line antimalarial drug in Africa [Fogh et al., 1979; E k a n e m , 1985; Salako and A d e r o u n m u , 1987] since 1946, additional maj or public health problems have b e e n created for these countries. Because of this, it has b e c o m e necessary to e m p l o y alternative antimalarial drugs, such as amodiaquine, sulfadoxine/pyrimethamine (Fansidar ®) and even quinine without preliminary pharmacokinetic studies. Quinine has b e e n used in the treatment of malaria since 1630. D e s p i t e its increasing use in the t r e a t m e n t of uncomplicated, severe and/or complicated chloroquineresistant malaria in Africa, there have b e e n very few pharmacokinetic studies of this drug in Africans. In order to guide a rational dosage regimen, it is necessary that a thoro u g h evaluation of its disposition be u n d e r t a k e n in this ethnic group.

A study of the pharmacokinetics of quinine in healthy adult Africans has n o w b e e n u n d e r t a k e n as a first step towards achieving this goal.

Methods Ten male adult Nigerians, aged 21-30 y, took part in the studies. They were considered healthy on the basis of clinical history and examination and the results of haematological and biochemical tests, which were all normal. The study was carefully explained to the subjects and all voluntarily agreed to participate in it. None of the subjects had ever taken quinine before and none was on any regular medication. They were all non-smokers and took alcohol only occasionally. The study protocol was approved by the Ethical Committee of the College of Medicine, University of Ibadan.

Study design The subjects were randomly allocated to two groups of five. One group received oral quinine first, followed three weeks later by the intravenous dose. The order of administration was reversed in the second group, with the same time interval between the two doses. The oral dose of 600 mg quinine hydrochloride (Evans Medical Ltd, Speke, Liverpool), approximately 500 mg quinine base, was given with 300 ml water, at 08.00 h after an overnight fast. The subject remained fasting for a further 4 h after which food and water were allowed freely. Venous blood (5 ml) was collected from an antecubital vein before and 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, 36 and 48 h after taking the drug. Intravenous studies were also done after an overnight fast. Quinine hydrochloride (600 rag, approximately 500 mg base; Chemiefarma nv, Maarssen, Holland) was added to 0.9 % saline 500 ml and was infused over a period of 4 h. Venous blood 5 ml was collected before and at 0.25, 0.5, 0.75, 1,2, 3 and 4 h during the infusion. Further blood sampling was done 0.5, 1, 2, 3, 4, 5, 6, 20, 32 and 44 h after the infusion. Blood was collected into a heparinized container and was immediately centrifuged at 1200 g for 10 rain to separate the plasma and red blood cells, which were stored at - 20 °C until analysed. Saliva samples were taken at the same times that venepunctures were done. The samples were collected after rinsing the mouth with water and without any special stimulus to increase saliva flow. The saliva (5-10 ml) was immediately centrifuged and the clear fluid was removed and stored at - 20 °C until analysed.

172

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Time (h) l~g. 1. Plasma ( • ) red blood cell ( • ) and saliva ( [] ) concentrationtime curves after oral administration of 500 mg quinine (n = 10)

A 12-lead electrocardiogram was recorded before and i and 3 h after drug administration. Blood pessure and heart rate were monitored every 15 min starting from 0.25 h before to 2 h after drug administration, and thereafter hourly for a further 8 h. The subjects were also questioned about any subjective feelings during the first 12 h after drug administration.

Quinine assay Quinine in plasma, red blood cells and saliva was assayed by a specific HPLC with fluorescence detection method. Plasma lml and the internal standard 100 gl aqueous primaquine solution (100 gg/ml) were alkalinized with 2 N sodium hydroxide. The mixture was extracted with 5 ml diethylether and vortexed for 60 s. The organic layer was separated following centrifugation at 2,000 revolutions per minute for 10 min. O- 1 N-Hydrochloric acid 100 gl was added to 2 ml organic layer and the mixture vortexed for 50~50 s and centrifuged at 1000 g per minute for 10 minutes. The upper ether layer was removed and 20 gl quinine extract injected into the HPLC. The mobile phase comprised 0.2 M potassium diphosphate, acetonitrile and methanol in the ratio 80:10:10 (v/v). To 100 ml of this mixture was added 0.8 ml perchloric acid, giving a pH of 2.8. The column contained Bondapak C18, 3.9 x 300 ram. The fluorescence detector was set at 254 nm. The compounds eluted from the column in the order quinine metabolite, quinine, and primaquine. The flow rate was 1.2 ml. min- ~.The retention times for quinine and internal standard were 10 and 12 min, respectively. The lower limit of detection was 20 ng. ml-L Recoveries over the concentration range of 1 gg-ml- ~ to 10 gg- ml- ~ were 92-102 %. Coefficients of variation within sample were 5.5 % and 3.5 %, and between samples 4.5 % and 1.5 %, for concentrations of 10 ng. ml- 1and 10 gg. ml- 1, respectively. Calibration plots were linear (r = 0.9994) up to 5 gg. ml- ~. The clear saliva was processed in the same way as the plasma. Red cells were lysed with sterile water, centrifuged at 1200 g per minute for 10 min and the upper layer 1 ml processed in the same way as the plasma samples.

Pharrnacokinetic analysis A computer derived plasma, red cell and saliva concentration-time curve was plotted for each subject, from which, the peak concentration (Gn,x) and the time to peak concentration (tmax) for each subject were read.

The concentration-time data were analysed using the GPHARM non-linear programme, and assuming a two-compartment open model. The terminal half-life (tla) was calculated by linear regression of the log-linear terminal phase of the concentration-time curve containing five or more data points. The area under the concentrationtime curve (AUC) was calculated using the trapezoidal rule. The area to infinite time as is conventional, was added as CJfl, where Gn is the last value of the quinine concentration on the calculated flslope, and fl is the elimination rate constant. Plasma clearance was calculated from the equation CLp = f- D/AUC, where D is the dose given and f = 1 for the i. v. route. The apparent volume of distribution (Vz) was calculated from the equation Vz = CLp//3. The absorption rate constant (ka) was calculated by the method of residuals. Values are given in the text and Tables as means with (SD). Differences between means were tested for significance using Student's t test P < 0.05 was taken as significant.

Results

IntgaFenous study T h r e e of t h e 5 subjects in t h e g r o u p w h o r e c e i v e d o r a l q u i n i n e first f a i l e d to c o m e for the i n t r a v e n o u s study, so i. v. d a t a w e r e a v a i l a b l e for only 7 subjects. T h e m e a n plasma, r b c a n d saliva c o n c e n t r a t i o n - t i m e curves for t h o s e 7 subjects a r e s h o w n in Fig. 1. T h e p e a k c o n c e n t r a t i o n in all t h r e e fluids c o i n c i d e d with t h e e n d of t h e infusion at 4 h, a n d t h e r e a f t e r t h e c o n c e n t r a t i o n in t h e p l a s m a declined m o n o e x p o n e n t i a l l y . T h e declining p h a s e s of t h e c o n c e n t r a t i o n - t i m e curves for r b c a n d saliva a p p r o x i m a t e ly p a r a l l e l e d t h a t of p l a s m a , t h e r b c a n d saliva c o n c e n t r a tions b e i n g a p p r o x i m a t e l y half- a n d o n e - t h i r d , r e s p e c t i v e ly, of t h a t in p l a s m a . The pharmacokinetic parameters calculated from the p l a s m a c o n c e n t r a t i o n - t i m e d a t e a r e s u m m a r i z e d in T a b l e 1. T h e e l i m i n a t i o n half-life was 12.6 ( 2 . 5 ) h , the p l a s m a c l e a r a n c e 0.19 (0.03) 1. h -1. kg -1 a n d the v o l u m e of d i s t r i b u t i o n was 3.6 (1.0)1. kg -1. T h e e l i m i n a t i o n of q u i n i n e f r o m the r e d cells a n d saliva was similar to t h a t f r o m p l a s m a , w i t h a r e d cell half-life of 10.7 (2.3) h and a saliva half-life o f 12.3 (2.2) h. T h e r e was n o significant diff e r e n c e in t h e h a l f q i v e s in t h e t h r e e fluids.

Oralstudy O r a l c o n c e n t r a t i o n - t i m e d a t a w e r e a v a i l a b l e for all t e n subjects. A b s o r p t i o n was r a p i d with an a b s o r p t i o n r a t e c o n s t a n t of 1.3 ( 0 . 5 7 ) h -~ a n d a b s o r p t i o n half-life of 0.53 h. Q u i n i n e a p p e a r e d in t h e p l a s m a , r e d cells a n d saliv a w i t h i n 0.25 and 0.5 h, a n d r e a c h e d its p e a k c o n c e n t r a tion in i n d i v i d u a l subjects b e t w e e n 1 a n d 3 h. T h e r e a f t e r , t h e c o n c e n t r a t i o n d e c l i n e d b i e x p o n e n t i a l l y up to 48 h. T h e d e c l i n i n g p h a s e s of t h e r b c a n d saliva c o n c e n t r a t i o n t i m e curves w e r e a p p r o x i m a t e l y p a r a l l e l to t h a t in p l a s m a , with t h e r b c a n d saliva c o n c e n t r a t i o n s b e i n g a p p r o x i m a t e ly 2/5 a n d 1/4 r e s p e c t i v e l y t h a t in p l a s m a . The pharmacokinetic parameters derived from the o r a l p l a s m a c o n c e n t r a t i o n - t i m e curve a r e s u m m a r i z e d in T a b l e 2. T h e p l a s m a half-life was 11.5 (2.7) h. T h e e l i m i n a t i o n half-lives f r o m r b c a n d saliva w e r e also similar at 12.2 (2.2) a n d 12.5 (2.5) h, respectively. T h e r e was n o signific a n t d i f f e r e n c e b e t w e e n t h e values.

173 1. Plasma Pharmacokinetic parameters after i.v. infusion of 500 mg quinine (base) in 500 ml normal saline over 4 hours in normal adult Nigerians

Table

Sub- Weight Cma, tm~ t~/z ject ( k g ) gg/ml min (h)

V~ CLp AUC Extra1.kg-1 1 h 1. gg. polated kg -2 ml-~-h AUC(%)

1 2 3 4 5 6 7 mean (SD)

4.9 4.3 2.5 2.5 4.3 4.3 2.6 3.62 (1.0)

64.7 59.9 51.0 65.8 47.2 57.6 40.0 55.1 (9.5)

2.0 2.5 4.5 3.4 3.2 3.0 5.2 3.38 (1.1)

240 14.9 240 13.5 240 13.2 270 10.4 240 14.0 240 14.7 240 8.1 244 12.6 (11.3)(2.5)

0.23 0.22 0.13 0.16 0.21 0.20 0.22 0.19 (0.03)

42.1 48.3 83.6 48.0 64.5 51.5 73.5 50.8 (15.39)

3.6 2.7 2.2 3.6 3.1 3.4 1.8 2.9 (0.7)

Plasma pharmacokinetic parameters of quinine after single oral dose of 500 mg (base) in normal Nigerian adults

Table 2.

Sub- Weight Cnaax tmax tle ject (ks) (gg ml- 1) rain (h)

1 2 3 4 5 6 7 8 9 10 Ydean (SD)

64.7 59.9 51.0 65.8 47.2 57.6 64.7 40.0 51.7 59.4 56.2 (8.5)

2.5 11.3 2.5 2.9 4.3 2.7 4.6 2.2 3.4 2.8 3.8 (2.7)

90 120 120 150 150 150 60 120 180 120 126 (34)

aOral CLp 1.h -1kg -1

14.1 0.17 7.4 0.10 8.1 0.40 9.4 0.18 11.4 0.20 13.2 0.22 14.2 0.18 11.8 0.47 15.7 0.15 10.3 0.32 11.5 0.24 (2.7)(0.12)

a

AUC Extrapg. polated ml-l.h AUC(%) 52.8 98.2 27.5 48.5 59.8 43.9 49.4 27.3 17.1 29.7 45.4 (23.0)

16.9 26.5 4.7 7.4 5.5 8.0 9.8 6.3 10.6 8.9 10.5 (6.6)

a Fractional absorption of 0.88 used in calculation

The mean AUCs were 57.9 (16.2) ~tg.h.ml -~ for the i.v. and 51.2 (24.0) gg-h-m1-1 for the oral dose, giving a bioavailability of 88 %. Comparison of other pharmacokinetic parameters in the seven subjects gave a tl/2 of 12.5 (2.5) h for i.v. and 10.8 (2.6) h for oral. The difference between the two values was not significant. The oral clearance calculated from the relation CLp ; fD/AUC, where f = 0.88, was found to be 0.24 (0.12) 1. h -~ kg 1. This, too, was not significantly different from the clearance of 0.19 (0.03) 1. h ~ kg ~obtained from the i.v. data. Blood pressure and pulse monitored throughout the study remained within normal limits. Maximum QTc prolongation occurred at different times in different subjects, but was less than 10 % of the baseline value in all subjects. There was no subjective adverse effect.

Discussion

The present study was an investigation of the pharmacokinetics of quinine in healthy adult Africans done to obtain normal data against which the behaviour of the drug in patients with malaria and other diseases can be judged. Quinine is increasingly being used in Africa not only for

severe and complicated malaria but also for the mild and uncomplicated disease, because of the widespread chloroquine- and multi-drug resistant P.falciparum in the continent. Although it is generally recognized that a rational approach to dosage should be based on the known activity of a drug against local strains of the parasite, and on the pharmacokinetics of the drug in the affected population, the use of quinine in most African countries is based neither on any known pattern of in-vitro sensitivity of the parasites nor on any pharmacokinetic data obtained in the particular racial group. Instead, the dose of quinine, its frequency and the duration of treatment have largely been based on the experience in Thailand [WHO, 1986]. Yet, the sensitivity of the parasite to quinine may not be the same in Africa as in Thailand, and the pharmacokinetics of the drug may not be the same in the two racial groups: for example, in-vitro studies have shown that our P.falciparum isolates are inhibited at concentrations lower than those reported for isolates from Thailand [Salako, 1987]. The present results show that after an oral dose of approximately 10 mg-kg -1 quinine was rapidly and almost completely absorbed. It had a short half-life, relatively small volume of distribution and low plasma clearance compared to chloroquine. Similar results have been obtained in Thai subjects [White, 1985]. It can also be seen here that the pharmacokinetics of quinine was not affected by the route of administration. It is interesting that these findings are not only comparable to those from Thailand [White et al. 1982; 1983; White, 1985], in which a fluorimetric assay for quinine was used, but also to those from Malaysia [Jamaludin et al., 1988], in which the newer H P L C technique was employed here similar to that one used. Quinine was found to pass rapidly into red blood cells and to be eliminated from them at the same rate as from plasma. The concentration in rbc was 40-50 % of that in plasma and was the same whether the drug is given orally or i. v. It is noteworthy that quinine was measurable in saliva within a few minutes of oral administration, or of commencing the i. v. infusion, and that it was detectable there for up to 36 h after a 500 mg dose. The concentration of drugs in saliva is influenced by a number of factors, particularly plasma and saliva pH, protein binding and the pka of the drug, according to Equation [1]: 1 + 10- (pHs-pka) f ~ R = 1 + 10- (pHp pka) X fg

Eq. 1

where R is the saliva/plasma concentration ratio, pH~ the p H of saliva (usually 6-8), pHp the p H of plasma (normally 7.4), fp and fs the unbound fractions of the drug in plasma and saliva, respectively; for quinine fp = 0.8 and f~ = 1.0, and the pka of quinine is 8.5. Substituting these values in the above equation gives a value of R ranging from 18.6 for saliva of p H of 6.0 to 0.24 for saliva of p H 8.0. The relative steadiness of the saliva/plasma quinine concentration ratio at 0.25-0.33 here indicates the relative constancy of saliva p H during the sampling period. Indeed, the p H of saliva measured during the study were in the range 7.5 to 8.0. It is possible,

174 therefore, that, in spite of the theoretical potential for wide variability of the saliva/plasma quinine concentration ratio, the range in a given period of 24-48 h may be sufficiently narrow for the salivary quinine concentration to be used to predict the plasma concentration [Matin et al., 1974]. It could be used as a non-invasive method to measure compliance with treatment. Compliance is of greater importance during quinine treatment than for therapy with chloroquine, mefloquine or sulfadoxinepyrimethamine, because the latter drugs can be given in a 1-3 day in dosage regimen, quinine must be given for 7 days, and it is relatively easy to omit doses or stop treatment prematurely. This may lead to frequent recrudescences and even predispose to selection of resistant strains. Quinine is extensively bound to plasma proteins [Berlin et al., 1975; Mihaly et al., 1987] and the extent of protein-binding is increased in acute malaria, possibly as a result of the increased circulating concentration of c~-acid glycoprotein [Mansor et al., 1990]. In the present study, the salivary quinine concentration, which represents the unbound fraction, was approximately one-quarter to onethird of the plasma quinine concentration. This would suggest, with some qualifications, that in the subjects studied, quinine was probably 70 % protein-bound. In falciparum malaria, increased protein binding would be expected to lead to a reduced salivary quinine concentration relative to the plasma level. In conclusion, the study has demonstrated rapid and almost complete absorption of quinine from an oral dose and has confirmed the absence of a dangerously high blood level after a 4-hour i.v. infusion of 10 m g . k g - L It has also demonstrated the potential value of saliva for monitoring quinine levels during treatment. Acknowledgements. The study received support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases.

References Berlin CM, Stackman JM, Vessel ES (1975) Quinine-induced alteration in drug disposition. Clin Pharmacol Ther 18:670~579 Ekanem OJ (1985) Plasmodium falciparum falciparum malaria not responding to chloroquine in Nigeria. Trans Roy Soc Trop Med Hyg 79:141 Jamaludin A, Mohamad M, Navaratnam V, Selliah K, Tan SC, Wernsdoffer WH, Yuen KH (1988) Relative bioavailability of the hydrochloride, sulphate and ethyl carbonate salts of quinine. Br J Clin Pharmaco125:261-263 Mansor SM, Taylor TE, McGrath CS, Edwards G, Ward SA, Wirima J J, Molyneux ME (1990) The safety and kinetics of intramuscular quinine in Malawian children with moderately severe falciparum malaria. Trans Roy Soc Trop Med Hyg 84:482-487 Matin SB, Wan SH, Karam JH (1974) Pharmacokinetics of tolbutamide: prediction by concentration in saliva. Clin Pharmacol Ther 16:1052-1058 Mihaly GW, Ching MS, Klejn MB, Paull J, Smallwood RA (1987) Differences in protein binding of quinine and quinidine to plasma proteins. Br J Clin Pharmaco124:769-774 Salako LA, Aderounmu AF (1987) In-vitro chloroquine and mefloquine resistant Plasmodiurn falciparum in Nigeria. Lancet 2: 572573 Salako LA (1987) Quinine and malaria: The African experience. Acta Leidensia 55:167-180 White NJ, Looareesuwan S, Warrell DA, Warrell MJ, Bunnag D, Harinasuta T (1982) Quinine pharmacokinetics and toxicity in cerebral and uncomplicated falciparum malaria. Am J Med 73: 564-572 White NJ, Chanthavanich R Krishna S, Bunch C, Silamut K (1983) Quinine disposition kinetics. Br J Clin Pharmaco116:399-404 White NJ (1985) Clinical pharmacokinetics of antimalarial drugs. Clin Pharmacokinet 10:181-215 WHO Malaria Action Programme (1986) Severe and Complicated Malaria. Trans Roy Soc Trop Med Hyg 80 [Suppl]: 1-50 Prof, L. A. Salako Department of Pharmacology and Therapeutics University of Ibadan Ibadan Nigeria