Pharmacokinetics of melarsoprol in uninfected vervet monkeys

Acta Tropica, 58(1994)35-49 © 1994 Elsevier Science B.V. All rights reserved 0001-706X/94/$07.00

35

ACTROP 00414

Pharmacokinetics of melarsoprol in uninfected vervet monkeys Christian Burri a, James D. Onyango b, Joanna E. Auma b, E.M.E. B u r u d i b, R e t o B r u n a'* aSwiss Tropical Institute (STI), Socinstr. 57, P. 0. Box, 4002 Basel, Switzerland, bKenya Trypanosomiasis Research Institute (KETRI), Muguga, Kenya (Received 12 April 1994; revision received and accepted 5 July 1994)

The level of the trypanocidal drug melarsoprol was determined in serum and cerebrospinal fluid (CSF) of six healthy vervet monkeys after intravenous application of the drug following a standard treatment schedule and a recently suggested alternative protocol. The maximum serum levels measured were about 3 Ixg/ml. A three-compartment model was used to analyze the serum data. The mean residence time calculated for melarsoprol in serum was 18 h, the volume of distribution was 3.6 l/kg and the clearance was 3.5 ml/min*kg. In the CSF the drug levels were generally very low, not exceeding 55 ng/ml, and the adaptation of the drug levels was found to be very slow. The comparison of the drug concentrations required to eliminate trypanosomes in vitro and the drug concentrations reached in the CSF during treatment revealed that the latter might be insufficient in some cases to eliminate all trypanosomes from this site. The peak serum levels during alternative application of the drug were lower compared to those during empirical treatment. No evidence for drug cumulation in the body was found. The results of this study are compared with recent pharmacokinetic data from human patients, and discussed in the context of the problem of relapses and reactive encephalopathy occurring after treatment of sleeping sickness. Key words: Arsenical; Melarsoprol; Pharmacokinetics; Trypanosomiasis (African); Trypanosoma brucei ssp.; Vervet monkey

Introduction Human African trypanosomiasis (sleeping sickness), caused by infection with Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense, is reported from 36 countries south of the Sahara. About 50 million people are living at risk of contracting the disease, and about 25000 new cases are reported annually (WHO, 1986). This number is clearly an underestimate, since reporting is poor and many affected areas are not accessible (Kuzoe, 1993). Chemotherapy of human trypanosomiasis is still unsatisfactory. In the absence of suitable alternatives the melaminylphenyl-arsenoxide melarsoprol remains the drug most commonly used for treatment of the late stage sleeping sickness (Fairlamb et al., 1992). Melarsoprol is usually very effective, yet relapses are reported to occur in 1-17% of the treated cases (Wellde et al., 1989; Pepin et al., 1989). Serious side effects like *Corresponding author. Fax: + 41 61 271 86 54. SSDI 0 0 0 1 - 7 0 6 X ( 9 4 ) 0 0 0 4 9 - 2

36 vertigo, vomiting and diarrhoea are common during treatment. The worst adverse reaction, a reactive encephalopathy, occurs in 5-10% of the patients treated, with a fatal outcome in 1-5% (Kuzoe, 1993). The cause of the encephalopathy is unknown, but it is commonly accepted that an immune phenomenon is involved in the reaction (Hailer et al., 1986; Jennings, 1993). All treatment schedules currently in use were developed empirically (Berman and Fleckenstein, 1991). Therapy consists of several series of three to four injections on consecutive days with an interval of about one week between the series. The doses administered increase either during the course of therapy or within the single series. The schedules vary depending on the trypanosome subspecies responsible for the infection, the country (WHO, 1986) and the local habits of the hospital. It is being discussed whether the doses usually given are appropriate. Some workers consider that they are subcurative and thus make reactive encephalopathy more probable. Trypanosomes cleared from the blood but persisting in the CNS are believed to cause an immunological reaction and to trigger this adverse reaction (Hunter and Kennedy, 1992; Jennings, 1993). Therefore some authors advocate a more aggressive treatment (Hunter and Kennedy, 1992; Jennings, 1993). Others believe that treatment is already too aggressive. Pepin stated that treatment could be overcurative, leading to high amounts of trypanosomal antigens which may bind to brain cells and attract antibodies or T-cells (Pepin and Milord, 1991). On the other hand, they stated that the starting doses might be insufficient for complete elimination, and that the maximum doses used might be to high (Milord and Pepin, 1992). Friedheim suggested investigating therapy schedules with constant, lower doses (Friedheim and Distefano, 1989). Reactive encephalopathy was also reported after the application of other trypanocidal drugs like DFMO and nifurtimox, indicating that this severe side effect may not be due to inherent drug toxicity of melarsoprol (Van Nieuwenhove, 1992). The pharmacokinetic properties of melarsoprol in human patients were recently investigated, and an alternative treatment schedule consisting of ten consecutive doses was suggested on the basis of computer simulations (Burri et al., 1993). It was reported that the brain might be the crucial compartment of the body for therapy, since relapses could be demonstrated to originate from this site (Jennings et al., 1979). In our recent investigations the cerebrospinal fluid (CSF) concentrations of melarsoprol were found to be generally very low compared to the serum levels, and in up to a third of all the patients no drug could be detected in the CSF at all. The interpatient variability of the CSF levels was large. No correlation could be shown between the serum levels and those of the CSF. It was hypothesised that the adaptation of the CSF levels to the serum levels was slow because of the chemical properties of the drug. Detailed pharmacokinetic evaluation of the CSF-data was not possible owing to the limited number of samples (Burri et al., 1993). Sample collection from humans is restricted by ethical considerations to the two to three timepoints when the CSF of patients is routinely checked for any remaining trypanosomes. The aim of the study was to compare the WHO protocol (1986) for treatment of African trypanosomiasis in Zambia and Kenya with an alternative treatment schedule (Burri et al., 1993). The study was performed in uninfected monkeys to exclude the influence of inflammatory processes on the penetration of the drug into the CSF.

37 Materials and methods

Drug The commercially available 3.6% solution of melarsoprol (Arsobal ®) in propylene glycol was obtained from Specia (Rh6ne-Poulenc-Rorer-Doma, Paris, France). The small amounts to be injected (20-50 I.tl) were diluted with an equal amount of propylene glycol prior to use to improve the precision of the application.

Animals Six uninfected, adult male vervet monkeys (Cercopithecus aethiops) weighing 4.1-6.2 kg were used for the experiments. The animals were kept individually and fed on vegetables, maize and concentrates. The animals were checked daily for signs of drug toxicity and side effects (e.g. behaviour, fur, skin, weight, temperature). The packed cell volume was determined throughout the study.

Drug application and sampling The monkeys were divided into two groups of three animals. The first group was treated according to the schedule for treatment of human African trypanosomiasis for Zambia and Kenya (WHO, 1986). The schedule was slightly modified to reduce the duration of the study in order to decrease the stress for the monkeys (see Table 1). The second group was treated with ten daily consecutive doses of 2.2 mg/kg body weight according to the alternative treatment schedule proposed by Burri et al. (1993). The detailed treatment schedules are given in Table 1. For application of the drug and sample collection the monkeys were anaesthetised with ketamine at a dose of 10 mg/kg. The drug was applied intravenously using either the brachial or iliac veins. The blood samples were collected from the iliac veins, and the CSF samples by lumbar puncture. The sample size was 1.5 ml of whole blood and 0.3-0.5 ml of CSF. Venous blood samples for analysis of melarsoprol levels in the serum of the empirically treated group were drawn 0.25, 0.5, 1, 3, 6, 12, 24, 48, 72 and 120 h after the last injection of the first and third series and 0.25, 24, 48 and 120 h after the last injection of the second series. Additional samples were collected 24 h after the injections one and two of each series in order to determine the trough levels of melarsoprol. Blood samples from the alternatively treated group were taken 24 h after every second injection for determination of the trough levels and 0.25, 0.5, 1, 3, 6, 12, 24, 48, 72 and 120 h after the last injection of the treatment. CSF samples were collected from the group of empirically treated monkeys 24 h after the first, second and the third injection of series one and three, 24 h after the second and 24 and 48 h after the third injection of series two and additionally 120 h after the last application of the drug. For the group of alternatively treated animals the timepoints of collection were 24 h after the first and subsequently after every second injection. Additionally CSF samples were collected 24, 72 and 120 h after the last injection.

38 TABLE 1 One group of monkeys was treated according to the WHO schedule for treatment of human African trypanosomiasis in Zambia and Kenya (empirical schedule); the second group was treated according to the alternative schedule proposed by Burri et al. (1993) Empirical schedule Day of treatment

Alternative schedule Dose

Day of treatment

Dose

0 1 2

0.36mg/kg 0.72mg/kg 1.10mg/kg

0 1 2

2.20mg/kg 2.20mg/kg 2.20mg/kg

10 11 12

1.40mg/kg 1.80mg/kg 2.20mg/kg

3 4 5

2.20mg/kg 2.20mg/kg 2.20mg/kg

20a 21a 22

3.60mg/kg 3.60mg/kg 3.60mg/kg

6 7 8

2.20mg/kg 2.20mg/kg 2.20mg/kg

30b 31b 32b

3.60mg/kg 3.60mg/kg 3.60mg/kg

9

2.20mg/kg

aThe doses for these injections were changed from 2.20 and 2.80 mg/kg to 3.60mg/kg, diverging from the original WHO schedule. bThis course of injections was not applied to the monkeys. All samples were immediately stored at - 2 0 ° C at K E T R I for a maximum of three weeks, and at - 8 0 ° C after being transported to the STI after completion of the sampling.

Sample analysis Melarsoprol concentrations in serum and cerebrospinal fluid (CSF) were determined in triplicate using a biological assay according to Burri and Brun (1992). A T. b. rhodesiense clone (STIB 704 BABA/2) was cultivated in microtiter plates for 72 h with twofold dilutions of melarsoprol. The minimum inhibitory concentration ( M I C ) was determined by microscopical examination. The serum or CSF samples were incubated under the same conditions and the M I C was determined. The test parameters were elaborated using 50 samples of pooled monkey serum, spiked with melarsoprol at known concentration. The limit of detection was 13 ng/ml, and the coefficient of variation, describing the precision of the method, was 23% for the complete range of concentrations. Aliquots of 50 gl were used for determination of melarsoprol in serum and 100 gl for CSF. The parameter measured by the bioassay is trypanocidal activity. Therefore no statement can be made about whether melarsoprol or its metabolites are present.

Pharmacokinetic evaluation For pharmacokinetic analysis the Topfit software package (Heinzel et al., 1993) was used.

39 For evaluation of the model the Akaike information criterion was used (Yamaoka, 1978). A mamillary model assuming exclusive elimination from the central compartment was used. A three compartment model was found to best represent the data. The formula Cp = A,e-~t + B,e-~t + C*e-~ was fitted to the concentration versus time values determined in serum. The reliability of fit for the curves of best fit to the determined serum values for the single animals was 0.83-0.98 (rZ). The pharmacokinetic parameters were calculated using standard methods (Gibaldi and Perrier, 1982). For derivation of pharmacokinetic values, the serum concentration versus time data for single animals were used. For estimation of the curve of best fit and calculation of the pharmacokinetic parameters of the CSF data, a fourth compartment representing the CSF was added to the previous model. The parameters of melarsoprol in serum, previously obtained by fitting the three compartment model to the serum values, were fixed for the subsequent iteration of the CSF data. The data set for the CSF was weighted with the factor 0.1, because the range of concentrations was very close to the limit of detection, and the number of determinations was limited. The pooled CSF concentration versus time data for all the monkeys in each group were used for derivation of pharmacokinetic values. The concentration versus time curve of melarsoprol in the CSF after application of the drug following the empirical schedule was simulated using the pharmacokinetic parameters estimated from the alternative schedule.

Results Serum

After application of the drug following the standard (empirical) treatment schedule the maximum serum concentrations were 1.7-3.1 ~tg/ml (median 1.8 Ixg/ml) after the first, 2.4-3.6 ~tg/ml (median 2.7 pg/ml) after the second and 3.1-3.8 ~tg/ml (median 3.1 I.tg/ml) after the third series. After application of the drug using the alternative schedule (ten consecutive doses of 2.2 mg/kg) the maximum serum concentrations were observed after 15 min. The maximum concentrations of the drug varied from 2.4 to 3.1 ~tg/ml (median 2.8 ~tg/ml). The maximum trough concentrations were 32-53 ng/ml (median 40 ng/ml) during the first series, 61-86 ng/ml (median 64 ng/ml) during the second and 150-260 ng/ml (median 230 ng/ml) during the last series of injections following the empirical schedule. They were 90-170 ng/ml (median 130 ng/ml) before the last application of melarsoprol during the alternative schedule (see Table 2 and Fig. 1). The mean serum concentrations dropped from concentrations of about 3 lag/ml to values in the range of 0.3-0.7 ~tg/ml within the first 6 h after application. The terminal half life was calculated to be 25.8-32.6 h for the empirically treated group and 16.7-25.4 h for the alternatively treated monkeys. The difference between these terminal half life values was significant (p=0.05). The serum concentrations were below the limit of detection (13 ng/ml) 120 h after the last injection in all the series. In the case of the empirically treated group,

Injection 3

Injection 2

Injection 1

Injection 3

Injection 2

Injection 1

0.25 24 48 120

24

24

Series 2

0.25 0.50 1 3 6 12 24 48 72 120

24

24

Series 1 0

Time (h)

2.4 0.068 0.045 < 1 .d.

0.061

0.039

1.7 n.d. n.d. n.d. 0.31 0.41 0.19 0.043 0.096 < I.d.

0.053

0.024

0

272

Monkey no.

1.7 0.13 0.048 < 1.d.

0.086

0.1

1.8 1.2 0.77 0.39 0.38 0.17 0.072 0.4 0.16 < i.d.

0.032

0.016

0

274

3.6 0.096 0.032 < 1.d.

0.064

0.049

3.1 2 1 0.32 0.32 0.16 0.097 0.032 < I.d. < I.d.

0.04

0.024

0

277

Injection 10

Injection 9

Injection 7

Injection 5

Injection 3

Injection 2

Injection 1

0.25 0.50 1 3 6 12 24 48 72 120

24

24

24

24

0.25

24

0

Time (h)

3.1 2.7 1.2 0.77 0.43 0.24 0.11 0.04 0.024 < 1.d.

0.13

0.13

0.13

0.16

1.5

0.064

0

333

Monkey no.

2.8 2.4 1.8 0.83 0.4 0.18 0.11 0.07 0.023 < I.d.

0.09

0.09

0.14

0.15

2.4

0.13

0

340

2.4 2.1 1.10 0.9 0.76 0.4 0.17 0.05 0.039 0.016

0.17

0.21

0.2

0.17

0.70

0.12

0

352

Serum concentrations of melarsoprol (~tg/ml) in monkeys after multiple intravenous injection; monkeys 272, 274 and 277 were treated following the empirical treatment schedule, monkeys 333, 340 and 352 following the alternative schedule

TABLE 2

0.25 0.50 1 3 6 12 24 48 72 120

24

24

Series 3

3.1 2.4 1.9 1.0 0.68 0.35 0.22 0.083 0.039 0.02

0.15

0.096

3.8 3.8 2.6 1.5 0.77 0.58 0.41 0.12 0.056 < 1.d.

0.23

0.18

3.1 2.3 1.8 1.5 0.76 0.44 0.23 0.096 0.06 0.032

0.26

n.d.

< I.d.: Below the limit of detection of 13 ng/ml; n.d.: sample collection was not possible at this timepoint.

Injection 3

Injection 2

Injection 1

42 101

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i ill

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\A

LI! l i t /

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r}'@o '~\

lO-1

\\

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1 o -2

\

10 -3

0

\

\

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J

I

I

I

2

4

6

8

10

I

t

12

14

I

16

I

18

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\

\

I

20

I

22

t

24

I

26

I

28

days

Fig. 1. Representativeconcentration versus time curves of melarsoprolin serum. & are the concentrations measured in the serum of monkey 277 after treatment following the empirical schedule; the dashed line represents the curve of best fit to the values determined. • are the concentrations measured in the serum of monkey 333 after treatment following the alternative schedule; the solid line represents the curve of best fit to the values determined. this was three days before the treatment was resumed with the next series of injections. Total clearance (CL) was 2.9-4.0 ml/min*kg (median 3.6 ml/min*kg-1). The calculated mean residence time ( M R T ) was in the range of 13.4-20.3 h (median 18.7 h). N o difference could be observed between the two groups o f animals. The pharmacokinetic parameters are given in Table 3.

Cerebrospinalfluid (CSF) All drug concentrations determined in the CSF were very low and for some samples below the limit of detection o f 13 ng/ml. Traces o f melarsoprol could be detected in all samples, however, the exact concentration for the samples below 13 ng/ml could not be determined because only a reduction o f growth but not the MIC could be observed with these samples. Following application o f the empirical schedule the trough levels measured were in the range from below the limit of detection (13 ng/ml) to 16 ng/ml during the first series, 16-26 ng/ml during the second and 16-55 ng/ml during the third series. The trough CSF values during treatment following the alternative schedule (10 x 2.20 m g / k g ) were 16-49 ng/ml, and they did not reach a final plateau during the ten days o f consecutive application (see Fig. 2).

TABLE 3 Pharmacokinetic parameters of melarsoprol in the serum of vervet monkeys after multiple intravenous application of the drug Parameter

V,,(1/kg) CL(ml'minkg -~) MRT(h) tlm(h )

Monkey no. (empirical schedule)

Mean ± s

272

274

277

4.5 3.9 19.5 28.9

3.5 2.9 20.3 32.6

3.6 3.6 17.9 25.8

3.8±0.5 2.8±1.7 22.5±6.7 29.1±3.4

Monkey no. (alternative schedule)

Mean + s

333

340

352

3.7 4.0 15.4 17.4

2.9 3.6 13.4 16.7

3.5 3.0 19.7 25.4

3.4±0.4 3.5±0.5 16.1±3.2 19.8±4.8

44

10 2

t~

/ /

zx

101

"-.\

/fJ /J

C

'

/ /

~'\\

z~

/

~'\ \

l

l I

\\

\\

\l

I I

t "--\~

I'

t I

//

10 °

10-1

[I

0

t

I

i

I

I

I

I

I

i

I

2

4

6

8

10

12

14

16

18

20

[

22

i

i

24

26

f

28

days

Fig. 2. Concentration versus time curves of melarsoprol in cerebrospinal fluid (CSF). A are the means of concentrations measured in the CSF of all monkeys of the group treated follwing the empirical schedule. The dashed line represents the concentration versus time curve derived from simulation of the application of the drug on the basis of the pharmacokinetic parameters calculated from the alternatively treated group. The concentrations of most of the CSF samples collected during the first series were below the limit of detection (13 ng/ml) and are not shown in this figure (see Table 4). • are the means of concentrations measured in the CSF of all monkeys of the group treated following the alternative schedule. The solid line represents the curve of best fit to the values determined. T h e h a l f life o f m e l a r s o p r o l in the C S F , c a l c u l a t e d f r o m the d a t a o b t a i n e d with the a l t e r n a t i v e t r e a t m e n t schedule, was a b o u t 120 h. T h e exact value can n o t be c a l c u l a t e d since s t e a d y state between the central a n d the C S F c o m p a r t m e n t has n o t been achieved d u r i n g the s t u d y period. M a x i m u m C S F levels were c a l c u l a t e d to be r e a c h e d a b o u t 10 h after the a p p l i c a t i o n o f the drug.

Drug toxicity N o signs o f general d r u g toxicity were o b s e r v e d in either g r o u p o f animals. L o c a l s r e a c t i o n s like t h r o m b o p h l e b i t i s a n d cellulitis o c c u r r e d at the site o f injection. This local r e a c t i o n c o u l d c o m p l e t e l y be e l i m i n a t e d b y using two needles, one to w i t h d r a w the d r u g f r o m the vial a n d one with a n a r r o w g a u g e for injection.

Discussion In a recent p u b l i c a t i o n we p r e s e n t e d the first p r e l i m i n a r y p h a r m a c o k i n e t i c d a t a o f m e l a r s o p r o l in m a n . It was s h o w n t h a t n o a c c u m u l a t i o n o f the d r u g o c c u r r e d in the

....

24 120

24

24

i,

Series 3

24 48 120

24

Series 2

24 120

24

24

38 16

22

27

21 22 13

23

< 1.d. < I.d.

< 1.d.

< 1.d.

0

272

Monkey no.

30 18

n.d.

30

16 n.d. 18

16

13 13

< 1.d.

n.d.

0

274

55 16

50

35

26 13 13

16

16 13

< 1.d.

< 1.d.

0

277

Injection 10

Injection 9

Injection 7

Injection 5

Injection 3

Injection 1

< 1.d.: Below the limit of detection of 13 ng/ml; n.d.: no sample collection possible at this timepoint.

Injection 3

Injection 2

Injection 1

Injection 3

Injection 2

Injection 3

Injection 2

Injection 1

Series 1 0

Time (h)

29 25

120

32

31

16

16

12

13

0

333

Monkey no.

72

24

24

24

24

24

24

0

Time (h)

19

22

49

26

26

33

n.d.

n.d.

0

340

13

n.d.

46

42

42.00

35

15

13

0

352

Cerebrospinal fluid concentrations of melarsoprol (ng/ml) in vervet monkeys after multiple intravenous injection; monkeys 272, 274 and 277 were treated following the empirical treatment schedule, monkeys 333, 340 and 352 following the alternative schedule

TABLE 4

46 body and that the serum concentrations drop to almost zero between the series of application. The CSF levels were generally very low, never exceeding 10% of the serum levels. A slow adaptation of the CSF levels was predicted based on the chemical properties of the drug, however a kinetical evaluation of the CSF data was not possible due the limited number of samples which could be collected from human patients (Burri et al., 1993). An alternative treatment schedule consisting of 10 consecutive injections of melarsoprol was suggested based on computer simulations. To confirm the predictions and to compare the pharmacokinetics of the alternative to the one of the standard schedules, a monkey model was chosen. The vervet monkey model used to investigate the pharmacokinetical properties of melarsoprol, with special consideration of the CSF, yielded pharmacokinetic parameters in serum which can be compared with the values obtained from the human patients. The clearance (CL) was 3.5ml/kg*min in the monkeys and about 1 ml/min*kg in humans, the volume of distribution (Vss) was 3.6 l/kg and >2 1/kg, respectively, and the mean residence time (MRT) was 17.7 h in monkeys and about 44 h in humans. No other discrepancies of the disposition of the drug was found in either study and no evidence became apparent for non-linearity of the kinetics of melarsoprol. Therefore the kinetic data for man and monkeys may be compared under consideration of the faster rate of elimination (e.g. higher clearance) from the monkeys. The predictions about the concentration versus time curve for the alternative schedule made by computer simulation (Burri et al., 1993) could be confirmed by the results for the monkeys treated with the alternative protocol. After empirical application of the drug to the monkeys the minimum concentrations before resuming the treatment with the subsequent series were calculated by extrapolation to be 1 ng/ml after the first series, and 2.5 ng/ml after the second series, respectively (see Fig. 1). These results are in line with the observation in the human patients where the concentrations dropped to almost zero between the series. No difference was found between the mean residence time (MRT), volume of distribution (Vss) and clearance (CL) of the two groups of animals after standard (empirical) and alternative treatment. The terminal half life (tl/2) of melarsoprol in serum was found to be greater after the application of the drug following the empirical schedule, but the difference may be explained by the small sample number and the large coefficient of variation of the bioassay used for drug determination. The value of the terminal half life, which is calculated from few data points at the end of the curve, is less stable than the mean residence time (MRT) or the clearance (CL), which are calculated on the basis of all data points. The number of data points of the CSF concentrations after application of the drug following the empirical schedule was insufficient to estimate the pharmacokinetic parameters. Due to the short courses, given by the therapy schedule, only a limited number of CSF samples could be collected from this group of monkeys. CSF samples were collected for determination of trough concentrations only, since no information about the timepoint of the maximum CSF levels was available prior to this study. More frequent collection of CSF samples from the vervet monkeys is not possible because blood contamination of the samples may occur, if the timepoints of collection are spaced by less than 48 h. To obtain information about the course of the drug concentrations in the CSF despite of these limitations, the concentration versus time curve was simulated on the basis of the parameters obtained from the alternative schedule and the dosing table of the empirical schedule.

47 The trough CSF levels were found to be generally very low, never exceeding 55 ng/ml. The half life in the CSF was calculated to be about 120 h and maximum drug levels were observed about 10 h after drug application. These results confirm the hypothesised slow adaptation and elimination of the drug from the CSF in comparison to those in serum (Burri et al., 1993). 95% of the steady state levels of a given drug are reached after four half lives after multiple administration of the drug (Rowland and Tozer, 1989). However, the steady state was not yet reached at the end of the alternative treatment lasting for ten days, reflecting the high value of the half life. Despite the higher doses applied during the third series of the empirical treatment (3.6 mg/kg compared to 2.2 mg/kg for the alternative schedule), the CSF levels were not higher compared to those obtained after the alternative treatment. Drug application was interrupted long before steady state concentrations were reached in the CSF. A similar problem may be assumed when the schedule used in West-Africa is applied, which specifies three series of four increasing doses of melarsoprol (1.2, 2.4, 3.6, 3,6 mg/kg body weight each), spaced by 10 days. The in vivo drug concentrations determined by the pharmacokinetic investigations may be compared to in vitro concentrations found to be toxic for trypanosomes. Both the in vivo and the in vitro concentrations were measured using similar biological assays (Kaminsky and Brun, 1993; Burri and Brun, 1992). The in vitro determination of the minimum inhibitory concentrations (MIC) of melarsoprol for several T. brucei ssp. isolates yielded values between 2 and 40 ng/ml, using test durations of 72 h. In addition it was observed that the MIC is depending on the isolate used and the time of incubation. For example the MIC decreased from values of about 100 ng/ml for 8 h incubation to 10 ng/ml for 48 h for a given isolate (unpublished data). Comparing MIC values for trypanosomes and the CSF drug concentrations measured in this study, we can speculate that the levels of melarsoprol in the CSF might be insufficient to eliminate all trypanosomes from this site after application of the drug following the empirical schedule. This might especially be true for the first series, where the trough concentrations measured never exceeded 16 ng/ml. The use of the bioassay has the advantage, that the in vivo and the in vitro values can be compared. The parameter measured by this method is trypanocidal activity and the assay can be used for determination of the drug concentration in biological samples as well as for testing the sensitivity of trypanosome isolates cultivated under in vitro conditions (Burri et al., 1992). On the other hand, no statement can be made about whether melarsoprol or its metabolites are present and the result is calculated as melarsoprol. The coefficient of variation was 23%, which is higher than this of specific analytical methods eg. HPLC. Until very recently the bioassay used was the only method available for routine determination of melarsoprol in a large number of samples. An HPLC method will be available within a short period of time (Berger et al., 1994). A decrease of the activity of melarsoprol in biological samples during long term storage at -20°C was reported by Tierney and Goodwin (1977). Therefore, the stability of the samples stored at different temperatures was tested using melarsoprol spiked samples. No decrease of the activity was observed after 3 weeks at - 2 0 °, but after 6 weeks the concentration had dropped to 85% of the original value. No alteration of the concentration was observed in samples stored at -80°C for up to one year (unpublished data).

48 Uninfected monkeys were used for this study to avoid any influence of infection on the permeability of the blood brain barrier. The interindividual variability of the CSF levels was markedly smaller amongst the uninfected monkeys than that found in the human patients described in the previous study (Burri et al., 1993). In one third of.the samples collected from humans no melarsoprol could be detected with the biological assay, but the maximum levels measured in some single samples were much higher than those in the monkeys (108 ng/ml in man, 55 ng/ml in monkeys). These findings might indicate that a variable alteration of the blood brain barrier caused by the process of inflammation during infection influences the penetration of melarsoprol into this compartment. Vervet monkeys appear to be a suitable model to investigate the changes of the blood brain barrier during infection and the effect this has on the drug levels in CSF, as well as for comparison of different treatment schedules. The two main findings of this study may be summarised as follows: First, the prediction of the slow adaptation of the CSF levels could be confirmed. Second, the comparison of the drug concentrations needed to eliminate trypanosomes in vitro and the drug concentrations achieved in the CSF during treatment revealed that the latter might be insufficient in some cases to eliminate all trypanosomes from this site, especially during the first series of standard application of the drug. Additionally, the peak serum levels during alternative application were lower compared to those during empirical treatment. No evidence for drug cumulation in the body was found. The results of this study and the observation that subcurative treatment may lead to severe adverse effects may reflect the potential usefulness of the suggested alternative treatment schedule.

Acknowledgements This study was supported by the Swiss Directorate for Development Cooperation and Humanitarian Aid. The expert assistance in animal care and sample collection of Drs. A. Njue, S.M. Karanja and J.B. Githiori is gratefully acknowledged. We thank Dr. Welker, Hoffmann-La Roche Ltd., for his invaluable help with the evaluation of the data. We thank the director of K E T R I for allowing the use of the institute facilities for this study. The contributions of B. Cenni and J. Jenkins, in critical reading of the manuscript, are highly appreciated.

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