Metrifonate in healthy volunteers: interrelationship between pharmacokinetic properties, cholinesterase inhibition and side-effects.

Metrifonate in healthy volunteers: interrelationship between pharmacokinetic properties, cholinesterase inhibition and side-effects Y. Aden-Abdi,12T. Viiiln,' 0. Ericsson,' L.L. Gustafsson,' & M.-L. Dahl-Puustinen' Metrifonate concentrations in plasma, its inhibition of blood cholinesterase, and side-effects were studied in 16 healthy volunteers who received a single oral dose of 2.5, 5, 7.5 or 15 mg/kg in a randomized double- blind study (4 subjects for each dose). Metrifonate was determined by a gas chromatographic method. Peak plasma levels were reached within 2 hours; the half-life in plasma, oral clearance, and normalized CM., and AUCs did not differ significantly between the four groups. Plasma cholinesterase (BuchE) was inhibited to low levels in all subjects, while erythrocyte cholinesterase (AchE) was affected in a dose- dependent fashion. The occurrence of side-effects correlated strongly with peak plasma levels but not with maximum AchE inhibition or with increase in salivation. This study shows that the absorption of metrifonate was not significantly different for doses between 2.5 and 15 mg/kg. The plasma levels of this drug correlated with the occurrence of side-effects. Introduction Metrifonate (2, 2, 2-trichloro-1-hydroxyethyl dimethyl phosphonate) is used in the treatment of schisto- somiasis caused by Schistosoma haematobium, an endemic disease in many tropical countries (1). As it is cheap, it could be used to control the disease at a mass level, provided that a simpler dosage regimen than the present one (3 doses of 7.5 mg/kg at two- week intervals) can be tested and implemented (2,3). To design a proper dosage regimen of metri- fonate, more knowledge about its pharmacokinetics and a better understanding of its pharmacological actions are necessary. Although Nordgren et al. (4,5) measured the plasma levels of metrifonate and its transformed product, dichlorvos, little is known about its absorption, distribution, and elimination in man. No studies have been carried out to investigate the relationship between the plasma levels and side- effects of metrifonate, and its action on cholinesterase inhibition in healthy volunteers. This study was designed to investigate the phar- macokinetics of metrifonate and the interrelation- ships between side-effects, plasma drug levels, and blood cholinesterase inhibition using different doses in the range of 2.5-15 mg/kg. ' Department of Clinical Pharmacology, Karolinska Institute, Hud- dinge University Hospital, S-141 86 Huddinge, Sweden. Requests for reprints should be sent to Dr Y. Aden-Abdi at this address. 2 Department of Pharmacology, Medical Faculty, Somalia National University, Mogadishu, Somalia. Reprint No. 5128 Materials and methods In this randomized double-blind study, the volun- teers were allocated to one of four treatment groups for a single oral dose of metrifonate: group A (2.5 mg/ kg), group B (5 mg/kg), group C (7.5 mg/kg), and group D (15 mg/kg). The drug was given at 08 h 00 after an overnight fast and was swallowed with a cup of water under supervision. Three hours later a standardized meal of meatballs with sauce, potatoes, salad, bread and butter, milk and ice cream was served to all subjects who were required to stay under observation for 8 hours. Subjects. Sixteen Swedish, healthy, male volunteers participated in the study. They were aged 20-49 years (mean, 31 + 9 years) and weighed from 62 to 90 kg (mean, 74.9 + 8 kg). All were non-smokers and, prior to the study, were passed as healthy by physical examin- ation and clinical laboratory tests. The volunteers were not allowed to take any drugs during the 7 days preceding the trial. The study was approved by the ethical committee at Huddinge University Hospital, Huddinge, Sweden. Drug used. Metrifonate (Bilarcilg, Bayer AG, Lever- kusen, Federal Republic of Germany) in 100-mg tablets, divisible into four parts, were purchased from the International Dispensary Association (IDA), Amsterdam, Holland. All doses were rounded to the nearest 25 mg. The number of tablets were matched by adding similar placebo tablets produced by ACO Lakemedel AB, Solna, Sweden. Blood sampling. Whole blood (10 ml) was drawn into Bulletin of the World Health Organization, 66 (6): 731-736 (1990) © World Health Organization 1990 731 Y. Aden-Abdi et al. heparinized test tubes from an indwelling cannula in the antecubital vein at 0, 10, 20, and 30 minutes, at 1, 1.5, 2, 2.5, 4, 8 and 24 hours, and at 7 and 10 days after drug administration. Immediately after sam- pling, 100p1 of whole blood was haemolysed with 1.9 ml of distilled water for blood cholinesterase analysis. After about 10 minutes the remainder of the samples were centrifuged for 10 minutes. Since metri- fonate is unstable in neutral and alkaline solutions (6, 7), 1 ml of the plasma was acidified with the same volume of 0.74 mol/l of phosphoric acid immediately after centrifugation for metrifonate measurements. All samples were stored at -18 °C and were analysed within a month. Adverse reactions. Blood pressure and pulse rates were measured after 5 minutes of rest while supine, and after 1 minute while standing at 8, 9.30, 10.30, and 16 h on dayo (day preceding the treatment) and on the treatment day. The volunteers were asked about side-effects from a check list at 0, 2, 4, 8 and 24 hours after drug administration. The intensity of the symptoms was graded as mild (+), moderate (+ +) or severe (+ + +) according to the description of the volunteers. Time of onset and duration of the symp- toms were also recorded. Any spontaneous com- ments by the volunteers were noted and evaluated similarly. Drug analysis. The concentrations of metrifonate were determined using gas chromatography with nitrogen- phosphorus sensitive detection (NPD) after extrac- tion of the plasma samples with chloroform. All samples were run in duplicate. The limit of determi- nation was 0.8 umol/l. Details of the method will be published elsewhere. Determination of chollnesteras activity. Plasma cholin- esterase (3.1.1.8; BuchE) and erythrocyte cholines- terase activities (3.1.1.7; AchE) were measured with a slight modification of the method developed by Augustinsson et al. (8), which is based on a spectro- photometric determination of the hydrolysis of pro- pionylthiocholine. The total cholinesterase activity was first measured in haemolysed whole blood. A second measurement after adding a selective inhi- bitor of BuchE (Astra 1397, 10-(a-diethylamino- propionyl)-phenothiazine HCI) gave the activity of AchE. The difference between these two measure- ments gave BuchE activity. All measurements were run in duplicate and the reaction was allowed to proceed for 2 minutes. Salivation. The saliva flow was measured as spon- taneous whole-mouth salivation on day0 and on the treatment day at 8, 9.30, 12 and 16 h. The volunteers swallowed saliva before collection, then 3 preweighed cotton rolls (Celluron®, Hartman, Heideheim/ Brentz, Federal Republic of Germany) were placed buccally on both sides in the lower vestibulum and sublingually for 2 minutes. The procedure was re- peated 3 times at intervals of 2 minutes. The amount of saliva secreted was weighed in mg (9, 10). Data analysis. Peak plasma concentrations Cmax) and the times at which concentrations peaked (tmax) were obtained from the plasma concentration vs time curves. The half-lives (t,/2) were determined by linear regression of the terminal log plasma concentration vs time curve. The AUCG 8 was calculated by the linear trapezoidal rule while the remaining area was calculated by AUCG_8 = Ch/K. The apparent oral clearance (Cl0) was determined by dividing the dose given by the total area under the plasma concentra- tion vs time curve. Comparison of the pharmacokinetic parameters between the groups was performed using the non- parametric Kruskal-Wallis test, while the inter- relationships between side-effects, peak plasma drug levels, maximum cholinesterase inhibition and increase in salivation were tested with the Spearman correlation rank test (11). The percentage increase in saliva AUC was calculated by comparing the area under the salivation curve (AUC,,,,) for the treatment day to that for day,. The cholinesterase activity was calculated as the percentage of the pretreatment value. Percentage inhibition values were found by subtracting the percentage activities from 100. Results Pharmacokinetic data for the four groups are given in Table 1. The plasma half-lives, oral clearance and normalized Cma,p, and AUC0 were not significantly different between the groups (P>0.60). Metrifonate AUCs were proportional to the doses, withl no evidence of dose-dependent kinetics (Fig. 1; r=0.99). Individual plasma concentrations vs time curves are shown in Fig. 2. BuchE was inhibited to low levels by all doses, while AchE was affected in a dose-dependent fashion. Maximum cholinesterase inhibition values were obtained during the first 2.5 hours for most of the volunteers. However, a few individuals obtained their maximum cholinesterase inhibition 4 or 8 hours after drug administration. The recovery of BuchE was quicker than that of AchE. Ten days after drug intake, mean BuchE activities of the 4 groups were 82+15%, 63+34%, 50+2% and 48 +2% of the pre- treatment values, respectively. Corresponding AchE WHO Bulletin OMS. Vol. 68. 1990.732 Pharmacokinetics of mbtrifonate In healthy volunteers Table 1: Age, weight and pharmacokinetic data (mean + SD) of the four groups (4 subjects In each group) Group A Group B Group C Group D (2-5 mg/kg) (5 mg/kg) (7.5 mg/kg) (15 mg/kg) P value' Age (years) 33+11 28+7 32+12 33+8 Weight (kg) 77+9 77+7 72+13 75+3 t,,, (range in h) 0.33-2 0.33-2 0.17-1.5 0.17-1 C,.. (pmo1/l)t 18.5+5.6 15.6+4.7 17.9+6.6 30.0+24.1 >0.6 t,,2 (h) 2.21 +0.21 2.17+0.53 2.16+0.21 2.21 +0.22 >0.9 AUC0.._ (,umol.h/l)6 61.6+11.2 53.5+11.4 58.1+9.8 66.1+26.2 > 0.67 AUCg-,, (% of total) 34.4+9.1 13.2+7.4 8.4+2.5 7.9+1.12 CIO (I/h/kg) 0.50+0.08 0.56+0.11 0.52+0.08 0.49+0.15 >0.6 ' Statistical calculations were done with the Kruskal-Wallis test. b Normalized to 7.5 mg/kg. Fig. 1. The AUC0,- of the 16 sublects vs dose. 250- 200- E 1 50 E C.)0 100 so _1 - a 2.5 5 7.5 Dose (mg/kg) values were 89 ± 11%,90 ±20%,97 ± 6% and 70+2% respectively. Mean increase of salivation of the four groups were -0.3+22.3%, 21.5±25.6%, 22.0+ 17.2%, and 13.5 +4.8% respectively. The peak plasma levels of metrifonate, maxi- mum cholinesterase inhibition values, and side-effects are given in Table 2. Eleven subjects complained of side-effects such as nausea, vomiting, abdominal 0 colic, diarrhoea, headache and dizziness; most were mild or moderate and disappeared within four hours. However, one volunteer from group D (15 mg/kg) showed pronounced side-effects which started 10WHO 90532 minutes after drug intake and persisted during the 15 day. There was a strong relationship (P <0.001) bet- ween peak plasma drug levels and the occurrence of Fig. 2. Log plasma concentration vs time curves of metrifonate for the four dosage groups. Group A (2.5 mg/kg) 150 100] 1 , 20 8 io c s. I 2- I1- o.s - I' . 1 2 3 4 5 6 7 8 Time after dose (h) Group C (7.5 mg/kg)4_ 1 2 3 4 5 6 7 a Time after dose (h) I 8 1 2 3 4 5 6 7 a Time after dose (h) 150* Group D 100* (15 mg/g) 50. 20- 10. 5. 0.5 - wFj0 90s3 1 2 3 4 5 6 7 6 Time after dose (h) WHO Bulletin OMS. Vol. 68. 1990. 150 100 o 50 E ,, 20- 8 *o- ac -9 5 0.5 150 1001 Group B (5 mg7kg) 0 E A Li a 50 20 10 5 2- 0.5- 733 Y. Aden-Abdi et al. Table 2: Comparison of the Individual Cm,., maximum chollnesterase Inhibition, salivation, and occurrence of sIde- effects In the 16 subjects Maximum inhibition (%) Type of side-effects" liv tio C... ('*mol/l) BuchE AchE (% increase) N V AC D DZ HA Group A (2.5 mg/kg): 8.3 97 24 32 + 4.6 72 28 -16 6.9 82 19 -14 4.9 93 20 -3 Group B (5 mg/kg): 12.5 92 23 7 8.6 81 19 -2 + 13.5 91 39 56 +++ ++ 7.1 98 28 25 + Group C (7.5 mg/kg): 20.8 98 37 8 + 11.1 88 27 16 25.4 88 37 17 + 14.3 100 33 47 + Group D (15 mg/kg): 131.3 91 68 17 +++ +++ ++ +++ ++ 38.3 100 48 18 + + 43.7 100 54 8 ++ ++ ++ 25.1 100 46 11 + + + No. of subjects reporting side-effects 3 1 7 3 4 2 ' N, nausea; V, vomiting; AC, abdominal colic; D, diarrhoea; DZ, dizziness; HA, headache; mild (+), moderate (+ +), severe (++ +). side-effects. However, such a relationship was either weak (P = 0.05) in the case of AchE or absent in that of BuchE and increase in salivation (P> 0.05). None of the other parameters that were investigated, i.e., blood pressure and pulse rate, changed significantly during the study. Discussion The results of this study show that metrifonate was quickly absorbed in all doses between 2.5 and 15 mg/ kg, with no significant variability between individuals in AUC, t,,2 and oral clearance, and with no evidence of dose dependence. The small variability found in the study is consistent with the reports (4) that metrifonate is mainly eliminated non-enzymatically via dichlorvos. Marked variability has usually been found in the kinetics of drugs that are metabolized enzymatically (12). Pharmacokinetic data of metri- fonate in healthy volunteers have not been reported before, but our data are similar to those reported earlier in patients with schistosomiasis (4, 5). The blood cholinesterase inhibition profile in this study is also similar to those reported previously (4,5,13). BuchE was rapidly inhibited to very low levels by all doses, but AchE decreased in a dose- dependent fashion (Table 2). The recovery of the enzyme was quicker for BuchE than for AchE which is in agreement with earlier findings (4,5, 13). The side-effects of metrifonate that are described in the literature have been extracted from therapeutic trials (2-5,13) on schistosomiasis patients. Neverthe- less, the type of side-effects reported in this study with healthy volunteers are similar to those reported pre- viously (2-5,13). There was a strong relationship between peak plasma drug levels and the occurrence of side-effects. However, such a relationship was either weak (P= 0.05) in the case of AchE or absent in that of BuchE and increase in salivation (P>0.05). Plestina et al. (13) studied the relationship between the side-effects of metrifonate and blood cholines- terase inhibition and did not find any correlation. However, the range between the doses used in that study was narrow, and the blood sampling schedule was not optimal to give an accurate assessment of relationships. One of the volunteers with high plasma levels of metrifonate and severe adverse reactions was found to be a slow hydroxylator of debrisoquine (14). The possibility that there is a link between his debri- soquine hydroxylation phenotype and his high plas- ma levels of metrifonate has not yet been confirmed (unpublished work). Six subjects, 3 slow and 3 rapid hydroxylators of debrisoquine (including the above- WHO Bulletin OMS Vol. 68. 1990.734 Pharmacokinetics of metrifonate In healthy volunteers mentioned subject), were given 2.5 mg/kg of metri- fonate and their pharmacokinetic parameters were not significantly different. Thus, the high plasma levels found in this subject and the fact that he was a slow hydroxylator of debrisoquine might be a coin- cidence. In conclusion, although metrifonate is presumed to be a prodrug, this study documents for the first time that its peak plasma levels and cholinesterase inhibition could be correlated with the occurrence of side-effects. No significant interindividual variability in t112, oral clearance, and normalized C., and AUCs, and no evidence of dose-dependency seem to exist in the kinetics of this drug. Acknowledgements This study was supported by training grants to Dr Yakoub Aden-Abdi and by institutional grants to the Department of Clinical Pharmacology, Huddinge University Hospital, Huddinge, from SAREC (Swedish Agency for Research Cooperation with developing countries, grant Nos 9.49 SAREC/1.SOM.01 and S/2.SOM.12). We thank Dr H. Emil- sson (ACO Lakemedel AB, Solna, Sweden) for preparing the placebo tablets, Margareta Rais and Christina Alm for skilled technical assistance and Ms J. Grunstein for expert secretarial assistance. Rlsumi Corr6latlon entre les propritbs pharmaco- cin6tiques, 1'effet Inhibiteur sur la cholines- t6rase et les effets secondaires du m6trifo- nate chez des volontaires en bonne sante Le metrifonate est un compose organophosphore utilise dans le traitement de la bilharziose a Schis- tosoma haematobium. La presente etude visait a elucider la pharmacocinetique du medicament et les correlations entre les effets secondaires, les concentrations plasmatiques, l'inhibition de la cholinesterase sanguine (ChE) et la secretion salivaire. Un protocole d'essai randomise en double aveugle a ete applique a 16 volontaires de sexe masculin, en bonne sante, ages de 20 a 49 ans (moyenne, 31+9 ans) et pesant de 62 a 90 kg (moyenne, 74,9+8 kg), qui ont recu une dose orale unique de 2,5, 5, 7,5 ou 15 mg/kg de metrifonate; 10 ml de sang ont ete preleves au moment de I'administration du medicament, puis au bout de 10, 20 et 30 minutes, 1, 1,5, 2, 2,5, 4, 8 et 24 heures, 7 jours et 10 jours. lmmediatement apres le prelevement, 100 p1 de sang ont ete hemolyses avec 1,9 ml d'eau distillee pour le dosage de la cholinesterase sanguine. Dix minutes plus tard, le reste des prelevements a ete centrifuge pendant 10 minutes. Un ml de plasma a ete melange avec un egal volume d'acide phosphorique a 0,74 mmol/l. Tous les echantillons ont ensuite 6te conserves a -18 °C et analyses dans un delai d'un mois. Les effets secondaires ont ete evalues 'a 'aide d'une liste de contr6le 0, 2, 4, 8 et 24 heures apres la prise du medicament. L'intensite des symp- tomes a ete notee selon une echelle comportant trois niveaux: legere (+) moderee (+ +) et forte (+ + +). La totalite de la salive a ete recueillie a 8 heures, 9 h 30, 12 heures et 16 heures, le jour du traitement et la veille. Le m6trifonate a ete dose par chromatographie gazeuse et la cholinesterase par spectrophotometrie. Le metrifonate a ete rapidement absorbe chez tous les sujets, la concentration plasmatique atteignant son maximum de 0,17 h a 2 h apres la prise du medicament (moyenne, 0,75+0,61 h). La demi-vie plasmatique moyenne, la clairance buc- cale apparente, la concentration plasmatique maxi- male (Cmax) et I'aire sous la courbe (AUC) pour les 16 sujets ont ete respectivement de 2,19+ 0,29 h, 0,52+0,11 I/h, 20,5+10,3 pmol/l et 59,8+ 14,7 pmol.h/l. L'aire sous la courbe etait propor- tionnelle a la dose, ce qui donne a penser que la cinetique est independante de la dose. On n'a pas constate de variabilite interindividuelle sig- nificative. L'inhibition a ete dose-dependante tant pour la cholinesterase plasmatique (BuchE) que pour la cholinesterase erythrocytaire (AchE), mais de facon plus marquee pour cette derniere. L'in- hibition a ete rapide, mais le retour a la normale a ete lent. Dix jours apres la prise du medicament, I'activite de la cholinesterase plasmatique s'etab- lissait a 82+15%, 63+34%, 50+2% et 48+2% respectivement des valeurs anterieures au traite- ment pour les quatre doses. En ce qui concerne la cholinesterase erythrocytaire, les valeurs cor- respondantes etaient de 89+11%, 90 + 20%, 97+6% et 70+2%. Les pourcentages moyens d'augmentation de la secretion salivaire ont ete de 0,3+22,3%, 21,5+25,6%, 22,5+17,2% et 13,5+4,8% respectivement. Onze sujets se sont plaints d'effets secondaires tels que nausees, vomissements, douleurs abdominales, vertiges et cephalees. On a observe une forte correlation entre la survenue des effets secondaires et la concentration plasmatique maximale du metri- fonate (P<0,001); par contre, la correlation avec l'inhibition maximale de la cholinesterase eryth- rocytaire a ete faible (P=0,05) et il n'y a eu aucune correlation significative avec l'inhibition maximale de la cholinesterase plasmatique (P>0,05). Cette etude a montre que la pharmaco- WHO Bulletin OMS. Vol. 68. 1990. 735 Y. Aden-Abdi ot al. cinetique du metrifonate etait independante de la dose et presentait une faible variabilitb interindivi- duelle. Les effets secondaires presentaient une correlation plus etroite avec la concentration plas- matique maximale du medicament qu'avec le degre maximal d'inhibition de la cholinesterase sanguine. References 1. Jordan, P. & Webbe, G., ed. Schistosomiasis: epidemiology, treatment and control. London, William Heinemann, 1982. 2. Aden-AbdI, Y. & Gustafeson L.L. A simplified dosage regimen of metrifonate in the treatment of Schistoma haematobium in Somalia. European journal of clin- ical pharmacology, 32: 437-441 (1987). 3. Aden-AbdI, Y. & Gustafseon, L.L. Field trial of efficacy of a simplified and standard metrifonate treatment of Schistosoma haematobium. European journal of clini- cal pharmacology, 37: 371-374 (1989). 4. Nordgren, I. et al. Plasma levels of metrifonate and dichlorvos during treatment of schistosomiasis with Bilarcil. American journal of tropical medicine, 29: 426-430 (1980). 5. Nordgr.n, I. et al. Levels of metrifonate and dichlorvos in plasma and erythrocytes during treat- ment of schistosomiasis with Bilarcil. Acta phar- macologica et toxicologica, 49 (suppl. V): 79-86 (1981). 6. Metcalf, R.L. et al. Toxic action of Dipterex and DDVP to the house fly. Journal of economic entomology, 52: 44-49 (1958). 7. Mlyamoto, J. Non-enzymatic conversion of Dipterex into DDVP and their inhibitory action on enzymes. Botyu-Kagaku, 24: 130-137 (1959). 8. Augustinsson, K.B. et al. A new approach to deter- mining cholinesterase activities in samples of whole blood. Clinica chimica acta, 89: 239-252 (1978). 9. Peck, R.E. The SHP test-an aid in the detection and measurement of depression. Archives of general psychiatry, 1: 35-40 (1959). 10. Nordin, C. et al. Little anticholinergic effect of E-10- hydroxynortriptyline compared with nortriptyline in healthy subjects. Clinical pharmacology and thera- peutics, 41: 97-102 (1987). 11. Daniel, W.W. Applied nonparametric statistics. Bos- ton, Houghton Mifflin, 1978. 12. Sj6qvist, F. et al. Fundamentals of clinical phar- macology. In: Speight, T.M., ed. Drug treatment, 3rd edition. Sydney and New York, Adis Press, 1987. 13. Plestina, R. et al. Effect of metrifonate on blood cholinesterases in children during the treatment of schistosomiasis. Bulletin of the World Health Organ- ization, 46: 746-759 (1972). 14. Steiner, E. et al. Polymorphic debrisoquine hydroxy- lation in 757 Swedish subjects. Clinical phar- macology and therapeutics, 44: 431-435 (1988). 736 WHO Bulletin OMS. Vol. 68.1990.