University of Nigeria Virtual Library

Transcription

1 Serial No University of Nigeria Virtual Library Author 1 Author 2 Author 3 Title I. U. ASUZU I. E. ODIKA S. M. ANIKA THE CHEMOTHERAPEUTIC EFFICACY OF DIMINAZENE ACETURATE AND LITHIUM CHLORIDE AGAINST RELAPSE INFRECTION OF TRYPANOSOMA BRUCEI IN RATES Keywords. Description Category Publisher TROPICAL MEDICIEN AND PARASITOLOGY VETERINARY PHYSIOLOGY AND PHARMACOLOGY THIEME MEDICAL PUBLISHERS INC., NEW YORK Publication Date JUNE, 1995 Signature

2 No. 2 Volume 46 June 1995 Tropical Medicine and Parasitology Official Organ of Deutsche Tropenmedizinische Gesellschaft Editors R. Garms, Hamburg R. Korte, Eschborn K. D. Walter, Hamburg Editorial Board H. J. Bremer, Heidelberg D. W. Biittner, Hamburg H. J. Diesfeld, Heidelberg B. 0. L. Duke, Lancaster R. D. Horstmann, Hamburg S. H. E. Kaufmann, Ulm A. A. Kielrnann, Nairobi D. Mehlitz, Berlin K. Mott, Geneva F. P. Schelp. Berlin Georg Thieme Verlag RiidigerstraBe 14 D Stuttgart Postfach D Stuttgart Reprint 0 Georg Thieme Verlag Stuttgart. New York Reprint with the permission of the publishers only Thieme Medical Publishers. Inc., 381 Park Avenue South New York, NY 10016

3 The chemotherapeutic efficacy of diminazene aceturate and lithium chloride against relapse infection of Trypanosoma brucei brucei in rats 99 - I. E.-Odika *, I. U. Asuzn2, S. M. AnikaZ ' Department of Veterinary Physiology and Pharmacology, Usmanu Danfodiyo University Sokoto; Department of Veterinary Physiology and Pharmacology, University of Nigeria, Nsukka, Nigeria Abstract The chemotherapeutic efficacy of diminazene aceturate (Berenil@) and lithium chloride (LiCl) in relapse infection of trypanosomiasis was investigated in rats experimentally infected with Trypanosoma brucei brucei. The study showed that the combination of diminazene aceturate at (7 mg/kg) and LiCl (10~g/kg) appeared more effective therapeutically than diminazene aceturate, or diminazene aceturate and LiCl and dexamethasone group, as more of the rats in the diminazene aceturate and LiCl treated-group remained aparasitaemic for longer days (60 days). Relapse parasitaemia occurred on days 10 and 12 in diminazene aceturate (7 mg/kg); diminazene aceturate (7 mg/ kg) and LiCl (10 pg/ kg) plus dexamethasone (1 mg/kg) treated group respectively, while relapse parasitaemia did not occur in the diminazene aceturate and LiCl treated group until day 20. Histopathological examination of the brain did not show any signs of inflammatory reaction in the diminazene aceturate and LiCl and dexamethasone treated group. However lesions associated with meningoencephalitis, such as cellular infilteration of mononuclear cells, perivascular cuffings and perivascular congestion and oedema were observed in the diminazene aceturate; diminazene aceturate and LiCl treated groups. Introduction Trypanosomiasis is a serious disease of various species of animals and man. It is an important parasitic disease in Africa where tremendous economic damages have been made in Iivestock Industry.. Drug resistance is one of the serious problems that complicate treatment of many infectious diseases and in the case of trypanosomiasis, has stimulated the search for chemotherapeutic control of the disease. Although much work has been carried out on the eficacy of many trypanocides in various species of animals (Wil- Accepted 29 November 1994 son, 1958; Fairclough, 1963; Verma et al., 1973; Wilson et al., 1975), the effective drug control of the disease is still problematic. Poor distribution of trypanocidal drug (diminazene aceturate) to the infected and intracellular sites, diminished activity of the drug in animals with suppressed immune system, under-dosage and lodgement of the parasite in the cryptic site (brain) are among the host related factors that influence the trypanocidal drug resistance (Hawking, 1963). This pharmacokinetic problem, i.e. poor distribution of drug may play important role in relapse phenomenon where re-infection occurs after chemotherapy. Relapse infection may be due to the fact that the parasites that localize in the cryptic site evade the trypanocidal effect of the drug (diminazene aceturate) mainly due o its large molecular size which prevents it from crossing the blood brain barrier. This limits its effectiveness, accounting for the numerous reported cases of relapse after chemotherapy with the drug. Therefore certain reversible breakdown of the blood brain barrier might have therapeutic use in permitting greater entry of the drug into the brain. In this regard, hyperosmolar solutions like lithium chloride and sucrose have been used for the osmotic opening of the blood brain barrier (Allison et al., 1976; Spatz et al., 1976). As a strategy to combat the pharmacokinetic problem, a study on the effect of increasing the permeability of blood brain barrier (BBB) to diminazene aceturate using hyperosmolar agents (lithium chloride and sucrose) was carried out in rats. Materials and methods Experimental animals: Twenty eight adult albino rats (Wistar Breed) of both sexes (males and females) and average weight (120g) were used in the experiment. They were obtained from the Nigeria Institute for Trypanosomiasis Research (NITR) Vom Plateau State, Nigeria. The rats were fed poultry chick mash (Bendel feed and flour Mill LTD Ewu. Nigeria) and water was provided ad-libitum. They were maintained in a tsetse fly proof house. Dypanosomes: Trypanosoma brucei brucei (strain Ghoko OTITOINITR) also obtained from NITR was used for the infection of the experimental rats. Trop. Med. Pariisitol. 46 (1995) O Georg Thieme Verlag Stuttgart. New York

4 100 Trop. Med. Parasitol. 46 (1995) - I. E. Odika, I. U. Asuzu, S. M. Anika Days O 1 t 2 0 Fig. 1 Mean PCV (%) values of infected animals before and after treatment. Treatment?(bold) ; Relapse?: Diminazene aceturate; A Diminazene aceturate + LiCI; 0 Diminazene aceturate + LICI + dexamethasone; lnfected untreated control. (i) (ii) Drugs and Ctwmicals: Diminazene aceturate ereni nil^, Hochst Farbwerke AC, Frankfurt. Germany) was used at a dosage of 7 rng/kg - - body - weight administcrcd intran~uscularly (im). Lithium chloride (LiCl)(BDH Chemicals LTD Poole, England) was used at a dosage of 10 bg/kg im. Experimenlalgroups: 'ltventy eight rats used in the experiment were inoculated with approximately 1.25 x 106 7: b. brr~cei (strain Ghoko OT/70/NITR, in I ml of phosphate buffer glucose saline) intraperitoneally (ip). When parasitaemia was established the rats were divided into 4 treatment groups of 7 rats each. Croup QI Qz Q3 Q4 Drug treatment Treated with diminazene aceturate alone 7 mg/kg, i.m. Treated with diminazene aceturate (7 mg/kg) + Lithium chloride (10 pg/kg), i.m. Treated with diminazene aceturate 7 mg/ kg + Lithium chloride 10 yg/ kg plus dexamethasone (I mg/ kg) ism. Infected, untreated control. Treatment was initated in all the groups, except the control (Q4), fourteen days post inoculation when infection was well established on assessment of parasitaemia by "wet rapid matchingn method of Lumsden and Herbert (1976) and Buffy coat technique (Murray et al., 1977). The dexamethasone added was to prevent inflammatory reactions usually associated with either diminazene accturate and lithium chloride therapy or extravasation of the parasites in the brain. At the end of the experimental period, rats from diminnzene aceturate, diminazene aceturate and lithium chloride, diminazene aceturate and lithium chloride plus dexamethasone and control groups were sacrificed and their brains were collected for histopathological studies. Clinical signs, packed cell volume (PVC), relapse parasitaemia and rectal temperature were monitored, in the course of the expcrimcnts. Days after treatment Fig. 2 Cumulatrve relapses (%) of 7: b. brucei infection in rats. Diminazene aceturate; A Diminazene aceturate + LiCI; + Diminazene aceturate + LC1 + Dexamethasone: Infected controls. Results Group QZ: Infected rats treated with diminazene aceturate alone The infected rats became positive for trypanosomes 4 days after inoculation and showed the following clinical signs: rough hair coat, decreased packed cell volume (PCV), anorexia, emaciation and pyrexia (which was intermittent). Twentv four hours after the in- Jection of berenil at 7 mg/kg i&, the clinical signs were reversed until relapse infection occurred. The effects of diminazene aceturate treatment on 7: b. brucei infected rats are shown in Figs. 1 and 2. Trypanosomes were not detectable in the blood of the infected rats 24 hours after diininazene aceturate treatment. The PCV increased steadily with the commencement of treatment. Relapse parasitaemia occurred after 10 days of aparasitaemia following treatment with diminazene aceturate, but the PCV declined progressively from day 19 post treatment. N- though there was observable fall in mean rectal temperature, there was no marked difference from that of control. Between the 10th and 40th day of treatment relapse parasitaemia occurred in 100% of the rats treated with diminazene aceturate. The cerebral alterations in the diminazene treated rats after 40 days were those of cellular infiltration of a few mononuclear cells. In the cerebral cortex there was no major histopathological lesion except perivascular oedema. Group Q2: Infected rats treated with diminazene aceturate and lithium chloride The clinical signs of infection like in Group Q1 disappeared after 24 hours following treatment with diminazene aceturate (7 mg/kg) and lithium chloride (10 yg/kg), im. The effects of diminazene aceturate and lithium chloride treatment are shown in Figs. 1 and 2. Trypanosomes cleared from the blood stream of the infected rats after 24 hours of treatment. The PCV increased

5 Chemotherapeutic ejj%cacy ofdiminazene and lithium chloride Trop. Med. Para.sito1. 46 (1 995) rapidly and steadily bul declined after relapse infection occurred. Relapse parasitaemia did not occur in this group until 20 clays after treatment (Fig. 2). By 60th day of treatment relapse parasitaemia occurred in 4 out of the 7 rats representing 57.1 percent as against 100 percent in diminazene aceturate treated-rats. The mean rectal temperature following treatment did not show much difference from LhaL of control and diminazene aceturate treated groups. The histopathological changes observed in diminazene aceturate and lithium chloride treated group, after 60 days, included presence of scanty macrophages and perivascular oedema. Group Qj: Injected rats trealed with diminaz6ne crcelurale and lithium chloride plus dexn~nethasone Figs. 1 and 2 show the effects of diminazene aceturate and lithium chloride plus dexamethasone in Z 6. 6rucei infected rats. The clinical signs like rough hair coat, dullness, decreased packed cell volume, (PCV) and pyrexia observed prior to treatment with diminazene aceturate (7 mg/kg) and lithium chloride (10pg/ kg) plus dexamethasone (1 mg/kg) did not clear until 72 hours after treatment. Similarly trypanosomes did not clear from the blood stream of the rats until after 72 hours of treatment. The packed cell volume like in other groups increased progressively following treatment. However the PCV declined (Pig. 1) after the onset of relapse, 19 days post treatment. Relapse parasitaemia occurred 12 days after treatment, and between the 12th and 50th day of treatment it occurred in 6 out of the 7 rats representing 86 percent. Cerebral alterations were not observed in any of the sections of the brain. 50 days post treatment. Group Q4: Infecled, untreated control The clinical signs earlier observed in the 7: b. Brucei infected rats persisted in this group. The rats showed progressive parasitaemia, fall in PCV and pyrexia which resulted in early death (Figs. 1 and 2). The meningeal area of the brain showed typical signs of meningoencephalitis after 18 days of infection. The following cerebral alterations were observed; cellular infiltration of mononuclear cells, perivascular cuffing~, perivascular congestion and oedema. Discussion Four days after the experimental rats were infected with 'l: b. brucei organisms they showed a gradual loss of condition which include rough hair coat, anaemia and lethargy which are similar to those reported earlier in acute cases of 'l: 6. brucei infection in dogs and mice (Losos and Ikede, 1972; Anika et al., 1987). When the infection was treated with diminazene aceturate alone, diminazene aceturate with LiCI, and a combination of diminazene aceturate, LiCl and Dexamethasone, the parasite cleared from the blood-stream (aparasitaemia) in all the treated groups before relapse occurred at varying lengths of time depending on the drug or drug combination used for treatment. The treatment with diminazene aceturate and LiCl seemed to be more effective when compared to the groups treated with diminazene aceturate alone or combination of diminazene aceturate and LiCl plus dexamethasone. 7: b. brucei were cleared from the blood of rats treated with diminazene aceturate alone, and diminazene aceturate and LiCl after 24 hours, whereas clearance of parasitaemia did not occur until after 72 hours in rats treated with diminazene aceutrate and LiCl plus dexamethasone. This could be associated with the anti-inflammatoiy effect of dexamethasone which could induce immunosuppression in the rats. This agrees with the work of Anika et al. (1987) which showed that dexamethasone has a catalytic effect on the course of infection in animals inoculatedwith this strain of trypanosome. Relapse parasitaemia was observed in the infected and diminazene aceturate treated-rats on day 10 following treatment. While it did not occur until day 12 in the diminazene aceturate and LiCl plus dexamethasone treated-groups and day 20 in the diminazene aceturate and LiCl group respectively (Pig. 2). By day 34, relapse had occurred in all the 7 rats in the diminazene aceturate treated group, 4 out of 7 in the diminazene aceturate and LiCl treated group and G out of 7 rats in the diminazene aceturate and LiCl plus dexamethasone treated-rats. Infact 3 rats in the diminazene aceturate and LiCl treated group remained aparasitaemic and without any clinical signs of infection for more than 60 days post treatment, representing 42.8 percent survival. The delay of relapse parasitaemia observed in the diminazene aceturate and LiCl treated group may be due to the effective distribution of diminazene aceturate in the various organs and increased concentration of diminazene aceturate in the brain caused by LiCl (Odika, 1993). In all the treated groups the packed cell volume (PCV) of the infected rats improved in contrast to the PCV of the infected, untreated control which continued to fall until all the rats died (Fig. 1). The mean rectal temperature before and after treatment did not show any marked difference from that of control. In the present study, the histopathological changes such as perivascular cuffings, cellular infiltration of mononuclear cells, and perivascular oedema and congestion observed in control group (Q4) were similar to those described earlier (Chirinwami et al., 1988). The low level of cellular infiltration and histo~athologicalesions demonstrated in the diminazene acet"rate an: LiCl group compared to the control (untreated) group was due to the effect of increased therapeutic concentration of diminazene aceturate in the brain which prevented cerebral trypanosomiasis and the associated histopathology. The absence of microscopic cerebral alterations in the dimin-

6 Pop. Med. Parasitol. 46 (1995) - I. E. Odika. I. U. Asuzu. S. M. Anika azene aceturate and LiCl plus dexamethasone treated group appeared to be attributed to the dexamethasone which prevented inflammatory reaction due to either extravasation of the trypanosome or presence of diminazene aceturate or LiCl in the brain. In conclusion the combination of diminazene aceturate (7 mg/kg) and LiCl (10pg/kg) did not completely prevent relapse in 'I: 6. brucei infected rats but improved the therapeutic efficacy of diminazene aceturate by significantly delaying the incidence and occurrence of relapse parasitaemia. The present study shows that the increase in brain concentration of diminazene aceturate by LiCl enhanced the chemotherapeutic efficacy of diminazene aceturate in cerebral trypanosomiasis, a phenomenon that will require further investigation. Acknowledgement The study was supported by the EEC Lome 111 Project for control of Animal trypanosomiasis in Nigeria. References Allison, J. H., M. E. Blisner: Increased brain myo-inositol-lphosphate in lithium treated rats. Biochem. Biophy. Res. Commun. 68 (1976) Anika, S. M., S. N. Shetty, I. U. Asrrzu, A. B. Chime: Effects of some trypanocides and anti-inflammatory agents in experimental Tnypanosoma brucei infection in mice. Zariya Veterinarian 2 (1987) 9-15 Chirinwami. B., E. A. E. Van March, J. M. Bruche~; I? ~:IMulumba, M. Wery, I? L. J. Gigase: Light microscopic ncuropathology of long term experimental 7'rypanosoma brucei gambiense infection in rats. hi. Soc. Med. Trop. 68 (1988) Fairclough, R.: A comparison of metamidium, samorin, bereni! and ethidium under field condition in Kenya. Veterinary Record 75 (1963) Ifawking, E: Drug resistance of i7ypanosoma congolense and other trypanosomes to quinapyramine, phenanthridines, berenil and other compound in mice. Ann. Trop. Med. Parasit. 57 (1963) Losos, G. J., S. 0. Ikede: Review of Pathology of diseases in domestic and laboratory animals cased by nypanosoma congolense, 7: vivax, 7: brucei, 7: rhodesiense and 7: gambiense. Veterinary Pathology 9 (1972) 1-71 Lumsden. W H. R., W J. Herbert, G. J. C. Hardy: In vivo prophylactic activity of berenil against trypanosomiasis in mice. Veterinary Record 77 (1976) Murray, M.. I? K. Murray. W I. M Mclntyre: An improved Parasitological technique for the diagnosis of African trypanosomiasis. Trans. Roy. Soc. Trop. Med. Hyg. 71 (1977) Odika, I. E.: The effects of increasing the brain concentration of diminazene aceturate in relapse infection of trypanosomiasis in rats. M.Sc. Thesis. University of Nigeria, Nsukka (1993) Spatz, M., A. M. Rap, S. I. Rapoport, I. Klatzo: Effect of hypertonic solutions and of HgC12 on the uptake of '4~-glucose analogue by rabbit brain. In: Rapoport. S. I. (ed.): Blood- Brain-Barrier in Physiology and Medicine. Raven Press. New York (1976), pp Verma, B. B., 0. I? Gautam, I? D. Malik: Diminazene aceturate in the treatment of experimental Dypanosoma evansi infection in buffalo calves. Veterinary Record 93 (1973) Wilson. S. G.: Animal trypanosomiasis in Northern Nigeria. In: Symposium on Animal Trypanosomiasis, Luanda CCTA publication No. 45 (1958) Wilson, A. J., J. G. Roux, J. Paris. C. R. Davidson, A. R. Gray: Observations on a herd of beef cattle maintained in a tse-tse area. I: Assessment of chemotherapy as a method for the control of trypanosomiasis. Trop. Animal Hlth Production 7 (1975) Dr. I. U. Asuzu Department of Veterinary. Physiology and PharmacoIogy ~ ~niversit~ of Nigeria PMB 011. Nsucca Nigeria