Microsoft word - who4-bourdichon-dr. kayer fu berlin diminazene gegen theil.
Development of a new drug carrier system for the use of the trypanocidal drug combination "Trypan" for the treatment of Malaria and African Trypanosomiasis in humans based on the nanosuspension technology A.J., Bourdichon Department Of Research and Development of Pharmaceutical Drugs Schinkelstr.13 22303 Hamburg N.W.N. Maina Kenya Trypanosomiasis Research Institute, Muguga PO.Box 363, Kikuyu, Kenya N. Mwinuka Ministry of Agriculture, Livestock Department PO.Box 1247, Songea-Ruvuma, Tanzania S.J. Akbar Dubai Camel Hospital PO.Box 9220, Dubai, United Arab Emirates Z. Xichen Department of Parasitic Diseases, Changchun University of Agriculture and Animal Science, China H. Uemura Institute of Tropical Medecine, Nagasaki University, Japan M. Fazil Animal Hospital Mombasa O. Kayser R.H. Müller Freie Universität Berlin Institut für Pharmazie Kelchstraße 31 12169 Berlin Introduction It is well known that developing an effective drug for chemotherapy of Protozoan Infections and particulary effective against the Human African trypanosomiasis, is a difficult task. The same active substances have been used for the past 30 years. Immunity against diminazene and other trypanocidal substances have recently registered growing frequency in Western and Eastern Africa. During the last few years we have developed a trypanocidal drug with prolonged effectiveness, which is still active after 90 days. Ideally, the new drug should possess the capacity of curing sleeping-sickness and even malaria in humans. Physical chemistry and combination regimens
In the formulation of TRYPAN has been combined, Diminazene-di-aceturate (diamidinophenyltriazene diaceturate tetrahydrate), Phenazone and Procaine. Diminazene is an aromatic diamidine dirived from Surfen C (Jensch.1958). The molecule is market as the diaceturate salt and consists of two amidinophenyl moieties linked by a triazene bridge, p,p-diamidinodiazo-aminobenzene diaceturate tetrahydrate. The trypanocidal drug „TRYPAN“ is patented in Germany and registered in Europe and in several countries in Africa. Original use
TRYPAN has a painless, antipyretic and long-lasting effect. According to the results of the several tests which we recently received from Tanzania, Ethiopia, United Arab Emirates (UAE) , Kenya (treatment of T.evansi in camels), Benin, Central African Republic, the TRYPAN has been judged as beeing the most effective Trypanocidal drug against infections with Tryp. congolense,Tryp.vivax,Tryp.brucei, Tryp.evansi, Piroplasmosis (Piroplasma motasi and cabali) or Theileria annulata and develop a protective action against Babesia . Moreover, in vitro and in vivo tests in mice have demonstrated the effectiveness of Trypan against two strains of the human malaria parasite P.falciparum which is resistant to chloroquine and against Leishmania donovani. Therapeutic activity of TRYPAN against Trypanosomiasis in domestic livestock Ndokouif, F., and Ndoma, F., from the Animal Health Authorities in the Central African Republic (1994) have demonstrated (A) the preventive chemotherapeutical effect of Isometamidium Chloride and TRYPAN over 110 days; and (B) trypanocidal infections on the cattle treated with TRYPAN were not detected during the three last parasitological controls. The tests have demonstrated the effectiveness of TRYPAN against Babesia. The tests were carried out on Zebu-Mbororos-cattle, they are porters of several kinds of Trypanosoma. The animals were reared on a farm situated in humid bush savannah area, where several kinds of hematophac flies breed enormously : Glosina fusca, Glosina fuscipes and - Tabanide. The obvious heaviness of the flies in the area was about 60 flies per day and drap. This means a certain risk in view of special sorts of Trypanosoma and Babesia. A blood test of a capillary of an ear was catched into two-micro test tubes prepared with Heparin and afterwards sealed with Plasticin. The blood tests made in duplicate had served to determine the result of centrifuged sediment and the sorts of the Trypanosoma by following the methode of Max Murray. Distribution of blood had been made to prove the hemoparasites with GIEMSA-colour methode. Twenty head of cattle chosen as porters of Trypanosoma were devided into 4 groups: - Group Berenil, - Group Veriben, - Group Isometamidium Chloride and - Group Trypan. The preventive- chemotherapeutical effect of the products has been controlled every month during 110 days hematoscopies with the treated animals. The results show that TYPAN has unlike Isometamidium Chloride proved to be more effective in the chemoprophylasis in the Central African republic, longer than the prologing effectiveness of both products, TRYPAN at unique dose (3.5 mg/kg B.W.) controlled with a healing rate of 60% within hrs and a healing rate of 100 % after 12 hrs. It´s effectiveness against Babesia in comparison to other diminazene is quite strong (Report on the Use of Trypanocidal Drug „TRYPAN“, J.Protozool.Res., 8, 258-262 (1998).
H., Assogba, from the PHARNAVET / ADIVET (National Veterinary Pharmacy) in Benin, (1995) , has demonstrated that the effectiveness of all trypanocidal products based on Diminazene and TRYPAN is similar after 2 and 3 days but the reinfestation comes back after 7 to 10 days. TRYPAN unlike other products, the reinfestation comes back later after 80 days, days with a rate of 25 %. TESTS Material and Methods: This research was carried out with Zebu-Mbororos-cattle on a farm during 110 days and the preventive-chemotherapeutical effect of the product has been controlled after 7 days, 10 days, 30 days and every month. Seventy (70) cattle chosen as porters of Trypanosoma were devided in 5 groups: Unlike the others Diminazene,with TRYPAN the reinfestation came back after 80 days with a rate of 25% (Report on the Use of the Trypanocidal Drug „TRYPAN“, J.Protozool.Res., 8, 258-262 (1998). F., Fagbohoun,T., Yokossi, Abdoulay Bo Bata, Rabou Seko, I., Xaouagou, (Laboratory for Veterinary Diagnostics from Bihicon/Benin), have demonstrated the preventive and curative chemotherapeutical effects of TRYPAN and that it´s broad activity permits with only one administration to eliminate all T.vivax and T.congolense with a rate of 100% after 24 hrs. (J.Protozool.Res., 8, 258-262 (1998). Akbar, S.J.*. Munawar,G.* , Ul-Haq,A* , Khan, S.M.** , Khan, M.A.** (1998) demonstrated trypanocidal activity in serum from camels treated with Trypan at a dose of 3.5 mg/kg b.w.TRYPAN was given to 3 infected camels, animals showed recovery based on the clinical and buffy-coat examination. No side-effects were observed in the treated camels.After 2 1/2 months one camel relapsed. This study was conducted to assess the efficacy of three trypanocidal drugs, viz. Melarosamine, Diminazene and Quinapyramine, in the treatment of experimental Trypanosoma evansi infection. A total of 16 animals of both sexes, aged about two years were divided into four groups. Each group comprised four camels. One group was kept as negative control while the remaining three groups were inoculated with T.evansi subcutaneously using dose of 0.02 ml containing 5 million Trypanosomes. The mean Packed Cell Volume (PCV) of infected animals dropped from 29±3 to 21,35±6.5 before treatment. The mean value of neutrophils, eoinophils, basophils, lymphocytes and manocytes were 32.7%, 1.80%, 1.06%, 62.4% and 1.30%, respectively. Value of mean total protein was 8.92g/dl while Albumin and Globulin were 2.62 g/dl and 6.20 g/dl respectively. Within group I, II and III one camel was kept as infected non-treated control. Cure rates of 66.66% for Diminazene (TRYPAN) and Melarosamine and 3.3% for Quinapyramine were recorded. (J.Protozool.Res., 8,249-252 (1998). Petrovsbili and Khamev (1977) on a group of 25 camels infected with trypanosomiasis found that 5 mg/kg i.m. Diminazene was effective in eliminating the infection and caused no adverse effect. Similar observations have been recorded by Raisinghani and Lotha (1980).Le ngoc, My., (1999) has demonstrated that a single dose of 3.5 mg/kg b.w., TRYPAN cure T. evansi infected buffaloes and it did cause side effects. Zhang Xichen, (1999), has demonstrated the effectiveness of TRYPAN against T.sergenti . Ten cows infected with Theileria sergenti were treated with the combination of TRYPAN at the dosage of 3.5 mg/kg B.W. or 15 ml/300 kg B.W. Nine of ten cows recovered four days after treatment. However, only five of ten cows infected with
Theileria sergenti and treated with free Diminazene at the dosage of 7 mg/kg B.W. were recovered four days after the treatment. N., Mwinuka (1999), has demonstrated that cattle infected with E.C.F and treated with the combination of TRYPAN and OTC (Oxytetracycline 20%) were 100 % cured after 2-3days and after 3-4 months they were not reinfected. The same results is obtained with TRYPAN against T.congolense. In late stage infected animals recovered after 2-4 days and 4 months after the treatment the treated animals were not reinfected. Z., Xichen (1999), has demonstrated that TRYPAN and Liposomal Diminazene is more effective that unencapsulated Diminazene against T.b.evansi in mice.Three hundred week-old mice, weighing 22-24 g were randomly divided into six groups of 50 mice each. Group 1, 2, 3, were pretreated with 0.2 ml saline solution , 0.2 ml diminazene solution (5mg/ml ) and 0.2 ml liposomal diminazene suspension (5 mg diminazene per ml). The experiment was carried out 3 different doses that are 79.5, 68.2 and 56.8 mg/kg b.w. for Diminazene Liposomes and 159 mg/kg b.w., 95 mg/kg b.w., and 31 mg/kg b.w. All injections were given intraperitoneally. The result showed that only the group of mice treated with diminazene liposomes of the dose of 69.2 mg/kg b.w. can prevent the challenging with 10.000 T.b. evansi . All mice treated with diminazene liposomes of the dose of 69.2 mg/kg b.w.were alive and all mice treated with unencapsulated diminazene died within 5 days. The results demonstrated that, when encapsuled within liposomes, diminazene liposomes was more effective that unencapsulated diminazene against T.b.evansi. Why is T. b.evansi highly susceptible to loposomal diminazene? The mechanism is not clear at present, but several studies have suggested that positively charged liposomes could fixed on the negatively external surface of Trypanosoma organisms. Others experiments are doing so as to demonstrate the effectiveness against other protozoa with the encapsulated diminazene combination of the drug „TRYPAN“ with Liposomes and Solid Lipid Nanosuspension. Therapeutic activity against sleeping sickneess in humans
Diminazene acturate is currently marketed under the trade names Azidine* , Berenil*, Ganaseg*, Veriben*, as both a trypanocide and babesiacide for domestic livestock. For all animals the general i.m.dose is 3.5 mg/kg b.w. The causing agents of Gambian and Rhodesian sleeping sickness are T.b.gambiense and T.rhodesiense, respectively. Although Diminazene diaceturate is not produced for the application to man it has already been often administered as medication for advanced stages of T. b. rhodesiense and T. b.gambiense infections (Hutchinson and Watson, 1962) and T.b.rhodesiense (Abaru and Motovu, 1981) . When administered to people, the compound is generally given as a deep i.m. injection using a 2% (w/v) solution in sterile 5% (w.v.) glucose. The regimen used is either 7 daily injections of the drug at a dose of 2 mg/kg b.w. , or 3 doses of 5 mg/kg b.w. given at one or more days interval . In vitro testing of Trypan against malaria P.falciparum Fazil, M.A., (1998), has reported that Diminazene is also used (illegally) by nomadic tribes in Kenya. However, No side-effects were observed . Tuner, P. , Würzburg University, (1998), Haruki Uemura , Nagasaki University, (1998) and Kaminsky, R. , Swiss Trop.Institute (1999) have demonstrated the effectivinesse of the combination of TRYPAN in vitro tests against P.falciparum.
The in vitro testing of TRYPAN against two strains of the human malaria parasite Plasmodium falciparum HB3 and DD2 which is resistant to chloroquine demonstrated that the lowest concentration used was 60 ng/ml and in both strains tested this concentration was extremely effective 5-6 time more effective as Chloroquine at killing the parasites (or inhibiting their growth) (Turner, P., 1998). Haruki Uemura , Nagasaki University, (1998) has demonstrated that TRYPAN is effective at the concentration of higher than 1.57 µg/ml, but below 157 ng/ml. The tests have shown more effectiviness; IC50S are around 5 ng/ml for chloroquine sensitive strain HB3 and 50 ng/ml for chloroquine Dd2. For experiment they dissolved all the contents in the bag 2.36 g in 15 ml of steriled water. It is used as a stock solution of 0.157 g/ml (157 mg/ml). They prepared 100 times dilution to the concentration of 1.57 g/l. From this solution, the series of 10 times dilutions were tested for checking effect of the parasites growth. They used FCR strain of P.falciparum and started at 0,3% parasitemia and 20% of hematocrit. They changed every 24 hrs and checked parasitemia also (Haruki Uemura, 1998). Kaminsky, R. , Swiss Tropical Institute, (1999) has demonstrated that TRYPAN is effective against P. falciparum, T. b. rhodesiense and T. b. brucei at the concentration of 7.75 ng/ml. In vitro testing against Leishmania donovani
Gicheru, M., (1999) has demonstrated the effectiveness of the combination of TRYPAN against Leishmania donovani. Three series of Leishmania donovani growth inhibition were tested in vitro . Each test started with 2 Million promastigotes per ml in a total of 5 mls. Two concentration of TRYPAN (3.5ug/ml and 7ug/ml) were included in the culture. In the controll culture no drug was included (0 ug/ml) . Schneiders drosopthilla insect media with 15% Foetal bovine serum was the growth medium. The results results indicates that the two concentrations (3.5 µg/ml and 7 µg/ml) of the TRYPAN tested were inhibitory to the growth of Leishmania promastigotes. The parasites were found to be dead between 7 th and 11 th day of the culture. Combination regimens
Many of the trypanocidal compounds used for human trypanosomiasis have a low therapeutic index. Drugs toxicity is therefore often observed. In attempt to reduce drug dose, and therefore the incidence of toxicity, Diminazene has been combined with a variety of compounds to dertermine if this has an additive or synergistic effect (Jennings et al., 1980; Jennings, 1993, Zweygarth and Röttcher, 1987). Lastly combination with procaine and phenazone has been shown to enhance the therapeutic activity of diminazene against T.evansi, T.brucei gambiense, T.vivax, T.congolense and P.falciparum. Since both procaine and phenazone are anti-inflammatory antipyretic and anaesthetic compounds, their co-administration with diminazene may increase the bioavailability of the trypanocide. Toxicity and residues
Treatment of livestock with standart therapeutic doses diminazene aceturate (3.5 - 7.0 mg/kg b.w.) rarely results in signs of toxicity. Furthermore, since diminazene´s therapeutic index in most animal species is relatively large, cattle, for instance, can tolerate i.m. doses as high as 21.0 mg/kg b.w. without exhibiting signs of systemic toxicity (Faireclough, 1963).
It is known that applying Diminazene to camels or dogs can be toxic to the animal. This Knowledge, however, is based upon high-dosed injection of 7.5 mg to 20.0 mg/kg b.w. and more. Diminazene is also relatively toxic in dogs. Experimentally, i.m. administration of doses in excess of 10.0 mg/kg b.w., once or repeatedly, results in severe signs of distrubance in the gastrointestinal tract, respiratory, musculoskeletal and nervous systems in both camels (Homeida et al., 1981) and dogs (Losos and Crokett, 1969). Diminazene is succeffully applied to treat camels in India with a dosage of 1.25 mg/kg b.w. and 5 mg/kg b.w., curing infections caused by T.evansi (Raisinghani and Lodha, 1980). Diminazene is extensivily distributed in the body of treated animals. Residues of the compound may persist for several weeks, principally in the liver and kidneys,and also, to a lesser extent. in gastrointestinal tract, lungs, muscle, brain and fat (Gilbert.1983; Kellner et al.,1985; Murilla and Kratzer,1989; Oneyeyili and Anika,1991). After treating lacting goats intravenously with 2 mg diminazene base/kg b.w. The maximum concentration of diminazene in milk (1.68µg/ml) was detected at 4 h (Aliu et al., 1984). A milk to plasma ratio of approximately 0.45 was maintened at equilibrum, Trace amounts of diminazene (0.05 µg/ml) were present for up to 72 h following treament.The ADI for Diminazene is established at 0-100 µg/kg b.w. MRLs in cattle are: muscle 500 µg/kg; liver 12,000 µg/kg; kidney 6000 µg/kg; fat - no MRL allocated; milk 150 µg/l (WHO,1995). Although diminazene has not been formally evaluated for its toxicity in man (Apted, 1980) . However, in 17 cases reported by Hutchinson and Watson (1962), no local toxicity was observed. Naomi, Maina., 1999) has demonstrated in a preliminary report the efficacy and toxicity of Trypan in five infected camels. No toxic effects were observed in the treated camels. Moreover , the in vivo test in mice has demonstrated that Trypan is slightly less toxic than the other diminazene formulation. Diminazene is also used (illegally) by the nomadic tribes in Kenya for the treatment of malaria with any side effect. They recover from these symptoms in 24 hours (Fazil, M.A., 1998). Yoshimura, H.,(Toxicol Lett 1999 Nov; 54(1):55-9), has demonstrated that diminazene diaceturate is not teratogenic in rats. Diminazene diaceturate was dissolved in deionized water and administered to pregnant rats by oral gavage once daily on days 8-15 of pregnancy at dose levels of 0, 100, 250, 500 and 1000 mg/kg. On day 21 of pregnancy, the dams were killed and the number of implants, resorptions and live fetuses counted. All fetuses were examined by routine teratological method. A significant increase in fetal resorption and decrease in fetal body weights were found at the 1000 mg/kg dose. No significant increase in the incidence of anomalous fetuses was observed in external, skeletal and internal examinations even at the maternally toxic dose. Trypano-resistance
Another problem we are also concerned with is the problem of trypano-resistance. Trypano-resistance has been documented over the last 30 years. A report by Njogu, Dolan, A.R., Wilson, B., and Sayer, P.D. 1985, report of trypano-resistance in Boran cattle, and in 1987 by Reiter, Büttner, I. M. and Seitz, A., or the report by Zhang, Giroud, C. 1992 and Balltz on the in vitro sensitivity of Trypanosoma evansi. Report by Andrew, S.,.Peregrine, Maureen, A.Gray and Shamshudeen, K.Molloo. Nicholaus T.
Mwinuka, Trypano-resistance in Tanzania (1998) in vivo sensitivity of T.congolense (ADRI).
Why do we need a new drug carrier system?
Colloidal drug carriers such as liposomes and biodegradable nanoparticles are easily taken up by phagocytic cells and accumulate in the organs of the reticuloendothelial system. Therefore, they hold promise as carriers for the treatment of certain intracellular infections with antibiotics that would normally not find easy access to intracellular sites. Both drug carriers are efficiently taken up by macrophages and in in vitro and in vivo models both proved helpful in the treatment of intracellular pathogens like Leishmania, Candida, Mycobacterium and Listeria. However, despite enormous research in the last twenty years in this field, no commercial pharmaceutical liposome product is on the market, with the exception of Ambisome. The main reasons are low physical stability, low shelf life time, insufficient oral administration, traces of toxic monomers, low serum stability, reduced circulation half-times, and high costs in scaling up production.
Despite these faults, the idea of colloidal drug carriers - targeting for uptake by endocytic processes - remains a valuable idea for drugs against intracellular pathogens. Accordingly, we developed related drug delivery systems: nanosuspensions and solid lipid nanoparticles (SLN). To validate their potential value, we incorporated well known reference agents like bupravaquone, atovaquone and amphotericin B in different solid lipid formulations and prepared well defined nanosuspensions. All these drug formulations were incubated with L. donovani-infected murine macrophage cultures and compared with the respective drug activities in aqueous solution. Viability of host cells and intracellular survival of parasites were determined by the respective MTT metabolism (Kiderlen and Kaye, 1990).
Results: All drugs showed improved antiparasitic activities for the formulated drug in comparison with the free drug and no significant host toxicity. For example, the EC50 values for amphotericin B were reduced more than 10 fold when given as nanosuspension (EC50 = 0.25 µg/ml vs. EC50Nano = 0.019 µg/ml). Light microscopy demonstrated strong uptake of the colloidal drug formulations by macrophages. Drugs incorporated in SLN were also more effective than aqueous solutions: Atovaquone activity was increased from EC50 = 0.51 µg/ml to EC50Nano = 0.24 µg/ml, buparvaquone from EC50 = 0.78 µg/ml to EC50Nano = 0.098 µg/ml, and amphotericin B from EC50 = 0.25 µg/ml to EC50Nano = 0.11 µg/ml. Conclusions: Both nanosuspensions and SLN as drug delivery systems are of high interest for the formulation of antiparasitic drugs against intracellular pathogens. In comparison to liposomes we have an improved system that is easy and cost-effective to produce and simple to handle. Both carriers show improved physical and chemical stability, and in vivo studies suggested enhanced bioavailability. Nanosuspensions also appear attractive for absorption of poorly soluble drugs (e.g. atovaquone) from the gastro-intestinal tract (GIT). As a general feature, nanoparticles tend to stick to the intestinal wall. This adhesivenes should increase absorption and dissolution time in the GIT, thereby enhancing uptake and bioavailability. These factors may also prove advantageous for drug delivery against parasites residing in the gut lumen or in intestinal cells (e.g. Cryptosporidium, helminths).
How to produce nanosuspensions ?
The Research Groups under supervision of Prof. Dr. R. H. Müller and Dr. Kayser at the Free University are presently engaged in an ongoing search for suitable drugs and new innovative drug formulations which can combat this disease. Development of a nanosuspension for the formulation of the poorly soluble drug carrier system The drug powder of the diminazene base is dispersed in a surfactant solution by a high speed stirrer. The starting size of the drug powder should preferentially be as small as possible, that means the drug should be jet milled. If jet milled drug quality is not available, a special pressure program can be run on the homogeniser to achieve a first size reduction of the coarse powder. Applied pressures range from about 100 bar to a maximum of 1500 bar, in some machines a maximum of 2000 bar can be reached (Rannie 56 and 118). The drug suspension is pressed through a very small gap in a size range of about 25 µm. The diameter of the cylinder, which contains the stock suspension is 3.0 cm. That means there is a reduction in diameter from 3.0 cm to just about 25 µm which leads to a high streaming velocity of the suspension. According to the Bernoulli equation the dynamic pressure increases and simultaneously the static pressure decreases. In the gap the static pressure decreases below the boiling pressure of water, that means the water boils leading to the formation of gas bubbles, which implode when the suspension leaves the gap and normal air pressure is reached again ( = cavitation). The implosion (cavitation) forces are sufficiently high to disintegrate the drug microparticles to nanoparticles. Additional disintegration effects are the high shear forces in the gap and particle collision (similar to jet mill). In many cases it is not sufficient that the suspension passes the homogeniser just once, typically multiple cycles are required. Depending on the hardness of the drug, the desired mean particle size and the required homogeneity of the product 3, 5, or 10 homogenisation cycles are run. Characterisation of stable nanosuspension (particle size, zeta-potential)
The production parameters of nanosuspensions affect the mean particle size of the bulk population and the content of particles in the micrometer range. The two production parameters affecting product quality are the homogenisation pressure and the number of cycles. The mean diameter of the bulk population will be determined by photon correlation spectroscopy (PCS; Zetasizer, Malvern Instruments, UK). PCS yields a mean diameter (Z-Average) and a polydispersity index ranging from 0 for a perfectly monodisperse particle population to 0.500 for a relatively broad size distribution. Emulsions for parenteral nutrition typically possess a polydispersity index between 0.100 to about 0.250. Monodispers polystyrene standard latex particles have a polydispersity index in the range of 0.010 to about 0.050. PCS is limited to the size range of about 3 nm to 3 µm, that means particles above 3 µm are not detected. Therefore for the analysis of microparticles laser diffraction was employed (Coulter LS 230, Coulter Electronics, Germany). Laser diffraction yields a volume distribution. As characterisation parameters diameters 50 %, 90 %, and 99 % are currently used, that means 50 %, 90 %, and 99 % of the particles are below the given size value, respectively. It should be noted, that laser diffraction (LD) data are volume based, the
PCS mean diameter is light intensity weighted size (Z-Average). Therefore the PCS mean diameter and the diameter 50 % from the LD are not identical, LD data are generally higher.
Determination of long term stability (aggregation, particle size, temp, time, microbiology)
All batches are subject of long term investigations to characterise physical and microbiological quality over a period of 6 months. Particle size is determined after storage of reference batches at 4°C, room temperature, and 40°C after 1, 2 ,4 ,8 , 12, and 24 weeks following the procedure as described under 3.2.2. Microbiological contamination is determined by plating out samples on standard agar media (Mueller-Hinton), incubation at 37°C over night and visually check for bacteria growth.
Development of stable and useable hydrogels (Carbophil, PVP, 10 Polymer- Derivatives) Commercial available polymers (Carbophil, PVP, 10 Polymer-Derivatives) will be used to prepare hydrogels with an amount of 0.1, 0.5, and 1.0% (M/M) of the polymer in water. All hydrogels will be characterised for their physical, especially rheological behaviour. Interactions and contraindications will be investigated to validate the formulation as a stable drug delivery device. In the second step, interactions of the final drug formulation (nanosuspension incorporated in the hydrogel) will be determined by visual and technological parameters (e.g. sedimentation, rheology, phase transfers). References : 1. F.Ndokouif F.Ndoma from Animal Health Authorities in Central African Republic.(1994)“ Efficacy of Trypanocidal drugs , Trypan , Berenil, Veriben, Trypamidium, in Central African Republic“ . ( J.Protozool.Res., 258-262(1998). 2. H., Assogba, Pharnavet/ADIVET in Benin. (1995), Curative and Prophylactic effects of Trypanocidal drugs, Trypan, Veriben , Berenil .(J.Protozool.Res.,258-262(1998). 3. Austin., Fagbohoun, Efficacy of Trypanocidal drugs, "Breeding Project Promotion Atacora", Natitingou/ in Benin . (1991). 4. Efficacy of Trypanocidal drugs, Trypan, Cymerlarsan,, Triquin, on experimentally induced Trypanosomiasis in Racing Camels .(1998).Akbar*, S.J., Munawar, G, Ul.Haq, A, Khan, S, M.A., Dubai Camel Hospital, PO.BOX 9220, Dubai. United Arab Emirates.(J.Protozool.Res., 8, 249-252(1998). 5. Curative and Prophylactic effect of TRYPAN in infected cattle. Fazil, M.A., Animal Hospital Mombasa - PO.Box 88432 Mombasa / Kenia.(1998). 6. Clinical Report about the prophylactic effect of TRYPN against T.congolense and the curative and prophylactic effect of TRYPAN in combination with Tetracycline against (E.C.F) T.parva, in infected cattles. Mwinuka, Nicholaus.,T.,. Livestock Department, Songea-Ruvuma / Tanzania.(1998).(J.Protzool.Res., 258-262 (1998). 7. Turner P., Würzburg University,Germany (1998). TRYPAN in vitro tests against P.falciparum . 8. Haruki Uemura, Nagasaki University , Japan. TRYPAN in vitro tests against malaria P.faciparum and in vivo test in malaria berghei mouse model. 9. Walter., R.D. , Bernhard-Nocht-Institut für Tropenmedizin, Hamburg (1998) TRYPAN and BERENIL in vitro tests against malaria P.faciparum. 10. Abatan, M.O.(1991) Combination therapy of trypanosomiasis using diminazene and non-steroidal anti-inflammatory drugs. J.Chemother. 232.235.
11.Anika, S.M. and Onyeyili, P.A. (1989) Effects of trypanosomal infections on the pharmacokinetics of diminazene aceturate in dogs. Trop.Med.Parasitol 40. 419-421. 12. Bailey, N.M. (1968) Oral Berenil in the treatment and prophylaxis of human trypanosomiasis. Trans. R. Soc.Trop.Med. Hyg. 62.122. 13. Hutchinson, M.P. and Watson, H.J.C. (1962) Berenil in the treatment of Trypanosoma Gambiense infection in man. Trans. R.Soc. Trop.Med. Hyg. 56. 227-230. 14. Jennings, F.W. Urquhart, G.M., Murray, P.K., and Miller, B.M., (1980) "Berenil" and nitromidazole combination in the treatment of Trypanosoma brucei infections with central nervous system involvement. Int. J.Parasitol. 10, 10 - 32. 15. Abaru, D.E., Liwo, D., and Okori, E.E. (1984) Restrospective long-term study of effects of Berenil by follow-up of patients treated since 1965. Tropenmed. Parasitol. 35. 148 150. 16. Abaru, D.E.and Matovu, F.S. (1981) Berenil in the early stage human trypanosomiasis cases. In : Proceedings, 17th meeting, International Scientific Concil for Trypanosomiasis Research and Control, Arusha, Tanzania OAU STRC Publication 112 pp. 194 198 18. Le ngoc, My. , (1999)( National Institute of Veterinary Research, Hanoi). Trypan in the treatment against T.evansi in infected buffaloes in Vietnam. 19. Kaminsky, R. , (1999) In vitro assays WHO-screening , Trypan in vitro test against P.faciparum, T.b.rhodesiense, T. cruzi, L.donovani and Berenil vs TRYPAN tested on T.b.brucei STIB 920. 20. A., J., Bourdichon, Report on the Use of the Trypanocidal Drug „TRYPAN“ (J.Protozool.Res., 8,258-262(1998) 21. A., J., Bourdichon, The use of the trypanocidal drug „TRYPAN“ in chemotherapy for Trypanosomiasis in domestic livestock and sleeping sickness in humans and experimentally in vitro tests against malaria P.falciparum . (International Colloquium, Institute of Tropical Medicine, Antwerp, Belgium (1998). 22. Z., Xichen, (1999), TRYPAN and Diminazene Liposomes against T.b.evansi in mice. 23. Kiderlen, A. F. and P. M. Kaye (1990). “A modified colorimetric assay of macrophage activation for intracellular cytotoxicity against Leishmania parasites.” J Immunol Methods 127: 11-8.
24. Yoshimura, H., National Veterinary Assay Laboratory, Tokyo, Japan (Toxicol Lett 1990 Nov; 54(1) : 55-9) 25. N.W.N., Maina , Kenya Trypanosomiasis Research Institute, Muguga, KETRI, Kenya (1999). „ The safety and efficacy of Trypan ® (Diminazene aceturate) in treatment of camel trypanosomiasis“. A preliminary report.
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