Microsoft word - who4-bourdichon-dr. kayer fu berlin diminazene gegen theil.

Published http://www.fao.org/paat/Who4-ant.doc

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.

Source: http://www.tropmed.ag/material/who4-trypan.pdf