TECHNOLOGICAL STUDY OF PREPARING DICLOFENAC GEL / Монголын эм зүй, эм судлал

Introduction: It is required to develop drug formulations and increase local preparations substituting the import preparations. The utilization of a new drug formulation of diclofenac gel is an example of a medicine which is encouraged to be prepared locally. Diclofenac gel is a cyclooxygenase inhibitor, which is a non-steroidal anti-inflammatory drug (NSAID) taken to reduce inflammation and as an analgesic reducing pain in certain conditions. It is important to develop technology and standard for diclofenac gel type of medicine in Mongolia.In 2010, domestic manufacturers produced about 30- 40 percentage of demand in Mongolia. However, there are many manufacturers that produce the same type of medicines. This shows that there is a need for local manufacturers to meet local demand. It is an indication of urgency in bringing in new technologies, new drug formulations, activating drug research and scientific work in domestic drug industry.Based on research, in 2011, non-steroidal anti- inflammatory drugs were the most sold drugs, which had a market share of 29.2%. Also in 2010, non- steroidal anti-inflammatory drugs of gel formulation had a market share of 130 million Tugriks.In 2011, there were nine different manufacturers of diclofenac gel have been registered to Mongolian Medicine Registrar/LICEMED/. They are:-Clafen 1%-20.0 produced in Antibiotice Ltd, Romania-Feloran 1%-60.0 produced in Bаlkanpharma Troyan AD, Poland-Almiral1%-10.0/25.0 produced in Medochemie Ltd, Cyprian-Olfen 1%-20.0 produced in Mepha Ltd, Switerland-Voltaren 1%-20.0/50.0 produced in Novartis pharma,Switzerland-Dicloran Plus 1%-30.0 produced in Unique pharmaceutical laboratories, India-DiclofenacAcri 1%-30.0 produced in Acrihin Ltd, Russia-Diclovit 1%-20.0 produced in Nijpharm, Russia-Diclomol 1%-20.0 produced in Win Medicare Ltd, India.These gels are packaged between 20-60 grams and the are price ranges from 2,700-12,000 Tugriks.In the world, nowadays diclofenac gel is most commonly produced.Diclofenac gel is colorless, and it penetrates the affected area quickly and effectively. The gel has an advantage of delivering full drug concentration to the affected area quickly, while it is convenient and pleasant to use.Purpose of study: The purpose of study’s to develop a Diclofenac gel technology for the first time in Mongolia. In order to reach my purpose, which owe as follows:• To determine convenient ingredients and gel baseof gel formulations• To develop a layout explaining gel manufacturingtechnology• To formulate raw ingredients based on the developed of the gel• To develop an evaluation and quality control systemfor producing gelMaterials and methods: Research work was done at the Monos School of Pharmacy and Drug Research Institute.Triethanolamin and Glycerin from Tsetsuuh LLC, Diclofenac sodium, hydroxypropylmethylcellulose, and menthol from Monos Pharm LLC, Carbomer 940, Ultrez-21 and Propylenglycol from Monos Cosmetic LLC were used for this research.To determine the drug ingredient: To determine gel formulations, primary and secondary ingredients were chosen carefully to maintain chemical compatibility and stability.Developed gel formulation was checked forquality control, which includes drug content, pH,appearance/Homogeneity/, and viscosity by Russian Pharmacopoeia XI and Mongolian Primary Pharmacopoeia. Permeability studies and skin irritation test was performed by USP.Result: First, carbopol concentrations of 0.5%, 1.0%, 1.5% and 1.2%, Ultrez-21 concentrations of 0.5%,0.8%, and 1%, and hydroxypropylmethylcelluloseconcentrations of 3%, 4%, 5% were prepared as gel formulations.Of those, carbopol concentrations of 1.2% and 1.5%, and Ultrez-21 concentrations of 0.8% and 1.0% were relatively better compared to other concentrations in terms of meeting drug content, appearance, pH level, and viscosity requirements. 0.2 gram of Menthol was added to provide comfort and pleasant odor when gel is applied as well as providing release of diclofenac sodiumFinal products were compared to similar diclofenac gels in the market in terms of pH level, drug content, skin irritation, and release of active substance. Of those final products, Ultrez-21 concentration of 1% resulted to be the most similar to the diflofenac gels in the market that satisfy the qualities mentioned above.Conclusion:1. The Ultrez-21 1% is determined to be the best gelling agent and 0.2 gram of Menthol is determined to be the best amount for increasing drug permeability.2. Developed technological procedure for preparingdiclofenac gel.3. To determine criteria for quality control, gel drug content, appearance, pH and viscosity were shown to be the most convenient measures.

Expression of truncated Babesia gibsoni thrombospondin-related adhesive proteins in Escherichia coli and evaluation of their diagnostic potential by enzyme-linked immunosorbent assay.

Among the previously established enzyme-linked immunosorbent assays (ELISAs), an ELISA using the full length of a recombinant thrombospondin-related adhesive protein of Babesia gibsoni (rBgTRAPf) is considered as the most sensitive diagnostic method for the detection of an antibody to B. gibsoni in dogs. However, the expression of rBgTRAPf in high concentration is poor and, thus, limits its usefulness as a diagnostic antigen. To improve its expression level, we have truncated BgTRAPf into two fragments having either an N- or a C-terminus (BgTRAPn or BgTRAPc, respectively). The expression of BgTRAPc protein in Escherichia coli yielded adequate recombinant protein. The specificity and sensitivity of ELISAs with the truncated proteins were determined using dog sera experimentally infected with B. gibsoni and specific pathogen-free (SPF) dog sera. A total of 254 field dog sera were examined by the ELISA with rBgTRAPn, rBgTRAPc, and rBgTRAPf as well as by an indirect fluorescent antibody test (IFAT). The specificity of rBgTRAPc was the highest (97.15%), and its kappa value was more (0.8003) than rBgTRAPn (0.7083). With a sufficient level of expression as well as higher specificity and reliable sensitivity, rBgTRAPc appears to be a potential candidate antigen for the serodiagnosis of B. gibsoni infection in dogs.

A trypanosome species isolated from naturally infected Haemaphysalis hystricis ticks in Kagoshima Prefecture, Japan.

Common arthropod vectors for trypanosomes are flies, fleas and bugs. This study reports on an unknown trypanosome species isolated from naturally infected Haemaphysalis hystricis ticks, hereby, referred to as Trypanosoma KG1 isolate. The parasite has been successfully cultured in vitro with L929 or HEK 293T cell line as feeder cells. This trypanosome cannot survive in vitro without feeder cells. Following experimental infections of ticks, the trypomastigote-like and the epimastigote-like forms of this trypanosome could be detected by Giemsa-stained smears of the midgut and salivary glands of Ornithodoros moubata ticks which were made to feed on a culturing medium containing Trypanosoma KG1 isolate through an artificial membrane. Trypanosoma KG1 isolate could also be detected from Giemsa-stained smears of the haemolymph up to 30 days post-inoculation into the O. moubata haemocoel. Trypanosoma KG1 isolate cannot be propagated in laboratory animals including mice, rats, rabbits and sheep. A phylogenetic tree constructed with the 18S rRNA gene indicates that Trypanosoma KG1 is a member of the stercorarian trypanosomes.

Babesia parasites develop and are transmitted by the non-vector soft tick Ornithodoros moubata (Acari: Argasidae).

Ornithodoros moubata ticks were fed on blood infected with Babesia equi. However, the parasites were quickly cleared as evidenced by the disappearance of B. equi-specific ribosomal RNA from the ticks. We hypothesized that if the Babesia parasite can escape midgut-associated barriers a non-vector tick can become infected with Babesia. To test this hypothesis, B. equi parasite-infected blood from in vitro culture was injected into the haemocoel of ticks. B. equi-specific rRNA was surprisingly detected 45 days after injection even in the eggs. Babesia-free dogs were infested with O. moubata ticks that were infected by inoculation with B. gibsoni-infected red blood cells. Parasitaemia and antibody production against Bg-TRAP of B. gibsoni increased gradually. These results indicate that O. moubata may be a useful vector model for Babesia parasites and also a very important tool for studies on tick immunity against Babesia parasites and tick-Babesia interactions.

Diagnosis of equine piroplasmosis in Brazil by serodiagnostic methods with recombinant antigens.

Serum samples from horses in the States of Sao Paulo and Mato Grosso do Sul, Brazil were examined for diagnosis of equine piroplasmosis by both the latex agglutination test (LAT) and enzyme-linked immunosorbent assay (ELISA) with recombinant antigens. Of the 47 samples analyzed, 38 (81%) and 42 (90%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 35 (75%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread and therefore a cause for serious concern in the States of Sao Paulo and Mato Grosso do Sul, Brazil.

Detection of antibodies to Hypoderma lineatum in cattle by Western blotting with recombinant hypodermin C antigen.

The cDNA encoding the entire mature hypodermin C (HC) of Hypoderma lineatum was cloned and expressed in Escherichia coli as a glutathione S-transferase fusion protein using pGEX vector. The recombinant HC protein (rHC) was tested by Western blotting to detect antibodies to H. lineatum in cattle. Western blotting with rHC as antigen clearly differentiated between H. lineatum-infested cattle sera and normal cattle sera. Forty-six out of forty-eight serum samples from cattle in Central Mongolia were positive, whereas all 30 serum samples from cows in Hokkaido, Japan, were negative by Western blotting. The result of Western blotting was identical to that of a previously developed enzyme-linked immunosorbent assay. These data demonstrated that Western blotting, with rHC expressed in E. coli, might be a useful method for the diagnosis of cattle hypodermosis.

Detection of Babesia caballi and Babesia equi in Dermacentor nuttalli adult ticks.

Ticks play an important role in human and veterinary medicine particularly due to their ability to transmit protozoan pathogens. In this study we have demonstrated that polymerase chain reaction (PCR) and nested PCR methods enabled detection of Babesia caballi and Babesia equi in field isolates of Dermacentor nuttalli adult ticks from Mongolia. Primers specific for 218 bp fragment merozoite antigen 1 (EMA-1) gene of B. equi successfully amplified products from all samples of D. nuttalli adult ticks while primers for the 430 bp fragment product from BC48 gene of B. caballi amplified products from seven of the 54 samples. Using PCR and nested PCR methods we have found mixed infections with B. equi and B. caballi in the tick vector. The amplified DNA fragment from D. nuttalli ticks was inserted into the EcoRV site of pBluescript SK and sequenced. The sequence of the 430 bp fragment was completely identical to the nucleotide sequence of the USDA strain of B. caballi. These results suggest that D. nuttalli may play an important role as a vector of both B. caballi and B. equi and also may be important in maintaining endemicity of equine piroplasmosis in Mongolia.

Molecular evidence of Babesia caballi (Nuttall and Strickland, 1910) parasite transmission from experimentally-infected SCID mice to the ixodid tick, Haemaphysalis longicornis (Neuman, 1901).

Molecular evidence that suggests the possible role of the ixodid tick, Haemaphysalis longicornis and its eggs in the transmission of equine Babesia caballi parasites is presented herein. Using polymerase chain reaction (PCR) to assay for DNA in parasites, presumably acquired by ticks that were allowed to feed on splenectomized-SCID mice, experimentally exposed to in vitro-cultivated B. caballi, we have obtained positive bands that corresponded to the expected B. caballi-specific 430bp gene fragment in 50% of female ticks used, and in 75 and 25% of eggs and larval progeny, respectively. Also, parasite DNA was detected in ticks, eggs and larvae as late as the 16th to the 20th day post-host infestation. Present findings support to the potential role of H. longicornis in the transmission of B. caballi parasites. Its capability, however, to successfully transmit the infection to horses under natural conditions in the field needs to be further ascertained. To our knowledge, this is the first documented study incriminating H. longicornis as a most and likely biological vector of equine babesias.