Chemical and bioassay techniques to authenticate quality of the anti-leishmanial drug miltefosine.

Miltefosine, an effective oral treatment of visceral leishmaniasis (VL), was selected in May 2005, by the governments of India, Nepal, and Bangladesh for the elimination of VL. However, abnormally high treatment failure rates reported in patients in Bangladesh, given a miltefosine generic product ("Miltefos", Popular Pharmaceuticals Ltd.) during 2008, led the World Health Organization (WHO) to procure this formulation for quality testing. Proton ((1)H) and phosphorous ((31)P) nuclear magnetic resonance (NMR) analyses of the Miltefos™ capsules did not give the peaks defined for Impavido®, the quality assured VL treatment product from Aeterna Zentaris. Contents of capsules of Impavido® yielded expected peaks for miltefosine (m/z 408.33 for the protonated parent ion and m/z 183.99 plus m/z 124.8 the fragment ions) that were absent in the Miltefos™ capsules. Furthermore, testing using an in vitro Leishmania donovani intracellular amastigote-macrophage model, yielded EC50 values of between 2.55 and 4.06 µg/mL and 3.02 to 5.92 µg/mL for extracts from the Impavido® capsules and the miltefosine standard, respectively. Lack of significant anti-leishmanial activity of Miltefos™ capsules was identified in this assay even at concentrations up to 100 µg/mL. Capsules of Miltefos™ were classified as falsified (absence of stated active pharmaceutical ingredient) by three methods-NMR and mass spectrometry analysis and bioassay.

Anti-leishmanial activity of plant-derived acridones, flavaglines, and sulfur-containing amides.

Visceral and cutaneous leishmaniases are an important public health problem in endemic geographic regions in 88 countries worldwide, with around 12 million infected people. Treatment options are limited due to toxicity and teratogenicity of the available drugs, response problems in HIV/Leishmania co-infections, and upcoming resistances. In this study, we investigated the anti-leishmanial activity of 13 plant-derived compounds in vitro aiming to find new drug candidates. Toxicity of the compounds was evaluated in human primary hepatocytes, and hemolytic activity was examined in freshly isolated erythrocytes. Two acridones, 5-hydroxynoracronycine and yukocitrine, two flavaglines, aglafoline and rocaglamide, and the sulfur-containing amide methyldambullin showed promising anti-leishmanial activities with 50% effective concentrations (EC50s) of 34.84, 29.76, 7.45, 16.45, and 6.29 µM, respectively. Hepatotoxic activities of 5-hydroxynoracronycine, yukocitrine, and methyldambullin were significantly lower compared to miltefosine and lower or equal compared to artesunate, whereas the ones of rocaglamide and aglafoline were slightly higher compared to miltefosine and significantly higher compared to artesunate. None of the compounds showed hemolytic activity.

Detection, quantifications and pharmacokinetics of toltrazuril sulfone (Ponazuril) in cattle.

Toltrazuril sulfone (Ponazuril) is a triazine-based anti-protozoal agent with highly specific actions against apicomplexan group of organisms, which are undergoing intensive investigation. Toltrazuril sulfone may have clinical application in the treatment of Neospora. caninum and other protozoal infections in cattle. To evaluate absorption, distribution, and elimination characteristics of toltrazuril sulfone in cattle, a sensitive validated quantitative high-pressure liquid chromatography method for toltrazuril sulfone in bovine biological fluids was developed. After a single oral dose of toltrazuril sulfone at 5 mg/kg (as 150 mg/g of Marquis; Bayer HealthCare, Shawnee Mission, KS, USA), samples from six cows showed good plasma concentrations of toltrazuril sulfone, which peaked at 4821 ng/mL +/- 916 (SD) at 48 h postadministration. Thereafter, plasma concentration declined to 1950 ng/mL +/- 184 (SD) at 192 h after administration with an average plasma elimination half-life of approximately 58 h. Following oral dose of toltrazuril sulfone, the observed peak plasma concentrations were in relatively close agreement ranging from the lowest 3925 ng/mL to the highest of 6285 ng/mL with the mean peak plasma concentration being 4821 ng/mL. This study shows that toltrazuril sulfone is relatively well absorbed after oral dose in cattle. These results are therefore entirely consistent with and support the reported clinical efficacy of toltrazuril sulfone in the treatment of experimentally induced clinical cases of N. caninum and other protozoal-mediated bovine diseases.

Speciation of antimony (III) and antimony (V) using hydride generation for meglumine antimoniate pharmaceutical formulations quality control.

The pentavalent antimonies, mainly the meglumine antimoniate, are recommends as first-choice medicines for leishmaniasis therapy. In this work we described the development of formulations of meglumine antimoniate injectable medication, as well as the analytical methodology used in the selective determination of Sb(III) and Sb(Total) by hydride generation - inductively coupled plasma atomic emission spectrometry (HG-ICP-AES) and ICP-AES, respectively. On that purpose the analytical methodology was developed focusing on the HG-ICP-AES technique. The formulations using propylene glycol/water as vehicles in a 20:80 proportion were more appropriate for subsequent use in industrial scale. These formulations also showed a lower variation on Sb(III) percentage, no need of buffer solution to stabilize the formulation and no influence of the autoclaving in the quality of the product. The results of the development of the analytical methodology point out the proposed method as an efficient alternative for the determination of Sb(III) in the presence of large quantities of Sb(V) in injectable solutions of meglumine antimoniate, in a selective, linear, accurate and precise manner. In addition, the method showed a low limit of quantification, less interference of the matrix, and more resilience than batch techniques proposed in the Brazilian Pharmacopeia.

Speciation of antimony (III) and antimony (V) using hydride generation for meglumine antimoniate pharmaceutical formulationsquality control

The pentavalent antimonies, mainly the meglumine antimoniate, are recommends as first-choice medicines for leishmaniasis therapy. In this work we described the development of formulations of meglumine antimoniate injectable medication, as well as the analytical methodology used in the selective determination of Sb(III) and Sb(Total) by hydride generation - inductively coupled plasma atomic emission spectrometry (HG-ICP-AES) and ICP-AES, respectively. On that purpose the analytical methodology was developed focusing on the HG-ICP-AES technique. The formulations using propylene glycol/water as vehicles in a 20:80 proportion were more appropriate for subsequent use in industrial scale. These formulations also showed a lower variation on Sb(III) percentage, no need of buffer solution to stabilize the formulation and no influence of the autoclaving in the quality of the product. The results of the development of the analytical methodology point out the proposed method as an efficient alternative for the determination of Sb(III) in the presence of large quantities of Sb(V) in injectable solutions of meglumine antimoniate, in a selective, linear, accurate and precise manner. In addition, the method showed a low limit of quantification, less interference of the matrix, and more resilience than batch techniques proposed in the Brazilian Pharmacopeia.

Repeated high dose imidocarb dipropionate treatment did not eliminate Babesia caballi from naturally infected horses as determined by PCR-reverse line blot hybridization.

Imidocarb treatment of horses infected with Babesia caballi is supposed to eliminate the infection, but data on the efficacy of this treatment is scarce. The study presented here concerns four Paso Fino horses, which were imported into the island of Curacao on the basis of a piroplasmosis negative complement fixation test (CFT). Upon re-testing with an indirect fluorescent antibody test immediately after arrival in Curacao, two horses appeared to have antibodies to B. caballi and all horses had antibodies to Theileria equi. Subsequent testing with polymerase chain reaction combined with a reverse line blot yielded positive results for both agents in all four horses. Treatment with five consecutive doses of imidocarb dipropionate (4.7 mg/kg BW im q 72 h), temporarily resulted in negative results, but B. caballi and T. equi were detected again in the samples taken at 6 and 18 weeks after completion of the treatment. These results confirm that the CFT is not a suitable test for pre-import testing and that even high dose treatment with imidocarb may not be capable of eliminating B. caballi and T. equi infections from healthy carriers.

A policy for leishmaniasis with respect to the prevention and control of drug resistance.

At the moment no country has a policy designed to control or prevent drug resistance in leishmaniasis. The risk of resistance is high in areas of anthroponotic visceral leishmaniasis, for example North Bihar, India, where the rate in some areas is 60%. Post-epidemic Sudan is also at risk. Zoonotic areas in which HIV co-infection is common could also be at risk as sandflies can become infected from co-infected individuals. Many factors determine the choice of drug for the treatment of visceral leishmaniasis, and drug resistance may not be the over-riding priority. In anthroponotic areas reduction in transmission through public health measures will be important, but the use of two drugs in combination should be seriously considered. Pharmacokinetic and other features of the drugs available, relevant to their use in combination are discussed and tentative suggestions made concerning trials of possible combinations. These include miltefosine plus paromomycin and allopurinol plus an azole. Lessons may be learnt from the experiences of similar problems in malaria, leprosy and tuberculosis. Guidelines are offered for the introduction of policies to use drugs in combination, which differ between anthroponotic and zoonotic areas of transmission.

Determination of the activity of pyrimethamine, trimethoprim, sulfonamides, and combinations of pyrimethamine and sulfonamides against Sarcocystis neurona in cell cultures.

Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome in horses from the Americas and is usually caused by infection with the apicomplexan parasite, Sarcocystis neurona. The activities of pyrimethamine, trimethoprim, sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfamethazine, and sulfathiazole were examined against developing S. neurona merozoites in bovine turbinate cell cultures. A microtiter plate host cell lesion based assay was used to determine the effects of agents on developing merozoites. A cell culture flask assay was used to determine if selective concentrations of the agents killed or only inhibited development of S. neurona. Pyrimethamine was coccidiocidal at 1.0 microg/ml and trimethoprim was coccidiocidal at 5.0 microg/ml. None of the sulfonamides had activity when used alone at 50.0 or 100.0 microg/ml. Combinations of sulfonamides (5.0 or 10.0 microg/ml) with 0.1 microg/ml pyrimethamine demonstrated improved activity.

Efficacy of fenbendazole for treatment of giardiasis in calves.

OBJECTIVE: To determine efficacy of fenbendazole for treatment of giardiasis in calves. ANIMALS: Twenty male and 15 female Holstein calves (100 to 180 kg), naturally infected with Giardia sp. PROCEDURE: In vitro fenbendazole susceptibility and resistance development was determined for a ruminant Giardia isolate by use of an adherence assay. Calves were treated as follows: group 1, a single administration of 5 mg of fenbendazole/kg of body weight; group 2, a single administration of 10 mg of fenbendazole/kg; group 3, 5 mg of fenbendazole/kg, every 24 hours for 3 days; group 4, 10 mg of fenbendazole/kg, every 24 hours for 3 days; group 5, 20 mg of fenbendazole/kg, every 24 hours for 3 days; group 6, 0.833 mg of fenbendazole/kg, every 24 hours for 6 days; and group 7, saline solution. Fecal Giardia cysts were counted on days -3 through -1 and 1 through 7, 9, 11, 13, 21, and 28 by use of sucrose gradient concentration and staining with a fluorescent monoclonal antibody. RESULTS: The 50% adherence inhibition concentration was 0.024 +/- 0.002 microgram/ml, and resistance could not be detected after 5 weeks of continuous culture at sublethal concentration of fenbendazole (0.01 microgram/kg). Fenbendazole was 100% effective in eliminating cysts from the feces within 6 days for calves in treatment groups 2-6. Reinfection was observed in some calves within the 28-day study period. CONCLUSIONS: Fenbendazole is effective in the elimination of Giardia infections in calves, but repeat treatments may be required in reinfected animals. CLINICAL RELEVANCE: Fenbendazole is an effective and economical treatment for Giardia-associated diarrhea and growth rate reduction in calves.

Therapy for feline dermatoses.

This article compiles information on various therapies used in feline dermatology. Information on the following therapeutic agents and devices is discussed: antibiotics, antifungals, antileprosy drugs, antiparasiticides, antivirals, antihistamines, behavior modification drugs, fatty acids, progestogens, steroids, immunomodulating drugs, chemotherapeutic/immunosuppressive agents, retinoids, mechanical devices, hyposensitization, immunotherapy, food elimination trials, hypoallergenic diets, and miscellaneous topical agents such as polyhydroxydine solution, tar, and benzocaine-containing creams.

Antimony oxidation states in antileishmanial drugs.

Chemical methods specific for the determination of the levels of trivalent antimony (Sb+3) and pentavalent antimony (Sb+5) were used to investigate proprietary formulas used to treat leishmaniasis. Trivalent antimony was determined by differential pulse polarography, whereas Sb+5 was determined by iodine titration. Proprietary formulas based on N-meglumine antimoniate (Glucantime) were analyzed in detail. The results showed Sb+3 in all ampules of Glucantime. In formulations said to contain either 85 or 100 mg of Sb+5/ml, we found both forms of antimony. The amount of Sb+3 ranged from 10.5 to 15.8% (10.06-18.96 mg of Sb/ml). These findings raise issues on product stability and standardization and may help to clarify resistance to antimonial drugs and the reducing effect of tissue on Sb+5.

Treatment of chronic experimental Trypanosoma cruzi infections in mice with MK-436, a 2-substituted 5-nitroimidazole.

The antiprotozoal drug 3-(1-methyl-5-nitroimidazol-2-yl)-3a, 4,5,6,7,7a-hexahydro-1,2-benzisoxazole (MK-436) is highly efficacious for treating mice chronically infected with different strains of Trypanosoma cruzi. The compound was administered by gavage in two daily doses of 250 mg per kg body weight to 130 mice that had been infected for 90 to 400 days with either type II or III strains of T. cruzi. The following parasitological cure tests were carried out: xenodiagnosis, haemoculture, and inoculation of blood into newborn mice. Indirect immunofluorescence tests and histopathological studies were also performed. The results indicate that the drug is highly efficacious against chronic infection caused by both type II (cure rate, 90%) and type III strains (cure rate, 95.7%). Histopathological examinations showed complete clearance of the cardiac and muscular lesions in 36% of the mice infected with type II strains and a decrease in the intensity and extension of the lesions in mice infected with type III strains. Indirect immunofluorescence tests were persistently positive for all the mice 3-6 months after the treatment.

Treatment of chronic experimental Trypanosoma cruzi infections in mice with MK-436, a 2-substituted 5-nitroimidazole

The antiprotozoal drug 3-(1-methyl-5-nitroimidazol-2-yl)3a, 4,5,6,7,7a-hexahydro-1,2-benzisoxazole (MK-436) is highly efficacious for treating mice chronically infected with different strains of Trypanosoma cruzi. The compound was administered by gavage in two daily doses of 250 mg per kg body weight to 130 mice that had been infected for 90 to 400 days with either type II or III strains of T. cruzi. The following parasitological cure tests were carried out: xenodiagnosis, haemoculture, and inoculation of blood into newborn mice. Indirect immunofluorescence tests and histopathological studies were also performed

The results indicate that the drug is highly efficacious against chronic infection caused by both type II (cure rate, 90 percent) and type III strains (cure rate, 95.7 percent). Histopathological examinations showed complete clearance of the cardiac and muscular lesions in 36 percent of the mice infected with type II strains and a decrease in the intensity and extension of the lesions in mice infected with type III strains. Indirect immunofluorescence tests were persistently positive for all the mice 3-6 months after the treatment(AU)