Clofazimine, a Promising Drug for the Treatment of Babesia microti Infection in Severely Immunocompromised Hosts.

BACKGROUND: Persistent and relapsing babesiosis caused by Babesia microti often occurs in immunocompromised patients, and has been associated with resistance to antimicrobial agents such as atovaquone. Given the rising incidence of babesiosis in the United States, novel drugs are urgently needed. In the current study, we tested whether clofazimine (CFZ), an antibiotic used to treat leprosy and drug-resistant tuberculosis, is effective against B. microti. METHODS: Mice with severe combined immunodeficiency were infected with 107B. microti-infected erythrocytes. Parasites were detected by means of microscopic examination of Giemsa-stained blood smears or nested polymerase chain reaction. CFZ was administered orally. RESULTS: Uninterrupted monotherapy with CFZ curtailed the rise of parasitemia and achieved radical cure. B. microti parasites and B. microti DNA were cleared by days 10 and 50 of therapy, respectively. A 7-day administration of CFZ delayed the rise of parasitemia by 22 days. This rise was caused by B. microti isolates that did not carry mutations in the cytochrome b gene. Accordingly, a 14-day administration of CFZ was sufficient to resolve high-grade parasitemia caused by atovaquone-resistant B. microti parasites. CONCLUSIONS: Clofazimine is effective against B. microti infection in the immunocompromised host. Additional preclinical studies are required to identify the minimal dose and dosage of CFZ for babesiosis.

Five-Stage Approach for a Systematic Screening and Development of Etravirine Amorphous Solid Dispersions by Hot-Melt Extrusion.

The aim of this study was to develop a fast, effective, and material sparing screening method to design amorphous solid dispersions (ASDs) of etravirine to drive more effectively the development process, leading to improved bioavailability (BA) and stability. A systematic step-by-step approach was followed by combining theoretical calculations with high-throughput screening (HTS) and software-assisted multivariate statistical analysis. The thermodynamic miscibility and interaction of the drug in several polymers were predicted using Hansen solubility parameters (δ). The selected polymers were evaluated by HTS, using solvent evaporation. Binary compositions were evaluated by their solubilization capacity and physical stability over 2 months. JMP 14.0 was used for multivariate statistical analysis using principal components analysis. Extrusion was performed in Thermo Scientific HAAKE MiniLab II, and extrudates were characterized by assay, related substances, dissolution, and physical state (polarized light microscopy (PLM), Raman spectroscopy, and X-ray powder diffraction (XRPD)). A short stability study was performed where milled extrudates were exposed to 25 °C/60%RH and 40 °C/75%RH for 3 months. Through thermodynamic predictions, five main polymers were selected. The HTS enabled the evaluation of 42 formulations for solubilization capacity and physical stability. The three most promising compositions were selected for hot-melt extrusion (HME) tests. In general, a good correlation was found among the results of theoretical predictions, HTS, and HME. Poly(vinylpyrrolidone) (PVP)-based formulations were shown to be easily extrudable, with low degradation and complete amorphicity, whereas in Soluplus, the drug was not miscible, leading to a high crystalline content. The drug release rate was improved more than two times with PVP, and the manufactured ASD was demonstrated to be stable physically and chemically. A fast and effective screening technique to develop stable ASDs for a poorly soluble drug was successfully developed as applied to etravirine. The given method is easy to use, requires a low amount of drug, and is fairly accurate in predicting the amorphization of the drug when formulated. The success of HME formulation development of etravirine was undoubtedly enhanced with this high-throughput tool, which led to the identification of extrudates with improved biopharmaceutical properties. The structural characterization performed by PLM, XRPD, and Raman spectroscopy demonstrated that the HME prototype was essentially amorphous. The unexpected stability at 40 °C/75%RH was correlated with the presence of molecular interaction characterized by Raman spectroscopy.

Drug targeted virtual screening and molecular dynamics of LipU protein of Mycobacterium tuberculosis and Mycobacterium leprae.

The lipolytic protein LipU was conserved in mycobacterium sp. including M. tuberculosis (MTB LipU) and M. leprae (MLP LipU). The MTB LipU was identified in extracellular fraction and was reported to be essential for the survival of mycobacterium. Therefore to address the problem of drug resistance in pathogen, LipU was selected as a drug target and the viability of finding out some FDA approved drugs as LipU inhibitors in both the cases was explored. Three-dimensional (3D) model structures of MTB LipU and MLP LipU were generated and stabilized through molecular dynamics (MD). FDA approved drugs were screened against these proteins. The result showed that the top-scoring compounds for MTB LipU were Diosmin, Acarbose and Ouabain with the Glide XP score of -12.8, -11.9 and -11.7 kcal/mol, respectively, whereas for MLP LipU protein, Digoxin (-9.2 kcal/mol), Indinavir (-8.2 kcal/mol) and Travoprost (-8.2 kcal/mol) showed highest affinity. These drugs remained bound in the active site pocket of MTB LipU and MLP LipU structure and interaction grew stronger after dynamics. RMSD, RMSF and Rg were found to be persistent throughout the simulation period. Hydrogen bonds along with large number of hydrophobic interactions stabilized the complex structures. Binding free energies obtained through Prime/MM-GBSA were found in the significant range from -63.85 kcal/mol to -34.57 kcal/mol for MTB LipU and -71.33 kcal/mol to -23.91 kcal/mol for MLP LipU. The report suggested high probability of these drugs to demolish the LipU activity and could be probable drug candidates to combat TB and leprosy disease.

Fusarihexins A and B: Novel Cyclic Hexadepsipeptides from the Mangrove Endophytic Fungus Fusarium sp. R5 with Antifungal Activities.

Two novel cyclic hexadepsipeptides, fusarihexin A (1: ) and fusarihexin B (2: ), and two known compounds, cyclo-(L-Leu-L-Leu-D-Leu-L-Leu-L-Val) (3: ) and cyclo-(L-Leu-L-Leu-D-Leu-L-Leu-L-Ile) (4: ), were isolated from the marine mangrove endophytic fungus Fusarium sp. R5. Their chemical structures were elucidated on the basis of spectroscopic data and Marfey's analysis. In an in vitro bioassay, fusarihexin A (1: ) remarkably inhibited three plant pathogenic fungi: Colletotrichum gloeosporioides (Penz.) Sacc., which causes anthracnose in many fruits and vegetables, Colletotrichum musae (Berk. and M. A. Curtis) Arx, which causes crown rot and anthracnose in bananas, and Fusarium oxysporum Schlecht. f. sp. lycopersici (Sacc.) W. C. Snyder et H. N. Hansen, which causes Fusarium wilt and fruit rot in tomatoes. Fusarihexin B (2: ) strongly inhibited C. gloeosporioides and C. musae. The compounds were more potent than carbendazim, which is widely used as an agricultural and horticultural fungicide worldwide.

A high-throughput phenotypic screen identifies clofazimine as a potential treatment for cryptosporidiosis.

Cryptosporidiosis has emerged as a leading cause of non-viral diarrhea in children under five years of age in the developing world, yet the current standard of care to treat Cryptosporidium infections, nitazoxanide, demonstrates limited and immune-dependent efficacy. Given the lack of treatments with universal efficacy, drug discovery efforts against cryptosporidiosis are necessary to find therapeutics more efficacious than the standard of care. To date, cryptosporidiosis drug discovery efforts have been limited to a few targeted mechanisms in the parasite and whole cell phenotypic screens against small, focused collections of compounds. Using a previous screen as a basis, we initiated the largest known drug discovery effort to identify novel anticryptosporidial agents. A high-content imaging assay for inhibitors of Cryptosporidium parvum proliferation within a human intestinal epithelial cell line was miniaturized and automated to enable high-throughput phenotypic screening against a large, diverse library of small molecules. A screen of 78,942 compounds identified 12 anticryptosporidial hits with sub-micromolar activity, including clofazimine, an FDA-approved drug for the treatment of leprosy, which demonstrated potent and selective in vitro activity (EC50 = 15 nM) against C. parvum. Clofazimine also displayed activity against C. hominis-the other most clinically-relevant species of Cryptosporidium. Importantly, clofazimine is known to accumulate within epithelial cells of the small intestine, the primary site of Cryptosporidium infection. In a mouse model of acute cryptosporidiosis, a once daily dosage regimen for three consecutive days or a single high dose resulted in reduction of oocyst shedding below the limit detectable by flow cytometry. Recently, a target product profile (TPP) for an anticryptosporidial compound was proposed by Huston et al. and highlights the need for a short dosing regimen (< 7 days) and formulations for children < 2 years. Clofazimine has a long history of use and has demonstrated a good safety profile for a disease that requires chronic dosing for a period of time ranging 3-36 months. These results, taken with clofazimine's status as an FDA-approved drug with over four decades of use for the treatment of leprosy, support the continued investigation of clofazimine both as a new chemical tool for understanding cryptosporidium biology and a potential new treatment of cryptosporidiosis.

A rapid screening assay for identifying mycobacteria targeted nanoparticle antibiotics.

Antibiotic resistance is a serious problem. Nanotechnology offers enormous potential in medicine, yet there is limited knowledge regarding the toxicity of nanoparticles (NP) for mycobacterial species that cause serious human diseases (e.g. tuberculosis (TB) and leprosy). Mycobacterial diseases are a major global health problem; TB caused by Mycobacterium tuberculosis (Mtb) kills up to 2 million people annually and there are over 200 000 leprosy cases each year caused by Mycobacterium leprae (M. leprae). Few drugs are effective against these mycobacteria and increasing antibiotic resistance exacerbates the problem. As such, alternative therapies are urgently needed but most current assays used to assess the effectiveness of therapeutics against mycobacteria are slow and expensive. This study aimed to develop a rapid, low-cost assay which can be used for screening the antimicrobial properties of compounds against pathogenic mycobacteria and to assess the toxicity of three NP (silver [Ag], copper oxide [Cu(II)O], and zinc oxide [ZnO]) against a green fluorescent protein reporter strain of Mycobacterium avium subspecies paratuberculosis, a slow growing, pathogenic mycobacterial species causing paratuberculosis in ruminants. Fluorescence was used to monitor mycobacterial growth over time, with NP concentrations of 6.25-100 µg/mL tested for up to 7 days, and a method of data analysis was designed to permit comparison between results. Mycobacterial sensitivity to the NP was found to be NP composition specific and toxicity could be ranked in the following order: Ag > Cu(II)O > ZnO.

[EXPERIMENTAL STUDY OF SPECIFIC ACTIVITY OF 1.3-DIAZINON-4 COM- POUND PYaTd1 DERIVATIVE IN VIVO].

AIM: Study anti-leprosy activity of.a 1.3-diazinon-4 compound derivative under the labora- tory code PYaTd1 on the model of intra-plantar infection of mice and evaluate the character of its antibacterial effect. MATERIALS AND METHODS: Study of specific activity was carried out in vivo on the experimental model of leprosy, proposed by Shepard C.C., that assumes execution of intraplantar infection of mice with a suspension of mycobacteria, produced from lepromas or autopsy tissue of a non-treated leprosy infected, or from tissues of experimental mice, previously infected with Mycobacterium leprae from non-treated patients. The study was carried out on 120 CBA line-mice infected with M.leprae (VIII passage) from patient M; Dapsone and PYaTdl compound were administered to animals next day after the infection with forage at a dose of 25 mg/kg for 4.5, 6, 9 and 11 months. The mice were split into 3 groups: control (infected.without treatment), com- parison (infected, receiving.dapsone), experimental (infected, receiving PYaTdl). After.the control term the mice were euthanized under chloroform anesthesia. Suspensions for quantification of mycobacteria were prepared from paw pads. Smears were stained by Ziehl-Nilsson. RESULTS: After 4.5 months the intensity of infect reproduction under, the effect of dapsone and PYaTd1 was reduced compared with control by 18 - 25 times. After a 6-mont course - by 50 - 75% and after 9 months - by 85 - 90%. After 11 months in mice that had received PYaTd1, an intensive suppression of microorganism reproduction as observed: the yield in paws was 70 times lower than in control. In the group that had received dapsone, a reduction of the number of mycobacteria by 20 - 25 times was detected, it was significantly less effective than under the conditions of PYaTd1 admnistration. CONCLUSION: A novel 1.3-diazinon- 4 derivative under the code PYaTd1 can actively supress reproduction of-M. leprae, that gives evidence regarding its specific anti-mycobacterial activity and determines perspectives of its further studies.

The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae.

Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.

Immunomodulatory properties of thalidomide analogs: pomalidomide and lenalidomide, experimental and therapeutic applications.

Thalidomide has a broad spectrum of anti-cancer activity. Antitumor activity of thalidomide may be related to a number of known properties, including anti-tumor necrosis factor (TNF)-α and T-cell costimulatory and antiangiogenic activities. The therapeutic potential of thalidomide provided motivation to develop more effective derivatives with considerably reduced toxicity. Thalidomide's immunomodulatory (IMiDs) analogs (lenalidomide, CC-5013; CC-4047, ACTIMID) represent a novel class of compounds with numerous effects on the immune system. Some of these analogs are thought to mediate the anticancer and anti-inflammatory effects observed in humans. Thalidomide is currently approved for the treatment of dermal reaction to leprosy and is currently in phase III trials for multiple myeloma (MM). IMiDs inhibit the cytokine's tumor necrosis factor-α (TNF-α), interleukins (IL) 1ß, 6, 12, and granulocyte macrophage-colony stimulating factor (GM-CSF). The repression of the tumor necrosis factor-a (TNF-α) expression is the crucial factor of many of the anti-inflammatory properties of thalidomide. The mechanisms underlying many of the anti-inflammatory properties of thalidomide, including its ability to co-stimulate T cells, still remain unclear. Some recent patent are also summarized in this review.

Anti-inflammatory activity of superoxide dismutase obtained from Debaryomyces hansenii on type II collagen induced arthritis in rats.

INTRODUCTION: Rheumatoid arthritis is an autoimmune inflammatory disease of unknown etiology, free radicals have been implicated in the genesis and perpetuation of damage in this pathology. OBJECTIVE: To evaluate the anti-inflammatory effect of Cu,Zn-superoxide dismutase (SOD) obtained from two different sources (bovine erythrocytes, Be-SOD, and Debaryomyces hansenii, Dh-SOD) with Type II Collagen-induced Arthritis model in rats. MATERIAL AND METHODS: Arthritis was induced by repeated injection of a porcine type II collagen-incomplete Freund adjuvant suspension on the back of Dark Augui (DA) rats. Arthritis was clinically evaluated throughout the study. Body weight was determined at three different times. Two different doses for each treatment (Be-SOD, Dh-SOD) were tested: 100 and 1,000 U/kg. At the end of the trial (day 28), histological analyses of the most inflamed ankle joint, as well as serum anti-collagen antibodies, were determined. RESULTS: Both sources of SOD decreased, although to a different extent, the incidence and severity of the disease. Arthritis score was lower in all treatments, except for the low dose of Be-SOD. Groups receiving either source of SOD showed a significant weight increase compared to the placebo group. Histological damage was similar in all groups. Only the group that received the highest dose of Dh-SOD showed a significant lower antibody titer; nevertheless, no correlation appears to derive from arthritis score and antibody titer. CONCLUSION: Our findings suggest that, although unable to counteract the arthritis syndrome, SOD may still be beneficial due to its anti-inflammatory activity. In the case of Dh-SOD, the best effect was observed at the highest dose tested.

Antileprosy activity and preliminary toxicological characteristics of some dialkyldithiocarbamate derivatives.

Antileprosy activity of dialkyldithiocarbamate derivatives was studied in experiments on mice infected with M. leprae into paw pads. We found that 2-diethyldithiocarbamoyl-3-cyano-5-nitropyridine is the most promising antileprosy agent; it effectively suppresses multiplication of M. leprae and is well tolerated under conditions of chronic animal experiment.

Rifampicin inhibits the retinal neovascularization in vitro and in vivo.

Rifampicin, an antibacterial drug widely used in the treatment of tuberculosis and leprosy, has recently been reported to have anti-oxidative and anti-apoptotic effects. However, its anti-angiogenic effect has not been investigated. We examined its anti-angiogenic effect on tube formation and proliferation by human umbilical vein endothelial cells (HUVECs) in vitro and on retinal neovascularization in a murine oxygen-induced retinopathy model in vivo. In addition, we explored the potential mechanisms for its anti-angiogenic effect. Rifampicin significantly suppressed HUVEC tube formation and proliferation, and its effects appeared to be mediated at least in part through inhibition of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Retinal neovasuclarization was induced in neonatal mice by returning the retina to normoxia (21% O2) after exposure to hyperoxia (75% O2) from postnatal day 7 (P7) to P12. Rifampicin was given subcutaneously at 20mg/kg once a day from immediately after hyperoxia (P12) to P16. At P17, flat-mounted retinas were prepared and evaluated for pathological and physiological angiogenesis. Rifampicin significantly suppressed retinal neovascularization (versus vehicle treatment), but revascularization of the capillary-free area did not differ between vehicle and rifampicin treatment. Rifampicin has anti-angiogenic effects in vitro and in vivo, and may be useful as an anti-angiogenic agent in the treatment of retinal neovascularization diseases.

Study of antileprosy activities of some dialkyldithiocarbamate derivatives.

Effects of some dialkyldithiocarbamate derivatives on multiplication of M. leprae were studied in infected mice. Compounds significantly suppressing M. leprae proliferation were selected. By antibacterial activity one of these compounds was superior to dapsone, the main antileprosy drug.

Anti-inflammatory and antinociceptive activities of an acid fraction of the seeds of Carpotroche brasiliensis (Raddi) (Flacourtiaceae).

Carpotroche brasiliensis is a native Brazilian tree belonging to the Oncobeae tribe of Flacourtiaceae. The oil extracted from its seeds contains as major constituents the same cyclopentenyl fatty acids hydnocarpic (40.5%), chaulmoogric (14.0%) and gorlic (16.1%) acids found in the better known chaulmoogra oil prepared from the seeds of various species of Hydnocarpus (Flacourtiaceae). These acids are known to be related to the pharmacological activities of these plants and to their use as anti-leprotic agents. Although C. brasiliensis oil has been used in the treatment of leprosy, a disease that elicits inflammatory responses, the anti-inflammatory and analgesic activities of the oil and its constituents have never been characterized. We describe the anti-inflammatory and antinociceptive activities of C. brasiliensis seed oil in acute and chronic models of inflammation and in peripheral and central nociception. The mixture of acids from C. brasiliensis administered orally by gavage showed dose-dependent (10-500 mg/kg) anti-inflammatory activity in carrageenan-induced rat paw edema, inhibiting both the edema by 30-40% and the associated hyperalgesia. The acid fraction (200 mg/kg) also showed significant antinociceptive activity in acetic acid-induced constrictions (57% inhibition) and formalin-induced pain (55% inhibition of the second phase) in Swiss mice. No effects were observed in the hot-plate (100 mg/kg; N = 10), rota-road (200 mg/kg; N = 9) or adjuvant-induced arthritis (50 mg/kg daily for 7 days; N = 5) tests, the latter a chronic model of inflammation. The acid fraction of the seeds of C. brasiliensis which contains cyclopentenyl fatty acids is now shown to have significant oral anti-inflammatory and peripheral antinociceptive effects.

Anti-inflammatory and antinociceptive activities of an acid fraction of the seeds of Carpotroche brasiliensis (Raddi) (Flacourtiaceae)

Carpotroche brasiliensis is a native Brazilian tree belonging to the Oncobeae tribe of Flacourtiaceae. The oil extracted from its seeds contains as major constituents the same cyclopentenyl fatty acids hydnocarpic (40.5 percent), chaulmoogric (14.0 percent) and gorlic (16.1 percent) acids found in the better known chaulmoogra oil prepared from the seeds of various species of Hydnocarpus (Flacourtiaceae). These acids are known to be related to the pharmacological activities of these plants and to their use as anti-leprotic agents. Although C. brasiliensis oil has been used in the treatment of leprosy, a disease that elicits inflammatory responses, the anti-inflammatory and analgesic activities of the oil and its constituents have never been characterized. We describe the anti-inflammatory and antinociceptive activities of C. brasiliensis seed oil in acute and chronic models of inflammation and in peripheral and central nociception. The mixture of acids from C. brasiliensis administered orally by gavage showed dose-dependent (10-500 mg/kg) anti-inflammatory activity in carrageenan-induced rat paw edema, inhibiting both the edema by 30-40 percent and the associated hyperalgesia. The acid fraction (200 mg/kg) also showed significant antinociceptive activity in acetic acid-induced constrictions (57 percent inhibition) and formalin-induced pain (55 percent inhibition of the second phase) in Swiss mice. No effects were observed in the hot-plate (100 mg/kg; N = 10), rota-road (200 mg/kg; N = 9) or adjuvant-induced arthritis (50 mg/kg daily for 7 days; N = 5) tests, the latter a chronic model of inflammation. The acid fraction of the seeds of C. brasiliensis which contains cyclopentenyl fatty acids is now shown to have significant oral anti-inflammatory and peripheral antinociceptive effects.

Use of morphostructural reaction of blood serum for toxicological evaluation of drugs.

We proposed a simple and economic method for determination of general toxic effects of drugs consisting in evaluation of serum morphology by polarization light microscopy.

Thalidomide in multiple myeloma.

Thalidomide--removed from widespread clinical use by 1962 because of severe teratogenicity--has anti-angiogenic and immunomodulatory effects, including the inhibition of TNF alpha. It has returned to practice as an effective oral agent in the management of various disease states including erythema nodosum leprosum, for which it was FDA-approved in 1998, and more recently certain malignancies, including multiple myeloma. Whilst the mechanism of action of thalidomide remains incompletely understood, considerable insight has been generated by extensive preclinical studies in multiple myeloma. Moreover, clinical trials both as a single agent and in combination have confirmed benefit in relapsed and refractory disease. Thalidomide's role in treating newly diagnosed patients is currently under study and it is now established as an important therapeutic option in the treatment of multiple myeloma.

Biodiversity of killer activity in yeasts isolated from the Brazilian rain forest.

The occurrence of killer activity against a panel composed of 22 industrially and (or) medically important yeasts was investigated in 438 yeast and yeast-like cultures belonging to 96 species, isolated from different environments of the Brazilian rain forest. Altogether, 26% of ascomycetes, 56% of basidiomycetes, and 42% of yeast-like cultures exhibited killer activity against at least one of the panel yeasts. More than 15 species never reported before as toxin producers were found, with Pseudozyma antarctica, Trichosporon asteroides, and Geotrichum klebahnii, showing the broader activity spectra. Plasmid curing did not cure the killer phenotypes of Candida maltosa, Debaryomyces hansenii, G. klebahnii, Tr. asteroides, Cryptococcus laurentii, and Ps. antarctica.