Repurposing chlorpromazine and its metabolites for antituberculosis drug discovery

Includes bibliographical referencesNew chemotherapeutics are urgently needed to combat Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). The development of compounds that could potentiate the activity of known antimycobacterial drugs is a relatively unexplored approach to new TB drug discovery. This study aimed to generate metabolites of chlorpromazine (CPZ), a phenothiazine with demonstrated in vitro activity against Mtb, and to investigate their potential utility in combination with anti-TB drugs. 7-HydroxyCPZ (M2), CPZ-N-oxide (M3), CPZ sulfoxide (M1), nor-CPZ (M5), nor-CPZ sulfoxide (M6b) and CPZ-N-S-dioxide (M4b) were generated from CPZ using various biotransformation systems and identified by Liquid Chromatography - Mass Spectrometry (LC/MS). The identity of M2 was confirmed with reference to a 7-hydroxyCPZ standard. M3, M1, M5, M6b and M4b were synthesized de novo and used to identify the metabolites generated in the biotransformation samples. Individually, CPZ and its metabolites (M2, M3, M5) were weakly active (MIC99 >50μM) against M. smegmatis (Msm) and Mtb while M1, M6b & M4b did not exhibit a MIC99 even at very high concentrations. Generally, an improvement in activity was observed where CPZ or its metabolites were used in combination with known anti-TB drugs. The combinations that exhibited a fractional inhibition concentration index (FICI) of < 0.5 were defined as synergistic. A combination of M2 and spectinomycin (SPEC) exhibited the highest synergism against Msm (FICI 0.19) and Mtb (FICI 0.13). In vitro assays established that CPZ and M2 are bactericidal against Mtb whereas M3 and M5 are bacteriostatic on their own. In combination assays, the use of RIF with M3 and M5, bedaquiline (BDQ) with M2, and SPEC with M3 were bactericidal. At 140μM, CPZ and M1, M2, M3 treated samples exhibited a 2-fold up-regulation of the cydA (Rv1623c) gene which encodes an essential subunit of the cytochrome bd-type menaquinol oxidase in Mtb. The same observation was made for RIF/M2 and RIF/M5 treated samples. These results suggest that the metabolites retain the mechanism of action (MoA) as the parental CPZ. The Mtb 16S rRNA gene, rrs (MTB000019) was identified as the biological target for SPEC. This brought into perspective the underlying mechanisms at play when SPEC is used in combination with CPZ, its metabolites or other drugs, against mycobacteria. This study establishes the utility of combination assays in confirming the active metabolite(s) of known drugs and provides proof of concept data to support follow-up investigations of CPZ and its metabolites as potential compounds for novel combination therapies for anti-TB drug development

Antinociceptive effect of Pentas lanceolata and Ximenia americana medicinal plants used to treat malaria traditionally in Kenya

Background: Pain being one of the many symptoms experienced in the course of an infection, this study was designed to investigate antinociceptive activity of two medicinal plants used to treat malaria traditionally. In traditional health practice there is usually a misunderstanding on whether the plants clear the disease causing organism or they cure the pain as one of the most common symptoms. The plants were selected based on their ethnomedical literature as a means of establishing a distinction between their antinociceptive and bio-activities. Objective: To determine antinociceptive effect of Pentas lanceolata and Ximenia americana. Materials and Methods: The aerial parts of P. lanceolata and the stem bark of X. americana were collected from Kiangombe forest and Kerio Valley in Embu and Elgeyo Marakwet Counties respectively. The collection was done with the help of an experienced taxonomist. The plant parts were dried, and extracted with methanol and aqueous solvents. Analgesic activity was determined by the tailflick and formalin test techniques in male albino mice. The positive control used in the experiment was acetylsalicylic acid (ASA) at 100 mg/kg. Results: Antinociceptive activity of the plant extracts in the tail flick assay was time and dose dependent, except in the case of P. lanceolata methanol extract. In the formalin test, the extracts significantly (p&lt;0.05) reduced the time spent in pain behavior in both the early and late phases Conclusion: The results of this study support the use of these plants to manage pain and imply other pharmacological benefits to the host other than parasiticidal effect in malaria treatment. Keywords: Medicinal plants, tail-flick, formalin test, antinociceptiv

In vivo Antifertility and Safety Profiles of Kenyan Moringa oleifera Lam. (Moringaceae) extracts

Background: Unsustainable high population growth rate coupled with many women dying of complications of unsafe abortion, due to a large number of unwanted pregnancies, has been a challenge in many parts of the world especially in developing countries. This indicates that new and alternative contraceptive methods that are safe, cheap and convenient are needed. Moringa oleifera Lam (Moringaceae) was selected for this study based on previous studies that indicated antifertility effect in rats, of the aqueous extract of the roots and the stem bark. Objective: To establish the antifertility properties of M. oleifera. Methodology: The aerial parts, seeds, root bark and twigs were extracted using methanol, petroleum ether, dichloromethane and ethyl acetate. In vivo antifertility evaluations in Swiss female mice, acute and sub-chronic toxicity and phytochemical studies were carried out on M. oleifera extracts. Results: The ethyl acetate extract of the seeds of M. oleifera demonstrated reversible antifertility effect at 800mg/kg. Physiological tests carried out on mice revealed that the extract arrested the estrus cycle either at the diestrus or the proestrus phase by prolonging them. Acute and chronic toxicity evaluation of the extract at 800mg/kg established the safety at the tested concentration. Thin layer chromatography (TLC) of the extract revealed the presence of terpenoids, steroids and fluorescent compounds, which may be responsible for the antifertility effect that was observed. Conclusion: The findings validate the ethnomedicinal use of M. oleifera seeds through the establishment of its safety, and the antifertility properties that make the extract a potential source of an alternative herbal contraceptive through further studies and development. Key words: Moringa oleifera, antifertility effect, estrus cycle, toxicity, phytochemical profile

Pyrethrins In Soil and Water From Selected Pyrethrum Growing Areas In Nakuru County, Kenya

Introduction: Pyrethrum also known as Chrysanthemum is a plant from which compounds known as pyrethrins are derived. The pyrethrins have&nbsp; been used for many years as insecticides. Incidentally due to their high instability they have slowly been replaced by synthetic pyrethroids.&nbsp; Pyrethrins are generally regarded as safe compared to the pyrethroids. However, the amounts released into the environment have not been well documented especially in pyrethrum growing regions.Objective:&nbsp; The aim of the study was to determine the concentration of pyrethrins that come from pyrethrum plants and released into the&nbsp; environment through their use as insecticides, thus, into drinking water and soil, in pyrethrum growing regions in Kenya.Methodology: Quantification to amounts of pyrethrins from pyrethrum plants, in soil and water bodies in and around pyrethrum farms in Kiambogo and Naivasha (Nakuru County). The study was carried out using High Performance Liquid Chromatography (HPLC). Water samples (0.5L) were collected from the following water bodies: rivers, streams, dams, wells and boreholes near or within pyrethrum farms.Conclusion: It was established that, the quantity of pyrethrins present in water and soil samples werebelow detectable levels within the WHO recommended range. Hence safe for the environment,more so for the farmers and the people living around pyrethrum farms. Key words: Pyrethrum, pyrethrins, chromatography, water, soil

Activités antimycobactériennes, Cytotoxicité et Screening phytochimique des extraits de trois plantes du Kenya

peer reviewedTuberculosis (TB), an airborne disease, is among the ten leading deadly diseases worldwide. Despite the efforts of WHO and its partners to eradicate it, it is still a public health issue especially with the rise of multi-drug resistant tuberculosis (MDR-TB) and extensively drug- resistant tuberculosis (XDR-TB). Commiphora species (Burseraceae family) are known in the Kenyan traditional medicine to treat respiratory diseases including TB. In the search of new anti-TB alternative drugs, plant materials from Commiphora mildbraedii Engl. (root bark and stem bark), Commiphora edulis (Klotzsch) Engl. (stem bark and leaves) and C. ellenbeckii Engl. (Stem bark and leaves) were tested for antimycobacterial activity, cytotoxicity and phytochemistry. 100 g of the powdered plant materials were macerated using the serial method with solvents of increasing polarity. Aqueous extraction was carried out by decoction. The microbroth dilution method was used to determine the antimycobacterial activity (MIC) against a model Mycobacterium smegmatis ATCC607 while the cytotoxicity evaluation (CC50) was carried out using the MTT assay. The most active extract was fractionated using preparative TLC and fractions were analysed by GC-MS. Thirty extracts were obtained from the 6 different plant materials and eleven of them exhibited the antimycobacterial activity with the methanolic extracts of the stem and root bark of C. mildbraedii, and the aqueous extract of the C. ellenbeckii leaves exhibiting high activities (MIC= 0.39, 0.78 and 0.78 mg/L respectively). The MTT assay showed no or low cytotoxicity. The GC-MS analysis of the preparative TLC fractions from the methanolic extract of C. mildbraedii revealed the presence of 42 compounds belonging to 10 different classes of phytochemicals. Lup-20(29)-en-3-one and o-xylene were the most abundant. Except o-xylene and α-terpineol, all the compounds were detected for the first time in the Commiphora genus. These findings justify the ethnomedicinal uses of Commiphora species in TB treatment

Antimalarial activities and toxicity levels of selected medicinal plants used in Kenya

Background: Resistance development to antimalarial drugs necessitates the look at traditional medicinal plants as sources of novel compounds that could have the otential to be developed into new antimalarial therapies. Four medicinal plants used in Kenya to treat malaria were investigated. Objective: To determine the in vitro and in vivo antimalarial activity and safety of four medicinal plants used in Kenya to treat malaria. Materials and Methods: Ximenia americana, Sericocomopsis hilderbrandtii, Pentas lanceolata and Fuerstia africana were collected from their habitat, dried, and extracted with methanol and aqueous solvents. In vitro antiplasmodial activity carried out using Plasmodium falciparum, In vivo antimalarial activity using Plasmodium berghei ANKA strain in Swiss albino mice. Cytotoxicity was carried out using MTT assay on VeroE99 cell lines, acute toxicity was investigated in Swiss albino mice. Results: All extracts had good in vitro activity against D6 strain of Plasmodium falciparum with IC50&lt;20µg/ml.  Aerial parts of Fuerstia africana methanol extract had the highest in vitro activity.  Seven extracts showed good in vivo activity with chemosuppresion &gt;30% while three demonstrated low activity. Fuerstia africana was moderately cytotoxic. Except for Ximenia americana water extract, all the extracts were safe with LD50 &gt; 5000mg/Kg. Conclusion: Results of this study support medicinal use of these plants and indicate that useful compounds can be isolated for further exploitation, formulation and use. Keywords: Medicinal plants, antiplasmodial activity, cytotoxicity, acute toxicit

Antimicrobial activity of organic total extracts of three Kenyan medicinal plants

In recent years, drug resistance to human pathogenicbacteria and fungi has increasingly been reported all over the world (Levy and Marshall, 2004; WHO 2004). Consequently, the increasing prevalence of multidrugresistant strains of microorganisms raises an urgent need to search for new sources of antimicrobial agents (Sieradzki et al, 1999) alongside other strategies such as regulated and rational use of antibiotics (Hernandez, 2005). The vast majority of traditionally used medicinal plants have not been adequately evaluated. This study was therefore undertaken to screen organic extracts obtained from three Kenyan medicinal plants for antibacterial and antifungal activity as a basis for further phytochemical studies. The plants to be studied were selected on the basis of ethnopharmacological reports of their use in traditional medicine; this approach is generally considered effective in the discovery of new bioactive agents from higher plants (Kloucek et al, 2005)

In vivo antifertility activity and phytochemical screening of selected Kenyan medicinal plants

Background: Medicinal plants are reported in folklore to play a role as fertility control agents. Very few studies have been carried out to confirm the safety and efficacy of medicinal plants used as anti-fertility agents. Objective: To establish anti-fertility activity, safety, effect on genital organs and estrous cycle, and phytochemical profile of total extracts from Terminalia brownii, Ximenia americana, Bridelia micrantha, Rhoicissus revoilii, and Ocimum masaiense. Methodology: Extracts of water and organic solvents were administered to female mice at a dose of 800 mg/kg orally for antifertility tests and at a dose of 0 to 5000mg/kg orally for acute toxicity test. Phytochemical screening was done using thin layer chromatography. Results: The leaf water extracts of the Bridelia micrantha, Ximenia americana showed a reversible anti-fertility effect while ethyl acetate extracts of the stem bark of Terminalia brownii had an irreversible anti-fertility effect. The bioactive extracts had an effect on the estrus cycle and had different phytochemical compounds with no  signs of toxicity. Discussion: The plant extracts tested exhibited antifertility activity, suggesting potential alternative to the current birth control methods. Compounds such as steroids, terpenoids, alkaloids, saponins and flavonoids present in the bioactive extracts may have contributed to the anti-fertility activity. Key words: Anti-fertility; Bridelia micrantha; Terminalia brownii; Ximenia American

Esterase phenotyping in human liver in vitro: specificity of carboxylesterase inhibitors

Esterases may play a major role in the pre-systemic or systemic clearance of drugs with functional groups amenable to hydrolysis, particularly in case of ester prodrugs. To understand the processes involved in the elimination of such drugs and to predict potential drug-drug interactions, it is necessary to determine the involved esterases in in vitro experiments (enzyme phenotyping). However, the tools currently available for this purpose are relatively scarce. The work presented in this communication aimed at determining the selectivity of known esterase inhibitors for carboxylesterases 1 and 2 (CES1 and CES2) in the human liver to clarify their suitability for esterase phenotyping. The main result was that eserine, when used at concentrations around 10 µM, is a highly specific CES2 inhibitor (besides being an inhibitor of cholinesterases), whereas other esterase inhibitors turned out less selective. When used together with tacrine (inhibitor of cholinesterases but not CES) and EDTA (inhibitor of paraoxonases), the involvement of the main drug hydrolyzing esterases in the clearance of a drug candidate can be elucidated. A second approach to esterase phenotyping, also shortly presented here, is based on data from recombinant (or isolated) esterases, together with relative activity factors, relating their activities to those of the same enzymes in subcellular fractions from the human liver. These two approaches will help to characterize the hydrolytic metabolism of drug candidates in a similar manner as practiced routinely for the oxidative metabolism by cytochrome P450 enzymes

Experimental chemotherapy with Allium sativum (Liliaceae) methanolic extract in rodents infected with Leishmania major and Leishmania donovani

Background & objectives: Several plant products have been tested and found to possess antileishmanialactivity. The present study was undertaken to establish whether methanolic extract ofAllium sativum Linn has antileishmanial activity in comparison to standard drugs.Methods: Methanolic extract of A. sativum bulbs was screened for in vitro and in vivo antileishmanialactivity against Leishmania major strain (NLB 145) and L. donovani strain (NLB 065). Pentostam®and Amphotericin B® were used as standard drugs. BALB/c mice and golden hamsters(Mesocricetus auratus) were used in in vivo studies on L. major and L. donovani respectively.Results: The extract exhibited very low cytotoxicity (IC50 >450 μg/ml) against Vero cells. Theextract had significantly better (p <0.001) leishmanicidal activity against both species (IC50 34.22μg/ml to L. major, 37.41 μg/ml to L. donovani) than Pentostam. However, the activity wassignificantly lower (p <0.001) than that of Amphotericin B against both the species. At aconcentration of 250 μg/ml, the extract induced the production of 60 μM of nitric oxide, a ten-foldup-regulation in activated macrophages. The multiplication indices for L. major amastigotes treatedin 100 μg/ml were significantly different (p <0.05). Treatment with the extract, daily for 28 daysled to a significant reduction (p <0.05) in footpad swelling in BALB/c mice; similar activitynoticed in the treatment with standard drugs. The Leishman-Donovan Units (LDU) for the extracttreated animals were significantly higher (p <0.05) than those of standard drugs, but lower comparedto the negative control.Interpretation & conclusion: Since the mechanism of action for the methanolic extract is apparentlyimmunomodulatory, garlic compounds could be purified and tried as complementary medicine inthe management of leishmaniases