Protective Effect of Hainosankyuto, a Traditional Japanese Medicine, on Streptococcus pyogenes Infection in Murine Model

BACKGROUND: Streptococcus pyogenes (S. pyogenes) causes various serious diseases including necrotizing fasciitis and streptococcal toxic shock syndrome. One serious problem observed recently with S. pyogenes therapy is attenuation of the antibiotic effect, especially penicillin treatment failure and macrolide resistance. Hainosankyuto, a traditional Japanese medicine based on ancient Chinese medicine, has been used for treatment of infectious purulent diseases in Japan. In this study, we investigated the protective and therapeutic efficacy of Hainosankyuto against S. pyogenes-skin infection. METHODOLOGY/PRINCIPAL FINDINGS: A broth microdilution method revealed that Hainosankyuto did not show a direct anti-bacterial effect against S. pyogenes. Force-feeding Hainosankyuto to infected mice for 4 consecutive days increased the survival rate and reduced the size of local skin lesions compared with mice fed PBS. Although we did not find the significant recovery of survival rate in Hainosankyuto administration only after S. pyogenes infection, the sizes of ulcer lesion were significant smaller after Hainosankyuto administration compared with mice fed PBS. No difference was observed in the anti-bacterial effect of Hainosankyuto between macrolide-susceptible and -resistant strains. Blood bactericidal assay showed that the survival rate of S. pyogenes using the blood from Hainosankyuto-treated mice was lower than that using the blood from untreated mice. We also found increased levels of IL-12, IFN-γ and a decreased level of TNF-α in the serum of S. pyogenes-infected mice treated with Hainosankyuto. Mouse peritoneal macrophage from Hainosankyuto-treated mice had significant phagocytic activity and increased mRNA levels of IL-12, IFN-γ and decreased mRNA level of TNF-α compared with control macrophage. CONCLUSIONS/SIGNIFICANCE: Hainosankyuto increased survival rate after S. pyogenes infection and up-regulated both blood bactericidal activity and macrophage phagocytic activity through modulation of inflammatory cytokines. Our data also suggest Hainosankyuto may be useful for the treatment of S. pyogenes infection more prophylactically than therapeutically

How can natural products serve as a viable source of lead compounds for the development of new/novel anti-malarials?

Malaria continues to be an enormous global health challenge, with millions of new infections and deaths reported annually. This is partly due to the development of resistance by the malaria parasite to the majority of established anti-malarial drugs, a situation that continues to hamper attempts at controlling the disease. This has spurred intensive drug discovery endeavours geared towards identifying novel, highly active anti-malarial drugs, and the identification of quality leads from natural sources would greatly augment these efforts. The current reality is that other than compounds that have their foundation in historic natural products, there are no other compounds in drug discovery as part of lead optimization projects and preclinical development or further that have originated from a natural product start-point in recent years. This paper briefly presents both classical as well as some more modern, but underutilized, approaches that have been applied outside the field of malaria, and which could be considered in enhancing the potential of natural products to provide or inspire the development of anti-malarial lead compounds

Resistance of a Rodent Malaria Parasite to a Thymidylate Synthase Inhibitor Induces an Apoptotic Parasite Death and Imposes a Huge Cost of Fitness

BACKGROUND: The greatest impediment to effective malaria control is drug resistance in Plasmodium falciparum, and thus understanding how resistance impacts on the parasite's fitness and pathogenicity may aid in malaria control strategy. METHODOLOGY/PRINCIPAL FINDINGS: To generate resistance, P. berghei NK65 was subjected to 5-fluoroorotate (FOA, an inhibitor of thymidylate synthase, TS) pressure in mice. After 15 generations of drug pressure, the 2% DT (the delay time for proliferation of parasites to 2% parasitaemia, relative to untreated wild-type controls) reduced from 8 days to 4, equalling the controls. Drug sensitivity studies confirmed that FOA-resistance was stable. During serial passaging in the absence of drug, resistant parasite maintained low growth rates (parasitaemia, 15.5%±2.9, 7 dpi) relative to the wild-type (45.6%±8.4), translating into resistance cost of fitness of 66.0%. The resistant parasite showed an apoptosis-like death, as confirmed by light and transmission electron microscopy and corroborated by oligonucleosomal DNA fragmentation. CONCLUSIONS/SIGNIFICANCE: The resistant parasite was less fit than the wild-type, which implies that in the absence of drug pressure in the field, the wild-type alleles may expand and allow drugs withdrawn due to resistance to be reintroduced. FOA resistance led to depleted dTTP pools, causing thymineless parasite death via apoptosis. This supports the tenet that unicellular eukaryotes, like metazoans, also undergo apoptosis. This is the first report where resistance to a chemical stimulus and not the stimulus itself is shown to induce apoptosis in a unicellular parasite. This finding is relevant in cancer therapy, since thymineless cell death induced by resistance to TS-inhibitors can further be optimized via inhibition of pyrimidine salvage enzymes, thus providing a synergistic impact. We conclude that since apoptosis is a process that can be pharmacologically modulated, the parasite's apoptotic machinery may be exploited as a novel drug target in malaria and other protozoan diseases of medical importance

Plasmodium berghei: lack of antimalarial activity of an analogue of folate precursor, 2,4-diamino-6-hydroxymethylpteridine in a mouse model.

It was earlier hypothesized that the malarial parasite may convert precursors of folate analogues to synthesize de novo inhibitors toxic to itself, but not to the mammalian cell. It was suggested that one such analogue, 2,4-diamino-6-hydroxymethylpteridine (DAP) may be converted to aminopterin (AMP), a known dihydrofolate reductase inhibitor. In the present study, we evaluated the ability of DAP to inhibit proliferation of Plasmodium berghei NK65 in mice, with(out) folinic acid rescue. Cumulative dosages of DAP ranging from 0.1 to 20mg/kg bw. administered either orally or intraperitoneally showed no suppression of parasite growth, or gave mild activities that were not statistically significant (P>0.05). Our findings do not seem to support the hypothesis of selective de novo metabolism of DAP to AMP by the malarial parasite

Differential establishment and survival of<i>Hymenolepis diminuta</i>in syngeneic and outbred rat strains

ABSTRACTExperimentalHymenolepis diminutainfection was carried out in inbred strains of rats (F344/N. JAR-2. LOU/M, TM, DA and DA-bg/bg) and outbred Wistar rats. All strains became infected with this cestode, but clear strain-dependent variation in the susceptibility toH. diminutainfection was observed. Marked differences in worm persistence and worm weight were found at 6 weeks post-infection in TM and DA rats. These strains would be useful to clarify the interactions betweenH. diminutaand its rat host.</jats:p

Plasmodium berghei: lack of antimalarial activity of an analogue of folate precursor, 2,4-diamino-6-hydroxymethylpteridine in a mouse model.

It was earlier hypothesized that the malarial parasite may convert precursors of folate analogues to synthesize de novo inhibitors toxic to itself, but not to the mammalian cell. It was suggested that one such analogue, 2,4-diamino-6-hydroxymethylpteridine (DAP) may be converted to aminopterin (AMP), a known dihydrofolate reductase inhibitor. In the present study, we evaluated the ability of DAP to inhibit proliferation of Plasmodium berghei NK65 in mice, with(out) folinic acid rescue. Cumulative dosages of DAP ranging from 0.1 to 20mg/kg bw. administered either orally or intraperitoneally showed no suppression of parasite growth, or gave mild activities that were not statistically significant (P>0.05). Our findings do not seem to support the hypothesis of selective de novo metabolism of DAP to AMP by the malarial parasite

Chloroquine efficacy in Plasmodium berghei NK65-infected ICR mice, with reference to the influence of initial parasite load and starting day of drug administration on the outcome of treatment.

We examined whether the initial number of parasites inoculated and the starting day of medication post-infection influenced the antimalarial efficacy of chloroquine (CQ) against Plasmodium berghei NK65 infection in ICR mice. Male ICR mice were inoculated intraperitoneally with 1 x 10(5), 1x10(6), 1 x 10(7), 1 x 10(8) P. berghei NK65-parasitized erythrocytes (pRBC). In the treated group, all mice received an oral dose of 20 mg/kg of CQ base for 4 days starting on day 0 after infection. From day 3, Giemsa-stained thin blood smears from tail vein blood were used to assess parasitemia. Mice in the untreated control in each group showed a progressive increase in parasitemia leading to death. Treatment of mice, inoculated with 1 x 10(5), 1 x 10(6) and 1 x 10(7) pRBC, with CQ showed a marked effect. All the mice survived during the experiment. During the observation period, malaria parasites could not be detected on microscopic examination. Conversely, mice inoculated with 1 x 10(8) pRBC showed little response to CQ treatment, and all mice showed a progressive increase in parasitemia and ultimately died. In another experiment, mice infected with 1 x 10(3) and 1x 10(5) pRBC were treated with an oral four-day dosage of 20 mg/kg of CQ base from days 2, 3 or 4 post-infection. Treatment of mice, inoculated with 1 x 10(3) pRBC, with CQ from days 2 and 3 showed a marked effect. All mice survived during the experiment. However, treatment from day 4 showed a limited derease in parasitemia and all the mice ultimately died. On the other hand, treatment from day 2 showed a marked effect against 1 x 10(5) P. berghei NK65-infected mice, but treatment from days 3 or 4 was only slightly effective and all the mice died with an increasing parasitemia. The present results indicate that in in vivo antimalarial drug-assay systems, several factors, sush as initial parasite load and starting time of treatment may influence the drug response in the host

Novel Rational Drug Design Strategies with Potential to Revolutionize Malaria Chemotherapy

Efforts to develop an effective malaria vaccine are yet to be successful and thus chemotherapy remains the mainstay of malaria control strategy. Plasmodium falciparum, the parasite that causes about 90% of all global malaria cases is increasingly becoming resistant to most antimalarial drugs in clinical use. This dire situation is aggravated by reports from Southeast Asia, of the parasite becoming resistant to the “magic bullet” artemisinins, the last line of defense in malaria chemotherapy. Drug development is a laborious and time consuming process, and thus antimalarial drug discovery approaches currently being deployed largely include optimization of therapy with available drugs—including combination therapy and developing analogues of the existing drugs. However, the latter strategy may be hampered by crossresistance, since agents that are closely related chemically may share similar mechanisms of action and/or targets. This may render new drugs ineffective even before they are brought to clinical use. Evaluation of drug-resistance reversers (chemosensitizers) against quinoline-based drugs such as chloroquine and mefloquine is another approach that is being explored. Recently, evaluation of new chemotherapeutic targets is gaining new impetus as knowledge of malaria parasite biology expands. Also, single but hybrid molecules with dual functionality and/or targets have been developed through rational drug design approach, termed as “covalent bitherapy”. Since desperate times call for radical measures, this review aims to explore novel rational drug-design strategies potentially capable of revolutionizing malaria therapy. We thus explore malaria apoptosis machinery as a novel drug target, and also discuss the potential of hybrid molecules as well as prodrugs and double prodrugs in malaria chemotherapy

Effects of anthelmintics on the development of eggs of Angiostrongylus costaricensis in vitro

Effects of the anthelmintics, pyrantel and levamisole, on egg development of Angiostrongylus costaricensis were studied in vitro. After 7 days, about 80% of eggs developed to first-stage larvae in Ham's F-12 medium with 10% foetal calf serum under 5% CO2. Significant inhibition of development was caused by pyrantel (10-9–10-8 g ml-1) and levamisole (10-9–10-8 g ml-1) (Mann-Whitney U-test; P&lt;0.05), and none of the eggs developed to first-stage larvae in higher concentrations of these anthelmintics (10-7 g ml-1). Furthermore, incubation with these drugs at 10-8 g ml-1 for at least 3 h or at 10-4 g ml-1 for 1 h caused irreversible effects on egg development.</jats:p