In vitro and in vivo anti-Leishmania activity of polysubstituted synthetic chalcones

The in vitro screening of 43 polysubstituted chalcones against Leishmania amazonensis axenic amastigotes, led to the evaluation of 9 of them in a macrophage-infected model with the two other most infectious Leishmania species prevalent in Peru (L. braziliensis and L. peruviana). The five most active and selective chalcones were studied in vivo, resulting on the identification of two chalcones with high reduction parasite burden percentages.     

In vitro and in vivo anti-Trypanosoma cruzi activity of a novel nitro-derivative

Nitroarylidenemalononitriles and their cyanoacetamide derivatives with remarkable anti-epimastigote properties, were synthesized attempting to obtain new 3,5-diamino-4-(5'-nitroarylidene)-4H-thiadiazine 1,1-dioxide derivatives, which in previous reports had shown anti-Trypanosoma cruzi activity. Tests to evaluate the cytotoxicity of compounds were performed on J774 macrophages. 5-nitro-2-thienyl-malononitrile (5NO2TM), was the only product which maintained a high anti-epimastigote activity at concentrations in which it was no longer cytotoxic, thus it was assayed against intracellular amastigotes. Its anti-amastigote activity was similar to that of nifurtimox. Afterwards in vivo toxicity and anti-chagasic activity were determined. A reduction in parasitemia was observed.     

In vitro and in vivo activity of ribavirin against Andes virus infection

Pathogenic hantaviruses are a closely related group of rodent-borne viruses which are responsible for two distinct diseases in humans, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (HPS, otherwise known as hantavirus cardiopulmonary syndrome, HCPS). The antiviral effect of ribavirin against Old World hantaviruses, most notably Hantaan virus, is well documented; however, only a few studies have addressed its inhibitory effect on New World hantaviruses. In the present study, we demonstrate that ribavirin is highly active against Andes virus (ANDV), an important etiological agent of HPS, both in vitro and in vivo using a lethal hamster model of HPS. Treatment of ANDV infected Vero E6 cells with ribavirin resulted in dose-dependent reductions in viral RNA and protein as well as virus yields with a half maximal inhibitory concentration between 5 and 12.5 μg ml(-1). In hamsters, treatment with as little as 5 mg kg(-1) day(-1) was 100% effective at preventing lethal HPS disease when therapy was administered by intraperitoneal injection from day 1 through day 10 post-infection. Significant reductions were observed in ANDV RNA and antigen positive cells in lung and liver tissues. Ribavirin remained completely protective when administered by intraperitoneal injections up to three days post-infection. In addition, we show that daily oral ribavirin therapy initiated 1 day post-infection and continuing for ten days is also protective against lethal ANDV disease, even at doses of 5 mg kg(-1) day(-1). Our results suggest ribavirin treatment is beneficial for postexposure prophylaxis against HPS-causing hantaviruses and should be considered in scenarios where exposure to the virus is probable. The similarities between the results obtained in this study and those from previous clinical evaluations of ribavirin against HPS, further validate the hamster model of lethal HPS and demonstrate its usefulness in screening antiviral agents against this disease.     

Anti-inflammatory activity of licochalcone A isolated from Glycyrrhiza inflata

Licochalcone A was isolated from the roots of Glycyrrhiza inflata and evaluated for its anti-inflammatory activity in xylene-induced mice ear edema and carrageenan-induced paw edema tests. At the same time, the inhibition of prostaglandin biosynthesis by licochalcone A was also studied in lipopolysaccharide (LPS)-induced mouse macrophage cells. At 5 mg/ ear, licochalcone A showed remarkable effects against acute inflammation induced by xylene, and at the doses of 2.5, 5, 10 mg/kg (p.o.), licochalcone A reduced significantly paw edema induced by carrageenan compared to the control at the fourth hour. Both COX-2 activity and expression were significantly inhibited by licochalcone A at all the test doses. Therefore, licochalcone A could be a useful compound for the development of new anti-inflammatory agents.     

In vitro osteogenic differentiation of human ES cells

Since their isolation in 1998, human embryonic stem (hES) cells have been shown to be capable of adopting various cell fates in vitro. Here, we present in vitro data demonstrating the directed commitment of human embryonic stem cells to the osteogenic lineage. Human ES cells are shown to respond to factors that promote osteogenesis, leading to activation of the osteogenic markers osteocalcin, parathyroid hormone receptor, bone sialoprotein, osteopontin, cbfa1, and collagen 1. Moreover, the mineralized nodules obtained are composed of hydroxyapatite, further establishing the similarity of osteoblasts in culture to bone. These results show that osteoblasts can be derived from human ES cultures in vitro and provide the basis for comparison of adult and embryonic-derived osteogenesis, and for an investigation of potential applications for hES cells in orthopaedic tissue repair.     

In vitro and in vivo dimerization of human endonuclease III stimulates its activity

Human endonuclease III (hNTH1), a DNA glycosylase with associated abasic lyase activity, repairs various mutagenic and toxic-oxidized DNA lesions, including thymine glycol. We demonstrate for the first time that the full-length hNTH1 positively cooperates in product formation as a function of enzyme concentration. The protein concentrations that caused cooperativity in turnover also exhibited dimerization, independent of DNA binding. Earlier we had found that the hNTH1 consists of two domains: a well conserved catalytic domain, and an inhibitory N-terminal tail. The N-terminal truncated proteins neither undergo dimerization, nor do they show cooperativity in turnover, indicating that the homodimerization of hNTH1 is specific and requires the N-terminal tail. Further kinetic analysis at transition states reveals that this homodimerization stimulates an 11-fold increase in the rate of release of the final product, an AP-site with a 3'-nick, and that it does not affect other intermediate reaction rates, including those of DNA N-glycosylase or AP lyase activities that are modulated by previously reported interacting proteins, YB-1, APE1, and XPG. Thus, the site of modulating action of the dimer on the hNTH1 reaction steps is unique. Moreover, the high intranuclear (2.3 microM) and cytosolic (0.65 microM) concentrations of hNTH1 determined here support the possibility of in vivo dimerization; indeed, in vivo protein cross-linking showed the presence of the dimer in the nucleus of HeLa cells. Therefore, it is likely that the dimerization of hNTH1 involving the N-terminal tail masks the inhibitory effect of this tail and plays a critical role in its catalytic turnover in the cell.     

In vitro and in vivo antiherpetic activity of three new synthetic brassinosteroid analogues

Brassinosteroids are a novel group of steroids that appear to be ubiquitous in plants and are essential for normal plant growth and development. It has been previously reported that brassinosteroid analogues exert an antiviral activity against herpes simplex virus type 1 (HSV-1) and arenaviruses. In the present study, we report the chemical synthesis of compounds (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one (2), (22S,23S)-5alpha-fluoro-3beta-22,23-trihydroxystigmastan-6-one (3), (22S,23S)-3beta,5alpha,22,23-tetrahydroxy-stigmastan-6-one (4) as well as their antiherpetic activity both in a human conjunctive cell line (IOBA-NHC) and in the murine herpetic stromal keratitis (HSK) experimental model. All compounds prevented HSV-1 multiplication in NHC cells in a dose dependent manner when added after infection with no cytotoxicity. Administration of compounds 2, 3, and 4 to the eyes of mice at 1, 2, and 3 days post-infection delayed and reduced the incidence of HSK, consisting mainly of inflammation, vascularization, and necrosis, compared to untreated, infected mice. However, viral titers of eye washes showed no differences among samples from treated and untreated mice. Since the decrease in the percentage of mice with ocular lesions occurred 5 days after treatment had ended, we suggest that brassinosteroids 2, 3, and 4 did not exert a direct antiviral effect in vivo, but rather may play a role in immune-mediated stromal inflammation, which would explain the improvement of the clinical signs of HSK observed.     

In vitro and in vivo investigations on the antitumour activity of Chelidonium majus

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In vitro and in vivo activity of an antibacterial peptide analog against uropathogens

The alarming rate of bacterial resistance induction highlights the clinical need for antimicrobial agents that act by novel modes of action. Based on the activity profile, the general tissue distribution and renal clearance of peptide-based drugs, we hypothesized that our newly developed pyrrhocoricin derivative would be able to fight resistant uropathogens in vitro and in vivo. Indeed, the Pip-pyrr-MeArg dimer killed all 11 urinary tract infection-related Escherichia coli and Klebsiella pneumoniae strains we studied in the sub-low micromolar concentration range. Almost all control antibiotics, including the currently leading trimethoprim-sulfametoxazole combination for urinary tract infection, remained without considerable activity against two or more of these bacterial strains. In a mouse ascending urinary tract infection model with E. coli CFT073 as pathogen, two doses of intravenous, subcutaneous or oral treatment with the Pip-pyrr-MeArg derivative reduced the bacterial counts in the kidneys, bladder and urine to varying levels. Statistically significant elimination or reduction of bacteria compared to untreated animals was observed at dual intravenous or subcutaneous doses of 0.4 or 10mg/kg, respectively. Serial passage of the same E. coli strain in the presence of sublethal doses of the designed peptide failed to generate resistant mutants. The Pip-pyrr-MeArg dimer showed no toxicity to COS-7 cells to the highest 500microM concentration studied.     

In vitro and in vivo antimycobacterial activity of antiinflammatory drug, diclofenac sodium

The non-steroidal antiinflammatory drug diclofenac sodium exhibited remarkable inhibitory action against both drug sensitive and drug resistant clinical isolates of Mycobacterium tuberculosis, as well as other mycobacteria. This agent was tested in vitro against 45 different strains of mycobacteria, most of which were inhibited by the drug at 10-25 microg/ml concentration. When tested in vivo, diclofenac, injected at 10 mg/kg body weight of a Swiss strain of white mice, could significantly protect them when challenged with a 50 median lethal dose of M. tuberculosis H37 Rv102. According to Chi-square test, the in vivo data were highly significant (P<0.01).     

In vitro and in vivo antitumour activity of a chimeric anti-CD19 antibody

Mouse monoclonal antibodies to CD19 detect an antigenic determinant expressed exclusively on the surface of B lymphocytes, and have previously been shown to be potentially useful therapeutic reagents for human B cell lymphoma. We report the production and characterization of a mouse/human chimeric antibody, cCD19, with potent in vivo antitumour activity. The genes encoding the variable domains for heavy (V_H) and light (V_L) chains were subcloned into eukaryotic expression vectors containing human constant region genes (IgG1 and ), and co-transfected into non-secreting Sp2/0 mouse myeloma cells. Intraperitoneal administration of cCD19 produced inhibition of growth of subcutaneous CD19⁺ Sultan human B lymphoma tumours inscid/scid mice. When the antibody was administered 18 and 20 days after subcutaneous tumour inoculation, an approximately 30% reduction in tumour size was noted by day 29. cCD19 faithfully mimicked the in vitro binding characteristics of mCD19 as (a) the chimeric antibody was shown by flow cytometry to bind exclusively to cell lines that expressed CD19, (b) cCD19 was able to inhibit the binding of mCD19 on CD19⁺ cells completely and (c) the affinity of binding of the two antibodies was not significantly different [K _a=(2.03±1.5)×10⁸]. In biodistribution studies, up to 14.8% of the total injected antibody dose per gram of tissue was localized in CD19⁺ Sultan tumours at 24 h approximately, 14.4% was present in the tumors at 48 h and about 13.7% at 72 h. These levels were comparable to mCD19 administered in the same fashion. cCD19 conjugated to idarubicin was specifically and strongly cytotoxic to CD19⁺ cells cultured in vitro, and demonstrated an IC₅₀ of 0.17 M, similar to that of mCD19 (0.32 M) and approximately 14-fold greater than the IC₅₀ of free idarubicin. The specific cytotoxic capacity of cCD19 and its likely reduced immunogenicity suggest that it may potentially be of use in the treatment of refractory B cell lymphoma in humans.     

In vitro and in vivo immunomodulatory activity of sulfated polysaccharides from Enteromorpha prolifera

Water-soluble sulfated polysaccharides extracted from Enteromorpha prolifera and fractionated using ion-exchange chromatography (crude, F₁, F₂ and F₃ fractions) were investigated to determine their in vitro and in vivo immunomodulatory activities. The sulfated polysaccharides, especially the F₁ and F₂ fractions, stimulated a macrophage cell line, Raw 264.7, inducing considerable nitric oxide (NO) and various cytokine production via up-regulated mRNA expression. The in vivo experiment results show that the sulfated polysaccharides (the crude and F₂ fractions) significantly increased Con A-induced splenocyte proliferation, revealing their potential comitogenic activity. In addition, IFN-γ and IL-2 secretions were considerably increased by the F₂ fraction without altering the release of IL-4 and IL-5. This implies that the F₂ fraction can activate T cells by up-regulating Th-1 response and that Th-1 cells might be the main target cells of the F₂ fraction. These in vitro and in vivo results suggest that the sulfated polysaccharides are strong immunostimulators.     

In vivo and in vitro activity and mechanism of action of the multidrug cytarabine-L-glycerylyl-fluorodeoxyuridine

Multidrugs have the potential to bypass resistance. We investigated the in vitro activity and resistance circumvention of the multidrug cytarabine-L-fluorodeoxyuridine (AraC-L-5FdU), linked via a glycerophospholipid linkage. Cytotoxicity was determined using sensitive (A2780, FM3A/0) and resistant (AG6000, AraC resistant, deoxycytidine kinase deficient; FM3A/TK-, 5FdU resistant, thymidine kinase deficient) cell lines. Circumvention of nucleoside transporter and activating enzymes was determined using specific inhibitors, HPLC analysis and standard radioactivity assays. AraC-L-5FdU was active (IC50: 0.03 microM in both A2780 and FM3A/0), had some activity in AG6000 (IC50: 0.28 microM), but no activity in FM3A/TK(-) (IC50: 18.3 microM). AraC-nucleotides were not detected in AG6000. 5FdU-nucleotides were detected in all cell lines. AraC-L-5FdU did not inhibit TS in FM3A/TK(-) (5%). Since phosphatase/nucleotidase-inhibition reduced cytotoxicity 7-70-fold, cleavage seems to be outside the cell, presumably to nucleotides, and then to nucleosides. The multidrug was orally active in the HT-29 colon carcinoma xenografts which are resistant toward the single drugs.     

Gene therapy for bone formation: in vitro and in vivo osteogenic activity of an adenovirus expressing BMP7

Bone morphogenetic proteins (BMPs) are well-established agents for inducing orthotopic and ectopic bone formation. However, their clinical usefulness as regenerative agents may be limited by a short in vivo half-life and low specific activity. BMP gene therapy is an alternative route for exploiting the bone-inductive activity of this class of molecules. To test the feasibility of this approach, we examined the osteogenic activity of AdCMV-BMP7, an adenovirus containing BMP7 cDNA under control of the CMV promoter that was constructed using Cre/lox recombination (Hardy et al. [1997] J. Virol. 71:1842-1849). Adenovirus vectors were shown to readily infect a wide variety of cell types in vitro including osteoblasts, fibroblasts, and myoblasts. COS7 cells transduced with AdCMV-BMP7 produced high levels of BMP-7 (approximately 0.5 microg/10(6) cells). Furthermore, transduction of C2C12 murine myoblast cells with AdCMVBMP-7 suppressed the muscle phenotype and induced in vitro osteoblast differentiation. To test its in vivo biological activity, AdCMV-BMP7 was mixed with a bovine bone-derived collagen carrier (10(8) plaque-forming units virus/site) and was implanted into mouse muscle and dermal pouches. In both cases, an ossicle containing cortical and trabecular bone and a clearly defined marrow cavity formed at the site of virus implantation within 4 weeks. These data demonstrate that AdCMV-BMP7 transduced cells produce biologically active BMP-7 both in vitro and in vivo and show that gene therapy by direct viral transduction using a virus/matrix implant may be a viable route for stimulating bone regeneration.     

In vitro and in vivo activity of soluble cross-linked uricase-albumin polymers: A model for enzyme therapy

The possibility of using soluble cross-linked enzyme-albumin polymers as a means of enzyme therapy for the treatment of certain enzyme deficiency diseases is investigated. The hyperuricemic Dalmatian coach hound is used as an experimental animal and the enzyme uricase (urate oxidase) as the administered enzyme. Chemically cross-linking uricase with an excess of canine albumin yields a soluble enzyme polymer that is significantly more heat stable and resistant to proteolytic activity than the native enzyme. Intravenous administration of similar amounts of enzyme in the native or polymeric form indicated that the “solubilized” enzyme survived in the circulation for a longer period of time (clearance half-time of 26 hours as opposed to 4 hours for the native enzyme) and was more effective in lowering plasma uric acid levels for longer periods. In vivo administration of the native enzyme lowered uric acid levels by about 35% with a return to normal levels with a half-time of about 24 hours. Subsequent injections of native uricase proved less effective and produced a severe hypersensitivity reaction following the third injection. No such adverse reactions or decreased activity of the administered “solubilized” uricase-albumin polymers were observed. The plasma uric acid levels were decreased by about 40% and only after 48 hours did the substrate levels begin to rise towards their resting levels.