In vitro and in vivo evaluation of the antimalarial MMV665831 and structural analogs

Antimalarial candidates possessing novel mechanisms of action are needed to control drug resistant Plasmodium falciparum. We were drawn to Malaria Box compound 1 (MMV665831) by virtue of its excellent in vitro potency, and twelve analogs were prepared to probe its structure–activity relationship. Modulation of the diethyl amino group was fruitful, producing compound 25, which was twice as potent as 1 against cultured parasites. Efforts were made to modify the phenolic Mannich base functionality of 1, to prevent formation of a reactive quinone methide. Homologated analog 28 had reduced potency relative to 1, but still inhibited growth with EC₅₀ ≤ 200 nM. Thus, the antimalarial activity of 1 does not derive from quinone methide formation. Chemical stability studies on dimethyl analog 2 showed remarkable hydrolytic stability of both the phenolic Mannich base and ethyl ester moieties, and 1 was evaluated for in vivo efficacy in P. berghei-infected mice (40 mg/kg, oral). Unfortunately, no reduction in parasitemia was seen relative to control. These results are discussed in the context of measured plasma and hepatocyte stabilities, with reference to structurally-related, orally-efficacious antimalarials.     

In vitro and in vivo evaluation of O-alkyl derivatives of tramadol

Tramadol is a centrally acting opioid analgesic structurally related to codeine and morphine. O-Alkyl, N-desmethyl, and non-phenol containing derivatives of tramadol were synthesized to probe their effect on metabolic stability and both in vitro and in vivo potency.     

Synthesis and evaluation of phenylequine for antimalarial activity in vitro and in vivo

Synthesis of the potent antiplasmodial 4-aminoquinoline, phenylequine (PQ), is reported for the first time. PQ and the two analogues show increased efficacy in moving from the chloroquine sensitive D10 to the chloroquine resistant K1 strain in vitro. The in vivo efficacy of PQ, and salts thereof, have been determined in Plasmodium berghei ANKA and Plasmodium yoelii. Phenylequine hydrochloride has shown an ED₅₀ of 0.81 in P. yoelii (cf chloroquine ED₅₀ = 1.31).     

In vitro and in vivo evaluation of polymethylene tetraamine derivatives as NMDA receptor channel blockers

The biological activities of six symmetrically substituted 2-methoxy-benzyl polymethylene tetraamines (1–4) and diphenylethyl polymethylene tetraamines (5 and 6) as N-methyl-d-aspartate (NMDA) receptor channel blockers, were evaluated in vitro and in vivo. Although all compounds exhibited stronger channel block activities in comparison to memantine in Xenopus oocytes voltage clamped at ?70 mV, only compound 2 (0.4 mg/kg intravenous injection) decreased the size of brain infarction in a photochemically induced thrombosis model mice at the same extent of memantine (10 mg/kg intravenous injection). Other compounds (1, 3, 4, 5 and 6) did not decrease the size of brain infarction significantly due to the limited injection doses. The present study suggests that compound 2 could represent a valuable lead compound to design low toxicity polyamines for clinical use against stroke.     

In vivo active antimalarial isonitriles

Building on the lead from antimalarial isonitriles 1-4 of marine origin, several easily accessible synthetic isonitriles were assessed for their antimalarial activity against Plasmodium falciparum (in vitro) and multidrug resistant Plasmodium yoelii in Swiss mice model (in vivo). Isonitrile 11 has shown promising activity in both these assays.     

In vivo and in vitro genotoxic evaluation of indorenate

5-Methoxytryptamine, beta-methylcarboxylate hydrochloride (indorenate) is a new antihypertensive serotonin derivative. We evaluated its genotoxic activity using the mouse bone marrow and cytogenetic test and the human lymphocyte culture cytogenetic assay. As endpoints we measured chromosomal aberrations, sister-chromatid exchanges and cellular proliferation kinetics. Our results agree in both systems showing that indorenate is a non-genotoxic agent in these assays.     

In vitro antimalarial activity and molecular docking analysis of 4-aminoquinoline-clubbed 1,3,5-triazine derivatives

Aims: Present report describes the in vitro antimalarial activity and docking analysis of seven 4‐aminoquinoline‐clubbed 1,3,5‐triazine derivatives on pf‐DHFR‐TS. Methods and Results: The antimalarial activity was evaluated in vitro against chloroquine‐sensitive 3D7 strain of Plasmodium falciparum. Compounds were docked onto the active site of pf‐DHFR‐TS using docking server to explicate necessary structural requirements for antimalarial activity. Conclusion: Title molecules demonstrated considerable bioactivity against the malaria parasite. Docking analysis revealed deep engulfment of the molecules into the inner groove of pf‐DHFR‐TS active site by making stable ligand–receptor posses. Hydrophobic interaction was identified as the only major interacting force playing a role between ligand–receptor interaction and minor with hydrogen bonds. Signi?cance and Impact of the study: The study provided the novel insight into the necessary structural requirement for rationale‐based antimalarial drug discovery.     

Synthesis and antimalarial evaluation of novel isocryptolepine derivatives

A series of mono- and di-substituted analogues of isocryptolepine have been synthesized and evaluated for in vitro antimalarial activity against chloroquine sensitive (3D7) and resistant (W2mef) Plasmodium falciparum and for cytotoxicity (3T3 cells). Di-halogenated compounds were the most potent derivatives and 8-bromo-2-chloroisocryptolepine displayed the highest selectivity index (106; the ratio of cytotoxicity (IC(50)=9005 nM) to antimalarial activity (IC(50)=85 nM)). Our evaluation of novel isocryptolepine compounds has demonstrated that di-halogenated derivatives are promising antimalarial lead compounds.     

In vitro and in vivo antimalarial activity of peptidomimetic protein farnesyltransferase inhibitors with improved membrane permeability

A series of protein farnesyltransferase inhibitor ester prodrugs of FTI-2148 (17) were synthesized in order to evaluate the effects of ester structure modification on antimalarial activity and for further development of a farnesyltransferase inhibitor with in vivo activity. Evaluation against P. falciparum in red blood cells showed that all the investigated esters exhibited significant antimalarial activity, with the benzyl ester 16 showing the best inhibition (ED₅₀ = 150 nM). Additionally, compound 16 displayed in vivo activity and was found to suppress parasitemia by 46.1% at a dose of 50 mg kg⁻¹ day⁻¹ against Plasmodium berghei in mice. The enhanced inhibition potency of the esters is consistent with improved cell membrane permeability compared to that of the free acid. The results of this study suggest that protein farnesyltransferase is a valid antimalarial drug target and that the antimalarial activity of these compounds derives from a balance between the hydrophobic character and the size and conformation of the ester moiety.     

In vitro and in vivo evaluation of hydrophilic dendronized linear polymers

Rigid-rod dendronized linear polymers consisting of a poly(4-hydroxystyrene) backbone and fourth-generation polyester dendrons were evaluated in vitro and in vivo to determine their suitability as drug delivery vectors. Cytotoxicity assays indicated that the polymers were well tolerated by cells in vitro. Biodistribution studies of the polymers in both nontumored and tumored mice revealed that as for random coil linear polymers, renal clearance was a function of polymer size, with significant urinary excretion observed for a 67 kDa dendronized polymer. High accumulation in organs of the reticuloendothelial system was exhibited by a dendronized polymer with a very high molecular weight (M(n) = 1740 kDa), but was not as significant for smaller polymers with M(n) = 67 kDa and M(n) = 251 kDa. The rank order for tumor accumulation of the polymers on a percent injected dose per gram tumor basis was 251 kDa approximately 1740 kDa > 67 kDa. These data will help guide the selection of highly functionalizable rigid-rod dendronized polymers with pharmacokinetic properties appropriate for use as drug carriers.     

In vitro cytotoxicity evaluation of some substituted isatin derivatives

A range of substituted 1H-indole-2,3-diones (isatins) were synthesized using standard procedures and their cytotoxicity evaluated against the human monocyte-like histiocytic lymphoma (U937) cell line in vitro. SAR studies identified C(5), C(6), and C(7) substitution greatly enhanced activity with some di- and tri-halogenated isatins giving IC(50) values <10 microM. Of the 23 compounds tested, four were selected for further screening against a panel of five human cancer cell lines. These compounds, in general, showed greater selectivity toward leukemia and lymphoma cells over breast, prostate, and colorectal carcinoma cell lines. The most active compound, 5,6,7-tribromoisatin (2p), was found to be antiproliferative at low micromolar concentrations and also activated the effector caspases 3 and 7 in a dose-dependent manner. These results indicate that di- and tri-substituted isatins may be useful leads for anticancer drug development in the future.     

Synthesis of three classes of rhodacyanine dyes and evaluation of their in vitro and in vivo antimalarial activity

Selected members of three classes of rhodacyanine dyes, [0, 0]-, [1, 0]-, and [0, 0, 0]-rhodacyanines, were synthesized and their in vitro antimalarial activities against Plasmodium falciparum K1 (chloroquine-resistant strain) as well as their in vivo activities against P. berghei in mice were determined. The novel [0, 0, 0]-rhodacynines, 3e and 3h, possessing a benzothiazole moiety, were shown to have highly promising antimalarial activities in vivo. Moreover, the [0, 0, 0]-rhodacyanines were found to be orally bioavailable.     

In vitro and in vivo evaluation of topical formulations of Spantide II

The purpose of this study was to develop and evaluate topical formulations of Spantide II, a neurokinin-1 receptor (NK-1R) antagonist, for the treatment of inflammatory skin disorders. Spantide II lotion and gel was formulated with and without n-methyl-2-pyrrolidone (NMP) as a penetration enhancer. The release of Spantide II from gels was evaluated using microporous polyethylene and polypropylene membranes in a Franz Diffusion cell setup. In vitro percutaneous absorption of Spantide II from lotion and gel formulations was evaluated using the above setup by replacing the membranes with hairless rat skin. The in vivo anti-inflammatory activity of Spantide II formulations was evaluated in an allergic contact dermatitis (ACD) mouse model. Among different gels studied, PF127 gel showed highest (70-fold) release of Spantide II compared with hydroxypropyl methylcellulose (HPMC) and hydroxypropyl cellulose (HPC) gels. Lotion and gel formulations with or without NMP showed no detectable levels of Spantide II in the receiver compartment of the Franz diffusion cell until 24 hours. However, Spantide II showed significant retention in epidermis and dermis from lotion and gel formulations at 24 hours. The dermal levels increased ≈3.5- and 2-fold when the lotion and gel formulations contained NMP as compared with the formulation with no NMP (P<.05). The in vivo studies indicated that Spantide II formulations with NMP were effective in significantly reducing ACD response, similar to dexamethasone (0.5 mM). In conclusion, Spantide II was stable as a topical formulation and delivered to target skin tissue (epidermis and dermis) for the treatment of ACD. In addition this study supports the role of cutaneous neurosensory system in modulating inflammatory responses in the skin. Published: October 31, 2005     

Synthesis and antimalarial activity of urenyl Bis-chalcone in vitro and in vivo

Some novel derivatives of Bis-chalcone were synthesized and characterized by their physical and spectral data. All the synthesized compounds were subsequently screened for in vitro globin hydrolysis, β-hematin formation, and murine Plasmodium berghei, using chloroquine as the reference drug. Most of the synthesized compounds exhibited mild to moderate susceptibilities toward the parasite in comparison with the standard. The most active antimalarial compound was 1,1-Bis-[(3′,4′-N-(urenylphenyl)-3-(3″,4″,5″-trimethoxyphenyl)]-2-propen-1-one 5, with a percentage of inhibition of heme polymerization of 87.05?±?0.77, and this compound increased the survival time after infection, reduce the parasitemia and delay the progression of malaria.     

In vitro cytotoxicity of some natural and semi-synthetic isocoumarins from Paepalanthus bromelioides

Numerous natural compounds have a potential for therapeutic applications, but may have to be chemically modified to alter toxic side effects. We investigated structural parameters that could affect the cytotoxicity of isocoumarins similar to 9,10-dihydroxy-5,7-dimethoxy-1H-naphtho(2,3c)pyran-1-one (paepalantine 1). Paepalantine 1 has antimicrobial activity, as well as significant in vitro cytotoxic effects in the McCoy cell line. Two other natural and two semi-synthetic isocoumarins with similar structures obtained from the capitula of Paepalanthus bromelioides were tested on the same cell line by the neutral red assay. Substitution of the 9 and/or 10-OH group made these compounds less cytotoxic.     

In vivo and in vitro evaluation of cell-mediated immunity in patients with paracoccidioidomycosis

The cellular immune response to specific and nonspecific agents was investigated. both in vivo and in vitro, in 19 patients with paracoceidioidomycosis. In addition, the immunologic study of an investigator aceidentally inoculated with P. brasiliensis was included in this study. Nearly half of the patients showed depressed cell-mediated immune responses, as evaluated by intradermal tests with an antigenic preparation from P. brasiliensis (P.b.Ag.), ubiquitous antigens, and by the ability to develop sensitization to 2,4-dinitrochlorobenzene. A similar proportion of impaired responses was observed when the patients' lymphocytes were cultured with phytohemagglutinin (PHA). C'. albicans antigen and P.h.Ag. A factor was detected in the plasma of some patients which reduced the ability of patients' and normal lymphocytes to undergo blastic transformation. A positive correlation was found between the ability to develop delayed cutaneous hypersensitivity reactions to P.b.Ag. and other ubiquitous antigens, normal in vitro responsiveness to PHA and the absence of humoral blastogenic inhibitory factor. The inhibition of leukocyte migration, but not lymphocyte transformation, correlated positively with delayed hypersensitivity. The percentage of T lymphocytes was significantly reduced in the group of patients, being the absolute number and percentage of B cells bearing receptors tor complement normal. Two polar immunological patterns emerged. One characterized by positiveness in the skin test to P.b.Ag. and lack of significant abnormalities in cellular immunity, and another anergic to P.b.Ag., with cell mediated immunity severely depressed. Between the two polar groups, there were patients with intermediary patterns of immune response. This paper also includes the results obtained with the administration of transfer factor and levamisole to some of the patients.     

In vitro and in vivo evaluation of proximal tubular acidification in aging rats

The normal aging process is accompanied by a progressive deterioration of renal function. We studied the kinetics of proximal tubular acidification of young (3 mo) and aging (22 mo) rats using in vivo and in vitro techniques. Blood acid-base parameters were similar in both groups. The maximum velocity of the Na(+)/H(+) exchange (NHE) in brush-border membrane vesicles (BBMV) showed a 72% decrease in aging compared with young rats, whereas the Michaelis constant remained unchanged. The NHE3 isoform of the Na(+)/H(+) exchanger was detected in BBMV by Western blot in both groups, and a decrease of 90% in the abundance was observed in aging rats. Micropuncture experiments with simultaneous luminal and peritubular perfusion with phosphate Ringer and continuous measurement of intratubular pH showed an acidification rate constant 34% smaller in aging compared with young rats. Proton flux was 48% lower in aging than in young rats. The present results suggest that proximal tubular acidification is impaired with aging.     

In vitro and in vivo toxicity evaluation of the freshwater cyanobacterium Heteroleiblenia kuetzingii

Cyanobacteria are prokaryotic organisms characterized by their ability to produce secondary metabolites with different biological activities. The aim of this work was to evaluate the in vitro and in vivo toxicity of the cosmopolitan freshwater cyanobacterium H. kuetzingii. An extract from H. kuetzingii and cyanobacterial growth media were assessed for presence of intracellular and extracellular toxins by in vitro tests using primary cell cultures from mouse kidney and fibroblasts, cell lines A549 and 3T3, a fish cell line RTgill-W1 as well as by a traditional in vivo mouse bioassay. The presence of toxicity was compared with the ELISA and HPLC data for corresponding cyanotoxins. In vitro tests showed pronounced cytotoxicity of the cyanobacterium extract and growth medium in which H. kuetzingii released potential extracellular toxic compounds as the mammalian cells were significantly more sensitive to exposure compared to the fish cells. Histopathological analyses of the liver and kidneys of treated mice showed pathological changes such as leukocyte infiltration and necrosis, changes in the proximal and distal convoluted tubules, lack of differentiation of Bowman’s space, enlarged Bowman’s capsules and massive hemorrhages. ELISA and HPLC analyses confirmed the presence of saxitoxins and microcystins at low concentrations. In addition, the histological analyses suggest that H. kuetzingii produces other, yet unknown toxic metabolites. Monitoring efforts are therefore required to evaluate the potential hazard for the freshwater aquatic systems and possible public health implications associated with this cyanobacterium.     

In vitro and in vivo evaluation of oridonin-loaded long circulating nanostructured lipid carriers

The purpose of this study was to develop poly(ethylene glycol)-coated nanostructured lipid carriers (PEG-NLC) for parenteral delivery of oridonin (ORI) to prolong drug circulation time in blood. Oridonin-loaded PEG-NLC (ORI-PEG-NLC) consisting of PEG(2000)-stearate, glycerol monostearate and medium chain triglycerides were prepared by emulsion-evaporation and low temperature-solidification technique. Oridonin-loaded NLC (ORI-NLC) were also prepared as control. ORI-PEG-NLC were observed by transmission election microscope and the morphology was in rotiform shape. The mean particle size of ORI-PEG-NLC was 329.2 nm and entrapment efficacy was 71.18%. The results of differential scanning calorimetry and X-ray diffraction revealed a low-crystalline structure of ORI and verified the incorporation of ORI into the nanoparticles. In vitro drug release of ORI-PEG-NLC exhibited biphasic drug release patterns with burst release initially and prolonged release afterwards. Pharmacokinetic analysis showed that the mean residence time of ORI-PEG-NLC was prolonged and AUC (area under tissue concentration-time curve) value was also improved compared with ORI-NLC and ORI solution. In conclusion, ORI-PEG-NLC could be a potential carrier to get prolonged retention time of oridonin in blood.     

In vitro and in vivo evaluation of the subtype-selective muscarinic agonist PD 151832

PD 151832 is a potent partial muscarinic agonist that displays a high level of functional selectivity for the muscarinic m1 receptor subtype, as evidenced by its selective stimulation of PI turnover and cellular metabolic activity in transfected Hm1-CHO cells at concentrations that produce minimal stimulation of other cloned human muscarinic receptors. PD 151832 enhanced the amplification of Hm1-transfected NIH-3T3 cells at concentrations lower than those required to produce similar effects in Hm2 or Hm3-transfected cells. The functional m1 selectivity of PD 151832 is consistent with its improvement of mouse water maze performance at doses far lower than those required to produce peripheral parasympathetic side effects.