Toufan Parman

Novel arylimidamides for treatment of visceral leishmaniasis.

ABSTRACT Arylimidamides (AIAs) represent a new class of molecules that exhibit potent antileishmanial activity (50% inhibitory concentration [IC50], <1 μM) against both Leishmania donovani axenic amastigotes and intracellular Leishmania, the causative agent for human visceral leishmaniasis (VL). A systematic lead discovery program was employed to characterize in vitro and in vivo antileishmanial activities, pharmacokinetics, mutagenicities, and toxicities of two novel AIAs, DB745 and DB766. They were exceptionally active (IC50 ≤ 0.12 μM) against intracellular L. donovani, Leishmania amazonensis, and Leishmania major and did not exhibit mutagenicity in an Ames screen. DB745 and DB766, given orally, produced a dose-dependent inhibition of liver parasitemia in two efficacy models, L. donovani-infected mice and hamsters. Most notably, DB766 (100 mg/kg of body weight/day for 5 days) reduced liver parasitemia in mice and hamsters by 71% and 89%, respectively. Marked reduction of parasitemia in the spleen (79%) and bone marrow (92%) of hamsters was also observed. Furthermore, these compounds distributed to target tissues (liver and spleen) and had a moderate oral bioavailability (up to 25%), a large volume of distribution, and an elimination half-life ranging from 1 to 2 days in mice. In a repeat-dose toxicity study of mice, there was no indication of liver or kidney toxicity for DB766 from serum chemistries, although mild hepatic cell eosinophilia, hypertrophy, and fatty changes were noted. These results demonstrated that arylimidamides are a promising class of molecules that possess good antileishmanial activity and desirable pharmacokinetics and should be considered for further preclinical development as an oral treatment for VL.

Lead optimization of 3-carboxyl-4(1H)-quinolones to deliver orally bioavailable antimalarials.

Malaria is a protozoal parasitic disease that is widespread in tropical and subtropical regions of Africa, Asia, and the Americas and causes more than 800,000 deaths per year. The continuing emergence of multidrug-resistant Plasmodium falciparum drives the ongoing need for the development of new and effective antimalarial drugs. Our previous work has explored the preliminary structural optimization of 4(1H)-quinolone ester derivatives, a new series of antimalarials related to the endochins. Herein, we report the lead optimization of 4(1H)-quinolones with a focus on improving both antimalarial potency and bioavailability. These studies led to the development of orally efficacious antimalarials including quinolone analogue 20g, a promising candidate for further optimization.

Toxicogenomics and Metabolomics of Pentamethylchromanol (PMCol)-Induced Hepatotoxicity

Pentamethyl-6-chromanol (PMCol), a chromanol-type compound related to vitamin E, was proposed as an anticancer agent with activity against androgen-dependent cancers. In repeat dose-toxicity studies in rats and dogs, PMCol caused hepatotoxicity, nephrotoxicity, and hematological effects. The objectives of this study were to determine the mechanisms of the observed toxicity and identify sensitive early markers of target organ injury by integrating classical toxicology, toxicogenomics, and metabolomic approaches. PMCol was administered orally to male Sprague-Dawley rats at 200 and 2000 mg/kg daily for 7 or 28 days. Changes in clinical chemistry included elevated alanine aminotransferase, total bilirubin, cholesterol and triglycerides-indicative of liver toxicity that was confirmed by microscopic findings (periportal hepatocellular hydropic degeneration and cytomegaly) in treated rats. Metabolomic evaluations of liver revealed time- and dose-dependent changes, including depletion of total glutathione and glutathione conjugates, decreased methionine, and increased S-adenosylhomocysteine, cysteine, and cystine. PMCol treatment also decreased cofactor levels, namely, FAD and increased NAD(P)+. Microarray analysis of liver found that differentially expressed genes were enriched in the glutathione and cytochrome P450 pathways by PMCol treatment. Reverse transcription-polymerase chain reaction of six upregulated genes and one downregulated gene confirmed the microarray results. In conclusion, the use of metabolomics and toxicogenomics demonstrates that chronic exposure to high doses of PMCol induces liver damage and dysfunction, probably due to both direct inhibition of glutathione synthesis and modification of drug metabolism pathways. Depletion of glutathione due to PMCol exposure ultimately results in a maladaptive response, increasing the consumption of hepatic dietary antioxidants and resulting in elevated reactive oxygen species levels associated with hepatocellular damage and deficits in liver function.

Effect of resveratrol on 17β-estradiol sulfation by human hepatic and jejunal S9 and recombinant sulfotransferase 1E1

The purpose of this study was to investigate the sulfation of resveratrol (3,5,4′-trihydroxystilbene) and its potential to exhibit drug-drug interactions via sulfation. The possible interaction of resveratrol with 17β-estradiol (E2), a major estrogen hormone and prototypic substrate for sulfate conjugation, was studied. Resveratrol and E2 are both known to undergo sulfate conjugation catalyzed by human sulfotransferases (SULTs). Resveratrol is a phytoestrogen with mixed estrogen agonist/antagonist properties that is being developed as a chemopreventive agent. The sulfate conjugation of E2 and resveratrol were studied individually using S9 fractions from human liver and jejunum as well as recombinant human SULT isoforms. The sulfation of E2 (3–20 nM) was then investigated in the presence of various concentrations (0, 0.5, 1, and 2 μM) of resveratrol using the two S9 preparations as well as recombinant SULT1E1, the major isoform responsible for E2 sulfation. Resveratrol inhibited E2 sulfation with estimated Ki values of 1.1 μM (liver), 0.6 μM (jejunum), and 2.3 μM (SULT1E1), concentrations that could be pharmacologically relevant. The results suggest that these phytoestrogens can potentially alter the homeostasis of estrogen levels. These findings also imply that resveratrol may inhibit the metabolism of other estrogen analogs or therapeutic agents such as ethinylestradiol or dietary components that are also substrates for SULT1E1.

Comparative Uterotrophic Effects of Endoxifen and Tamoxifen in Ovariectomized Sprague-Dawley Rats

Endoxifen (4-hydroxy-N-desmethyl-tamoxifen), one of the major active metabolites of tamoxifen, has substantially greater estrogen antagonist properties and antiproliferative effects in breast tumor cells than tamoxifen, a mixed estrogen agonist/antagonist. An associated risk of endometrial cancer and hyperplasia has been linked to the estrogen agonist properties of tamoxifen. We evaluated endoxifen using a classic uterotrophic effects method. Rats were given endoxifen or tamoxifen orally for 3 days. Estradiol was the positive control. Endoxifen and tamoxifen plasma levels exceeded those previously observed clinically. Uterine weight was 3-fold higher in the estradiol group than in the tamoxifen or endoxifen groups, which did not differ from vehicle controls. Tamoxifen and endoxifen caused a greater increase in luminal epithelial cell height than estradiol. Both tamoxifen and endoxifen produced an increase in the stromal BrdU labeling index (LI) that was ≤ estradiol and inversely related to dose, but did not affect luminal epithelial cell BrdU LI. As expected, estradiol increased luminal epithelial cell proliferation. These results indicate that endoxifen induces uterotrophic effects, but is less potent than estradiol in eliciting these effects. Given prior preclinical observations that endoxifen has superior antitumor activity than tamoxifen, the observations of similar uterine effects suggest that the endoxifen risk/benefit ratio may be superior to tamoxifen.

Lead Optimization of Antimalarial Propafenone Analogues

Previously reported studies identified analogues of propafenone that had potent antimalarial activity, reduced cardiac ion channel activity, and properties that suggested the potential for clinical development for malaria. Careful examination of the bioavailability, pharmacokinetics, toxicology, and efficacy of this series of compounds using rodent models revealed orally bioavailable compounds that are nontoxic and suppress parasitemia in vivo. Although these compounds possess potential for further preclinical development, they also carry some significant challenges.

Quantitative proteomics for cardiac biomarker discovery using isoproterenol-treated nonhuman primates.

To identify new cardiac biomarkers, a quantitative proteomic analysis has been performed on serum and heart tissue proteins from three species of nonhuman primates following isoproterenol (ISO) treatment. Three serum proteins—serum amyloid A (SAA), α-1-acid glycoprotein (A1AG), and apolipoprotein A-1 (Apo A1)—were consistently identified as changed and remained altered 72 h post dose in all three species post ISO treatment, indicating the potential of including these proteins in preclinical or clinical evaluation of drug-induced cardiac injury. Furthermore, proteomic analysis of heart tissue proteins following ISO treatment demonstrated detrimental effects on calcium signaling and energy generation in cardiac myocytes. It is worth noting that cardiac troponins were not identified in serum but were identified as altered in heart tissue lysate along with other cardiac-specific proteins. This strategy for cardiac biomarker discovery by proteomic screening of heart tissue proteins, followed by verification in serum samples using immunoassays or targeted mass spectrometry, could be applied in future biomarker studies.

Serum Biomarkers Reveal Long-term Cardiac Injury in Isoproterenol-treated African Green Monkeys

The assessment of cardiac toxicity is a major challenge in both drug development and clinical trials, and numerous marketed pharmaceuticals have been removed from the market due to unpredicted cardiac effects. Serum troponins are widely used indicators of cardiac injury; however, they are short-lived and have not been validated in preclinical animal models. In this study, we have used filter-aided sample preparation (FASP) and tandem mass tag (TMT) labeling to investigate serum protein alterations in isoproterenol-treated African green monkeys. Our results showed that the combination of FASP and TMT labeling provided highly reproducible and efficient sample preparation, which enables us to identify and quantify serum proteins with high confidence. We focused on the proteins that exhibit long-term alteration upon isoproterenol injection and discovered nine proteins exhibiting significant changes at 48 and 72 h postdosing. We further chose three proteins, serum amyloid A (SAA), frutose biphosphate aldolase A (FBAA), and fetuin A, for validation using enzyme-linked immunosorbent assay (ELISA). The serum concentration of SAA showed a ∼ 50 fold increase, while concentration of FBAA and fetuin A exhibited a significant decrease accompanying isoproterenol-induced cardiotoxicity. This work provides valuable insights for multimarker evaluation of long-term cardiac injury.

Identification of (+)-Erythro-Mefloquine as an Active Enantiomer with Greater Efficacy than Mefloquine against Mycobacterium avium Infection in Mice

ABSTRACT Infection caused by Mycobacterium avium is common in AIDS patients who do not receive treatment with highly active antiretroviral therapy (HAART) or who develop resistance to anti-HIV therapy. Mefloquine, a racemic mixture used for malaria prophylaxis and treatment, is bactericidal against M. avium in mice. MICs of (+)-erythro-, (−)-erythro-, (+)-threo-, and (−)-threo-mefloquine were 32 μg/ml, 32 μg/ml, 64 μg/ml, and 64 μg/ml, respectively. The postantibiotic effect for (+)-erythro-mefloquine was 36 h (MIC) and 41 h for a concentration of 4× MIC. The mefloquine postantibiotic effect was 25 h (MIC and 4× MIC). After baseline infection was established (7 days), the (+)- and (−)-isomers of the diastereomeric threo- and erythro-α-(2-piperidyl)-2,8-bis(trifluoromethyl)-4-quinolinemethanol were individually used to orally treat C57BL/6 bg+/bg+ beige mice that were infected intravenously with M. avium. Mice were also treated with commercial mefloquine and diluent as controls. After 4 weeks of treatment, the mice were harvested, and the number of bacteria in spleen and liver was determined. Mice receiving (+)- or (−)-threo-mefloquine or (−)-erythro-mefloquine had numbers of bacterial load in tissues similar to those of untreated control mice at 4 weeks. Commercial mefloquine had a bactericidal effect. However, mice given the (+)-erythro-enantiomer for 4 weeks had a significantly greater reduction of bacterial load than those given mefloquine. Thus, (+)-erythro-mefloquine is the active enantiomer of mefloquine against M. avium and perhaps other mycobacteria.

P2-448: Preclinical evaluation of CDD-0102, a selective M1 agonist with potential utility in Alzheimer's disease

Background: Agonists that selectively activate M1 muscarinic receptors might be useful in treating memory and cognitive deficits, while preventing the formation of amyloid plaques and neurofibrillary tangles associated with Alzheimer’s disease. 5-(3-Ethyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine trifluoroacetic acid (CDD-0102J) displays functional selectivity for M1 receptors and enhances memory function in animals with cholinergic deficits. The hydrochloride salt (CDD-0102A) also promotes alpha-secretase activity and reverses the apoptotic effects of Abeta in differentiated PC12 cells. Methods: CDD-0102A was evaluated in a series of studies to determine pharmacokinetic parameters, drug metabolism and toxicity. Results: Preclinical toxicology studies indicate that CDD-0102A is negative in bacterial mutagenicity, mammalian cell clastogenicity, and mouse micronucleus assays. Moreover, CDD-0102A does not inhibit the HEK-hERG channel current. In a 28-day repeat-dose toxicity study with CDD-0102A in male and female rats, excessive cholinergic stimulation was apparent at high doses yet no overt toxicities were observed. Dogs were more sensitive to CDD-0102A in a comparable study with dose-dependent increased frequency and severity of cholinergic symptoms, although no significant effects on cardiovascular and pulmonary safety parameters were observed. Studies in rats yielded linear pharmacokinetics with dose proportional increases in Cmax and AUC and an estimated t1/2 of 3-5 hr. Comparative drug metabolism studies indicated significant metabolism only in rabbit S9 preparations, with limited metabolism in liver microsomal and S9 preparations from mice, rats, dogs, monkeys and humans. Three potential metabolites were identified from rabbit liver microsomes. Conclusions: The preclinical data are being used in planning Phase I clinical studies of CDD-0102A in healthy adult volunteers. Taken together, the data suggest that CDD-0102A may be useful in treating the symptoms and some of the underlying causes of Alzheimer’s disease.