Tryptophan Substitution in CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) Introduces δ-Opioid Receptor Antagonism, Preventing Antinociceptive Tolerance and Stress-Induced Reinstatement of Extinguished Cocaine-Conditioned Place Preference.

The macrocyclic tetrapeptide CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) and its D-Trp isomer exhibit kappa opioid receptor (KOR) antagonism which prevents stress-induced reinstatement of extinguished cocaine-conditioned place preference. Here, we evaluated the effects of substitution of Trp and D-Trp on the peptides' opioid activity, antinociceptive tolerance, and the ability to prevent relapse to extinguished drug-CPP. Six analogs were synthesized using a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, efficacy in the [35S]GTPγS assay, metabolic stability in mouse liver microsomes, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance, and conditioned place preference (CPP) were also assessed in vivo, and the ameliorating effect of analogs on the reinstatement of extinguished cocaine-place preference was assessed. Substitutions of other D-amino acids for D-Trp did not affect (or in one case increased) KOR affinity, while two of the three substitutions of an L-amino acid for Trp decreased KOR affinity. In contrast, all but one substitution increased mu opioid receptor (MOR) affinity in vitro. The metabolic stabilities of the analogs were similar to those of their respective parent peptides, with analogs containing a D-amino acid being much more rapidly metabolized than those containing an L-amino acid in this position. In vivo, CJ-15,208 analogs demonstrated antinociception, although potencies varied over an 80-fold range and the mediating opioid receptors differed by substitution. KOR antagonism was lost for all but the D-benzothienylalanine analog, and the 2'-naphthylalanine analog instead demonstrated significant delta opioid receptor (DOR) antagonism. Introduction of DOR antagonism coincided with reduced acute opioid antinociceptive tolerance and prevented stress-induced reinstatement of extinguished cocaine-CPP.

Phenylalanine Stereoisomers of CJ-15,208 and [d-Trp]CJ-15,208 Exhibit Distinctly Different Opioid Activity Profiles.

The macrocyclic tetrapeptide cyclo[Phe-d-Pro-Phe-Trp] (CJ-15,208) and its stereoisomer cyclo[Phe-d-Pro-Phe-d-Trp] exhibit different opioid activity profiles in vivo. The present study evaluated the influence of the Phe residues' stereochemistry on the peptides' opioid activity. Five stereoisomers were synthesized by a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance development, and place conditioning were also assessed in vivo. All of the stereoisomers exhibited antinociception following either intracerebroventricular or oral administration differentially mediated by multiple opioid receptors, with kappa opioid receptor (KOR) activity contributing for all of the peptides. However, unlike the parent peptides, KOR antagonism was exhibited by only one stereoisomer, while another isomer produced DOR antagonism. The stereoisomers of CJ-15,208 lacked significant respiratory effects, while the [d-Trp]CJ-15,208 stereoisomers did not elicit antinociceptive tolerance. Two isomers, cyclo[d-Phe-d-Pro-d-Phe-Trp] (3) and cyclo[Phe-d-Pro-d-Phe-d-Trp] (5), did not elicit either preference or aversion in a conditioned place preference assay. Collectively, these stereoisomers represent new lead compounds for further investigation in the development of safer opioid analgesics.

Development of a robust, sensitive and selective liquid chromatography-tandem mass spectrometry assay for the quantification of the novel macrocyclic peptide kappa opioid receptor antagonist [D-Trp]CJ-15,208 in plasma and application to an initial pharmacokinetic study.

Selective kappa opioid receptor (KOR) antagonists may have therapeutic potential as treatments for substance abuse and mood disorders. Since [D-Trp]CJ-15,208 (cyclo[Phe-d-Pro-Phe-d-Trp]) is a novel potent KOR antagonist in vivo, it is imperative to evaluate its pharmacokinetic properties to assist the development of analogs as potential therapeutic agents, necessitating the development and validation of a quantitative method for determining its plasma levels. A method for quantifying [D-Trp]CJ-15,208 was developed employing high performance liquid chromatography-tandem mass spectrometry in mouse plasma. Sample preparation was accomplished through a simple one-step protein precipitation method with acetonitrile, and [D-Trp]CJ-15,208 analyzed following HPLC separation on a Hypersil BDS C8 column. Multiple reaction monitoring (MRM), based on the transitions m/z 578.1→217.1 and 245.0, was specific for [D-Trp]CJ-15,208, and MRM based on the transition m/z 566.2→232.9 was specific for the internal standard without interference from endogenous substances in blank mouse plasma. The assay was linear over the concentration range 0.5-500ng/mL with a mean r(2)=0.9987. The mean inter-day accuracy and precision for all calibration standards were 93-118% and 8.9%, respectively. The absolute recoveries were 85±6% and 81±9% for [D-Trp]CJ-15,208 and the internal standard, respectively. The analytical method had excellent sensitivity with a lower limit of quantification of 0.5ng/mL using a sample volume of 20µL. The method was successfully applied to an initial pharmacokinetic study of [D-Trp]CJ-15,208 following intravenous administration to mice.

Synthesis, structure-activity relationships, and biological studies of chromenochalcones as potential antileishmanial agents.

Antileishmanial activities of a library of synthetic chalcone analogues have been examined. Among them, five compounds (11, 14, 16, 17, 22, and 24) exhibited better activity than the marketed drug miltefosine in in vitro studies against the intracellular amastigotes form of Leishmania donovani. Three promising compounds, 16, 17, and 22, were tested in a L. donovani/hamster model. Oral administration of chalcone 16, at a concentration of 100 mg/kg of body weight per day for 5 consecutive days, resulted in >84% parasite inhibition at day 7 post-treatment and it retained the activity until day 28. The molecular and immunological studies revealed that compound 16 has a dual nature to act as a direct parasite killing agent and as a host immunostimulant. Pharmacokinetics and serum albumin binding studies also suggest that compound 16 has the potential to be a candidate for the treatment of the nonhealing form of leishmaniasis.

Anti-secretory and cyto-protective effects of peganine hydrochloride isolated from the seeds of Peganum harmala on gastric ulcers.

Gastroprotective mechanism of peganine hydrochloride isolated from Peganum harmala seeds was investigated. Peganine hydrochloride was evaluated against cold restraint (CRU), aspirin (AS), alcohol (AL) and pyloric ligation (PL) induced gastric ulcer models in rats. Potential anti-ulcer activity of peganine was observed against CRU (50.0%), AS (58.5%), AL (89.41%) and PL (62.50%) induced ulcer models. The reference drug omeprazole (10mg/kg, p.o.) showed 77.45% protection against CRU, 49.97% against AS and 69.42% against PL model. Sucralfate, another reference drug (500mg/kg, p.o.) showed 62.50% protection in AL induced ulcer model. Peganine significantly reduced free acidity (33.38%), total acidity (38.09%) and upregulated mucin secretion by 67.91%, respectively. Further, peagnine significantly inhibited H(+) K(+)-ATPase activity in vitro with IC50 of 73.47µg/ml as compared to the IC50 value of omeprazole (30.24µg/ml) confirming its anti-secretory activity.

Synthesis and insight into the structure-activity relationships of chalcones as antimalarial agents.

Licochalcone A (I), isolated from the roots of Chinese licorice, is the most promising antimalarial compound reported so far. In continuation of our drug discovery program, we isolated two similar chalcones, medicagenin (II) and munchiwarin (III), from Crotalaria medicagenia , which exhibited antimalarial activity against Plasmodium falciparum . A library of 88 chalcones were synthesized and evaluated for their in vitro antimalarial activity. Among these, 67, 68, 74, 77, and 78 exhibited good in vitro antimalarial activity against P. falciparum strains 3D7 and K1 with low cytotoxicity. These chalcones also showed reduction in parasitemia and increased survival time of Swiss mice infected with Plasmodium yoelii (strain N-67). Pharmacokinetic studies indicated that low oral bioavailability due to poor ADME properties. Molecular docking studies revealed the binding orientation of these inhibitors in active sites of falcipain-2 (FP-2) enzyme. Compounds 67, 68, and 78 showed modest inhibitory activity against the major hemoglobin degrading cysteine protease FP-2.

Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis IVA.

BACKGROUND: Treatments are being developed for an increasing number of mucopolysaccharidoses, and early diagnosis is expected to be necessary to maximize the benefits of therapy. Therefore, we developed an assay for N-acetylgalactosamine-6-sulfate sulfatase (GALNS), the enzyme deficient in mucopolysaccharidosis IVA (Morquio A syndrome), that is applicable for clinical diagnosis. METHODS: A novel substrate for GALNS was synthesized for a new enzyme activity assay that is based on tandem mass spectrometry and uses dried blood spots (DBSs) as the enzyme source. We optimized the assay conditions, including the substrate concentration, reaction pH, lead formate concentration, incubation time, punch size of the DBS, and mass spectrometer conditions. We also assessed inter- and intraassay variation. RESULTS: The assay uses either solid-phase or liquid-phase extraction before analysis by mass spectrometry. An evaluation of blood spots from 90 randomly chosen healthy newborns and 9 patients with Morquio A syndrome showed a well-defined interval between their respective enzyme activities. Inter- and intraassay imprecision was <10%. CONCLUSIONS: This tandem mass spectrometry assay requires a minimal number of sample-preparation steps, thus making it easy to implement. The assay has the potential to be adopted for early diagnosis of Morquio A syndrome. We believe this assay could be performed in a multiplex fashion with assays for other lysosomal enzymes.

Design and synthesis of substrates for newborn screening of Maroteaux-Lamy and Morquio A syndromes.

In continued efforts to develop enzymatic assays for lysosomal storage diseases appropriate for newborn screening laboratories we have synthesized novel and specific enzyme substrates for Maroteaux-Lamy (MPS VI) and Morquio A (MPS IVA) diseases. The sulfated monosaccharide derivatives were found to be converted to product by the respective enzyme in blood from healthy patients but not by blood from patients with the relevant lysosomal storage disease. The latter result shows that the designed substrates are highly selective for the respective enzymes.

Peganine hydrochloride dihydrate an orally active antileishmanial agent.

Protozoic infections caused by genus Leishmania pose an enormous public health threat in developing countries, compounded by the toxicity and resistance to current therapies. Under the aegis of our ongoing program on drug discovery and development on antileishmanial agents from plants, we carried out bioassay guided fractionation on Peganum harmala seeds which resulted in the isolation of 1 as an antileishmanial agent. 2D-NMR spectral data and single crystal X-ray crystallography data indicated 1 as peganine hydrochloride in dihydrated form. The compound 1 exhibited in-vitro activity against both extracellular promastigotes as well as intracellular amastigotes residing within murine macrophages in Leishmania donovani. Furthermore, 1 also exhibited in-vivo activity, 79.6 (+/-8.07)% against established VL in hamsters at a dose of 100mg/kgb.wt.

Antileishmanial activity mediated by apoptosis and structure-based target study of peganine hydrochloride dihydrate: an approach for rational drug design.

OBJECTIVES: The aim of this study was to resolve the putative pathway responsible for death induced by peganine hydrochloride dihydrate isolated from Peganum harmala seeds at cellular, structural and molecular level in Leishmania donovani, a causative agent of fatal visceral leishmaniasis. METHODS: The mode of action was assessed using various biochemical approaches including phosphatidylserine exposure, estimation of mitochondrial transmembrane potential and in situ dUTP nick end labelling staining of nicked DNA in the parasite. Molecular modelling and molecular dynamics studies were conducted with DNA topoisomerase I to identify the target of peganine hydrochloride dihydrate mediating apoptosis. Further, DNA topoisomerase I inhibition by peganine hydrochloride dihydrate was also assessed using an L. donovani topoisomerase I relaxation assay. RESULTS: Peganine hydrochloride dihydrate, besides being safe, was found to induce apoptosis in both the stages of L. donovani via loss of mitochondrial transmembrane potential. Molecular docking studies suggest that a binding interaction with DNA topoisomerase I of L. donovani (binding energy of -79 kcal/mol) forms a stable complex, indicating a possible role in apoptosis. The compound also inhibits L. donovani topoisomerase I. CONCLUSIONS: The compound induces apoptosis in L. donovani and inhibits DNA topoisomerase I.

Antidyslipidemic activity of Indigofera tinctoria.

Indigofera tinctoria is a perennial shrub, which belongs to the family Papilionaceae. As a part of our drug discovery program we have investigated the antidyslipidemic activity of the alcoholic extract from Indigofera tinctoria as well as its three other components, that is, chloroform, butanol and aqueous fractions in dyslipidemic hamsters that were fed a high fat diet. The chloroform fraction showed a significant decrease in the plasma triglycerides (TG, 52%) (P < 0.001), total cholesterol (TC, 29%) (P < 0.05), glycerol (Gly, 24%) and free fatty acids (FFA, 14%). This decrease was also accompanied by an increase in high density lipoproteins (HDL) by 9% and an increased HDL-C/TC ratio of 52% at the dose of 250 mg/kg of body weight.

Xyloccensin X and Y, two new limonoids from xylocarpus molluccensis: NMR investigation in mixture.

Two new limonoids, xyloccensin X (1) and xyloccensin Y (2), have been identified in mixture using NMR spectroscopy. Both limonoids were isolated in mixture from the fruit of the plant Xylocarpus molluccensis. The structures of these were proposed after extensive 2D NMR analyses in mixture.

Antidyslipidemic activity of furano-flavonoids isolated from Indigofera tinctoria.

Flavonoids appear to play a major role in reducing the risk of cardiovascular diseases by decreasing the blood lipid levels. In continuation of our drug discovery program on antidyslipidemic agents we have isolated three furano-flavones 1-3 and a rare flavonol glycoside 4 from the aerial parts of Indigofera tinctoria. Our results disclose that the treatment with diastereomeric flavonoid mixture 1 and 2 (80:20) significantly decreased the plasma triglycerides (TG) by 60%, total cholesterol (TC) 19%, glycerol (Gly) 13%, and free fatty acid (FFA) 25% accompanied with increase in high density lipoproteins-cholesterol (HDL-C) by 8% and HDL-C/TC ratio 36% in high fat diet (HFD) fed dyslipidemic hamsters at the dose of 50 mg/kg body weight. The flavonoid 3 has exhibited moderate antidyslipidemic activity.

4-hydroxyisoleucine an unusual amino acid as antidyslipidemic and antihyperglycemic agent.

Trigonella foenum-graecum, commonly known as fenugreek, is an annual herbaceous plant. From the seeds of T. foenum-graecum an unusual amino acid, 4-hydroxyisoleucine 5, has been isolated, which significantly decreased the plasma triglyceride levels by 33% (P<0.002), total cholesterol (TC) by 22% (P<0.02), and free fatty acids by 14%, accompanied by an increase in HDL-C/TC ratio by 39% in the dyslipidemic hamster model.

Synthesis of chromenochalcones and evaluation of their in vitro antileishmanial activity.

A large number of novel chromenochalcones were synthesized by pyridine-catalysed chromenylation of mono-chelated meta-dihydric acetophenones with the monoterpene, citral dimethyl acetal and subsequent Claisen-Schmidt condensation of the resultant acylchromenes with appropriate aromatic aldehydes. These chromenochalcones 1-19 were screened against in vitro extracellular promastigotes and intracellular amastigotes of Leishmania donovani. The most potent compound in this series was compound 9 with a pyridine ring-A, which showed 99% inhibition of promastigotes at 10 microg/ml, 82% at 0.25 microg/ml and 96% at 10 microg/ml concentration against amastigotes.