Effect of Tridax procumbens (Linn.) on bile duct ligation-induced liver fibrosis in rats.

The present study was undertaken to clarify whether methanolic extract of Tridax procumbens prevents liver fibrosis in rat. The hepatic fibrosis was induced by 28 days of bile duct ligation in rats. The 4-week treatment with Tridex procumbens reduced the serum aspartate aminotransferase (U L⁻¹), glutamate pyruvate transaminase (U L⁻¹), alkaline phosphatase (IU L⁻¹), lactate dehydrogenase (IU L⁻¹), total bilirubin (mg dL⁻¹), direct bilirubin (mg dL⁻¹) and hydroxyproline (mg gm⁻¹) content in liver and improved the histological appearance of liver section. The results of this study led us to conclude that T. procumbens can reduce the degree of hepatocellular damage and may become antifibrotic agent for liver fibrosis.

Hepatoprotective and antioxidant activity of Phaseolus trilobus, Ait on bile duct ligation induced liver fibrosis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Phaseolus trilobus Ait (Fabaceae) is extensively used by tribal people of Nandurbar district (Maharashtra, India) in the treatment of Jaundice and other liver disorders. AIM: of the present study was to assess the medicinal claim of Phaseolus trilobus as hepatoprotective and antioxidant. MATERIAL AND METHODS: The hepatoprotective activity of methanol and aqueous extract of Phaseolus trilobus was evaluated by bile duct ligation induced liver fibrosis and antioxidant activity was evaluated using in vitro and in vivo antioxidant models viz anti-lipid peroxidation assay, super oxide radical scavenging assay and glutathione estimation in liver. Liver function tests were carried out to detect hepatoprotective activity, which was further supported by histopathological examination. RESULTS: Methanol and aqueous extracts of Phaseolus trilobus reduced elevated level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin and hydroxyproline significantly (p<0.01) in bile duct ligated Wistar rats, proving hepatoprotective activity comparable with Silymarin. Both the extracts were found to reduce the elevated levels of serum thiobarbituric acid reactive substance (TBARS) and elevate superoxide scavenging radical activity proving antioxidant activity comparable with ascorbic acid. The reduced level of glutathione was found to be elevated in liver proving antioxidant activity comparable with Silymarin. CONCLUSION: Phaseolus trilobus posses hepatoprotective property and is effective in oxidative stress induced cholestatic hepatic injury.

Development and validation of a reversed-phase HPLC method for simultaneous estimation of ambroxol hydrochloride and azithromycin in tablet dosage form.

A simple, precise and accurate reversed-phase liquid chromatographic method has been developed for the simultaneous estimation of ambroxol hydrochloride and azithromycin in tablet formulations. The chromatographic separation was achieved on a Xterra RP18 (250 mm x 4.6 mm, 5 microm) analytical column. A Mixture of acetonitrile-dipotassium phosphate (30 mM) (50:50, v/v) (pH 9.0) was used as the mobile phase, at a flow rate of 1.7 ml/min and detector wavelength at 215 nm. The retention time of ambroxol and azithromycin was found to be 5.0 and 11.5 min, respectively. The validation of the proposed method was carried out for specificity, linearity, accuracy, precision, limit of detection, limit of quantitation and robustness. The linear dynamic ranges were from 30-180 to 250-1500 microg/ml for ambroxol hydrochloride and azithromycin, respectively. The percentage recovery obtained for ambroxol hydrochloride and azithromycin were 99.40 and 99.90%, respectively. Limit of detection and quantification for azithromycin were 0.8 and 2.3 microg/ml, for ambroxol hydrochloride 0.004 and 0.01 microg/ml, respectively. The developed method can be used for routine quality control analysis of titled drugs in combination in tablet formulation.

Molecular requirements of the human nucleoside transporters hCNT1, hCNT2, and hENT1.

Concentrative nucleoside transporters (CNTs) and equilibrative nucleoside transporters (ENTs) are important in physiological and pharmacological activity and disposition of nucleosides and nucleoside drugs. A better understanding of the structural requirements of inhibitors for these transporters will aid in designing therapeutic agents. To define the relative and unified structural requirements of nucleoside analogs for interaction with hCNT1, hCNT2, and hENT1, we applied an array of structure-activity techniques. Unique pharmacophore models for each respective nucleoside transporter were generated. These models reveal that hCNT2 affinity is dominated by hydrogen bonding features, whereas hCNT1 and hENT1 displayed mainly electrostatic and steric features. Hydrogen bond formation over 3'-OH is essential for all nucleoside transporters. Inhibition of nucleoside transporters by a series of uridine and adenosine analogs and a variety of drugs was analyzed by comparative molecular field analysis. Cross-validated r2 (q2) values were 0.65, 0.52, and 0.74 for hCNT1, hCNT2, and hENT1, respectively. The predictive quality of the models was further validated by successful prediction of the inhibition of a set of test compounds. Addition of a hydroxyl group around the 2-position of purine (or 3-position of pyrimidine) may increase inhibition to hCNT2 transporter; addition of hydroxyl group around the 2,7-position of purine (or the 3,5-position of pyrimidine) would increase the inhibition to hENT1 transporter. Utilization of these models should assist the design of high-affinity nucleoside transporter inhibitors and substrates for both anticancer and antiviral therapy.

Ontogenic and longitudinal activity of Na(+)-nucleoside transporters in the human intestine.

The objectives of our study were to identify the types of nucleoside transporters present in the human fetal small intestine and to characterize their developmental activity, longitudinal distribution, and transport kinetics compared with those present in the adult intestine. Nucleoside uptake by intestinal brush-border membrane vesicles was measured by an inhibitor-stop rapid filtration technique. Only the purine-specific (N1; hCNT2) and the pyrimidine-specific (N2; hCNT1) Na(+)-dependent nucleoside transporters were found to be present on the brush-border membranes of the enterocytes along the entire length of the fetal and adult small intestines. The activity of these transporters was higher in the proximal than in the distal small intestine. Both the N1 and N2 transporters found in the fetal intestine shared similar kinetic properties (Michaelis-Menten constant and Na(+)-nucleoside stoichiometry) to those in the adult intestine. During the period of rapid morphogenesis (11-15 wk gestation), no temporal differences were apparent in the activity of the N1 and N2 transporters in the fetal small intestine. These findings have implications for the absorption of drugs from the amniotic fluid by the fetus after maternal drug administration of nucleoside drugs such as the antivirals zidovudine and didanosine.

Structure-inhibitory profiles of nucleosides for the human intestinal N1 and N2 Na+-nucleoside transporters.

PURPOSE: To determine the structure-inhibitory profiles of nucleosides for the N1 and N2 Na+-nucleoside transporters of the human intestine. METHODS: The uptake of 3H-labeled prototypic substrates of the N1 (inosine) and N2 (thymidine) transporters into human intestinal brush border membrane vesicles was measured by a rapid filtration technique in the presence and absence of various uridine and adenosine analogs and antiviral and anticancer nucleoside drugs (100 and 1000 microM). RESULTS: In the ribose ring, the 3'-oxygen is required for inhibition of uptake of nucleosides by both the N1 and N2 transporters. The structural requirements for such inhibition differ with respect to modifications on the 5' position of the sugar ring or on the base. The N2 transporter is more tolerant to these substitutions than is the N1 transporter. The 6 position on uracil and the 8 position on adenine are critical for inhibition of uptake of nucleosides by both the N1 and N2 nucleoside transporters. CONCLUSIONS: These data are the first evidence that the binding site(s) of the human N1 and N2 transporters differ in their interaction with analogs of their common substrates, uridine and adenosine. Such studies can provide insight into the critical structural determinants of the substrate necessary for recognition by the Na+-nucleoside transporters of the human intestine.

Conformation of oligodeoxynucleotides associated with anionic liposomes.

There has been significant progress in the development of antisense therapeutics for a wide range of medicinal applications. Further improvement will require better understanding of cellular internalization, intracellular distribution mechanisms and interactions of oligodeoxynucleotides with cellular organelles. In many of these processes interactions of oligodeoxynucleotides with lipid assemblies may have a significant influence on their function. Divalent cations have been shown to assist cellular internalization of certain oligodeoxynucleotides and to affect their conformation. In this work we have investigated conformational changes of phosphorothioate oligodeoxynucleotides upon divalent cation-mediated interaction with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG) liposomes. For the sequences investigated here the native conformation underwent significant change in the presence of anionic DPPG liposomes only when divalent cations were present. This change is sequence-specific, ion-selective and distinct from previously reported changes in oligodeoxynucleotide structure due to divalent cations alone. The conformation of one oligodeoxynucleotide in the presence of calcium and DPPG yields circular dichroism spectra which suggest C-DNA but which also have characteristics unlike any previously reported spectra of liposome-associated DNA structure. The data suggest the possibility of a unique conformation of liposome-associated ODNs and reflect a surprisingly strong tendency of single-stranded DNA to retain a characteristic conformation even when adsorbed to a surface. This conformation may be related to cellular uptake, transport of oligodeoxynucleotides in cells and/or function.

Influence of divalent cations on the conformation of phosphorothioate oligodeoxynucleotides: a circular dichroism study.

Phosphorothioate oligodeoxynucleotides (ODNs) have been extensively investigated in vivo and in vitro for antisense control of gene expression. It has been shown that cellular uptake of phosphorothioate ODNs in some in vitro cell systems increases in the presence of divalent cations. In this work, we analyze the conformation of phosphorothioate ODNs and specific changes induced in it by various divalent cations using circular dichroism (CD) spectroscopy. CD data were obtained with several phosphorothioate ODNs in the absence and presence of the divalent cations Mg(2+), Ca(2+), Sr(2+), Ba(2+) and Mn(2+). All CD spectra indicated stable conformations of the ODNs in solution. The spectra were strongly dependent on ODN sequence and composition. Some ODNs such as T(23) and another with 'random' distribution of bases showed CD spectra characteristic of B-form DNA. Other ODNs which had at least three consecutive guanines in their sequences exhibited spectra characteristic of parallel G-tetraplexes. CD spectra of antisense ODNs exhibited specific responses to divalent cations. Changes in the conformation were not simply due to ionic strength effects. Mn(2+) diminished secondary structure in some ODNs. Group II divalent ions stabilized the parallel G-tetraplexes, and Mg(2+) generally had the weakest stabilizing efficiency. Each sequence/ion combination had a specific response so these effects cannot be generalized. These sequence-dependent, divalent ion-sensitive, and structurally unique solution conformations may be related to ion-mediated ODN uptake.

Expression of arylamine N-acetyltransferase in human intestine.

BACKGROUND: Arylamine N-acetyltransferases in humans (NAT1 and NAT2) catalyse the acetylation of arylamines including food derived heterocyclic arylamine carcinogens. Other substrates include the sulphonamide 5-aminosalicylic acid (5-ASA), which is an NAT1 specific substrate; N-acetylation of 5-ASA is a major route of metabolism. NAT1 and NAT2 are both polymorphic. AIMS: To investigate NAT expression in apparently healthy human intestines in order to understand the possible role of NAT in colorectal cancer and in the therapeutic response to 5-ASA. METHODS: The intestines of four organ donors were divided into eight sections. DNA was prepared for genotyping NAT1 and NAT2 and enzymic activities of NAT1 and NAT2 were determined in cytosols prepared from each section. Tissue was fixed for immunohistochemistry with specific NAT antibodies. Western blotting was carried out on all samples of cytosol and on homogenates of separated muscle and villi after microdissection. RESULTS: NAT1 activity of all cytosols was greater than NAT2 activity. NAT1 and NAT2 activities correlated with the genotypes of NAT1 and NAT2 and with the levels of NAT1 staining determined by western blotting. The ratio of NAT1:NAT2 activities showed interindividual variations from 2 to 70. NAT1 antigenic activity was greater in villi than in muscle. NAT1 was detected along the length of the villi in the small intestine. In colon samples there was less NAT1 at the base of the crypts with intense staining at the tips. CONCLUSIONS: The interindividual variation in NAT1 and NAT2 in the colon could affect how individuals respond to exposure to specific NAT substrates including carcinogens and 5-ASA.

Reduction of an eight-state mechanism of cotransport to a six-state model using a new computer program.

A computer program was developed to allow easy derivation of steady-state velocity and binding equations for multireactant mechanisms including or without rapid equilibrium segments. Its usefulness is illustrated by deriving the rate equation of the most general sequential iso ordered ter ter mechanism of cotransport in which two Na+ ions bind first to the carrier and mirror symmetry is assumed. It is demonstrated that this mechanism cannot be easily reduced to a previously proposed six-state model of Na+-D-glucose cotransport, which also includes a number of implicit assumptions. In fact, the latter model may only be valid over a restricted range of Na+ concentrations or when assuming very strong positive cooperativity for Na+ binding to the glucose symporter within a rapid equilibrium segment. We thus propose an equivalent eight-state model in which the concept of positive cooperativity is best explained within the framework of a polymeric structure of the transport protein involving a minimum number of two transport-competent and identical subunits. This model also includes an obligatory slow isomerization step between the Na+ and glucose-binding sequences, the nature of which might reflect the presence of functionally asymmetrical subunits.

Functional expression of human intestinal Na+-dependent and Na+-independent nucleoside transporters in Xenopus laevis oocytes.

We have shown previously that the human jejunal brush border membrane expresses both the N1 (cif) and the N2 (cit) Na+-dependent (concentrative) nucleoside transporters but not the Na+-independent (facilitative) nitrobenzylmercaptopurineriboside (NBMPR)-sensitive (es) transporter (Patil SD and Unadkat JD, Am J Physiol, 272: 1314-1320, 1997). In the present study, we have demonstrated that when Xenopus laevis oocytes are microinjected with human jejunal mRNA, four nucleoside transporters are expressed simultaneously, namely the N1 and N2 Na+-dependent nucleoside transporters and the es and the NBMPR-insensitive (ei) Na+-independent transporters. The expressed Na+-dependent nucleoside transporters showed substrate specificity identical to that previously described by us using jejunal brush border membrane vesicles (Patil SD and Unadkat JD, Am J Physiol, 272: 1314-1320, 1997). The expressed es and ei Na+-independent transporters demonstrated broad substrate selectivity with both purines and pyrimidines capable of inhibiting the uptake of guanosine and thymidine mediated by this transporter. The expressed Na+-dependent nucleoside transporters mediated the transport of their respective nucleoside substrates with a high affinity and a low capacity, whereas the es and the ei transporters mediated the transport of nucleosides with a low affinity and a high capacity. Collectively, these observations suggest that the Na+-independent nucleoside transporters are expressed in the basolateral membrane of the human jejunal epithelium. Based on these data, we hypothesize that the concentrative transporters in the brush border membrane and equilibrative transporters in the basolateral membrane are arranged in series in the human jejunal epithelium to allow efficient vectorial transport of nucleosides from the lumen to the blood. The simultaneous expression of four nucleoside transporters in X. laevis oocytes establishes a basis for molecular cloning of these four human nucleoside transporters.

Sodium-dependent nucleoside transport in the human intestinal brush-border membrane.

The objective of the study was to determine the identity and kinetic characteristics of nucleoside transporters present in the brush-border membrane of the human jejunum. With use of brush-border membrane vesicles, uptake of [3H]uridine was stimulated two- to threefold by an inwardly directed Na+ gradient and was inhibited by both 100 microM thymidine and 100 microM guanosine nucleosides, which serve as model substrates for purine (N1, cif) and pyrimidine (N2, cit) transporters, respectively. [3H]thymidine and [3H]guanosine transport exhibited an overshoot phenomenon only in the presence of a Na+ gradient. Na(+)-thymidine uptake was inhibited by 100 microM cytidine or thymidine but not by guanosine, inosine, formycin B, or hypoxanthine. [3H]guanosine uptake was inhibited by 100 microM inosine, guanosine, or formycin B but not by thymidine or cytidine. Both adenosine and uridine inhibited uptake of [3H]thymidine and [3H]guanosine to a similar extent, indicating that both N1, cif and N2, cit Na(+)-nucleoside transporters are expressed in human jejunum. Enhanced uptake of Na(+)-thymidine by an inside-negative potential difference generated by K+ and valinomycin provides evidence that nucleoside transport is rheogenic, involving net transfer of a positive charge. The Hill coefficient was unity for all three substrates, indicating a Na(+)-nucleoside coupling stoichiometry of 1:1. At saturating Na+ concentration (150 mM) the kinetic parameters (n = 3-4) Michaelis-Menten constant and maximum velocity for uridine, thymidine, and guanosine uptake were 4.15 +/- 1.79, 2.74 +/- 0.58, 12.02 +/- 1.34 microM and 25.93 +/- 7.38, 16.10 +/- 3.64, 63.92 +/- 10.23 pmol.mg-1.10 s-1, respectively. These results suggest that, in contrast to the human kidney that expresses the N4 nucleoside transporter, the human jejunum expresses both N1 and N2 Na(+)-nucleoside transporters.

Osteoclastoma like giant cell tumour of the thyroid--a variant of undifferentiated carcinoma--a case report.

A case of osteoclastoma like giant cell tumour of thyroid is reported in a patient who presented with dysphagia and swelling in the neck. The light microscopy showed numerous osteoclast-like giant cells embedded in mononuclear stromal cells, the overall appearance resembling that of giant cell tumour of bone.

Synthesis and antiviral properties of (+/-)-5'-noraristeromycin and related purine carbocyclic nucleosides. A new lead for anti-human cytomegalovirus agent design.

(+/-)-5'-Noraristeromycin (3) has been prepared in three steps beginning with the 2,3-O-isopropylidene derivative of (+/-)-(1 alpha, 2 beta, 3 beta, 4 alpha)-4-amino-1,2,3-cyclopentanetriol (7). Also prepared from the same starting material were the related hypoxanthine (4), guanine (5), and 2,6-diaminopurine (6) analogues. Compounds 3-6 were evaluated for antiviral activity against a large number of viruses with marked activity being observed for 3 towards vaccinia virus, human cytomegalovirus, vesicular stomatitis virus, parainfluenza (type 3) virus, measles virus, respiratory syncytial virus, reovirus (type 1), and the arenaviruses Junin and Tacaribe. None of the compounds showed cytotoxicity to the host cell monolayers used in the antiviral studies. Both 3 and 6 have been found to be inhibitors of S-adenosyl-L-homocysteine hydrolase (AdoHcy hydrolase), which likely accounts for their antiviral activity. Inhibition of AdoHcy hydrolase represents a new approach to human cytomegalovirus drug design that should be pursued. Also, the activity of 3 should be further scrutinized for the treatment of pox-, rhabdo-, paramyxo-, reo-, and arenavirus infections.

(+-)-carbocyclic 5'-nor-2'-deoxyguanosine and related purine derivatives: synthesis and antiviral properties.

Beginning with 3-cyclopenten-1-ylamine hydrochloride, the 5'-nor derivatives of carbocyclic 2'-deoxyguanosine (2), 2'-deoxyadenosine (3), and 2,6-diaminopurine 2'-deoxyribofuranoside (4) have been prepared. These compounds were evaluated for antiviral potential versus herpes simplex virus, varicella-zoster virus, cytomegalovirus, vaccinia virus, vesicular stomatitis virus, and human immunodeficiency virus and found to lack activity. Also, compounds 2-4 were virtually nontoxic toward the host (human diploid fibroblast ESM and HEL) cells. These biological properties may be due to the inability of 2-4 to be phosphorylated to the requisite nucleotide level that is likely to be necessary for biological activity by correlation to carbocyclic 2'-deoxyguanosine (1), which possesses significant antiviral properties as a result of conversion to its 5'-triphosphate derivative.

Folate analogues. 31. Synthesis of the reduced derivatives of 11-deazahomofolic acid, 10-methyl-11-deazahomofolic acid, and their evaluation as inhibitors of glycinamide ribonucleotide formyltransferase.

The Boon-Leigh procedure, involving condensation of a 6-chloro-5-nitropyrimidine (22) with an alpha-amino ketone (20 or 21) followed by reduction of the nitro group, cyclization, and L-glutamylation, led to the formation of 11-deazahomofolate (29) and its 10-methyl derivative (30). The corresponding (6R,S)-5,6,7,8-tetrahydro (4, 5) and 7,8-dihydro (31, 32) derivatives were prepared by catalytic hydrogenation. (6S)-11-Deazatetrahydrohomofolate was prepared from 29 by enzymatic reduction. Compounds 29 and 30 had little effect (IC50 greater than 2 x 10(-5) M) on Lactobacillus casei glycinamide ribonucleotide (GAR) formyltransferase but (6R,S)-11-deazatetrahydrohomofolate (4) is a potent inhibitor of this enzyme (IC50 = 5 x 10(-8) M). It is at least 100 times more inhibitory than 33, the 6S compound, indicating that the 6R component of the mixture having the unnatural configuration at C6 (34) is responsible for the potent inhibition. Compound 4 is a much weaker inhibitor of murine (L1210) and human (MOLT-4) leukemia cell GAR formyltransferases (IC50 greater than 1 x 10(-5) M). (6R,S)-11-Deaza-10-methyltetrahydrohomofolate (5) (IC50 = 1.1 x 10(-5) is 200 times weaker than 4 against L. casei GAR formyltransferase. However, 11-deaza-10-methyldihydrohomofolate (32) is more inhibitory (IC50 = 5.5 x 10(-7) M) than 5 or 30. None of the compounds showed inhibition of L. casei aminoimidazolecarboxamide ribonucleotide (AICAR) formyltransferase, dihydrofolate reductase, or thymidylate synthase. The dihydro derivatives 31 and 32 are 5% as active as dihydrofolate as substrates for L. casei dihydrofolate reductase. Compound 4 showed moderate inhibition of the growth of L. casei, Streptococcus faecium, MOLT-4 cells, and MCF-7 human breast adenocarcinoma cells.

Folate analogues. 32. Synthesis and biological evaluation of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid and related compounds.

The chemical synthesis of three close analogues (2-4) of N10-propargyl-5,8-dideazafolate (PDDF) is described. The quinazoline ring of 2 and 4 was constructed from the pivotal intermediate 9 in a novel and unambiguous manner during the final step of the synthesis under very mild conditions. 2-Desamino-2-methyl-N10-propargyl-5,8-dideazafolate (DMPDDF) (2) was a strong inhibitor of human and Lactobacillus casei thymidylate synthases, whereas 2-desamino-2-(trifluoromethyl)-N10-propargyl-5,8-didezafolate (3) and 2-desamino-2,3-dimethyl-N10-propargyl-5,8-dideazafolate (4) were only weak inhibitors of this enzyme. DMPDDF exhibited excellent growth inhibition of Manca human lymphoid leukemia and H35 hepatoma cells in culture. The inhibitor activities of 2 were 43- and 65-fold greater than that of PDDF, respectively, in these cell lines. H35R cells that are resistant to methotrexate by virtue of a transport defect were cross resistant to DMPDDF but not to PDDF. H35FF cells which have 70-fold greater amounts of thymidylate synthase compared to H35N cells were 130-fold resistant to DMPDDF. Furthermore, the toxicity of DMPDDF to H35 hepatoma cells could be completely reversed by thymidine, establishing its locus of action as thymidylate synthase. Transport studies in vitro established that DMPDDF effectively inhibits MTX influx into H35 hepatoma cells, whereas PDDF has no effect on MTX transport in this cell line. These data suggest that the greater activity of DMPDDF relative to PDDF is partly due to the ability of the former compound to enter cells via the MTX/reduced folate transport system. Enzyme inhibition data of 4 suggest that the presence of N3H in DMPDDF is essential for binding to thymidylate synthase.