Nucleosides 5(15): synthesis of novel 1,2,4-triazolo[3,4-c]-1,2,4-triazole nucleosides.

Ribosylation of 3-methylthio-5-phenyl-1,2,4-triazole (1) with ribose derivative 2 gave the protected 1,2,4-triazole-nucleoside 3, which reacted with hydrazine hydrate to afford the 3-hydrazino-1,2,4-triazole derivative 5. Reaction of 5 with aromatic aldehydes yielded the corresponding hydrazones 6, which cyclized under bromination in acetic acid to give 8. Debenzoylation of 8 afforded novel 1,2,4-triazolo[3,4-c]-1,2,4-triazole nucleosides 9.

In vitro studies of poorly absorbed drugs using porcine intestine in the ring model RIMO.

Two compounds, an aminobisphosphonate (A) and a steroid (B), showing a very large difference in lipophilicity (log PC (A) = O, log PC (B) > 8) but both revealing very poorly absorption in vivo of less than 1% were tested in respect of in vitro absorption and mucoadhesion using a model with porcine intestine named RIMO (ring model). Explanations for the poor absorption of the two compounds were investigated in order to propose methods for absorption enhancement. Moreover, absorption and mucoadhesion were measured for different parts of intestine with radiolabelled substances. Last but not least the inhibiting effect of calcium ions was investigated as well as enhancement by DTT (dithiothreitol), EDTA and bile. Results showed that mucus plays an important role in oral bioavailability. The aminobisphosphonate formed a complex with calcium ions which was insoluble in the mucus and resulted in a smaller amount of substance being absorbed. The very lipophilic steroid was kept back from the mucosa by the hydrophilic mucus, but addition of the solubilizer bile increased the absorption. The results were in good agreement with in vivo bioavailability data. Therefore, simplicity of the model and easy access of porcine intestine by slaughter houses make RIMO a valuable tool for investigations concerning the role of mucus and influences of diverse additives on absorption.

In vitro absorption studies with carvedilol using a new model with porcine intestine called BM-RIMO (Boehringer-Mannheim ring model).

Carvedilol (CAS 72956-09-3, Dilatrend) is a beta-blocker with additional vasodilating, antiproliferative and antioxidative properties. It is indicated for the treatment of high tension (HT), coronary artery disease (CAD) and congestive heart failure (CHF). Carvedilol has been investigated in numerous in vivo studies and thus comparisons of in vitro results to in vivo observations are possible. In this publication the results of studies on the in vitro absorption of carvedilol in a new model called BM-RIMO (Boehringer-Mannheim ring model) using porcine intestine are reported. In particular the influence of absorption enhancers and of pH and the existence of absorption windows were examined and the relevance of the obtained data for in vivo conditions was estimated. The main route of carvedilol absorption seemed to be transcellular. In vitro as well as in vivo absorption decreased within the intestine in the following order: jejunum > ileum > colon. The highest amount of in vitro absorption of carvedilol was achieved in the jejunum at a neutral pH. Enhancers such as bile and the mucoadhesive agent chitosan had opposite effects on the absorption of the compound. The results indicate that BM-RIMO is a simple, cheap and fast tool for the investigation of the influence of absorption enhancers, pH and different parts of intestine on absorption. For carvedilol the in vitro model tends to overestimate absorption in the colon, possibly because of the lack of faeces.

Aminoguanidine inhibits albuminuria, but not the formation of advanced glycation end-products in skin collagen of diabetic rats.

Aminoguanidine, an inhibitor of advanced glycation reactions in vitro, inhibits the development of diabetic complications in animal models of diabetes, suggesting that it acts by inhibition of advanced glycation reactions in vivo. However, effects of aminoguanidine on the formation of specific advanced glycation end-products (AGEs) in vivo have not been rigorously examined. Therefore, we studied the effects of aminoguanidine on the formation of pentosidine and N(epsilon)-(carboxymethyl)lysine (CML), measured by analytical chemical methods, in collagen of streptozotocin-diabetic Lewis rats at doses which ameliorated urinary albumin excretion, an index of diabetic nephropathy. At 12 weeks, diabetic animals had fivefold higher blood glucose, threefold higher glycated hemoglobin and fivefold higher collagen glycation, compared to metabolically healthy controls; pentosidine and CML in skin collagen were increased by approximately 30 and 150%, respectively. Administration of aminoguanidine, 50 mg/kg by daily intraperitoneal injection, significantly inhibited the development of albuminuria (approximately 60%, P < 0.01) in diabetic rats, without an effect on blood glucose or glycation of hemoglobin or collagen. Surprisingly, aminoguanidine failed to inhibit the increase in pentosidine and CML in diabetic rat skin collagen. Similar results were obtained in an independent experiment in which aminoguanidine was administered in drinking water at a dose of 0.5 g/l. We conclude that the therapeutic benefits of aminoguanidine on albuminuria may not be the result of inhibition of AGE formation.

Production of sequence specific polyclonal antibodies to human parathyroid hormone 1-37 by immunization with multiple antigenic peptides.

To generate site-specific antibodies to the N-terminal bioactive fragment of the parathyroid hormone hPTH 1-37, multiple antigenic peptide systems (MAP) for immunization were used. Two 10 residue fragments and a 14 residue fragment derived from knowledge of the secondary structure of hPTH 1-37 were selected to be synthesized as MAPs. Each peptide (hPTH 1-10, hPTH 9-18, and hPTH 24-37) was synthesized directly onto a branching heptalysine core matrix by automated solid phase synthesis. The hPTH 1-10 and the hPTH 24-37 MAP were highly immunogenic in rabbits. Ten polyclonal antisera obtained from rabbits were characterized by epitope mapping. Antigenic determinants were found as follows: 1) Sera K1-K3 raised to MAP 1-10 showed a predominant binding sequence at hPTH 1-5. 2) Sera K4-K6 raised to MAP 8-18 preferentially bound to residues 9-14. 3) Immunizing with hPTH 24-37 MAP led to antisera characterized as follows: serum K7 recognized residues 24-37, the sequence used for immunization, sera K8, K9 and K10 bound to residues 24-37 and 26-34. In summary, the favoured regions as deduced from the secondary structure of hPTH 1-37 were covered by the produced antibodies.

Synergistic effects of ularitide acetate with classical bronchorelaxants on guinea-pig tracheal smooth muscle.

Ularitide (CAS 118812-69-4, urodilatin) is a member of the family of the atrial natriuretic peptides. In the present study, the relaxant effects of ularitide acetate, isoproterenol (isoprenaline) hemisulfate, aminophylline, zaprinast, and different combinations between these drugs were investigated on methacholine chloride-precontracted guinea-pig tracheal smooth muscle. Ularitide acetate was a weaker bronchorelaxant than isoproterenol hemisulfate and aminophylline. Moreover the relaxation induced by ularitide acetate was reversible, while the relaxation induced by isoproterenol hemisulfate, aminophylline, and zaprinast was irreversible. Combinations between in each case two of these substances were overadditive, if the phosphodiesterase-inhibiting component was applicated before the combination partner. Their effects were only additive, if the combination partners were applicated simultaneously. All combinations between ularitide acetate and isoproterenol hemisulfate, aminophylline, or zaprinast respectively relaxed the tracheas irreversibly. These results suggest that ularitide acetate might be a novel partner for classical bronchorelaxants in potent bronchorelaxing combinations in the therapy of asthma bronchiale.

A new immunoenzymometric assay for bioactive N-terminal human parathyroid hormone fragments and its application in pharmacokinetic studies in dogs.

Advances in the treatment of clinical disorders of mineral in homeostatis and metabolic bone disease with intact parathyroid hormone 1-84 or one of the biologically active N-terminal fragments require a precise and sensitive measurement in serum. Therefore, a two-site immunoenzymometric assay for the quantitative determination of bioactive hPTH-1-37 (human parathyroid hormone) at picomolar concentrations was developed. Monoclonal antibodies (mAB) against hPTH-1-37 were raised by hybridoma cells in serum-free cell culture. Furthermore, sequence-specific polyclonal antibodies were obtained by immunisation of rabbits using multiple antigenic peptides (MAP) representing the conspicuous regions of the primary structure of hPTH-1-37. The polyclonal and monoclonal antibodies were characterised by epitope mapping. The combination of a monoclonal antibody (13C63/5) recognising hPTH fragment 16-24 with a polyclonal antibody (k2) showing a predominant binding sequence at hPTH-1-5 led to a sandwich assay specific for N-terminally intact and therefore biologically active hPTH. The validated assay ranging from 4 to 1000 pmol/l was applied to pharmacokinetic studies of hPTH-1-37. After s.c. administration of 30 mu g/kg in 5 beagles, the maximum serum concentrations of hPTH-1-37 ranging at 2139 +/- 857 pmol/l were observed 45 min after the injection. Clearance of the peptide calculated from the exponential disappearance curve was 32.0 +/- 9.1 ml/min/kg with a mean t1/2 of 37 +/- 10 min.

Comparison of the effects of ularitide acetate and other bronchorelaxing substances on the thrombin-induced permeability raise of human endothelial cell monolayers.

Endothelial cell contraction plays a pivotal role in vascular leakage. It increases the extravasation of fluid and macromolecules from the lumen into the interstitium. This is also true for bronchial edema. Previous studies have indicated that an elevation of intracellular adenosine-3',5'-cyclic monophosphate (cAMP) or guanosine-3',5'-cyclic monophosphate (cGMP), respectively, can counteract this vascular leakage by improving the endothelial barrier function in analogy to the relaxation of smooth muscle cells. To investigate the potential antiedemateous effects of ularitide acetate (CAS 115966-23-9), isoproterenol hemisulfate (CAS 6078-56-4), sodium nitroprusside (CAS 13755-38-9, SNP), aminophylline (CAS 317-34-0), and combinations of these compounds, their effects on thrombin-induced macromolecular permeability raise in relation to cGMP- or cAMP-levels, respectively, in a model of human umbilical vein endothelial cells (HUVECs) were examined. Ularitide acetate, isoproterenol hemisulfate, and SNP all increased the amount of cyclic nucleotides and decreased the raise in permeability in the following order of potency: isoproterenol hemisulfate > ularitide acetate > SNP. Aminophylline raised both cGMP- and cAMP-levels in a weaker amount and was not able to decrease the thrombin-induced permeability raise on its own. By way of contrast, preincubation of HUVECs with aminophylline resulted in a more than additive potentiation of the cGMP-levels and the permeability lowering induced by ularitide-acetate. These in vitro-data indicate that ularitide-acetate, especially in combination with phosphodiesterase (PDE) inhibitors, could probably have beneficial effects in bronchial permeability edema.

Diarylsulfonamides as selective, non-peptidic thrombin inhibitors.

Based on the structures of aminopyridine thrombin inhibitors (1), a series of aminoalkyl- and guanidinoalkyl-substituted diarylsulfonamides were prepared. The most potent derivative, N-[3-(4-guanidinobutoxy)-5-methyl-phenyl]-benzenesulfonamide (6c) had Ki = 0.18 microM for thrombin and did not inhibit trypsin, plasmin, or factor Xa. Comparison of the X-ray structures of the thrombin/1b and the thrombin/6c complexes revealed important aspects which govern the binding of such diarylsulfonamides to thrombin.

Potent free radical scavenging activity of propol isolated from Brazilian propolis.

We evaluated free radical scavenging activity of the water, methanol and chloroform extracts of propolis in 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and xanthine-xanthine oxidase (XOD) generated superoxide anion assay systems. The free radical scavenging activity guided fractionation and chemical analysis led to the isolation of a new compound, propol (3-[4-hydroxy-3-(3-oxo-but-1-enyl)-phenyl]-acrylic acid) from the water extract, which was more potent than most common antioxidants such as vitamin C and vitamin E (alpha-tocopherol) in these assay systems.

Dose proportionality studies of novel thiazolidinedione derivatives as potent antidiabetic agents in mice.

The determination of the plasma concentrations of the new oral antidiabetic agents BM 13.1246 ((+/-)-5-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)-ethoxy] benzyl]-2,4-thiazolidinedione), [sequence: see text] BM 13.1215 ((+/-)-5-[(5-methyl-2-phenyl-4-oxazolyl)-methyl-2- benzofuranyl-5-methyl]-2,4-oxazolidinedione), [sequence: see text] and BM 50.1050 ((+/-)-5[4-[2-(5-methyl-2-phenyl-4-oxazolyl)-ethoxy] naphthalyl]methyl-2,4-thiazolidinedione) [sequence: see text] in ob/ob mice plasma was performed by using liquid-liquid extraction and high-performance liquid chromatography with ultraviolet (270 nm) detection. The analytical procedures have recoveries of more than 80%, and a between-run precision of less than 4% for all analysed compounds. The pharmacokinetic behaviour, especially the dose proportionality, was investigated in ob/ob mice after repeated oral doses of 1 and 10 mg/kg, respectively. All compounds were absorbed quickly and attained maximum plasma concentrations within 2-5 h after administration. In the examined interval of dosing, an approximately proportional increase of the plasma levels for BM 13.1246 and BM 50.1050 was observed. After repeated oral doses the terminal half-lives are about 4 h for BM 13.1246, 8 h for BM 13.1215, and 6 h for BM 50.1050.

Pharmacokinetics of some new oral blood glucose-lowering agents in dogs.

The determination of the plasma concentrations of the new oral antidiabetic agents BM 17.0505 (2-(4-cyclopentylphenoxy)-7- (4-chlorphenyl)-heptanic acid), BM 13.1196 (2-(4-chlorphenyl)- heptanic acid), BM 13.1196 (2-(4-cyclopentylphenoxy)-7-(2-methoxy- phenyl)-heptanic acid: BM 13.1188 (2-(4-benzylphenoxy)-7-(2-methoxy- phenyl)-heptanic acid) and BM 13.1180 (2-(4-butylphenoxy)-5- (4-chlorphenyl)-pentanic acid) in dog plasma were performed by using liquid-liquid extraction and high-performance liquid chromatography with ultraviolet (220 nm) detection. The analytical procedures have recoveries of more than 90%, and a between-run precision of less than 5% for all analysed compounds. The pharmacokinetic behaviour, especially the dose proportionality, was investigated in dogs after a single oral dose of 5 and 50 mg/kg and repeated oral doses of 5 and 50 mg/kg, respectively. All compounds were absorbed quickly and attained maximum plasma concentrations within 1-3 h after administration. In the examined interval of dosing, a clear non proportional increase of the plasma levels was observed. After repeated oral doses the terminal half-lives are about 60-70 h (BM 17.0505), 80 h (BM 13.1196), 30 h (BM 13.1180) and 100-140 h (BM 13.1180).

Pharmacokinetics of the new oral blood glucose-lowering agent (-)-2-(4-tert.-butylphenoxy)-7-(4-chlorophenyl)-heptanic acid sodium salt in mice, rats and dogs.

The pharmacokinetic behavior of the alpha-activated carbonic acid (-)-2-(4-tert.-butylphenoxy)-7-(4-chlorophenyl)-heptanic acid sodium salt ((-)-BM 13.1074-Na) was examined in ob/ob mice, rats and dogs. By applying an enantioselective HPLC-method, the in vivo stability of the administered (-)-enantiomer could be demonstrated in all tested species. After oral administration the compound was absorbed quickly and maximum plasma levels were reached within 1 h (ob/ob mice and rats) and 3 h (dogs), respectively. In dose proportionally studies in ob/ob mice, with doses of 0.25 and 1 mg/kg, a clear non proportional-increase of the plasma levels was observed. The terminal half-lives of (-)-BM 13.1074 after multiple dosing are approx. 30 h in ob/ob mice, 9 h in rats and approx. 380 h in dogs. The average effective plasma concentration in ob/ob mice is found to be 43.5 mg/l; minimal toxic concentrations are 58.8 mg/l in rats and 105.6 mg/l in dogs, respectively.

Pharmacokinetics of the organic nitrates trans-N-(4-nitroxycyclohexyl)-urea in dogs and trans-N-(4-nitroxycyclohexyl)-acetamide in dogs and in man.

The pharmacokinetic behavior of the organic nitrates BM 12.1247 (trans-N-(4-nitroxycyclohexyl)-urea) and BM 12.1307 (trans-N-(4-nitroxycyclohexyl)-acetamide, CAS 137291-91-3) were examined in beagle dogs after oral and intravenous administration. To this end, a reliable and specific assay using capillary gaschromatography with electron capture detection (GC-ECD) was developed. BM 12.1247 showed its maximum plasma level after 2.2 h by oral application; the bioavailability was nearly 100%. The elimination half-life of 8.8 h p.o. Corresponded to the half-life after i.v. administration, concerning BM 12.1307, the elimination half-lives were still longer, they reached 11-13 h and bioavailability reached 68-70%; these results were confirmed by crossover tests. In a first application of healthy volunteers it was possible to show that the organic nitrate BM 12.1307 is eliminated much more slowly in man than in dog, the half-life of the compound in man being longer than the half-life in dog by a factor of 2.3.

Biotransformation of the organic nitrate trans-N-(4-nitroxycyclohexyl)acetamide in dogs.

The biotransformation of BM 12.1307 (trans-N-(4-nitroxycyclohexyl)acetamide, CAS 137291-91-3) in the dog was examined after oral and intravenous administration. For that purpose, the organic nitrate was synthesized as radioactive [14C]- and as [13C]-labeled compounds. The defined isotopic mixture was administered to the dogs. Within the examined period of 168 h, the elimination of BM 12.1307 and its metabolites via urine and feces amounted to 76.5% after oral application, and to 80.7% of the applied dose after intravenous application. The major amount of radioactivity was eliminated via urine (69.4% and 73.6% of the dose, respectively), whereas the fecal elimination was found to be negligible. Investigations of the urinary samples showed that the drug is metabolized to a high percentage trans-N-(4-Hydroxycyclohexyl) acetamide is the main metabolite; 73% of the radioactive compounds (after p.o.-administration and 69% after intravenous application could be identified as the alcohol of BM 12.1307; the amounts of the drug totalled 9% and 13%, respectively. The quantitative determination of BM 12.1307 in urine and plasma was performed by gas chromatography; the amount of the main metabolite excreted in urine was determined by high-pressure liquid chromatography. Trans-N-(4-hydroxycyclohexyl)-acetamide, N-(4-oxocyclohexyl)acetamide, and 3-acetamido-7-oxa-bicyclo [4.1.0]heptane were formed as metabolites. For the identification and characterization of the possible metabolic structures, these compounds were synthesized and used in comparison with the detected drugs.

Conventional and enantioselective determination of a new blood glucose-lowering agent in biological fluids using liquid-liquid extraction and high-performance liquid chromatography.

Two analytical methods are described for the determination of 2-(4-tert.-butylphenoxy)-7-(4-chlorophenyl)heptanoic acid sodium salt (I) in animal models (beagle dog and rat). Method 1 is conventional reversed-phase high-performance liquid chromatography on an octadecylsilane column with an eluent of acetonitrile-0.02 M potassium buffer (pH 3) (65:35, v/v). Method 2 is used for the enantioselective determination of I. This method uses a chiral column (Chiralcel OJ) with an eluent of n-hexane-2-propanol (95:5, v/v) containing 3 ml/l trifluoracetic acid. The analytical procedure has a recovery of more than 90%; within-run precision of less than 5.1%, and between-run precision of less than 4.3%.

Biotransformation and pharmacokinetics of the nitrate trans-2-amino-2-methyl-N-(4-nitroxycyclohexyl)-propionamide in dogs.

The biotransformation and the pharmacokinetic behavior of the organic nitrate trans-2-Amino-2-methyl-N-(4-nitroxycyclohexyl)-propionamide (BM 12.1179, CAS 129795-96-6) were examined in dogs. BM 12.1179 was predominantly eliminated by urinary excretion, and the unchanged molecule prevailed in urine as well as in plasma. By means of various mass spectroscopic methods, the chemical structures of the metabolites were elucidated. As metabolites trans-2-amino-2-methyl-N-(4-hydroxycyclohexyl)-propionamide and trans-2-amino-2-methyl-N-(4-oxocyclohexyl)-propionamide were formed. Urine levels of the main metabolite were determined by high-pressure liquid chromatography; plasma and urine levels of BM 12.1179 were determined by capillary gas chromatography. The absolute bioavailability of BM 12.1179 was 80-100%. The plasma protein binding was about 34% which is high in comparison to other organic nitrates. BM 12.1179 represents a long-acting organic nitrate in that it shows a slow reductive denitration, and a long elimination half-life of about 10 h.

Pharmacokinetics of the organic nitrates trans-2-amino-2-methyl-N-(4- nitroxycyclohexylmethyl)-propionamide in dogs, and of 4-(2-nitroxyethyl)-piperidine in dogs and in monkeys.

The plasma concentrations of the organic nitrates (nitric acid esters) trans-2-amino-2-methyl-N-(4-nitroxycyclohexylmethyl)-propionamide (BM 12.1200) and of 4-(2-nitroxyethyl)-piperidine (BM 12.1173, CAS 129999-77-5) were determined in dog plasma by capillary gas chromatography with electron capture detection (GC-ECD). Intra- and interassay variation coefficients of the gas chromatographic analysis lay between 2.9 and 8.8%; recoveries amounted to 50-62%. Both nitric acid esters were absorbed quickly and attained maximum plasma levels within 1 h. The total bioavailability of BM 12.1200 is > 55%, and that of BM 12.1173 is 63%; their elimination half-lives are, respectively, 4.2 h and 1.3-1.4 h. Thus, BM 12.1173 and the reference substance IS-5-MN (isosorbide-5-mononitrate) show corresponding elimination half-lives. These results regarding the pharmacokinetic parameters of BM 12.1173 were confirmed by cross-over application of BM 12.1173 and IS-5-MN to Macaca Arctoides. In comparison to all species which have been treated with IS-5-MN thus far, the shortest elimination half-life (t1/2 = 0.6 h) is found in monkeys.

[Spectroscopic properties of the partial beta-agonist doxaminol (BM 10.188) and its metabolites]. / Spektroskopische Eigenschaften des partiellen beta-agonisten Doxaminol (BM 10.188) und seiner Stoffwechselprodukte.

The biotransformation of the positively inotropically active compound N-methyl-N-(2-hydroxy-3-phenoxy-propyl)-11-(2-aminoethyl)-6,11- dihydrodibenz[b,e]-oxepine, neutral fumarate, (Doxaminol, racemic mixture of diastereomeres) in dogs is examined. The metabolits M1-M7 were isolated and their chemical structures identified by 1H-NMR, 13C-NMR and mass spectroscopic methods. 2-Hydroxy-3-phenoxypropionic acid, phenoxyacetic acid, 3-(4'-hydroxy)-phenoxy-1,2-propandiol and phenylacetic acid were formed by side chain oxidation of the parent molecule. Furthermore, the following conjugates were characterized: Doxaminol-O-glucuronide, 4'-hydroxydoxaminol-O-glucuronide, and 1-hydroxy-3-phenoxy-2-propyl sulfate.

Biotransformation of the partial beta-agonist doxaminol in dog.

The biotransformation of the positive inotropic compound doxaminol (N-methyl-N-(2-hydroxy-3-phenoxy-propyl)-11-(2-amino-ethyl)-6,11- dihydrodibenz[b,e]oxepine, neutral fumarate; BM 10.188) was examined in bastard shepherd dogs. Metabolic products, formed by oxidative cleavage of various side chain carbon atoms of the molecule, as well as conjugated complexes with glucuronic and sulfuric acid, were isolated from urine and plasma. As main metabolites 2-hydroxy-3-phenoxy-propionic acid and phenoxyacetic acid were formed. By means of 1HNMR and 13C-NMR spectroscopy and various mass spectroscopic methods, the chemical structures of the metabolites were elucidated.