Effects of parathyroid hormone and agonists of the adenylyl cyclase and protein kinase C pathways on bone cell proliferation.

The anabolic effect of parathyroid hormone (PTH) on bone is partly due to a stimulation of osteoblast proliferation. The PTH signal is transduced by the pathways of adenylyl cyclase (AC)/protein kinase (PK) A and phospholipase C/PKC/Ca++. There is still uncertainty about the relative contribution of the two pathways to the proliferative effects of the hormone. In our study, PTH(1-34), AC/PKA agonists, and phorbol 12-myristate-13-acetate (PMA, a PKC activator) stimulated cell proliferation in cultured mouse calvariae. In isolated osteoblasts, only PMA stimulated proliferation, whereas AC/PKA agonists and PTH(1-34) inhibited it. As already known, PTH in the presence of supramaximal concentrations of transforming growth factor-beta (TGF-beta) stimulated osteoblast growth; under these same conditions, AC/PKA agonists reproduced the stimulatory effect of PTH(1-34), whereas PMA became inhibitory. PTH(1-31), which stimulates AC without affecting PKC, acted similarly to the fully active PTH(1-34) in both calvaria and isolated osteoblasts. On the contrary, midregion fragments that activate only PKC stimulated calvaria cell proliferation faintly in comparison with PTH(1-34); no effect was seen in osteoblasts, either with or without TGF-beta. Our study shows that the effects of PTH on proliferation can be mimicked by agonists of the AC/cAMP pathway. Although PMA is indeed able to stimulate cell growth in tissue explants, its effects on isolated osteoblasts markedly diverge from those of PTH. We conclude that activation of the AC/PKA pathway is the main component of the proliferative effects of PTH.

Tetrapeptide tachykinin antagonists: synthesis and modulation of the physicochemical and pharmacological properties of a new series of partially cyclic analogs.

We report on the synthesis and the pharmacological properties of a new series of tachykinin antagonists based on the pseudopeptide pharmacophore cyclo[-Abo-Asp(D-Trp-Phe-N(Me)Bzl)-] which contains the 2-azabicyclo[2.2.2]octane-3(S)-carboxylic acid (Abo) residue. Variation of the substituents on the tryptophan indole nitrogen was shown to modulate water solubility and transport properties of the analogs as well as potency in classical in vitro response and binding assays. One water-soluble compound, 16, in which the substituent was 3-carbonylpropionate, strongly prolonged the reaction time in the mouse hot-plate test both after iv or oral administration and was devoid of degranulating activity in rat peritoneal mast cells.

A water-soluble, stable dipeptide NK1 receptor-selective neurokinin receptor antagonist with potent in vivo pharmacological effects: S18523.

The potassium salt of a chemically stabilized dipeptide, {1-[4-(1 H-tetrazol-5-yl)butyl]indol-3-yl}carbonyl-Hyp-Nal-N(methyl)-Bzl , (Hyp = (R)-4-hydroxy-L-proline; Nal = 3-L-(beta-naphthyl)-alanine), S18523, is described as a new water-soluble, potent and selective NK1 receptor antagonist. The low molecular weight antagonist (M(r) = 736) displays nanomolar potency (pA2 = 9.6) in the rabbit vena cava (NK1) bioassay and nanomolar affinity (pKi = 9.1) on the human NK1 receptor expressed by lymphoblastoma cells. It is devoid of mu-opiate affinity (Ki > 10(-4) M with respect to tritiated Tyr-DAla-Gly-MePhe-Gly-ol), has negligible calcium-channel affinity (estimated Ki = 2.6 x 10(-5) M, with respect to isradipine) and does not cause peritoneal mast-cell degranulation. S18523 has strong antinociceptive effects in three classical pain tests in vivo both by i.v. and p.o. routes. The dipeptide potently antagonizes bronchoconstriction provoked by exogenous substance P in the guinea-pig and acts longer than the non-peptide antagonist CP99994, when administered as aerosol. Finally, S18523 displays antiinflammatory properties, since it dose-dependently inhibits substance P-induced plasma extravasation both in the bladder (ID50 = 0.18 mg/kg i.v.) and bronchi (ID50 = 0.14 mg/kg i.v.) of the guinea-pig.

In vitro and in vivo pharmacology of S 16474, a novel dual tachykinin NK1 and NK2 receptor antagonist.

Since tachykinins released from lung sensory nerve endings are thought to play a role in inflammatory diseases of airways via NK1 and NK2 receptors, dual tachykinin NK1 and NK2 receptor antagonists may have a great therapeutic potential. In vitro, the cyclopeptide S 16474 (cyclo-[Abo-Asp(D-Trp(Suc0Na)-Phe-N-(Me)Bzl)]) bound to both human tachykinin NK1 and NK2 receptors expressed in two lines of transfected Chinese hamster ovary cells (IC50 values 85 nM and 129 nM, respectively), while showing a poor affinity for the rat tachykinin NK1 receptor. S 16474 inhibited the contractions induced by substance P in isolated rabbit vena cava (pA2 7.0) and by neurokinin A in rabbit pulmonary artery (pA2 5.6). In vivo in anaesthetized guinea-pigs, S 16474 was found to dose dependently inhibit the bronchoconstrictions induced by intravenously administered substance P, neurokinin A and capsaicin. Plasma extravasation evoked in bronchi by endogenously released tachykinins under vagus nerve stimulation was abolished by S 16474 (10 mu mol/kg i.v.). These results demonstrate clearly that S 16474 is a tachykinin receptor antagonist exhibiting, in vitro and in vivo, a dual inhibitory effect on NK1 and NK2 receptors.

An overview of assay methods for D-penicillamine.

The stability and biotransformation of D-penicillamine as it relates to assay development is discussed. A review of published literature on assays is presented with respect to blood/plasma levels of D-penicillamine in man and to statistical evaluation of assays. Also, the paper describes gas chromatographic assay for D-penicillamine disulfide and L-cysteine D-penicillamine disulfide in urine and plasma, as well as a new high performance liquid chromatography assay for D-penicillamine in plasma.

Estimation of the lower limit of quantitation, a method detection performance parameter for biomedical assays, from calibration curves.

A model for the lower limit of quantitation for biomedical chromatographic assays is proposed. It is based on the IUPAC definition for the limit of detection and can be estimated from assay calibration data. It has been applied to ten different sets of calibration data from various assays of drugs in biological matrices.

Drug level monitoring of antiasthmatic drugs.

In general assays pertaining to drug level monitoring (DLM) of antiasthmatic agents (except theophylline), published during the period 1978-1983, used mostly high-performance liquid chromatographic (HPLC) methodology (approximately 45%) with mass spectrometric (MS) based assays in second place (approximately 30%) followed by immunochemical techniques (approximately 25%). Whenever nanogram or subnanogram antiasthmatic drug concentrations had to be measured such as for the adrenergic stimulants or for the prophylactic agents, then both HPLC-and MS-based methodologies were employed with about equal frequency. The trend in DLM for the phosphodiesterase inhibitor class (theophyllines) seemed to be shifting towards the HPLC methodologies. In part, this was justified by the need for improved selectivity. This criterion appears to have been better satisfied by HPLC, but for all practical purposes the immunochemical methods are and will probably continue to prevail in the clinical laboratory setting until HPLC procedures become truly automated. In the case of DLM of corticosteroids used for the asthmatic, the situation is in our opinion still unclear. This is caused by the presence of endogenous corticosteroids and metabolites, the levels of which in man are known to vary. The current immunochemical procedures offer a facile but less selective option. The future for selective routine corticosteroid assays may well be in HPLC or gas chromatography coupled with MS.

Biotransformation of reproterol in the intestinal tract of the rat.

The major metabolite of reproterol (Broncho-spasmin) in rat feces is 2-[3-theophyllinyl(7)-propyl]-4,6,8-trihydroxy-1,2,3,4-tetrahydroisoquinoline. It was also found in the bile of orally or intravenously dosed rats in the form of glucuronides. The biotransformation of an oral dose of reproterol to this metabolite occurred mostly in the cecum-colon section of the intestinal tract. Experiments in antibiotic treated rats showed no significant effect of the treatment on the extent of metabolite formation. This metabolite was also formed by incubation of reproterol with cecum-colon homogenates under aerobic as well as anaerobic conditions. The pharmacological action after an oral dose must be attributed to reproterol absorbed as intact form, since the metabolite is inactive.

[Studies on the biotransformation of reproterol. Structure determination of the main metabolite (author's transl)]. / Untersuchungen zur Biotransformation von Reproterol. Strukturaufklärung des Hauptmetaboliten.

Biotransformation of 7-(3-[2-(3,5-dihydroxyphenyl)-2-hydroxy-ethylamino]-propyl)-theophylline (reproterol, Bronchospasmin), a beta 2-adrenergic drug recently introduced into therapeutic use, leads to the same main metabolite in animals and in man. By mass-spectroscopy and by synthesis its structure was shown to be tetrahydroisoquinoline derivative which is produced from reproterol by uptake of an additional carbon atom concomitant with cyclization.

A structural rationale for the design of water soluble peptide-derived neurokinin-1 antagonists.

Molecular models of a pharmacophore for NK1 neurokinin antagonists and of ligand-receptor complexes for the human NK1 G protein-coupled receptor are presented. The models develop a structural rationale for the discovery of the recently described highly potent peptidomimetic NK1 antagonists S18523 and S19752 which were designed to be water soluble. Water solubility was conferred on these compounds by introduction of an anionic butyl-tetrazole substituent on the scaffold of dipeptide-derived NK1 antagonist analogues. The models provide convincing evidence that the anionic butyl-tetrazole moieties of S18523 and S19752 protrude outside the membrane-spanning domain of the receptor and do not interfere significantly with the core of the antagonist binding site. It is emphasized that this result could only be obtained through the combination of the two modelling approaches. The result suggest a general way to modify the transport properties of the peptidomimetic antagonists without altering the receptor-binding interaction, and it outlines the potential of including the combination of pharmacophore models and crude models of receptor-ligand complexes early in the drug design process.

The formaldehyde-donating activity of N5,N10-methylene tetrahydrofolic acid in xenobiotic biotransformation.

Reaction of reproterol, (7-(3-[2-(3,5-dihydroxyphenyl)-2-hydroxyethylamino]propyl)theophyl line (I), with formaldehyde liberated in a solution of N5,N10-methylene tetrahydrofolic acid at pH 7.3 led to the formation of 7-[3-(4,6,8-trihydroxy-1,2,3,4-tetrahydroisoquinolinyl-2)propyl]th eophylline (II), but no such reaction was observed in a solution of N5-methyl tetrahydrofolic acid. Compound II was also formed by incubation of I with rat-liver subcellular fractions. The highest formaldehyde-donating activity was found in the soluble fraction but some activity was also observed with washed mitochondria and microsomes. Dimedone was transformed to methylene bisdimedone by incubation with the soluble fraction. The amount of formaldehyde liberated in the soluble fraction during a four hour incubation at 22 degrees C was 1-3.6 mumol/g of liver. Metaproterenol and terbutaline, structurally related to I, reacted with formaldehyde to yield the corresponding tetrahydro-isoquinolines. The rate of this chemical reaction for the three drugs correlated with the amount of the tetrahydroquinolines formed by incubation with the soluble fraction and in orally dosed rats.

Study on in vivo decarboxylation of D-penicillamine in rats.

Carboxy-labeled 14C-D-penicillamine was prepared and dosed at 50 mg/kg to Sprague-Dawley rats kept in metabolism cages. The recovery of 14C in excreta, tissue and expired air was 98.4%. The 14CO2 expired amounted only to 1.5% of the 14C-dose indicating that in vivo decarboxylation of the drug in rats is not a significant metabolic pathway.

Determination of the anticonvulsant felbamate in beagle dog plasma by high-performance liquid chromatography.

An automated internal standard method for the determination of felbamate in 0.1 ml of plasma from pediatric and adult beagle dogs was developed. Plasma proteins are precipitated with acetonitrile and after centrifugation the supernatant is directly injected on a Spherisorb ODS2, 3 microns, 150 mm x 4.6 mm I.D. column with 25% acetonitrile in aqueous phosphate buffer, pH 6.50, as mobile phase with ultraviolet detection at 210 nm. The run time is 10 min, the linear range is 0.150-150 micrograms/ml felbamate, and the lower limit of quantitation is 0.150 microgram/ml.

Pharmacokinetics of felbamate in pediatric and adult beagle dogs.

The relative bioavailability and pharmacokinetics of felbamate (FBM) after a single oral dose and after 10 once-daily oral doses of 60 mg/kg were investigated in adult and pediatric dogs of both sexes. The pediatric and adult dogs were aged 4-6 weeks and 1-2 years, respectively. Analysis of variance (ANOVA) was performed on the bioavailability parameters among all groups and between the first and last doses. No sex-related differences in bioavailability and pharmacokinetic parameters were observed. The bioavailability of FBM in pediatric dogs was significantly less as compared with that in adult dogs. Rapid overall elimination of the drug in pediatric dogs appears to be responsible for the lower bioavailability. The bioavailability of FBM after the last dose was also significantly lower than after the first dose for both age groups. No major differences in the rate constant of FBM absorption (ka) and volume of distribution at steady state (VSS) were observed between the two age groups. As with other clinically useful antiepileptic drugs (AEDs), higher doses of FBM may be required in pediatric populations to achieve optimum drug levels, assuming that age-related changes in FBM disposition will also be confirmed in humans.

Distribution of the anticonvulsant felbamate to cerebrospinal fluid and brain tissue of adult and neonatal rats.

The concentrations of felbamate (FBM) and its three metabolites were determined in plasma, cerebrospinal fluid (CSF), and brain tissue of adult and neonatal rats that received single oral doses of FBM at amounts varying from 250 to 2000 mg/kg for adults and from 100 to 500 mg/kg for neonates. The increase in plasma Cmax and AUC0-24 with the dose was less than proportional in both age groups. The highest plasma Cmax in adults dosed with 2000 mg/kg was 140.3 micrograms/ml; in neonates dosed with 500 mg/kg it was 257.0 micrograms/ml. The maximum brain concentrations for these dosages were 90.9 and 220.4 micrograms/g, respectively. The average brain/plasma, CSF/plasma, and brain/CSF partition coefficients for the drug were 0.64, 0.55, and 1.16 for adults and 0.83, 0.67, and 1.23 for neonates, respectively. No statistically significant change of the partition coefficients with time or dose was observed (p < 0.05). Very good linear correlations between FBM plasma concentrations and concentrations in CSF and brain tissue were obtained (r2 > 0.98). Only the 2-hydroxy metabolite was present in considerable amounts in plasma and brain tissue of the high-dose groups of both ages.

The disposition of 3-methyl-2,3-dihydro-9H-isoxazolo[3,2-b]-quinazolin-9-one (W-2451).

3-Methyl-2,3-dihydro-9H-isoxazolo[3,2-b]quinazolin-9-one was readily absorbed, metabolized and eliminated in rat, dog, monkey and man. The radioactivity elimination after i.v. or p.o. administration of W-2451-14C in rats was biphasic with corresponding half-lives of 1.8 and 8.7 hours. Plasma half-lives of W-2451 in the dog, rhesus monkey and man were 1.4, 1.2 and 3.2 hours, respectively. In the rat, excretion via the urine was predominant, no significant accumulation in tissue occurred. The only major metabolite found in rat and dog urine, rat plasma and in the rat liver 9000 g supernatant fraction was the 3-hydroxy derivative of the drug. The unsaturated compound with the double bond in the 2,3-position and other hydroxylated metabolites were also present. Very little free or conjugated anthranilic acid and 3-(o-carboxy-phenylimino)-4-methylisoxazolidine were found.

Felbamate metabolism in the rat, rabbit, and dog.

Two major and one minor metabolite of felbamate (FBM) as well as unchanged drug were isolated and identified by electron impact and chemical ionization mass spectrometry from rat and dog urine after dosing with [14C]FBM. The metabolites were 2-(4-hydroxyphenyl)-1,3-propanediol dicarbamate (p-OHF), 2-hydroxy-2-phenyl-1,3-propanediol dicarbamate, and 2-phenyl-1,3-propanediol monocarbamate. The metabolites and FBM were excreted mainly in urine, where their sum accounted to 81-94% of the radioactivity in hydrolyzed rat urine samples, 71-82% in rabbit urine samples, and 69-83% in dog urine samples. The amount of metabolites in the conjugated form was estimated to be 20-35% in rat, 20-30% in rabbit, and 10-20% in dog urine. The major biliary metabolite in all three species was p-OHF, whereas the amount of FBM was small. Metabolites found in dog feces were the same as those in the urine.

Felbamate pharmacokinetics in the rat, rabbit, and dog.

Rats, rabbits, and dogs were given single iv or single and multiple oral doses of felbamate ranging from 1.6-1000 mg/kg. Absorption of oral drug was complete in all species. The mean Cmax increased with dose from 13.9 to 185.9 micrograms/ml in rats, from 19.1 to 161.9 micrograms/ml in rabbits, and from 12.6 to 168.4 micrograms/ml in dogs. The tmax also increased with dose from 1-8 hr in rats, 8-24 hr in rabbits, and 3-7 hr in dogs. The plasma elimination half-life for the drug increased with dose from 2-16.7 hr in rats, 7.2-17.8 hr in rabbits, and 4.1-4.5 hr in dogs. A proportional increase in Cmax with dose was observed in all species up to 300-400 mg/kg doses. A biexponential equation fitted the drug plasma concentration vs. time data well. For multiple oral doses of 50 mg/kg or less, projected and observed steady-state concentrations agreed well. Animals dosed with [14C]felbamate eliminated most of the radioactivity in urine (58-87.7%), less in feces (7-23.7%), with considerable amounts in the bile. In rats, radioactivity was readily distributed into tissues and crossed the placenta and blood-brain barrier, but no accumulation in any tissue was observed. The volume of distribution was 131, 54, and 72% of body weight for rats, rabbits, and dogs, respectively. Binding of drug to rat, rabbit, and dog plasma proteins ranged from 22.4-35.9%. The overall plasma clearance of the drug for rats, rabbits, and dogs was 327, 52, and 108 ml.h-1.kg-1, respectively. Renal clearance of unchanged drug accounted for an estimated 20-35% and hepatic clearance due to metabolism for 65-80% of the overall clearance.