Pharmacological and toxicological effects of chronic porcine growth hormone administration in dogs.

The purpose of this study was to evaluate the pharmacological and toxicological effects of exogenous GH administration in normal adult dogs. Because porcine GH (pGH) is structurally identical to canine GH, pGH was selected for a 14-wk study in dogs. Thirty-two dogs (< 2 yr) were randomized to 4 groups (4 dogs/sex/group); 1 group was treated with the vehicle and 3 groups received pGH at 0.025, 0.1, or 1.0 IU/kg/day subcutaneously. Daily clinical signs and weekly body weights were recorded. Hematology, serum biochemistry, urinalyses, electrocardiograms, and ophthalmoscopic examinations were done. Serum GH, insulin-like growth factor-1 (IGF-1), insulin, thyroxine (T4), triiodothyronine (T3), and cortisol levels were determined. Necropsies were performed, organs weighed, and tissues were fixed and processed for light microscopic examination. Porcine GH caused increased body weight gain (p < or = 0.05) through the mid dose; the mean weight gains at study termination in mid- and high-dose groups were 2.8 kg and 4.7 kg, respectively, compared to 0.4 kg and 0.8 kg in control and low-dose groups, respectively. Dose-related increased weights of liver, kidney, thyroid, pituitary gland, skeletal muscle, and adrenal gland were noted. In pGH-treated dogs, increased skin thickness seen grossly correlated histologically with increased dermal collagen. There was no gross or histomorphological evidence of edema. There were dose-related increased serum IGF-1 levels (approximately 2-10-fold; p < or = 0.05) that correlated with the elevated serum GH levels in pGH-treated dogs. Also, increased serum insulin levels (p < or = 0.05) through the mid dose were seen throughout the study. In high-dose dogs, the insulin levels remained elevated over 24 hr postdose. The serum glucose levels in fasted dogs remained within the control range and there was no chronic hyperglycemia based on glycosylated hemoglobin levels. Renal glomerular changes, significant polyuria with decreased urine specific gravity, and increased serum insulin levels suggested that the dogs had early insulin-resistant diabetes. There was minimal or no biologically significant effect of pGH on serum T3, T4, and cortisol levels in dogs. Other serum biochemical changes in pGH-treated dogs included decreased urea nitrogen and creatinine, and increased potassium, cholesterol, and triglycerides. Significant increases in serum calcium and phosphorous levels and alkaline phosphatase activity (bone isozyme) correlated with the histological changes in bone. In pGH-treated dogs, there was a dose-related normochromic, normocytic, nonregenerative anemia. The changes described above, except for the anemia, are related to either anabolic or catabolic effects of high doses of GH. Based on this study, it is concluded that the dog is a good model in which to evaluate the safety of GH secretagogues as well as compounds with GH-like activity.

Pharmacokinetics of falintolol II. Absorption, distribution and elimination from tissues and organs following ocular administration and intravenous injection of falintolol in albino rabbits.

The absorption, distribution and elimination of falintolol maleate was studied in various ocular and extraocular tissues and organs following ocular instillation, intravenous injection of a 0.5% 14C-falintolol ophthalmic solution and repeated ocular instillations of a 1% non-labeled falintolol ophthalmic solution into albino New Zealand rabbits. Falintolol was distributed in all studied tissues and organs after both routes of administration. After ocular instillation, levels of total radioactivity were distinctly higher in ocular tissues than after intravenous injection. Thus, the level was 475 times more important in cornea, 72 times in aqueous humor and 36 times in iris and ciliary body after ocular instillation. On the other hand, levels of total radioactivity in extraocular tissues and organs were 30-50% higher after intravenous injection compared to ocular instillation of the same dose. Peak levels of total radioactivity were generally achieved between 30 min and 1 h after ocular instillation, while 1.5 h after intravenous injection an increase in the declining part of the curve occurred. This increase, characteristic of an enterohepatic reabsorption, was also observed in blood and plasma 1 h after intravenous injection. Urinary elimination was the major means of excretion since 79.6% of total radioactivity was found in urine 6 h after intravenous injection and 74.5% 12 h after ocular instillation. But after ocular instillation, only 5% was excreted as unchanged falintolol. Whatever the route of administration, after single or repeated application, no drug accumulation was evident.

Beta-adrenoceptor binding potencies of new aliphatic and alicyclic oxime ethers and their relevance to intraocular pressure control.

The selectivity and binding potency of a series of alkyliminoxypropanolamines characterized by the lack of an aromatic nucleus are studied using a new non-selective radioligand, (-)-(125I)-Iodocyanopindolol. The relationship between the effectiveness in lowering the intraocular pressure (IOP) in experimental hypertensive rabbit eyes and their capacity to bind to ciliary processes beta 2-adrenoceptors is discussed. The inhibition constant (Ki) and beta 2/beta 1 ratios indicate a beta 2-selectivity for the tested drugs. Cyclopropyl, dicyclopropyl, cyclopentyl and cyclohexyl derivatives displayed a potent binding to ciliary processes beta 2-adrenoceptors and lowered the IOP about -18%. These compounds induced a lowering in IOP equal to that produced by timolol and appear to be effective and safe beta-adrenergic antagonists in open-angle glaucoma therapy. Decreasing the size of the alkyl group of the oxime, removing the oxime function or modifying the beta-hydroxyl group from the side chain led to a significant decrease in beta 2-adrenoceptor binding and induced weak hypotensive ocular activity. Since the tested alkyliminoxypropanolamine series has very similar physicochemical characteristics and therefore, ruled out the differences in their ability to reach, through the cornea, the targeted ciliary processes, it was demonstrated that contrary to generally held views, the action of the new beta-antagonist series on IOP is related to their ability to antagonize ocular beta 2-adrenoceptors.

Effects of adrenergic agents on alpha-chymotrypsin-induced ocular hypertension in albino and pigmented rabbits: a comparative study.

Alpha-chymotrypsin-induced ocular hypertension in albino rabbits is widely used as an experimental model to screen potential antiglaucoma drugs. The present study compares the intraocular pressure (IOP) response following the ocular application of single or repeated adrenergic agents in conscious albino and pigmented rabbits. A single instillation of clonidine was not as effective in lowering the IOP in pigmented hypertensive rabbit eyes as in albino hypertensive eyes. Similarly, betaxolol moderately lowered the IOP in albino rabbits but induced a slight response when pigmented rabbits were used as an experimental model. Twice-a-day applications of betaxolol in pigmented hypertensive eyes permitted an identical level of IOP decrease to be reached, as observed in a one-day study in albino rabbits, after at least 6 days of treatment. It has been suggested that the pigmented layers of the iris-ciliary body may act as sites for topically applied antiglaucoma drugs. Non-specific binding could explain in part the frequent discrepancy observed between the preclinical results obtained in albino hypertensive rabbit eyes and clinical results obtained in glaucomatous human eyes.

Characterization of alpha 2-adrenergic receptors, negatively coupled to adenylate cyclase, in rabbit ciliary processes.

Inhibition of ciliary process adenylate cyclase was studied on rabbit membrane preparations. When considered individually, epinephrine, GTP and NaCl did not inhibit adenylate cyclase activity. On the other hand, when present together, epinephrine, GTP (10(-5) M) and NaCl (200 mM) acted synergistically to cause a 27% inhibition of basal activity. A similar inhibition was observed with 1-norepinephrine. Clonidine and BHT 920, two alpha 2-agents were found to be partial agonists causing 63% and 82% as much inhibition as epinephrine. Phenylephrine, an alpha 1-agonist did not inhibit adenylate cyclase activity at concentrations up to 10(-4) M. Yohimbine and phentolamine prevented the inhibition of adenylate cyclase by epinephrine, while prazosin was ineffective. Alpha 2-receptor selectivity in rabbit ciliary processes and their negative coupling to an adenylate cyclase via a NaCl-dependent GTP binding protein, Ni, is thus well established.

Transport of glutathione by intestinal brush border membrane vesicles.

Intestinal brush border membranes prepared from pig jejunum were shown to transport intact reduced glutathione into an osmotically responsive intravesicular space. At early incubation time (under 1 min.) GSH was poorly hydrolysed in its free amino acids. Primarily glutathione uptake was obtained against a concentration gradient (vesicle greater than medium) and was stimulated by Na+.

[Comparative study of the antiglaucomatous activity of Glauplex 2 and pilocarpine nitrate on alpha-chymotrypsin-induced experimental glaucoma]. / Etude comparative de l'activité antiglaucomateuse de Glauplex 2 et du nitrate de pilocarpine sur le glaucome expérimental à l'alpha-chymotrypsine.

The antiglaucomatous effects of Glauplex 2 and pilocarpine nitrate on alpha-chymotrypsine-induced experimental glaucoma were studied in 8 rabbits. Changes in intraocular pressure were measured over a period of 12 hours after a single instillation of Glauplex 2 or two instillations of 2.6 p. cent pilocarpine nitrate at t = 0 and t = 6 hours. The antihypertensive effect of a single instillation of Glauplex 2 was shown to be approximately equivalent to that of two instillations of 2.6 p. cent pilocarpine nitrate.

Human renal gamma-glutamyltransferase hydrolysis and transfer reactions with glutathione as substrate.

The hydrolysis and transfer reactions of purified human renal gamma-glutamyltransferase were studied in vitro with glutathione as substrate at pH and substrate concentration reflecting the physiological conditions. The pH optimum ranged from 7.48 to 8.44 for hydrolysis and 7.90 to 8.92 for transfer with glutamine as acceptor. The Michaelis constants for glutathione were 13 microM in hydrolysis and 58 microM in transfer reactions respectively. Inhibition of transfer occurred for glutathione concentrations above 0.4 mM. Various ions, urea, creatinine, uric acid and L-amino acids were shown to have no appreciable effect on both reactions except L-glutamine which acts as an activator on the hydrolysis activity. Taken together, our results, if they are transposable in vivo would be relevant of an enzyme acting like an hydrolase rather than like a transferase.

Purification by affinity chromatography and characterization of a neutral alpha-glucosidase from horse kidney.

A horse kidney neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) was purified about 580-fold with a yield of 33% by an affinity chromatography technique using the p-aminophenyl-beta-D-maltoside, a substrate derivative, as ligand. The purified enzyme, homogeneous in polyacrylamide gel electrophoresis, was a glycoprotein with a molecular weight of 280 000 as calculated by gel filtration and its isoelectric focusing points was found to be pH 4.1. The purified enzyme was able to hydrolyze various substrates having (alpha-1,2), (alpha-1,3), (alpha-1,4), and (alpha-1,6) glucosidic linkages. The V/Km ratio shows that the (alpha-1,4) linkages are the best substrates. The pKm of the purified enzyme determined at different pH values indicated that two ionizable groups with pK values 5.2 and 6.9 could be essential in the active site. Enzyme modification with cardodiimide abolished the maltase activity. The turanose, a substrate analogue, protected the enzyme against this inactivation.

Neutral alpha-glucosidase from human kidney. Molecular and immunological properties. Relationship with intestinal glucoamylase.

Some molecular properties of the purified neutral alpha-glucosidase from human kidney were studied. The enzyme is a glycoprotein with high molecular weight (315000-352000 according to the method used). Its sedimentation coefficient is 12.9S. It exhibits at least three peaks of activity in isoelectric focusing experiments. This heterogeneity appears to be related to sialic acid residues from the carbohydrate moiety. An anti-human renal alpha-glucosidase antiserum was raised from rabbit. The antiserum effect on human intestinal maltases was studied in immunodiffusion experiments. An identity pattern was observed between renal neutral alpha-glucosidase and intestinal glucoamylase. No precipitation occurred with intestinal sucrase. Renal neutral alpha-glucosidase and intestinal glucoamylase were both completely precipitated by the antiserum, their maltase activity being only slightly inhibited in the antigen-antibody complex. From their molecular and immunological properties a large homology appears between human renal alpha-glucosidase and intestinal glycoamylase.

[Catalytic properties of a neutral alpha-glucosidase from human kidney]. / Propriétés catalytiques de l'alpha-glucosidase neutre du rein humain

The catalytic properties of a neutral alpha-glucosidase purified to homogeneity from human renal cortex are described. The pH optimum was 6 (maltose and starch). It has a broad range of substrate specificity, hydrolysing di- and oligosaccharides with alpha (1 leads to 2), alpha (1 leads to 3), alpha (1 leads to 4) and alpha (1 leads to 6) linkages. Glucosidase action on maltosaccharides was associated with pronounced substrate inhibition at concentrations exceeding 0,5 mM. It also hydrolyses polysaccharides as starch and glycogen. The Km and Vmax values for the various substrates were determined. The enzymes exhibited intrinsic transglucosylase activity. It catalysed glucosyl-transfer reaction from maltose to itself (disproportionation). Mixed substrate inhibition studies, inhibition studies and heat inactivation are interpreted in terms of the existence of at least two interacting sites on the enzyme.