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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.

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We have identified and characterized potent and specific inhibitors of geranylgeranyl-protein transferase type I (GGPTase I), as well as dual inhibitors of GGPTase I and farnesyl-protein transferase. Many of these inhibitors require the presence of phosphate anions for maximum activity against GGPTase I in vitro. Inhibitors with a strong anion dependence were competitive with geranylgeranyl pyrophosphate (GGPP), rather than with the peptide substrate, which had served as the original template for inhibitor design. One of the most effective anions was ATP, which at low millimolar concentrations increased the potency of GGPTase I inhibitors up to several hundred-fold. In the case of clinical candidate L-778,123, this increase in potency was shown to result from two major interactions: competitive binding of inhibitor and GGPP, and competitive binding of ATP and GGPP. At 5 mM, ATP caused an increase in the apparent K d for the GGPP-GGPTase I interaction from 20 pM to 4 nM, resulting in correspondingly tighter inhibitor binding. A subset of very potent GGPP-competitive inhibitors displayed slow tight binding to GGPTase I with apparent on and off rates on the order of 10 6 M ؊1 s ؊1 and 10 ؊3 s ؊1 , respectively. Slow binding and the anion requirement suggest that these inhibitors may act as transition state analogs. After accounting for anion requirement, slow binding, and mechanism of competition, the structure-activity relationship determined in vitro correlated well with the inhibition of processing of GGPTase I substrate Rap1a in vivo.Inhibition of prenylation of Ras oncoproteins has long been considered an attractive approach to anti-cancer therapy of Ras-dependent tumors. Four isoforms of Ras are found in mammalian cells, Kirsten-Ras (Ki-Ras) 4A and 4B, Harvey-Ras (Ha-Ras), and N-Ras. Ki4B-Ras is the most frequently mutated isozyme in human cancer, ranging from 20% up to 90% in pancreatic cancers, whereas Ha-Ras mutations are found in a small percentage of bladder cancers. Most oncogenic mutations in Ras result in the elimination of the GTPase activity, locking Ras in its active, GTP-bound conformation. All Ras proteins undergo multiple post-translational modifications on their carboxyl termini that are required for membrane localization and biological activity. The COOH-terminal CAAX motif (where C is cysteine, A stands for aliphatic, and X for any amino acid) is recognized by farnesyl-protein transferase (FPTase), 1 which transfers a farnesyl group from farnesyl pyrophosphate (FPP) to the cysteine thiol. This prenylation step is followed by proteolytic clipping of the three terminal amino acids by a farnesyl-CAAX specific protease and subsequent methylation of the cysteine residue by a carboxymethyl transferase. Ha-Ras and N-Ras are further modified by palmitoylation which provides additional membrane interactions. A polylysine stretch in KiRas near the COOH terminus serves the same purpose. However, the prenylation step has been shown to be the single most critical modification for function of all Ras prote...