Glucokinase (GK) activation as a potential strategy to treat type 2 diabetes (T2D) is well recognized. Compound 1, a glucokinase activator (GKA) lead that we have previously disclosed, caused reversible hepatic lipidosis in repeat-dose toxicology studies. We hypothesized that the hepatic lipidosis was due to the structure-based toxicity and later established that it was due to the formation of a thiourea metabolite, 2. Subsequent SAR studies of 1 led to the identification of a pyrazine-based lead analogue 3, lacking the thiazole moiety. In vivo metabolite identification studies, followed by the independent synthesis and profiling of the cyclopentyl keto- and hydroxyl- metabolites of 3, led to the selection of piragliatin, 4, as the clinical lead. Piragliatin was found to lower pre- and postprandial glucose levels, improve the insulin secretory profile, increase β-cell sensitivity to glucose, and decrease hepatic glucose output in patients with T2D.
A semiautomated quantitative assay for rat serum alkaline phosphatase (ALP) isoenzyme determination was developed, incorporating selective precipitation of bone alkaline phosphatase (BALP) with wheat germ lectin and differential inhibition with levamisole for determination of intestinal alkaline phosphatase (IALP). The assays for each isoenzyme were linear over a broad range of activities. The within-run and between-run coefficients of variation were less than 11% for all 3 isoenzymes. Dilution of serum with saline results in an artifactual overestimation of BALP activity. Comparison of ALP and ALP isoenzyme activity in rats of various ages showed that BALP activity drops dramatically with increasing age of rats. IALP activity is greater in immature rats compared to that in mature rats. While there was no difference between male and female rats at 4 wk of age with regard to total ALP activity and activity of any of the isoenzymes, total ALP activity and activity of the individual isoenzymes were higher in males than in females at most ages over 4 wk. Gavage with corn oil resulted in increased serum IALP activity, and bile duct ligation resulted in increased liver alkaline phosphatase activity. This combined assay for the 3 ALP isoenzymes in rat serum is an efficient means of analysis of large numbers of samples and should increase markedly the specificity of serum ALP activity in identifying the target organ in toxicologic studies when serum ALP activity is increased.
Adrenal medullary hyperplasia and pheochromocytomas are induced in rats by a variety of non-genotoxic agents, and we have hypothesized that these agents induce lesions indirectly by stimulating chromaffin cell proliferation. Vitamin D3, which has not been previously associated with adrenal medullary proliferative lesions, is the most potent in vivo stimulus to chromaffin cell proliferation yet identified. The present investigation utilized the vitamin D3 model to prospectively test the relationship between mitogenicity and focal proliferative lesions in the adrenal medulla and to determine early events in the pathogenesis of these lesions. Charles River Crl:CD BR rats were treated with 0; 5000; 10,000; or 20,000 IU/kg/day of vitamin D3 in corn oil (5 ml/kg) by oral intubation. Rats were killed after 4, 8, 12, or 26 weeks of treatment, following a final week of labeling with bromodeoxyuridine (BrdU) using a mini-pump. Adrenal sections were double-stained for BrdU and phenylethanolamine-N-methyl transferase (PNMT) to discriminate epinephrine (E) from norepinephrine (NE) cells or for vesicular acetylcholine transporter (VAchT) to identify cholinergic nerve endings. Vitamin D3 caused a 4-5-fold increase in BrdU labeling at week 4, diminishing to a 2-fold increase by week 26. An initial preponderance of labeled E cells gave way to a preponderance of labeled NE cells. By week 26, 17/19 (89%) animals receiving the 2 highest doses of vitamin D3 had focal adrenal medullary proliferative lesions, in contrast to an absence of lesions in control rats. The lesions encompassed a spectrum including BrdU-labeled "hot spots" not readily visible on H and E sections, hyperplastic nodules, and pheochromocytomas. Lesions were usually multicentric, bilateral, and peripheral in location, and almost all were PNMT-negative. The lesions were not cholinergically innervated, suggesting autonomous proliferation. Hot spots, hyperplastic nodules, and pheochromocytomas appear to represent a continuum rather than separate entities. Their development might involve selective responses of chromaffin cell subsets to mitogenic signals, influenced by both innervation and corticomedullary interactions. A number of non-genotoxic compounds that induce pheochromocytomas in rats are known to affect calcium homeostasis. The results of this study provide further evidence to support the hypothesis that altered calcium homeostasis is indirectly involved in the pathogenesis of pheochromocytomas, via effects on chromaffin cell proliferation.
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