Peroxisome proliferator-activated receptor-gamma agonist, rosiglitazone, protects against nephropathy and pancreatic islet abnormalities in Zucker fatty rats.

Rosiglitazone (BRL 49653), a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist and potent insulin action-enhancing agent, was given in the diet (50 micromol/kg of diet) to male Zucker rats ages 6-7 weeks for 9 months (prevention group). In this treatment mode, rosiglitazone prolonged the time to onset of proteinuria from 3 to 6 months and markedly reduced the rate of its subsequent progression. Progression was also retarded when treatment was commenced (intervention group) after proteinuria had become established (4 months; ages 24-25 weeks). In either treatment mode, rosiglitazone normalized urinary N-acetyl-beta-D-glucosaminidase activity, a marker for renal proximal tubular damage, and ameliorated the rise in systolic blood pressure that occurred coincidentally with the development of proteinuria in Zucker fatty control rats. The renal protective action of rosiglitazone was verified morphologically. Thus in the prevention group there was an absence of the various indexes of chronic nephropathy that were prominent in the Zucker fatty control group, namely, glomerulosclerosis, dilated tubules containing proteinaceous casts, a loss of functional microvilli on the tubular epithelium, and varying degrees of chronic interstitial nephritis. An intermediate pathology was observed in the intervention group. Also, pancreatic islet hyperplasia, ultrastructural evidence of beta-cell work hypertrophy, and derangement of alpha-cell distribution within the islet were prominent features of Zucker fatty control rats, but these adaptive changes were ameliorated (intervention group) or prevented (prevention group) by rosiglitazone treatment. These data demonstrate that treatment of Zucker fatty rats with rosiglitazone produced substantial protection over a prolonged period against the development and progression of renal injury and the adaptive changes to pancreatic islet morphology caused by sustained hyperinsulinemia.

Effect of probucol on serum lipids, atherosclerosis and toxicology in fat-fed LDL receptor deficient mice.

Although numerous transgenic mouse models for atherosclerosis have been developed recently, little is known about their response to hypolipidaemic or anti-atherosclerotic agents. We investigated the effect of the known hypocholesterolaemic and anti-atherosclerotic drug probucol on serum lipids, lipoproteins and atherosclerosis in fat-fed low density lipoprotein (LDL) receptor deficient mice. Probucol at doses of 0.2 and 1% in the diet which are similar to those used in the mouse by other investigators reduced serum cholesterol by 26 and 37%, respectively. Probucol also reduced serum triglyceride levels by 33 and 47% at doses of 0.2 and 1%, respectively. The decrease in serum cholesterol and triglycerides was mainly due to a decrease of these lipids in VLDL and or chylomicrons. Despite these potentially beneficial changes in serum lipids atherosclerotic lesion areas in the aortic root were unchanged in the probucol treated mice. After 12 weeks treatment most of the mice receiving probucol had swollen feet and tails due to oedema. Histological examination of the base of the hearts from the probucol treated mice revealed lipid droplets within the reticuloendothelial and other interstitial cells. There was also an interstitial subacute inflammatory cell infiltration associated with the lipid deposition. The oedema induced by probucol could be the result of cardiac insufficiency due to interstitial lipidosis and inflammation in the base of the heart together with the extensive atherosclerotic lesions in the aortic sinus.

Acceleration of the development of diabetes in obese diabetic (db/db) mice by nicotinamide: a comparison with its antidiabetic effects in non-obese diabetic mice.

Destruction of pancreatic beta cells has been implicted in the progression to hyperglycemia in type 1 diabetes. While there is evidence of beta-cell loss in type 2 diabetes, its contribution to the development of the diabetic state is undecided. Nicotinamide has defensive effects against toxic insults to the pancreatic islets and confers protection in both human and animal models of type 1 diabetes, but its effects on type 2 diabetes are less well documented. This report describes a comparison of the outcome of chronic oral administration of nicotinamide on the development of diabetes in obese diabetic (db/db) and non-obese diabetic (NOD) mice models of type 2 and type 1 diabetes, respectively. Nicotinamide was administered in the diet (5 g/kg diet) for 12 (db/db) or 24 (NOD) weeks. Over the 12 weeks of the study, control diabetic (db/db) mice became progressively more hyperglycemic and glycosuric, while serum and pancreatic insulin levels decreased compared with those on day 0. In mice treated with nicotinamide, there was a pronounced acceleration in the development of hyperglycemia and glycosuria, as well as a decrease in pancreatic insulin levels, compared with time-matched controls. In addition, the morphology of the pancreatic islets of nicotinamide-treated diabetic (db/db) mice showed an enhanced islet disorganization. By comparison, in NOD mice, nicotinamide prevented the decline in serum and pancreatic insulin levels and maintained normal islet architecture and insulin content. Our data shows that in contrast to its preventative effects on the development of autoimmune diabetes in NOD mice, chronic nicotinamide administration to obese diabetic (db/db) mice markedly accelerated the progression of diabetes. The results of our study caution against the use of nicotinamide in insulin-resistant states, such as type 2 diabetes.

Potential benefit of inhibitors of advanced glycation end products in the progression of type II diabetes: a study with aminoguanidine in C57/BLKsJ diabetic mice.

Prolonged hyperglycemia in type II diabetic patients is linked both with diabetic complications and with further impairment of glucose homeostasis, possibly due to glucose toxicity of the beta cell. While the connection between the accumulation of extracellular advanced glycation end products (AGEs) and the development of complications is well established, it has only recently been suggested that intracellular glycation may be equally adverse and could be involved in the pathogenesis of glucose toxicity in vitro. Aminoguanidine is a recognized inhibitor of the formation of both extracellular and intracellular AGEs. In this study, we show that the development of diabetes, measured by increased water intake and concomitant midday blood glucose levels in type II genetically diabetic mice, is reduced by treatment with aminoguanidine at a dosage of 500 mg/kg/d for 12 weeks in the diet. In addition, at the end of the study, aminoguanidine reduced the decline in serum and pancreatic insulin levels and the degree of pancreatic islet morphological degeneration, all of which are associated with pancreatic insufficiency following prolonged hyperglycemia in this animal model. These results suggest that AGEs may be involved in the aggravation of type II diabetes in vivo and aminoguanidine may be beneficial in its treatment.

The Lack of Toxicity of Potassium ChannelActivators in Heart Cell Cultures.

High doses of the potassium channel activators (KCAs) BRL 44269, levcromakalin and pinacidil in a number of laboratory animal species cause a profound reduction in blood pressure which results in reflex tachycardia, ischaemia and myocardial necrosis. Thus, it is considered that the in vivo cardiac pathology seen with KCAs is an indirect effect as a consequence of excessive pharmacological effects rather than direct myocardial toxicity. This hypothesis was tested, in vitro, in the chick embryonic myocardial myocyte reaggregate (MMR) model system. Changes in spontaneous beating activity (SBA), leakage of lactate dehydrogenase (LDH) and cell morphology by light and transmission electron microscopy were used to assess toxicity. The MMRs were cultured for up to 24hr in a series of different concentrations of the three KCAs in the range 1-10,000mum. In addition to an untreated control, allylamine (50mum), a known direct acting myocardial cytotoxin, was used as a positive control. Incubation with allylamine caused clear evidence of toxicity and permanent cessation of SBA. In contrast, KCAs caused changes in SBA and significant toxicity was only seen at the highest concentration (10,000mum) of BRL 44269. These results are supportive of the view that KCA-induced cardiac pathology in vivo is due to an indirect pharmacological action rather than a direct, cytotoxic mechanism.

Study of the cardiotoxic potential of pharmaceutical compounds in chick myocardial myocyte reaggregate cultures.

Cardiotoxicity produced by doxorubicin in vivo is considered to be due to a direct effect on the myocardium and this is also a major component with toxicity of isoprenaline and digoxin. In the case of the cardiotoxicity produced at high doses by the antihypertensives hydralazine and pinacidil, an indirect mechanism operating by way of their exaggerated pharmacological effects is believed to be responsible. These compounds were examined for their cardiotoxic potential in vitro using chick myocardial myocyte reaggregate (MMR) cultures; allylamine HCl was used as a positive control. Cultures were incubated for up to 24 hr with each compound; parameters analysed were: spontaneous beating activity (SBA), lactate dehydrogenase (LDH) leakage and microscopic evidence of cytotoxicity. Allylamine, doxorubicin, digoxin and to a lesser extent isoprenaline were highly toxic to MMR cultures, as demonstrated by their effects on SBA, LDH leakage and morphology. Hydralazine showed very mild cytotoxicity at the highest concentrations with no LDH leakage; pinacidil was not cytotoxic but showed a dose-related inhibition of SBA. These results confirm the direct toxic action of doxorubicin and digoxin on myocardial cells and indicate that this is also an important mechanism in vivo for isoprenaline. The lack of any significant toxicity with hydralazine and pinacidil accords with an indirect mechanism based on their pharmacology.

Study of the effects of cardiovascular drugs in heart cell cultures.

Compounds that produce myocardial pathology in vivo can be separated into two main classes-those that are directly toxic to the myocardium and those that are considered to act by way of an indirect vascular or neurologically based mechanism. An in vitro model of myocardium without nervous or systemic influences can be used to differentiate between direct myocardial cytotoxic effects and indirect cardiac pathology arising in vivo from exaggerated vascular or neural pharmacological effects of a number of drugs. In this study direct-acting cardiotoxic compounds are distinguished from those causing cardiac pathology by indirect mechanisms by their different pattern of effects in chick embryonic myocardial myocyte reaggregates (MMRs) cultures. The toxicity of the direct-acting cardiotoxic drugs allylamine (positive control, 50 mum) and doxorubicin were compared with digoxin and isoprenaline, which show both direct and indirect mechanisms in vivo, and the indirectly acting hydralazine and pinacidil. Changes in spontaneous beating activity (SBA), leakage of lactate dehydrogenase (LDH) and cell morphology by light and transmission electron microscopy were used to assess toxicity. The MMRs were cultured for up to 24 hr in a series of concentrations of the five compounds in the range 0.1 to 10,000 mum. Allylamine, doxorubicin, digoxin and, to a lesser extent, isoprenaline were highly toxic to the MMRs, as shown by alterations in SBA, LDH leakage and cellular morphology. In contrast, hydralazine showed a very mild degree of toxicity at the highest concentrations in the absence of LDH leakage; treatment with pinacidil did not show any evidence of morphological degeneration but did cause a dose-related inhibition of SBA. These results are consistent with the view that doxorubicin and digoxin are directly toxic to myocardial cells and also suggests that this is an important mechanism in vivo for isoprenaline. The absence of a significant degree of toxicity with hydralazine and pinacidil is consistent with an indirect toxic mechanism.

Relevance of Chlamydia pneumoniae murine pneumonitis model to evaluation of antimicrobial agents.

A mouse model of Chlamydia pneumoniae pneumonitis was established in outbred MF1 mice immunosuppressed with cyclophosphamide. Following intranasal inoculation with 2.2 x 10(3) inclusion-forming units of C. pneumoniae TW-183 per mouse, chlamydiae were culturable from the lungs for at least 29 days. Progressive subacute pneumonitis with perivascular and peribronchial lymphoid cell hyperplasia was observed, and C. pneumoniae organisms were located in consolidated areas of tissue by immunocytochemistry. Mice were treated orally, commencing at 8 days after infection, with clinically achievable concentrations of amoxicillin-clavulanate or ciprofloxacin (three times daily for 7 days), ofloxacin, doxycycline, or erythromycin (twice daily for 7 days), or azithromycin (once daily for 4 days). Despite disparate antichlamydial activity in cell culture and different pharmacokinetic properties in infected animals, all treatments reduced the chlamydial load in the lungs (P < 0.05) when the loads were evaluated by culture at 1 and 10 days after the cessation of dosing, and this was reflected in the histopathological and immunocytochemistry scores. There was no significant difference between these treatments, and C. pneumoniae TW-183 was eradicated from the majority but not from all mice. These results confirm the limited clinical data available to date. In conclusion, a range of oral antimicrobial agents commonly used for the treatment of community-acquired respiratory infection was found to be efficacious in this experimental model of C. pneumoniae pneumonitis, which may therefore be of utility in chemotherapy and follow-up studies.

Rosiglitazone prevents the onset of hyperglycaemia and proteinuria in the Zucker diabetic fatty rat.

AIM: To investigate the potential of rosiglitazone, a highly potent agonist at the nuclear peroxisome proliferator activated receptor-gamma (PPAR-gamma), to prevent the development of diabetes in the Zucker diabetic fatty (ZDF) rat or to ameliorate the condition at a later stage of the disease. METHODS: Rosiglitazone (10 micromol/kg body weight daily) was given via the diet to ZDF rats from aged 6 weeks, before the onset of hyperglycaemia (Prevention group), or from aged 21 weeks after hyperglycaemia and proteinuria were established (Intervention group). Untreated ZDF rats and age-matched Zucker lean rats (ZL) served as controls and the experiment was terminated when the animals were aged 28 weeks. RESULTS: Whilst the combined ZDF control and Intervention groups were already hyperglycaemic (14.6 +/- 1.6 vs. ZL 5.7 +/- 0.1 mmol/l, mean +/- s.e.m.; p < 0.05), glycosuric and polydipsic at aged 11 weeks, and thereafter had a declining plasma insulin concentration, rosiglitazone Prevention treatment maintained normoglycaemia even at aged 27 weeks (3.7 +/- 0.3 mmol/l vs. ZL 3.0 +/- 0.3 mmol/l; NS). Intervention treatment at aged 21 weeks, however, failed to ameliorate the diabetes. These functional data were supported by determinations of pancreatic insulin content (microg/mg tissue as follows: ZL, 43.1 +/- 3.9; ZDF control (28 weeks) + ZDF Intervention control (21 weeks), 6.0 +/- 0.8; Prevention, 63.6 +/- 15.8; Intervention, 6.2 +/- 0.9) and by morphological, immunohistochemical and electron microscopical examination of pancreata at the end of the study. Thus, islets from rosiglitazone Prevention rats were similar to ZL rats, whereas ZDF controls and Intervention rats exhibited islets depleted of insulin, with a disorganized architecture and an ultrastructure indicative of work hypertrophy. ZDF control rats and Intervention rats, though not rosiglitazone Prevention rats, also exhibited marked proteinuria, indicative of renal glomerular damage. CONCLUSIONS: Our results demonstrate that in ZDF rats, rosiglitazone prevents the progression from insulin resistance to overt diabetes. These data provide a rationale for investigating whether treatment with rosiglitazone of patients with early signs of perturbed glucose metabolism (e.g. impaired fasting glucose (IGT)) may prevent the progression to type 2 diabetes and its associated complications.

Comparison of adipose tissue changes following administration of rosiglitazone in the dog and rat.

Rosiglitazone (BRL-49653-C), a thiazolidinedione, is a potent agonist for the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma). Thiazolidinediones have been reported to induce adipocyte differentiation in vitro and there is limited data on their effects in vivo. The objective of this study was to compare the effects of rosiglitazone on adipocyte differentiation between dogs and rats. Morphological (light and ultrastructural) and morphometric evaluations were conducted on perirenal adipose tissue from dogs that have been treated for 1 month with 0.4, 5, 60 mg/kg/day and rats treated for the same period with 80 mg/kg/day. There was a dose-related change in the phenotype of white adipose tissue in dogs, reflected by an increase in nuclear numerical density (up to threefold) and cytoplasmic area fraction (up to 2.1-fold). In addition, there was an enlargement of the nuclei and a reduction in the size of the white adipocyte lipid vacuoles. Ultrastructural changes included an increase in the number of mitochondria per adipocyte. In the rat, similar changes were seen in nuclear numerical density (1.5-fold increase) and cytoplasmic area fraction (2.2-fold increase). There were also increased numbers of mitochondria per cell in white adipocytes giving them similar numbers of mitochondria to brown adipocytes. In the brown adipocytes, there was a reduction in cytoplasmic area fraction with a corresponding increase in the size of the lipid filled vacuoles in other words there was a converging of the phenotypes of the white and brown adipose tissues.

Nabumetone, an effective anti-inflammatory agent, lacks gastrointestinal irritancy in the rat when dosed orally for one month: comparison with tiaprofenic acid and etodolac.

OBJECTIVE: To determine the effects of nabumetone, compared with tiaprofenic acid and etodolac, on anti-inflammatory efficacy and gastrointestinal irritancy in the rat when dosed orally for one month at a high anti-inflammatory dose. METHODS: Carrageenan paw edema was used as a model of inflammation. Gastrointestinal mucosal integrity was assessed by concurrently measuring ulcer formation. mucosal and tissue prostanoid production and plasma haptoglobin. Haemoglobin, present in the cecal contents, was used as a measure of blood loss. RESULTS: Nabumetone, tiaprofenic acid and etodolac inhibited inflammation. Etodolac induced marked gastrointestinal damage and blood loss whereas tiaprofenic acid caused only gastric damage. Nabumetone was found not to alter mucosal integrity. CONCLUSION: Nabumetone proved to be an effective anti-inflammatory agent that was devoid of gastrointestinal irritancy.

Staphylococcal osteomyelitis--a comparison of co-amoxiclav with clindamycin and flucloxacillin in an experimental rat model.

A rat model of Staphylococcus aureus osteomyelitis was used to compare treatment with co-amoxiclav, flucloxacillin and clindamycin. Co-amoxiclav (amoxycillin/clavulanic acid 200/50 mg/kg), flucloxacillin (200 mg/kg) and clindamycin (50 mg/kg) were injected subcutaneously tds for 28 days, commencing 14 days after infection. Eight days after cessation of treatment, high numbers of staphylococci were recovered from the infected tibiae of all control rats. All treatments, at clinically achievable concentrations, significantly (P < 0.05) reduced the bone bacterial titres. However, 50% of tibiae from co-amoxiclav-treated animals were sterile, compared with 17% and 25% from flucloxacillin- or clindamycin-treated animals respectively. Histopathological examination of tibiae reflected the bacteriological results, and showed that the severity of the osteomyelitis was greatly reduced in antibiotic-treated animals compared with non-treated controls. Twenty-eight days after cessation of therapy, bacterial counts from co-amoxiclav and clindamycin-treated animals remained significantly (P < 0.05) lower than those of non-treated controls, although the gross and microscopic appearance of clindamycin and flucloxacillin-treated tibiae suggested that recrudescence of the infection may have occurred. The results of this study demonstrated that co-amoxiclav was as effective as flucloxacillin and clindamycin in the treatment of an experimental chronic staphylococcal osteomyelitis.

Neutropenia does not prevent etodolac- or indomethacin-induced gastrointestinal damage in the rat.

Neutrophils have been implicated in the acute formation of gastric mucosal erosions induced by nonsteroidal antiinflammatory drugs. The aims of the present study were to determine, in rats, the role of neutrophils in the pathogenesis of etodolac- and indomethacin-induced gastrointestinal ulceration and blood loss. Both drugs caused gastrointestinal ulceration, which was associated with increased blood loss, a rise in plasma haptoglobin concentration, and a rise in the number of circulating neutrophils. A marked infiltration of neutrophils occurred only in ileal tissue. Pretreatment with a selective antineutrophil serum induced a significant neutropenia, which failed to inhibit either etodolac- or indomethacin-induced gastrointestinal ulceration and blood loss. A further study demonstrated that the antineutrophil serum did not prevent gastric erosions induced by indomethacin, but it inhibited carrageenan paw edema, which is dependent, in part, on neutrophil infiltration and activation. It is concluded that neutrophils do not contribute to gastrointestinal ulceration and blood loss induced by nonsteroidal antiinflammatory drugs. Furthermore, in contrast with previous studies, our results provide no evidence that neutrophils contribute to indomethacin-induced acute gastric erosion formation.

Effect of potent urease inhibitor, fluorofamide, on Helicobacter sp. in vivo and in vitro.

The therapeutic potential of urease inhibition of Helicobacter pylori has been studied by examining the effect of the potent urease inhibitor, fluorofamide (N-(diaminophosphinyl)-4-fluorobenzenamide), on urease activity and bacterial survival in vivo and in vitro. In culture, acid protection in H. pylori was shown to be due to changes in the pH of the medium brought about by the release of ammonia. Both the acid protection and the ammonia release were completely blocked by fluorofamide at low doses (ED50 = approximately 100 nM). However, fluorofamide was unstable under acidic conditions (T1/2 = 5.7 min at pH 2). Despite this, fluorofamide was the best available compound to test in vivo. In ferrets naturally infected with H. mustelae, a single dose (50 mg/kg, per os) of fluorofamide completely inhibited bacterial urease. In repeat dosing studies, fluorofamide (50 mg/kg per os, three times a day) was compared with the Helicobacter triple therapy regime (amoxycillin, metronidazole, and bismuth subcitrate). Fluorofamide failed to eradicate the H. mustelae infection, compared to 80% eradication with triple therapy. However, histological samples showed a profound reduction in bacterial numbers following fluorofamide treatment. A combination of fluorofamide and amoxycillin was dosed to ferrets (seven days of treatment with 50 mg/kg fluorofamide plus 10 mg/kg amoxycillin per os twice a day); however, this failed to eradicate the infection, despite there being a reduction in bacterial numbers in 3/5 ferrets after 21 days after dosing stopped. It was concluded that urease inhibitors (either alone or in combination with antibiotics) are unlikely to have therapeutic potential for Helicobacter pylori infections. This is probably because, in vivo, some bacteria (perhaps dormant forms) are not entirely dependent upon urease for survival. However, given the acid instability of fluorofamide, the possibility that more stable urease inhibitors might have therapeutic potential, cannot be excluded.

Anti-inflammatory and gastrointestinal effects of nabumetone or its active metabolite, 6MNA (6-methoxy-2-naphthylacetic acid): comparison with indomethacin.

6MNA, the active metabolite of the non-acidic anti-inflammatory drug nabumetone, was investigated using intravenous administration for effects on (a) carrageenan paw oedema and gastric irritancy compared to either oral nabumetone or both oral and intravenous indomethacin when given acutely and (b) gastrointestinal irritancy when given in repeat dosing studies. An oral dose of nabumetone or intravenous 6MNA produced effective anti-inflammatory activity together with significant inhibition of paw exudate PGE2. An anti-inflammatory oral dose of nabumetone or intravenous 6MNA produced minimal effects on gastric 6-keto-PGF1 alpha production, with an absence of gastric damage, in contrast to indomethacin. In repeat dose studies, 6MNA failed to induce gastrointestinal damage even at doses where general toxicity was evident. These results show that in the rat 6MNA, the active metabolite of nabumetone, is an effective anti-inflammatory drug but, even in very high intravenous doses, does not have the propensity to induce gastrointestinal damage.

Antiinflammatory and gastrointestinal effects of nabumetone or its active metabolite, 6-methoxy-2-naphthylacetic acid (6MNA). Comparative studies with indomethacin.

6MNA, the active metabolite of the nonacidic antiinflammatory drug nabumetone, was investigated using intravenous administration for effects on (1) carrageenan paw edema and gastric irritancy compared with either oral nabumetone or both oral and intravenous indomethacin when given acutely and (2) gastrointestinal irritancy when given in repeat dosing studies. Oral doses of nabumetone or intravenous 6MNA produced effective antiinflammatory activity together with significant inhibition of paw exudate PGE2. Antiinflammatory oral doses of nabumetone or intravenous 6MNA produced minimal effects on gastric 6-keto PGF1 alpha production with an absence of gastric damage, in contrast with indomethacin. In repeat dose studies, 6MNA failed to induce gastrointestinal damage even at doses where general toxicity was evident. These results show that in the rat, 6MNA is an effective antiinflammatory drug but even in very high intravenous doses does not have the propensity to induce gastrointestinal damage.