[[omega-(Heterocyclylamino)alkoxy]benzyl]-2,4-thiazolidinediones as potent antihyperglycemic agents.

A series of [(ureidoethoxy)benzyl]-2,4-thiazolidinediones and [[(heterocyclylamino)alkoxy]-benzyl]-2,4-thiazolidinediones was synthesized from the corresponding aldehydes. Compounds from the urea series, exemplified by 16, showed antihyperglycemic potency comparable with known agents of the type such as pioglitazone and troglitazone (CS-045). The benzoxazole 49, a cyclic analogue of 16, was a very potent enhancer of insulin sensitivity, and by modification of the aromatic heterocycle, an aminopyridine, 37, was identified as a lead compound from SAR studies. Evaluation of antihyperglycemic activity together with effects on blood hemoglobin content, to determine the therapeutic index, was performed in 8-day repeat administration studies in genetically obese C57 Bl/6 ob/ob mice. From these studies, BRL 49653 (37) has been selected, on the basis of antihyperglycemic potency combined with enhanced selectivity against reductions in blood hemoglobin content, for further evaluation.

An investigation of the beta-adrenoceptor that mediates metabolic responses to the novel agonist BRL28410 in rat soleus muscle.

The beta-adrenoceptor agonist BRL26830A selectively stimulates metabolic rate in the rat and this thermic effect is resistant to blockade by propranolol. These effects of BRL26830A are partly due to selective stimulation by its metabolite BRL28410, of brown adipocyte beta-adrenoceptors, these receptors being resistant to propranolol. To investigate whether the metabolic effects of BRL28410 in skeletal muscle are also mediated by atypical beta-adrenoceptors, the potencies of BRL28410 and isoprenaline were compared for beta-adrenoceptor mediated responses in rat stripped soleus muscle. In addition, pA2 values for antagonism by propranolol of these responses were determined. Isoprenaline had similar EC50 values for stimulation of lactate formation (4.3 X 10(-9) M) and inhibition of glycogen synthesis (3.4 X 10(-9) M) and these values are similar to its reported EC50 values for stimulation of atrial rate (beta 1-adrenoceptor-mediated) and relaxation of the uterus (beta 2-adrenoceptor-mediated). BRL28410 had similar EC50 values for stimulation of lactate formation (3.7 X 10(-6) M) and inhibition of glycogen synthesis (3.8 X 10(-6) M). These values are only about two-fold less than reported values for relaxation of the uterus, but ten-fold less than reported values for stimulation of atrial rate. The pA2 value of dl-propranolol for antagonism of the effect of isoprenaline on glycogen synthesis (8.38 +/- 0.13) was in the range expected for beta 1- or beta 2-adrenoceptors, but with BRL28410 as agonist the pA2 value was about one unit lower (7.39 +/- 0.11). The beta-adrenoceptors that mediate the metabolic effects of BRL28410 in soleus muscle therefore differ from those that mediate atrial rat, uterine relaxation and adipocyte lipolysis. In addition, the low pA2 value of dl-propranolol versus BRL28410 in rat soleus muscle, which contrasts with the normal pA2 value previously reported for guinea-pig trachea, suggests that beta 2-adrenoceptors in these two tissues can be differentiated with suitable pharmacological agents.

Ciglitazone is not itself thermogenic but increases the potential for thermogenesis in lean mice.

Chronic dietary administration of the oral hypoglycaemic ciglitazone (3 g/day for 14-28 days) to lean, non-diabetic CDl mice resulted in increased brown adipose tissue mitochondrial GDP binding and a marked increase in the thermic effect of the beta-adrenoceptor agonist BRL 26830A. However, ciglitazone was not itself thermogenic after an acute dose, nor did it raise resting metabolic rate during chronic dietary dosing.

Substrate supply for thermogenesis induced by the beta-adrenoceptor agonist BRL 26830A.

The nature of the substrate that fuels the thermogenic response to the novel beta-adrenoceptor agonist BRL 26830A has been investigated. Respiratory quotient measurements indicated that the increase in metabolic rate produced by BRL 26830A in rats was fuelled wholly by lipid. BRL 26830A also produced a marked reduction in the lipid content of total dissectable brown adipose tissue. The energy content of this lipid lost during the 4-h period after dosing was equivalent to approximately 50% of the thermogenic effect of the compound over the same period, suggesting that lipid stored in brown adipose tissue is a major initial fuel for BRL 26830A induced thermogenesis. However, marked depletion of brown adipose tissue lipid prior to administration of BRL 26830A had no effect on the subsequent thermogenic response to the compound. Oral administration of glucose altered the pattern of fuel utilization for resting metabolism, but thermogenesis was still fuelled mainly by lipid. Administration of methyl palmoxirate, which inhibits oxidation of long-chain fatty acids, completely prevented the thermic effect of BRL 26830A, suggesting that lipid is a necessary fuel for this process. These results do not support suggestions that carbohydrate is quantitatively important as a fuel for nonshivering thermogenesis.

Experimental manipulations of eating: advances in animal models for studying anorectic agents.

The material set out in this text has been designed to show the wide range of procedures which have the capacity to modify eating behavior--to produce hyper- or hypophagia, to alter the profile of eating patterns, or to adjust dietary preferences and selection. Accordingly, in investigating anorectic drugs it seems necessary to observe the effects of drug actions in a variety of experimental models. This strategy will provide a more complete description of the effect of a drug, will throw light on the mechanism of action, and will provide a more realistic base for predicting the effects of drugs in man.

Differences between the effects of noradrenaline and the beta-adrenoceptor agonist BRL 28410 in brown adipose tissue and hind limb of the anaesthetized rat.

In mature (450-600 g) 21 degrees C-acclimated male rats, anaesthetized with urethane, blood flow (measured by the radioactive microsphere technique) to brown adipose tissue (BAT) was determined during the infusion of the beta-adrenoceptor agonist BRL 28410 or noradrenaline bitartrate at doses chosen to give similar increases in whole body oxygen uptake. Blood flow to BAT during BRL 28410 infusion was only about one third of that found during noradrenaline infusion although increases in whole body thermogenesis were similar (55 and 77% for BRL 28410 and noradrenaline, respectively). This suggests that BAT may be less involved in the thermogenic response to BRL 28410 than to noradrenaline. In a separate experiment using slightly smaller rats (350-500 g) hind limb oxygen uptake was measured in situ using a venous bypass preparation. BRL 28410, at a dose having a maximum effect on whole body thermogenesis (53% increase), had no effect on oxygen delivery to the hind limb but significantly increased oxygen extraction by 33% (p less than 0.001). In contrast, noradrenaline, also at a dose that maximally increased whole body thermogenesis, led to a 35% decrease in oxygen delivery to the hind limb and no change in oxygen extraction. For the thermogenic beta-agonist BRL 28410 the hind limb, and presumably muscular tissue in general, may be contributing to thermogenesis.

Effects of serotonin antagonists on the performance of a simple food acquisition task in rats treated with fenfluramine isomers.

The effect of fenfluramine on food-rewarded runway behaviour was studied in rats that had reached stable performance levels in a three-trials test procedure. d-Fenfluramine was about twice as potent as 1-fenfluramine in its influence on all aspects of runway behaviour: starting speed, running speed and the number of pellets eaten in each trial. Blockade of peripheral serotonin receptor sites by pretreatment with xylamidine, at a dose (2 mg/kg) blocking serotonin-induced inhibition of food intake, was unable to counteract the decrease in runway performance that resulted from treatment with either 2.5 mg/kg d-fenfluramine or 5.0 mg/kg 1-fenfluramine. However pretreatment with metergoline (2 mg/kg), an antagonist that affects both central and peripheral receptor sites, improved the performance of rats given these doses of d- and 1-fenfluramine. It is concluded that both isomers of fenfluramine affect food-rewarded runway behaviour through a mechanism that involves the stimulation of central but not peripheral serotoninergic pathways.

Genetically obese C57BL/6 ob/ob mice respond normally to sympathomimetic compounds.

The suggestion that defective thermoregulatory thermogenesis in the genetically obese (ob/ob) mouse is due to a low thermic response to noradrenaline has been investigated using both noradrenaline and the longer-acting sympathomimetic compounds, ephedrine and BRL 26830A. Below thermoneutrality (23.5 degrees C) the metabolic rate of obese mice was lower than that of their lean littermates, whereas at a thermoneutral temperature (31 degrees C) the metabolic rate of the obese mice was as high as that of lean mice. This confirms the view that the ob/ob mouse has defective thermoregulatory thermogenesis. However, in C57BL/6 mice, this defect is not due to a failure to respond to noradrenaline, because at 31 degrees C the maximum thermic effects of noradrenaline, ephedrine and BRL 26830A were as high in obese as in lean mice and at 23.5 degrees C they were higher in obese than in lean mice. Furthermore, the response of brown adipose tissue to beta-adrenoceptor stimulation appears normal since noradrenaline caused a normal rise in brown adipose tissue temperature, and treatment with noradrenaline or BRL 26830A in vivo caused a normal increase in GDP binding by brown adipose tissue mitochondria. At 31 degrees C propranolol depressed metabolic rate equally in lean and obese C57BL/6 mice, whereas at 23.5 degrees C it depressed metabolic rate more in lean than obese mice. In contrast to C57BL/6 mice, Aston ob/ob mice showed a reduced thermic response to noradrenaline. These results suggest that defective thermoregulatory thermogenesis in the ob/ob mouse is primarily due to a reduced ability to raise sympathetic tone, but in some strains an additional failure in the thermic response to noradrenaline may develop.

Treatment of obesity with thermogenic beta-adrenoceptor agonists: studies on BRL 26830A in rodents.

Studies on BRL 26830A in rodents have shown that thermogenic beta-adrenoceptor agonists have potential for the therapy of obesity. BRL 26830A reduced body weight gain in ob/ob mice and fa/fa rats by reducing lipid accumulation. It had no effect on lean body mass. BRL 26830A did not reduce food intake, its anti-obesity effect being due to stimulation of energy expenditure. This thermic effect was enhanced in the obese animals by repeat dosing. BRL 26830A did not affect body weight gain in the lean counterparts of the obese animals because its thermic effect in lean animals was reduced by repeat dosing. Brown adipose tissue is an important site of BRL 26830A-induced thermogenesis. A single dose of BRL 26830A raised brown adipose tissue temperature, depleted brown adipose tissue lipid and unmasked GDP-binding sites in brown adipose tissue mitochondria. Repeat dosing caused hypertrophy of brown adipose tissue and improved cold tolerance in mice. In-vitro studies showed that the rat brown adipocyte beta-adrenoceptor does not fall into the beta 1/beta 2 classification and BRL 28410, which mediates the biological effects of BRL 26830A in vivo, selectively stimulated the brown adipocyte receptor. It is concluded that BRL 26830A achieves its anti-obesity effect by mimicking natural mechanisms involved in thermogenesis and the control of body weight.

Feeding and satiation observed in the runway: the effects of d-amphetamine and d-fenfluramine compared.

A paradigm involving feeding to satiety over the course of repeated trials in the runway was used to examine the effects of d-amphetamine (1.0, 1.5 mg/kg) and d-fenfluramine (2.0, 3.0 mg/kg). 1.0 mg/kg d-amphetamine was found to have no significant effect on running performance or feeding in the runway. 1.5 mg/kg d-amphetamine significantly reduced the total food intake during the test but had little impact during the first three trials. In contrast, d-fenfluramine, even at the lower dose and during the initial trials, significantly reduced running performance and feeding to levels normally associated with satiation in the non-drugged animals. The results are discussed in relation to the contrasting modes of action of amphetamine and fenfluramine on food intake.

Effects of anorectic drugs and prior feeding on food-rewarded runway behavior.

Two treatments that act through central catecholamine pathways and are normally found to be strongly anorectic (d-amphetamine, 1.25 mg/kg and diethylpropion, 5.00 mg/kg) failed to influence either latency to run or running velocity in single trial running for food reward. In contrast, d-fenfluramine (2.5 mg/kg), which normally has similar anorectic potency but acts via a serotoninergic mechanism, significantly increased latency and decreased running velocity. Prior feeding (30 min ad lib access to food) also decreased runway performance to a similar degree. Further studies, using a 3 trial procedure where rats were allowed to feed for 30 sec following each run, revealed that d-amphetamine (1.25 mg/kg), both with and without penfluridol pretreatment (2.5 mg/kg), failed to affect running velocity or the amount of food eaten. However, d-fenfluramine (2.5 mg/kg) and a postsynaptic serotonin receptor agonist, m-chlorophenylpiperazne (1.0, 2.0 mg/kg) led to a significant reduction in these measurements. Thus it appears that "serotoninergic" anorectic drugs, like the state induced by prefeeding, depress food-rewarded runway behavior whereas "catecholaminergic" anorectic agents lack such effect.

The development of obesity in preweaning obob mice.

1. The body compositions of obob and lean (ob+ and ++) mice at 10, 12, 17 and 28 d of age were investigated using a 'cold stress' test to identify the two groups. 2. At each of these ages the obob mice were found to contain significantly more fat than the lean. At 10 d 20% more fat was present and by 17 d the increase was 72%. The obob mice at 28 d contained nearly three times as much fat as the lean. 3. Carcass energy was significantly higher in obob mice at all ages investigated. 4. Other changes in body composition found in the 28 d obob mice, i.e. a reduction in total carcass nitrogen and water content, were already established in the 17-d-old mice but differences at 10 and 12 d were not apparent. 5. The livers of obob mice were significantly heavier than those from lean control mice at 28 d but no differences were detected at the earlier ages. 6. The results are discussed with reference to the early origin of obesity in obob mice.