Evaluation of Amides, Carbamates, Sulfonamides, and Ureas of 4-Prop-2-ynylidenecycloalkylamine as Potent, Selective, and Bioavailable Negative Allosteric Modulators of Metabotropic Glutamate Receptor 5.

Negative allosteric modulators (NAMs) of the metabotropic glutamate receptor 5 (mGlu5) hold great promise for the treatment of a variety of central nervous system disorders. We have recently reported that prop-2-ynylidenecycloalkylamine derivatives are potent and selective NAMs of the mGlu5 receptor. In this work, we explored the amide, carbamate, sulfonamide, and urea derivatives of prop-2-ynylidenecycloalkylamine compounds with the aim of improving solubility and metabolic stability. In silico and experimental analyses were performed on the synthesized series of compounds to investigate structure-activity relationships. Compounds 12, 32, and 49 of the carbamate, urea, and amide classes, respectively, showed the most suitable cytochrome inhibition and metabolic stability profiles. Among them, compound 12 showed excellent selectivity, solubility, and stability profiles as well as suitable in vitro and in vivo pharmacokinetic properties. It was highly absorbed in rats and dogs and was active in anxiety, neuropathic pain, and lower urinary tract models.

Reduced biliary sterol output with no change in total faecal excretion in mice expressing a human apolipoprotein A-I variant.

BACKGROUND/AIMS: Apolipoprotein (apo)A-I(M) (ilano), is a molecular variant of apoA-I(wild-type), associated with dramatically low HDL-cholesterol levels, but no increased risk for cardiovascular disease. In view of the present uncertainties on the role of apoA-I in liver cholesterol removal by way of bile acids and neutral sterols, and of the greater capacity of apoA-I(M) (ilano) to remove arterial cholesterol, biliary sterol metabolism was evaluated in transgenic mice expressing apoA-I(M) (ilano). METHODS: ApoA-I(M) (ilano) mice were fed a high-cholesterol/high-fat diet, and compared with human apoA-I(wild-type) mice. Plasma lipid levels, hepatic bile flow and composition, hepatic and intestinal cholesterol and bile acid content, and faecal sterol content were measured. Moreover, the expression of hepatic ABCA1, SR-B1 and that of hepatic and intestinal genes involved in bile acid metabolism were evaluated. RESULTS: The dietary treatment led to a strong elevation in HDL-cholesterol levels in A-I(M) (ilano) mice, associated with an increased expression of hepatic ABCA1. ApoA-I(M) (ilano) mice showed lower cholesterol output from the liver compared with apoA-I(wild-type) mice, in the absence of liver sterol accumulation. Faecal excretion of neutral sterols and bile acids was similar in the two mouse lines. CONCLUSIONS: In spite of a different response to the dietary challenge, with an increased ABCA1 expression and a lower hepatic cholesterol output in apoA-I(M) (ilano) mice, the net sterol excretion is comparable in the two transgenic lines.

Rosuvastatin does not affect human apolipoprotein A-I expression in genetically modified mice: a clue to the disputed effect of statins on HDL.

BACKGROUND AND PURPOSE: Besides a significant reduction of low-density lipoprotein (LDL) cholesterol, statins moderately increase high-density lipoprotein (HDL) levels. In vitro studies have indicated that this effect may be the result of an increased expression of apolipoprotein (apo)A-I, the main protein component of HDL. The aim of the present study was to investigate in vivo the effect of rosuvastatin on apoA-I expression and secretion in a transgenic mouse model for human apoA-I. EXPERIMENTAL APPROACH: Human apoA-I transgenic mice were treated for 28 days with 5, 10 or 20 mg·kg(-1) ·day(-1) of rosuvastatin, the most effective statin in raising HDL levels. Possible changes of apoA-I expression by treatment were investigated by quantitative real-time RT-PCR on RNA extracted from mouse livers. The human apoA-I secretion rate was determined in primary hepatocytes isolated from transgenic mice from each group after treatment. KEY RESULTS: Rosuvastatin treatment with 5 and 10 mg·kg(-1) ·day(-1) did not affect apoA-I plasma levels, whereas a significant decrease was observed in mice treated with 20 mg·kg(-1) ·day(-1) of rosuvastatin (-16%, P < 0.01). Neither relative hepatic mRNA concentrations of apoA-I nor apoA-I secretion rates from primary hepatocytes were influenced by rosuvastatin treatment at each tested dose. CONCLUSIONS AND IMPLICATIONS: In human apoA-I transgenic mice, rosuvastatin treatment does not increase either apoA-I transcription and hepatic secretion, or apoA-I plasma levels. These results support the hypothesis that other mechanisms may account for the observed HDL increase induced by statin therapy in humans.

Lupin (Lupinus albus) protein isolate (L-ISO) has adequate nutritional value and reduces large intestinal weight in rats after restricted and ad libitum feeding.

BACKGROUND: A protein isolate from white lupin (Lupinus albus; L-ISO) has potential as a novel human food ingredient, but its nutritional effects are unknown. METHODS: We evaluated protein quality and effects on body composition in rats of isoenergic diets of L-ISO, lactalbumin, or casein with both restricted (10-day) and ad libitum (28-day)intake. The diets were equivalent in protein per se, but supplementation was used to balance essential amino acid levels. RESULTS: In both studies, the rats consumed similar amounts of each diet, and no effect of diet on the gain:feed ratio was observed--though gain:N ratio and net protein utilization were slightly lower for the L-ISO diet. Lower large intestinal weights after the L-ISO than after the lactalbumin diet were observed in both studies. The L-ISO diet resulted in lowered body fat percentage in the 10-day study but in an elevated level in the 28-day study. Liver composition (DNA, RNA, glycogen, and fat) and plasma levels of some amino acids (His, Thr, Ala, Pro, Tyr, Val and Met) were affected by diet, but no effects on plasma lipid, glucose, or uric acid were observed. CONCLUSION: The L-ISO diet did not affect feed intake and has adequate nutritional quality in rats whilst modifying large intestinal weight in a potentially beneficial manner--suggesting potential for this protein in human nutrition.

Apolipoprotein A-I and the molecular variant apoA-I(Milano): evaluation of the antiatherogenic effects in knock-in mouse model.

No evidence of premature vascular disease is found in apolipoprotein A-I(Milano) (apoA-I(M)) human carriers, despite very low high density lipoprotein (HDL) cholesterol levels. Whether apoA-I(M) may impart a "gain of function" in atherosclerosis protection compared to wild-type apoA-I is hotly debated. To address this question, knock-in mice expressing human apoA-I or apoA-I(M) were crossed with atherosclerosis-susceptible mice expressing the human apoB/A-II transgene (h-B/A-II/A-I(Hu/Hu) and h-B/A-II/A-I(M)(Hu/Hu)). On a chow diet, h-B/A-II/A-I(M)(Hu/Hu) mice were characterized by low HDL cholesterol levels compared to h-B/A-II/A-I(Hu/Hu) mice (35.65+/-8.00 mg/dl versus 58.09+/-13.50mg/dl, respectively; p<0.005). Gender differences in response to high fat diet were observed in both h-B/A-II/A-I(M)(Hu/Hu) and h-B/A-II/A-I(Hu/Hu) lines. h-B/A-II/A-I(M)(Hu/Hu) females had higher total cholesterol levels compared to h-B/A-II/A-I(Hu/Hu) females (895.08+/-183.07 mg/dl versus 544.43+/-116.42 mg/dl; p<0.05) and developed larger atherosclerotic lesions (148,260+/-78,924 microm(2) versus 54,132+/-43,204 microm(2), respectively; p<0.05). On the contrary, no difference in mean lesion area was found between h-B/A-II/A-I(M)(Hu/Hu) and h-B/A-II/A-I(Hu/Hu) males (19,779+/-6,098 microm(2) versus 15,706+/-13,095 microm(2); p=0.685). Our data suggest that, in the atherosclerosis-susceptible human apoB/A-II mouse model, expression of the human apoA-I(M) gene does not have protective advantage over that of the apoA-I gene.

Targeted replacement of mouse apolipoprotein A-I with human ApoA-I or the mutant ApoA-IMilano. Evidence of APOA-IM impaired hepatic secretion.

Despite a pro-atherogenic profile, individuals carrying the molecular variant (R173C) of apolipoprotein (apo)A-I, named apoA-I(Milano) (apoA-I(M)), appear to be at reduced risk for cardiovascular disease. To develop an in vivo system to explore, in a controlled manner, the effects of apoA-I(M) on lipid metabolism, we have used the gene targeting technology, or "gene knock-in" (gene k-in), to replace the murine apoA-I gene with either human apoA-I or apoA-I(M) genes in embryonic stem cells. As in human carriers, mice expressing apoA-I(M) (A-I(M) k-in) are characterized by low concentrations of the human apolipoprotein and reduced high density lipoprotein cholesterol levels, compared with A-I k-in animals. The aim of the present study was to investigate the basic mechanisms of hypoalphalipoproteinemia associated with the apoA-I(M) mutation. ApoA-I and apoA-I(M) mRNA expression, as assessed by Northern blot analysis and quantitative real time reverse transcription-PCR, did not exhibit significant differences in either liver or intestine. Moreover, human apolipoprotein synthesis rates were similar in the k-in lines. When the secretion rate of the human apolipoproteins was assessed in cultured hepatocytes from the mouse lines, secretion from apoA-I(M)-expressing cells was markedly reduced (42% for A-I(M) k-in and 36% for A-I/A-I(M) k-in mice) as compared with that of A-I k-in hepatocytes. These results provide the first evidence that the hypoalphalipoproteinemia in apoA-I(M) human carriers may be partially explained by impaired apoA-I(M) secretion.