Effects of multiple ascending doses of the glucagon receptor antagonist PF-06291874 in patients with type 2 diabetes mellitus.

AIMS: To assess the pharmacokinetics, pharmacodynamics, safety and tolerability of multiple ascending doses of the glucagon receptor antagonist PF-06291874 in patients with type 2 diabetes mellitus (T2DM). METHODS: Patients were randomized to oral PF-06291874 or placebo on a background of either metformin (Part A, Cohorts 1-5: 5-150 mg once daily), or metformin and sulphonylurea (Part B, Cohorts 1-2: 15 or 30 mg once daily) for 14-28 days. A mixed-meal tolerance test (MMTT) was administered on days -1 (baseline), 14 and 28. Assessments were conducted with regard to pharmacokinetics, various pharmacodynamic variables, safety and tolerability. Circulating amino acid concentrations were also measured. RESULTS: PF-06291874 exposure was approximately dose-proportional with a half-life of ∼19.7-22.7 h. Day 14 fasting plasma glucose and mean daily glucose values were reduced from baseline in a dose-dependent manner, with placebo-corrected decreases of 34.3 and 42.4 mg/dl, respectively, at the 150 mg dose. After the MMTT, dose-dependent increases in glucagon and total glucagon-like peptide-1 (GLP-1) were observed, although no meaningful changes were noted in insulin, C-peptide or active GLP-1 levels. Small dose-dependent increases in LDL cholesterol were observed, along with reversible increases in serum aminotransferases that were largely within the laboratory reference range. An increase in circulating gluconeogenic amino acids was also observed on days 2 and 14. All dose levels of PF-06291874 were well tolerated. CONCLUSION: PF-06291874 was well tolerated, has a pharmacokinetic profile suitable for once-daily dosing, and results in reductions in glucose with minimal risk of hypoglycaemia.

Protection against multiply drug-resistant and geographically distant strains of Haemonchus contortus by vaccination with H11, a gut membrane-derived protective antigen.

H11 is a "hidden antigen" derived from adult Haemonchus contortus which is capable of conferring a high degree of protection against subsequent challenge. To be commercially useful, any vaccine based on this antigen must protect against field strains and drug-resistant worms. The results show that vaccination with H11 affords high levels of protection against the multiply drug-resistant (MDR) Lawes strain of Haemonchus contortus. Moreover, there was no difference in protection whether the H11 was extracted from drug-resistant or susceptible worms, indicating that multiple drug-resistance does not significantly alter the immunogenicity of H11. Ideally, a commercial vaccine based on this antigen should also be capable of protecting against strains from geographically separate areas, so that it is not necessary to manufacture "region-specific" vaccines. Vaccination with H11 purified from either Australian or U.K. Haemonchus contortus was effective in protecting against subsequent challenge with Australian larvae, indicating that one type of H11 should have world-wide application.

Vaccination of sheep against haemonchosis with H11, a gut membrane-derived protective antigen from the adult parasite: prevention of the periparturient rise and colostral transfer of protective immunity.

Pregnant ewes were immunised with a fraction highly enriched in the membrane glycoprotein antigen H11, isolated from the intestinal brush border of adult Haemonchus contortus. Immunity induced by immunisation was able to abolish almost completely (98-99%) the worm egg output from pregnant ewes challenged with ca. 10,000 infective larvae of H. contortus during the last trimester. Furthermore, lambs born and reared on vaccinated ewes had substantial antibody levels to H11 derived from maternal transfer. This antibody conferred moderate protection against a bolus challenge of ca. 3000 infective larvae of H. contortus in 5-week-old lambs.

Duration of protective immunity against ovine haemonchosis following vaccination with the nematode gut membrane antigen H11.

To establish for how long protective antibody levels may be maintained, lambs were vaccinated with the gut membrane antigen H11 and challenged with Haemonchus contortus 14, 84, 126 or 168 days later. Compared to controls, mean faecal egg counts of vaccinated lambs were reduced by 97 per cent, 99 per cent, 92 per cent and 86 per cent respectively. Total worm burdens at postmortem five weeks after infection were reduced by 87 per cent, 94 per cent, 92 per cent and 62 per cent respectively. In vaccinated lambs, antibody levels to H11 peaked at about 60 days after the first vaccination and were maintained for the duration of the experiment. There was evidence of secondary antibody responses to H11 following challenge.

Interactions between the beta-adrenergic agonist salbutamol and genotype on meat quality in pigs.

Salbutamol (2.7 ppm) fed to pigs between weaning and slaughter increased ADG (5%), dressing percentage (2%) and cross-sectional area of the longissimus (LD) muscle (14%). In fatter, White-line-sired pigs, but not in leaner, Meat-line-sired animals, it reduced backfat thickness (25%). Liver weight and glycogen concentration also were decreased. In neither genotype were there effects of Salbutamol on pH45, drip loss or reflectance value of the LD, but ultimate pH was elevated in semimembranosus (SM), adductor (AD) and supraspinatus (SS) muscles, resulting in reduced color saturation values. Total muscle heme pigment concentrations were reduced by 10 (SS) to 19% (LD and SM) and the intramuscular fat concentration of the AD, but not the LD, was reduced by 21%. Treated pigs had LD and SM muscles that when measured instrumentally were 15 and 8% tougher, respectively, after cooking, but the texture of the SS was not significantly altered by treatment. Salbutamol increased plasma lactate and creatine phosphokinase (CPK) concentrations. Compared with White-line-sired animals, Meat-line-sired pigs had a higher dressing percentage (2%) and LD muscles with larger cross-sectional area (13%). They also had higher circulating CPK levels.

The effects of the beta-adrenergic agonist salbutamol on meat quality in pigs.

The beta-adrenergic agonist Salbutamol was administered to pigs at 3 ppm in the feed between weaning and slaughter at 85 kg. Growth rate was not affected by Salbutamol. Treated pigs had a higher dressing percentage (2.6%) and produced carcasses that were less fat (17%) and had longissimus (LD) muscles of larger (11%) cross-sectional area. They also had smaller livers that contained less glycogen. The thinner backfat in treated animals was less firm and tended to separate from the underlying lean. However, these changes were attributable solely to the reduced fatness and there was no direct effect of Salbutamol. There were no differences in pH 45 min postmortem, percentage drip loss during storage or reflectance value of the LD between the two groups, indicating no greater propensity for Salbutamol-treated pigs to develop pale, soft, exudative muscle. However, treated pigs had higher final pH values in the muscles; this was reflected in slightly reduced hue and saturation values. These results suggest that the propensity of the pigs to develop dark, firm, dry meat was slightly increased. Salbutamol-treated pigs produced LD muscles that were slightly tougher (22%), had reduced concentration of heme pigments in the muscle, reduced plasma glucose and increased plasma creatine phosphokinase activity. Salbutamol improved lean meat yield but slightly increased the potential to produce dark, firm, dry meat and reduced tenderness.

Eating quality of meat from pigs given the beta-adrenergic agonist salbutamol.

Meat from pigs given the beta-adrenergic agonist Salbutamol was assessed by a trained taste panel. Overall, there were no significant effects of Salbutamol on panel scores for pork flavour, foreign flavour or overall acceptability. Although the differences were also not significant, meat from treated pigs was rated slightly tougher (P = 0·10) but more juicy (P = 0·14) than that from untreated animals. This concurred with higher instrumental values for toughness although the correlation between taste panel and instrumental texture measurements was low (r = 0·36, P < 0·001).

Influence of the beta-agonist salbutamol on claw horn lesions and walking soundness in finishing pigs.

The effects of feeding the beta-adrenergic agonist salbutamol to pigs at levels between 0.5 and 5.0 mg/kg feed for different periods were studied in six experiments. The feet and ability to walk of pigs weighing 30 to 100 kg were examined at intervals. False sand-crack, white-line and heel erosion lesions were classified as mild or severe, and corns and wall haemorrhages were also recorded. At each examination the pig was given a grade for the overall severity of its foot lesions. Salbutamol fed at 1.0 to 5.0 mg/kg feed, for as little as 21 to 28 days, increased the frequency (P < 0.05 to 0.001) and often the severity (P < 0.05) of the foot lesions, the higher doses tending to produce more severe lesions, and the overall foot grades deteriorated (P < 0.001). The effects on both sexes were similar. No changes were observed when 0.5 mg/kg was fed for 56 days. Despite the severity of many of the foot lesions, the pigs became lame in only one experiment. Electron microscopy indicated that salbutamol was interfering with horn production, but light microscopy revealed no changes in skin sections. These findings suggested that salbutamol was not directly affecting the function of keratinocytes. Supplementing the diet of the pigs with biotin and methionine did not delay, or prevent, the effects of salbutamol.

Regulation of glycolytic flux in the heart of the fetal guinea pig.

Functional glycolytic capacity and its regulation have been studied in the fetal guinea-pig heart during O2 deprivation in situ and in the Langendorff perfused heart. Anaerobic glycolytic flux, at 2 mumol/min per g wet wt. was similar in the 48-50 and 60-65 days fetal and adult guinea-pig heart, despite lower fetal phosphofructokinase activity. During O2 deprivation in situ and in the perfused heart glucose was the major substrate, with glycogen making a smaller contribution. Glycolytic capacity became more tightly regulated during fetal heart development. Thus at 48-50 days glycolysis was increased during O2 deprivation by substrate supply, but at 60-65 days activation of phosphofructokinase was required also. Low malate/aspartate cycle activity in the fetal heart was suggested by the absence of an increase in malate and alanine at the expense of aspartate. The large proportion of aerobic glycolytic flux converted to lactate concurred with this. Because of the low O2 consumption and relatively high aerobic glycolytic flux, the proportion of glycolytically-derived ATP was 3-4 fold higher in the fetal than adult heart, and may explain its functional resistance to O2 deprivation.

Metabolic events associated with the preparation of the fetus for independent life.

The metabolic changes late in fetal development that are essential for neonatal survival are discussed. In many species gluconeogenesis develops before birth but provides substrate for intracellular biosynthesis and not for glucose production because of low activities of glucose 6-phosphate translocase. At the time of glycogen deposition in species with a relatively mature brain at birth the translocase develops and glucagon and adrenaline can stimulate glucose production and synthesis to elevate blood glucose concentrations both pre- and postnatally. The other metabolic fuel accumulated before birth, fat, can also be mobilized prenatally and in fetuses that are relatively mature at birth it may be used as an alternative fuel. The fetal rat brain can oxidize fatty acids and the brain of fetuses such as that of the guinea-pig and man can oxidize ketone bodies before birth. The timing and degree of oxidation of ketone bodies relates to the timing of myelination and protects the brain against hypoglycaemia. These late changes in development are associated with a sharp increase in plasma cortisol and adrenaline concentrations and a high fetal insulin concentration.

The effects of salbutamol on blood pressure and heart rate in Large White and Pietrain-cross breeds of pig.

The effects of the beta 2-agonist salbutamol, on pig heart rate and blood pressure were evaluated at the expected commercially used concentration of 3 ppm in final feed, and at three times this level. Salbutamol was administered to pigs previously fed on control diet ('naive'), and in a second study to pigs fed 3 ppm salbutamol for 19 days ('acclimatized'). Heart rate and blood pressure were measured in conscious 30 kg pigs before (pre-ingestive), during (ingestive) and after ingestion (absorptive) of feed containing 3 or 9 ppm salbutamol. To assess any interaction with 'stress genotype' pigs, measurements were performed in Large White and Pietrain-cross breeds. The mean preingestive heart rates for the Large White and Pietrain-cross pigs were 127 and 109 beats/min, respectively. The corresponding figures for mean arterial blood pressure were 121 and 122 mmHg. The act of ingesting control feed caused heart rate to rise by 36-39% and blood pressure to increase by 17-27%. During the absorptive phase for 'naive' pigs fed 3 ppm salbutamol in the diet, blood pressure fell 5-11% and heart rate increased 20-24%, reflecting a classical baroreceptor mediated response. At 9 ppm the fall in blood pressure of 5-11% was similar to that at 3 ppm, while the rise in heart rate was larger at 31-38%. The magnitude of the changes at both 3 and 9 ppm was less than that evoked by the act of ingestion, with the exception of the heart rate response in Large Whites at 9 ppm, which was similar. The responses of the two breeds were comparable, indicating that salbutamol is unlikely to exacerbate the cardiovascular responses seen in potentially stress-susceptible pigs. The acute changes elicited by 3 ppm salbutamol during the absorptive phase in 'naive' pigs were abolished or substantially less in acclimatized pigs, implying a tachyphylaxis to the agonist's actions. This was confirmed by 9 ppm of salbutamol increasing heart rate of 'acclimatized' Large Whites to the same degree as 3 ppm in 'naive' counterparts. The desensitization in Pietrain-cross pigs was even more marked with no increase in heart rate produced by 9 ppm salbutamol following acclimatization. These data, combined with the rapid tachyphylaxis of response, indicate that salbutamol at the intended commercial inclusion of 3 ppm will not compromise the cardiovascular stability of the growing pig at rest.

Ultrastructural and enzymatic development of fetal guinea pig heart.

The activities of some enzymes of glycolysis, the citric acid cycle, and amino acid metabolism have been measured in the fetal guinea pig heart over the last third of gestation and correlated with heart ultrastructural development. There is little change in glycolytic enzyme activity except for a two- to threefold increase in phosphofructokinase activity. Mitochondrial content and enzyme activities are low in the early fetal heart, and, although content is similar in the late fetus and adult, enzyme activities increase twofold postnatally, indicating fetal heart mitochondria are incompletely developed. The activities of aspartate and particularly alanine aminotransferase are low in the fetal heart. Over the last third of gestation the myofibrillar content of the fetal myocyte increases twofold to the adult value by term. Associated with this is a fourfold rise in myofibrillar and sarcoplasmic reticulum Ca2+-ATPase activity. Na+-K+-ATPase activity is similar in the late fetal and adult heart but one-third lower in the early fetal heart.

Studies on the growth of the fetal guinea pig. The effects of ligation of the uterine artery on organ growth and development.

The effects of reduced maternal placental blood flow on the growth and development of the fetal guinea pig have been studied by unilateral ligation of the uterine artery at day 30 of pregnancy. Fetal guinea pigs were investigated about 20 or 30 days later. In about one-third of cases fetal death occurred, in another third fetuses less than 60% of normal weight were observed and in the remainder all fetuses were in the normal weight range. In the growth retarded fetuses prenatal growth occurred at about 50% of the rate in control. There was no postnatal 'catch up' as growth still remained lower than in controls. Restricted fetal growth affected particularly development of the visceral tissues in which case size declined in proportion to body weight. Brain and adrenal by comparison were less affected as their contribution to total body weight increased, but even so in the severely retarded fetuses the mass of both fell. The responses of the liver were in general consistent with a delay in the pattern of development. Thus DNA, RNA, protein and haematopoietic cell content changes occurred later than normal. In contrast an enhanced deposition of glycogen was apparent in the liver of the growth-retarded fetus. The results indicate some of the ways in which nutritional deprivation of the fetuses leads to reprogramming of growth and maturation of selected fetal tissues to allow non-essential changes to await more favourable times.

Studies on the growth of the fetal guinea pig. The effects of nutritional manipulation on prenatal growth and plasma somatomedin activity and insulin-like growth factor concentrations.

In guinea pigs between days 41-46 of pregnancy prenatal growth has been manipulated by alteration of nutritional state. Three methods were used. Uterine artery ligation at day 30 of pregnancy depressed fetal growth rate by greater than 50% and was associated with falls in plasma insulin, IGF-1, cortisol, thyroid hormone, glucose, acetate and free fatty acid concentrations and rises in that of IGF-2, glucagon and amino acids. Fetal plasma was inhibitory to sulphate incorporation into pig costal cartilage. Complete food withdrawal from pregnant guinea pigs for 2 days at days 43-44 of pregnancy caused mild fetal growth retardation and similar changes in plasma constituents, except in that plasma IGF-2 concentrations were now depressed and plasma was not inhibitory to sulphate incorporation into pig costal cartilage. Production of hypoglycaemia by 4-times-daily maternal injections of glucose between days 41-46 of pregnancy accelerated fetal growth rate. It also elevated fetal plasma concentrations of insulin, IGF-1, IGF-2, sulphation-promoting activity, thyroid hormones, glucose and free fatty acids and depressed that of glucagon and amino acids. Fetal growth rate during the experimental period showed a good correlation with plasma glucose, insulin and IGF-1 and, to a certain extent, with sulphation-promoting activity. It did not correlate closely with fetal plasma IGF-2 concentration. Hepatic glycogen concentrations showed a good correlation with plasma IGF-2 levels.

Induction of protective immunity in cattle against infection with Fasciola hepatica by vaccination with cathepsin L proteinases and with hemoglobin.

Two cathepsin L proteinases, cathepsin L1 and cathepsin L2, secreted by liver flukes may be involved in tissue penetration, nutrition, and protection from immune attack. To ascertain the immunoprophylactic potential of these proteinases, and of another molecule, liver fluke hemoglobin (Hb), we performed vaccine trials in cattle. In the first vaccine trial various doses of cathepsin L1 were tested. The mean protection level obtained was 53.7%. In a second vaccine trial cathepsin L1 and Hb elicited 42.5 and 43.8% protection levels, respectively, while a combination of the two molecules induced a significantly higher level of protection (51.9%). Cathepsin L2 was not examined alone; however, vaccination of cattle with a combination of cathepsin L2 and Hb elicited the highest level of protection (72.4%). The animals that received cathepsin L1-Hb or cathepsin L2-Hb showed reduced liver damage as assessed by serum glutamic dehydrogenase and gamma-glutamyl transferase levels. Furthermore, a reduced viability was observed for fluke eggs recovered from all vaccine groups. This anti-embryonation effect of vaccination was particularly evident in the group that received cathepsin L2-Hb where >98% of the eggs recovered did not embryonate to miracidia. Although all vaccine preparations induced high antibody titers which were boosted following the challenge infection, there was no correlation between antibody titers and protection. The results of these trials demonstrate that cathepsin Ls and Hb could form the basis of a molecular vaccine that would not only reduce parasite burden but would also prevent transmission of liver fluke disease.