Pharmacokinetics of single-dose sildenafil administered orally in clinically healthy dogs: Effect of feeding and dose proportionality.

Basic information related to the pharmacokinetics of sildenafil in dogs is scarce. This study aimed to describe the pharmacokinetic properties of oral sildenafil and determine the effect of feeding and dose proportionality. The effect of feeding on pharmacokinetics of sildenafil (1 mg/kg) was investigated using a crossover study with six dogs. In addition, the dose proportionality of sildenafil ranging 1-4 mg/kg was evaluated using five dogs in the fasted states. The plasma concentrations of sildenafil were determined using high-performance liquid chromatography, and pharmacokinetic parameters were calculated using a noncompartmental analysis. Sildenafil administrations were well tolerated in all studies. Feeding reduced the area under the curve extrapolated to infinity (AUCinf ) and the maximum plasma concentration (Cmax ) significantly. The elimination half-life (T1/2 ) did not differ between the fasted and the fed states. For dose proportionality, nonproportional increases in AUCinf and Cmax at 1-4 mg/kg doses were detected by a power model analysis.

Non-targeted analyses of animal plasma: betaine and choline represent the nutritional and metabolic status.

Simple liquid chromatography-mass spectrometry (LC-MS) was applied to non-targeted metabolic analyses to discover new metabolic markers in animal plasma. Principle component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) were used to analyse LC-MS multivariate data. PCA clearly generated two separate clusters for artificially induced diabetic mice and healthy control mice. PLS-DA of time-course changes in plasma metabolites of chicks after feeding generated three clusters (pre- and immediately after feeding, 0.5-3 h after feeding and 4 h after feeding). Two separate clusters were also generated for plasma metabolites of pregnant Angus heifers with differing live-weight change profiles (gaining or losing). The accompanying PLS-DA loading plot detailed the metabolites that contribute the most to the cluster separation. In each case, the same highly hydrophilic metabolite was strongly correlated to the group separation. The metabolite was identified as betaine by LC-MS/MS. This result indicates that betaine and its metabolic precursor, choline, may be useful biomarkers to evaluate the nutritional and metabolic status of animals.

Effects of metformin on plasma concentrations of glucose and mannose, G6Pase and PEPCK activity, and mRNA expression in the liver and kidney of chickens.

1. The objective of this study was to determine the effects of the gluconeogenesis inhibitor metformin on 21-d old chickens. The following parameters were measured in the liver and kidney: plasma glucose, plasma mannose, enzyme activities and mRNA expression levels of glucose-6-phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase (PEPCK). 2. Chickens were divided into two groups, and received either metformin (300 mg/kg body weight) or water. Plasma glucose and mannose concentrations were analysed by high performance liquid chromatography (HPLC). G6Pase and PEPCK activities were determined by glucose 6-phosphate and malic acid substrate methods, respectively. The expression levels of mRNA were determined by real-time PCR. 3. Plasma glucose and mannose reached their lowest concentrations 1 h after metformin administration. At 0·5 h-1 h after metformin administration, the enzyme activities and mRNA expression levels of G6Pase and PEPCK reached their lowest point in the kidney and their highest point in the liver. The decrease observed in the kidney may have been associated with reductions in both plasma glucose and mannose concentrations. 4. In conclusion, the effect of metformin on the kidney of chickens is similar to its effect in mammals. In contrast, no suppression of enzyme activity or mRNA expression was observed in chicken liver. Therefore, the mode of action of metformin, via AMPK activation, may be different in the chicken liver.

Serum glycated albumin: Potential use as an index of glycemic control in diabetic dogs.

Measurements of serum fructosamine, glycated hemoglobin, and glycated albumin (GA) complement serum glucose concentration for better management of diabetes mellitus (DM). Especially, the serum fructosamine test has long been used for diagnosing and monitoring the effect of treatment of DM in dogs. However, fructosamine tests are currently not performed in veterinary medicine in Japan. GA and fructoasmine levels have been shown to strongly correlate. However, the clinical implications of using GA remain to be elucidated. Therefore, the purpose of the current study was threefold: 1) Determine whether GA% is altered by acute hyperglycemia in normal dogs, simulating stress induced hyperglycemia; 2) Demonstrate that GA% does not dynamically change with diurnal variation of blood glucose concentration in diabetic dogs; and 3) Investigate whether GA% is capable of providing an index of glycemic control for 1-3 weeks in diabetic dogs as is the case with diabetic human patients. Our study demonstrated that serum GA% remains very stable and unaltered under acute hyperglycemic conditions (intravenous glucose injection) and in spite of diurnal variation of blood glucose concentration. Furthermore, serum GA% can reflect long-term changes (almost 1-3 weeks) in blood glucose concentration and the effect of injected insulin in diabetic dogs.

Clinical significance of plasma mannose concentrations in healthy and diabetic dogs.

Circulating levels of monosaccharides can act as a reflection of systemic glucose/ energy metabolism. Characteristic changes observed in these levels can be seen in patients with diabetes and other metabolic disorders. There have been a few reports describing the significance of mannose metabolism as an energy source under physiological and pathological conditions. However, the relationship between circulating levels of mannose and the pathophysiology of diabetes mellitus are unknown in dogs. This study examined circulating levels of mannose between healthy control and diabetic dogs and evaluated the clinical significance of mannose levels in dogs. Diabetic dogs demonstrated a higher circulating level of mannose in comparison to normal healthy control dogs. Plasma mannose was positively correlated with plasma glucose and fructosamine, respectively. Interestingly, plasma mannose levels were affected by plasma insulin levels. In the context of feeding and glucose tolerance tests, plasma mannose levels responded to changes in circulating insulin levels. Circulating plasma mannose levels decreased after feeding in both control and diabetic animals in spite of observed insulin level differences. However, when glucose tolerance tests were given, a positive correlation between mannose levels and insulin levels was observed. Therefore, plasma mannose levels obtained via glucose tolerance testing may be used as a new diagnostic method for evaluating insulin resistance or deficiency in diabetic dogs.

Plasma glucose, mannose, and non-esterified fatty acid concentrations in layer-type chickens.

1. The oral administration of glucose or dietary glucose reduces fasting plasma mannose concentrations in mammals. On the other hand, there have been no reports on plasma mannose levels in birds. We have analysed chicken plasma mannose and glucose by an original high-performance liquid chromatography (HPLC) method, together with plasma non-esterified fatty acid (NEFA) concentrations in chickens. 2. Plasma glucose concentrations of chickens did not differ among three different age groups (0, 18 and 150 d). However, the plasma mannose concentrations of chicks at the age of 0 d were higher than those of chickens at the ages of 18 and 150 d. 3. At the age of 18 and 150 d, plasma glucose concentrations were elevated and plasma mannose and NEFA concentrations were decreased after regular feeding, compared to fasting levels.

Changes in metabolite, energy metabolism related enzyme activities and peripheral blood mononuclear cell (PBMC) populations in beef heifers with two differing liveweight change profiles in New Zealand.

Metabolite and immunoreactive insulin (IRI) concentrations, energy metabolism related enzymes activities and peripheral blood mononuclear cell (PBMC) populations were measured in blood of pregnant Angus heifers with differing liveweight change profiles (gaining or losing), in New Zealand to investigate the meanings of those parameters in the restricted feeding beef heifers. Beef heifers losing liveweight (-412 g/day) showed significantly lower concentrations of plasma IRI, and higher concentrations of plasma free fatty acid (FFA) than heifers gaining liveweight (483 g/day). The cytosolic and mitochondrial malate dehydrogenase (MDH) activities and MDH/lactate dehydrogenase (M/L) ratio in leukocytes of the liveweight losing heifers were significantly higher than those the liveweight gaining heifers. Percentages of cluster of differentiation (CD) 3 positive cells and natural killer (NK) cells in PBMC decreased significantly in the liveweight losing heifers compared to those in the liveweight gaining heifers. Plasma IRI and FFA concentrations, leukocyte cytosolic and mitochondrial MDH activities and CD3 positive and NK cell populations may be useful markers to evaluate metabolic conditions and immunity in the restricted feeding beef heifers.

Plasma metabolite concentrations and hepatic enzyme activities in pregnant Romney ewes with restricted feeding.

Plasma metabolite concentrations and activities of enzymes related to energy metabolism in plasma, peripheral leukocytes and liver of pregnant Romney ewes with restricted feeding were measured to assess those metabolites and enzymes as indicators for evaluating metabolic conditions in the ewes. The body weights and plasma lactate concentrations of the low-feeding ewes (about 1.0 times maintenance) were significantly lower than those of the high-feeding ewes (about 1.5 times maintenance). There were no significant differences in plasma protein, glucose, triglyceride, cholesterol, pyruvate and immunoreactive insulin concentrations and plasma and leukocyte enzyme activities between both groups. Hepatic malate dehydrogenase activities were significantly higher and hexokinase activities were significantly lower in the low-feeding ewes than in the high-feeding ewes. Restricted feeding could maintain pregnancy and some plasma metabolites and peripheral leukocyte enzymes may be useful indicators for evaluating metabolic changes in ewes.

Simultaneous determination of serum mannose and glucose concentrations in dog serum using high performance liquid chromatography.

Serum mannose and glucose concentrations in dogs before and after eating a meal were determined simultaneously with a recently established HPLC method combined with a UV and fluorescence detection system of p-aminobenzoic acid ethyl ester (ABEE)-derivatized monosaccharides. In this newly established HPLC method, detection limits were 0.09 micromol/L for mannose and 0.04 mmol/L for glucose. Linearity of peak areas vs. amounts of mannose and glucose in the range of 0.27-320 micromol/L and 0.13-64 mmol/L were observed, respectively. The value of the glucose content measured by the HPLC method was in good agreement with that of the commonly used enzymatic method (control). Serum glucose concentrations in dogs 90 min after the meal were almost the same as those before the meal, whereas serum mannose concentrations decreased significantly after the meal. This HPLC method may be useful for determination of monosaccharides in animal blood.

Comparison of plasma metabolite concentrations and enzyme activities in beef cattle raised by different feeding systems in Korea, Japan and New Zealand.

Concentrations of metabolites and immunoreactive insulin (IRI) and activities of enzymes related to energy metabolism were measured in plasma of Korean and Japanese beef cattle, which were raised by the indoor feeding system programmed to feed larger amount of roughage in their growing periods and larger amount of concentrate diet in their finishing periods (Japanese feeding system), and grazing New Zealand beef cattle. By the Japanese beef grading system, Korean and Japanese beef cattle showed high beef quality score, average grade 3.3 and 3.6, respectively. The plasma free fatty acid and lactate concentrations and lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and aspartate aminotransferase (AST) activities in Korean beef cattle were significantly higher than those in Japanese beef cattle. The plasma lactate concentration in Korean beef cattle was 8.40 mmol/l, which was similar to the values observed in lactic acidosis. The higher activities of plasma LDH, MDH and AST may indicate slight liver damage by slightly acidotic conditions in Korean beef cattle. New Zealand beef cattle fed on pasture which they harvest by grazing showed significantly lower plasma glucose, cholesterol, lactate and IRI concentrations and enzyme activities than those in Korean and Japanese beef cattle fed on larger amount of concentrate diets. Plasma metabolite concentrations and energy metabolism-related enzyme activities may be good indicators for evaluating metabolic conditions of beef cattle raised by different feeding systems.

Changes in peripheral leukocytes enzymes activity and plasma metabolite concentrations in growing Holstein calves.

Plasma metabolite and immunoreactive insulin concentrations and activities of enzymes related to energy metabolism in peripheral leukocytes were measured in growing Holstein calves. A ratio of girth of abdomen divided by girth of thorax (A/T ratio) of calves was significantly elevated after weaning, and the A/T ratio maybe a good indicator to evaluate rumen development. Plasma glucose and free fatty acid concentrations were changed in calves accompanying change in feeding. Activities of lactate dehydrogenase with pyruvate as substrate (LDH-P) and hexokinase (HK) in cytosolic fractions of peripheral leukocytes decreased significantly after weaning the calves reflecting the change of energy source from milk replacer with high percentages of fat and glucose and lactose as absorbable carbohydrate to pelleted feed containing starch as less absorbable carbohydrate and roughage. Some peripheral leukocyte enzymes such as LDH and HK may be good indicators to evaluate changes in energy metabolism of growing calves.

Role of cathepsin D in prostatic cancer cell growth and its regulation by brefeldin A.

We investigated a possible relationship between brefeldin A (BFA), an antibiotic, and cathepsin D (Cat.D), a lysosomal protease, in prostate cancer proliferation. Effects of BFA (30 ng/ml) were examined on the growth of three human prostatic cancer cell lines, PC-3, DU-145, and LNCaP cells. Its effect on Cat.D in these cancer cells was assessed by Western blots and compared with Cat.D expressed in clinical prostate specimens (n = 55). BFA profoundly (> 70%) inhibited the growth of all three cancer cell lines. Western blots revealed that expression of procathepsin D (Pro.Cat.D) was markedly increased with BFA, whereas actively proliferating (control) cells greatly exhibited mature Cat.D. Analysis of prostate specimens then showed predominant Pro.Cat.D expression in non-cancerous tissues while also showing enhanced expression of mature Cat.D in all cancer specimens. Therefore, BFA-induced growth inhibition in prostatic cancer cells is associated with a blocking of Cat.D maturation (activation), suggesting a possible role of Cat.D in prostate cancer proliferation/development.

Glyoxalase I phenotype as a potential risk factor for prostate carcinoma.

OBJECTIVES: To elicit a possible link between glyoxalase I (Gly-I), a detoxifying enzyme, and the incidence of prostate cancer (PCa), we investigated Gly-I phenotypic expression in the prostatic tissue and red blood cells (RBCs) from patients with PCa. METHODS: Eighty-seven clinical specimens, including 42 PCa tissue samples, 20 RBC samples, and 25 matched pair (prostate and RBC) samples from patients at prostatectomy were examined. The Gly-I phenotypes in these specimens were assessed by nondenaturing starch-polyacrylamide gel electrophoresis. RESULTS: Of the 87 patients, 63 (72.4%) were white, 15 (17.2%) were black, and 9 (10.4%) were another ethnicity (eg, Hispanic, Asian, Indian). Three Gly-I phenotypes were detected in these specimens as fast, intermediate, and slow-moving bands on the gel. The fast phenotype was the most common form found in the white (34 [54%] of 63) and black (8 [53.3%] of 15) patients, but the third ethnic group was too small for proper analysis. To validate this finding, the data from the white patients were compared with the Gly-I phenotypic frequencies in U.S. populations. The data analysis confirmed that a higher incidence (54%) of the fast type in our white patients was statistically significant (P <0.0001) compared with its phenotypic frequency of 30.6% in the general U.S. white population. CONCLUSIONS: The significantly high frequency (P <0.0001) of the fast Gly-I phenotype was detected among patients with PCa, suggesting it is a potential risk factor for PCa. Whether its increased incidence in whites reflects the lack of sample numbers for other ethnic groups needs additional investigation.

Stable isotope-labeling studies on the oxidative coupling of caffeic acid via o-quinone.

The formation of ortho-quinone from ortho-diphenol is a key step in its dimerization. An NMR analysis of the oxidation of 3,4-dihydroxycinnamic acid (caffeic acid) by NaIO4 revealed the formation of 3-(3',4'-dioxo-1',5'-cyclohexadienyl) propenoic acid (o-quinone) prior to the formation of furofuran-type lignan 4,8-exo-bis (3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane-2,6-dione. Both electrolytic and enzymatic oxidation of caffeic acid also generated o-quinone. The yields of o-quinone from caffeic acid were quantified by NMR and HPLC analyses. A stable isotope-labeling study of the formation of lignans directly proved the random radical coupling of semiquinone radicals formed from a set of caffeic acid and o-quinone.

[Clinical efficacy of leuprolide acetate and combined treatment with estramustine for advanced prostate cancer].

BACKGROUND/PURPOSE: Twenty-two institutes have organized Keio University Prostate Cancer Study Group to study clinical efficacy and safety of Leuprolide acetate (Leuplin) for the treatment of advanced prostate cancer (clinical stage D1 and D2). Cotreatment of Leuplin and Estramustine phosphate disodium (Estracyt) has been performed to investigate its clinical efficacy. MATERIALS AND METHODS: One hundred and two cases of advanced prostate cancer were treated either with Leuplin alone (group I), Leuplin and Estracyt (group II) or Estracyt alone (group III). After 12 weeks treatment, clinical effects against subjective symptoms (pain, voiding difficulty, performance status and body weight), serum testosterone level, tumor size and serum PSA level were examined to investigate short-term effect of each treatment. The treatment had been continued for 24 months and the treatment effects including progression free survival and overall survival were analyzed. RESULTS: Clinical efficacy after 12 weeks treatment were examined among 97 cases (group I; 35 cases, group II; 36 cases, group III; 26 cases). The background of those patients in each group was statistically equal. Treatment effects against subjective symptoms and serum testosterone level statistically revealed no significant difference among 3 groups. Treatment effects against primary tumor, bone metastatic lesion, lymphnode metastatic lesion and serum PSA level were investigated and anti-tumor effect was characterized by total efficacy rate (complete remission rate plus partial remission rate) of each treatment group. Treatment efficacy rates for each lesion and PSA demonstrated no statistical difference among 3 treatment groups. Total efficacy rate of group I, II and III were 88.2%, 84.0% and 78.3%, respectively, which statistically revealed no significant difference. Total efficacy rate of each group after completing 24 months treatment was; group I 80.0%, group II 55.6% and group III 83.3%, which statistically showed no significant difference among 3 treatment groups. The median day for progression free survival of group I, II and III were 661, 731 and 517, respectively. The overall survival rate of group I, II and III after completing 24 months treatment were 77.5%, 83.0% and 72.4%, respectively. Both progression free survival rates and overall survival rates revealed no significant difference among 3 groups. Side effects during 24 months treatment were seen in 8.6% of group I, 47.2% of group II and 26.9% of group III, and these occurrence rates were significantly different among the groups (p = 0.0013). CONCLUSION: Although number of the cases had not been able to continue the treatment for their side effects, the statistical characterization demonstrated that cotreatment of Leuplin and Estracyt had no greater treatment effect than monotreatment of each drug.

Induction of apoptosis in human prostatic cancer cells with beta-glucan (Maitake mushroom polysaccharide).

PURPOSE: To explore more effective treatment for hormone-refractory prostate cancer, we investigated the potential antitumor effect of beta-glucan, a polysaccharide of the Maitake mushroom, on prostatic cancer cells in vitro. MATERIALS AND METHODS: Human prostate cancer PC-3 cells were treated with various concentrations of the highly purified beta-glucan preparation Grifron-D(R) (GD), and viability was determined at 24 h. Lipid peroxidation (LPO) assay and in situ hybridization (ISH) were performed to unravel the antitumor mechanism of GD. RESULTS: A dose-response study showed that almost complete (>95%) cell death was attained in 24 h with GD > or = 480 microg/mL. Combinations of GD in a concentration as low as 30 to 60 microg/mL with 200 microM vitamin C were as effective as GD alone at 480 microg/mL, inducing >90% cytotoxic cell death. Simultaneous use with various anticancer drugs showed little potentiation of their efficacy except for the carmustine/GD combination (approximately 90% reduction in cell viability). The significantly (twofold) elevated LPO level and positive ISH staining of GD-treated cells indicated oxidative membrane damage resulting in apoptotic cell death. CONCLUSION: A bioactive beta-glucan from the Maitake mushroom has a cytotoxic effect, presumably through oxidative stress, on prostatic cancer cells in vitro, leading to apoptosis. Potentiation of GD action by vitamin C and the chemosensitizing effect of GD on carmustine may also have clinical implications. Therefore, this unique mushroom polysaccharide may have great a potential as an alternative therapeutic modality for prostate cancer.

Adverse effects of oxidative stress on renal cells and its prevention by antioxidants.

BACKGROUND AND PURPOSE: Recent reports suggest that reactive oxygen species; e.g., hydrogen peroxide (H(2)O(2)), could be the primary cause of various drug-induced renal injuries. We investigated the effects of H(2)O(2) on renal cells to understand its mode of action and to explore cytoprotection from such a fatal injury. MATERIALS AND METHODS: Renal proximal tubular LLC-PK(1) cells were exposed to various concentrations of H(2)O(2), and cell viability was determined at specified times. Lipid peroxidation assay and Western blot analysis of heat shock proteins (Hsp70 and Hsp90) were performed to assess the cellular effects. RESULTS: The dose-response study showed that H(2)O(2) > or = 100 microM was severely cytotoxic. Even a 1-h exposure was sufficient to induce >95% cell death in 24 h. Lipid peroxidation was significantly (>50%) increased, while Hsp90, but not Hsp70, was partially degraded, to an approximately 85-kDa fragment, after a 3-h H(2)O(2) exposure. However, such cytotoxic cell death was remarkably ( approximately 90%) prevented by the antioxidants pyruvate or N-acetylcysteine (NAC), and Hsp90 remained intact. CONCLUSION: Hydrogen peroxide-induced renal cell death involves increased lipid peroxidation and partial degradation of Hsp90. Both pyruvate and NAC are capable of detoxifying H(2)O(2) to maintain cell viability and Hsp90 integrity. Acute renal injuries associated with oxidative stress might preventable by appropriate antioxidants.

Methylglyoxal-induced apoptosis in human prostate carcinoma: potential modality for prostate cancer treatment.

OBJECTIVE: To examine the cellular effects of methylglyoxal (MG), a toxic physiological metabolite, on human prostatic cancer PC-3 cells. METHODS: The effects of MG on cell growth and viability were evaluated first, and then its effects on the cell cycle and the glycolytic process were analyzed by Western blots and specific assays. Possible MG-induced apoptosis was also assessed by DNA analysis using agarose gel electrophoresis. RESULTS: MG > or =3 mM caused severe growth inhibition, resulting in nearly 100% cell death by 24h. The time course study revealed that expression of cyclin D(1), cdk2, and cdk4 was significantly (>50%) downregulated in 3 h of MG (3 mM) exposure, followed by the dephosphorylation of retinoblastoma protein by 6 h. Both the glyceraldehyde-3-phosphate dehydrogenase activity and the cellular lactate level were also reduced by approximately 50 and 80%, respectively, following 6-hour MG exposure. Induction of apoptosis by MG was indicated by partial degradation of poly(ADP-ribose) polymerase and further confirmed by discrete DNA fragmentation detected on an agarose gel. CONCLUSION: MG is capable of inducing apoptosis in prostatic cancer PC-3 cells, due primarily to a blocking of the cell cycle progression (G(1) arrest) and glycolytic pathway. Therefore, MG could be a potent apoptosis inducer, which may have a potential for prostate cancer treatment.