Safety, pharmacokinetics and use of the novel NK-1 receptor antagonist maropitant (Cerenia) for the prevention of emesis and motion sickness in cats.

The present study characterizes the safety, pharmacokinetics, and anti-emetic effects of the selective NK-1 receptor antagonist maropitant in the cat. Safety of maropitant was determined following 15 days of subcutaneous (SC) administration at 0.5-5 mg/kg. Maropitant was well tolerated in cats at doses that exceeded the efficacious anti-emetic dose range of the drug by at least a factor of 10 and adverse clinical signs or pathological safety findings were not noted at any dose.The pharmacokinetics of maropitant in cats were determined following single dose oral (PO), intravenous (IV) and SC administration. Maropitant had a terminal half-life of 13-17 h and a bioavailability of 50 and 117% when administered PO and SC, respectively. Efficacy was determined against emesis induced either by xylazine or by motion. A dosage of 1 mg/kg maropitant administered IV, SC or PO prevented emesis elicited by xylazine. The compound had good oral antiemetic activity and a long (24 h) duration of action. Maropitant (1.0 mg/kg) was highly effective in preventing motion-induced emesis in cats. These studies indicate that the NK-1 receptor antagonist maropitant is well tolerated, safe and has excellent anti-emetic properties in cats.

The safety of dirlotapide in dogs.

The safety of dirlotapide in dogs was evaluated in two studies with parallel designs. In an acute tolerance study, 24 beagles (six dogs per treatment) were treated orally once daily for 14 days with placebo or dirlotapide at 2.5, 5.0, or 10.0 mg/kg/day. In a margin-of-safety study, 38 overweight, neutered beagles were treated orally once daily for 3 months with dirlotapide at doses up to 0.5 mg/kg/day (six dogs), 1.5 mg/kg/day (12 dogs) and 2.5 mg/kg/day (six dogs). Control dogs received placebo at 0.3 mL/kg/day (10 dogs) and 0.5 mL/kg/day (four dogs). Results were similar for both studies, and no serious adverse events were observed. Dirlotapide was clinically well-tolerated in dogs at dosages up to 10 mg/kg/day for 14 days and 2.5 mg/kg/day for 3 months. Dirlotapide produced the expected decrease in food intake and body weight (up to 20-40%) without ill effects. Clinical, pathologic, and histopathologic findings were reversible and consistent with suppression of food intake and rapid weight loss produced by elevated dirlotapide dosages. In both studies, sporadic emesis and loose stools were observed in both placebo and dirlotapide-treated dogs. Incidence of emesis generally increased with dose and decreased with treatment time. Elevations in hepatic transaminase activity were seen in dogs treated with more than 1.5 mg/kg dirlotapide daily, but were not associated with clinical signs or microscopic evidence of hepatic degeneration or necrosis.

Biologic activity of dirlotapide, a novel microsomal triglyceride transfer protein inhibitor, for weight loss in obese dogs.

Dirlotapide is a novel microsomal triglyceride transfer protein inhibitor intended for the treatment and management of obesity in dogs. The biologic effects of dirlotapide, weight loss, decreased food intake, increased fecal fat, decreased serum cholesterol, and body composition, were evaluated in a controlled, blinded study. Sixteen obese beagles were randomized to treatment with placebo (n = 4) or dirlotapide (n = 12) following a 2-week acclimation period in which baseline data were collected. The dirlotapide dose, adjusted to produce weight loss for 3 months and then stabilize body weight for 1 month (weight management), produced a significant difference (expressed as a percentage of baselines) in weekly weight loss, food intake, fecal fat, serum cholesterol concentration, and body composition (measured by dual energy X-ray absorptiometry) compared with placebo treatment (P < 0.05). The initial dirlotapide dosage of 0.5 mg/kg (10 times the initial label dose) resulted in a high rate of weight loss (3.3% weekly) and anorexia, emesis, and loose stools for some dogs. A 25% dose reduction (mean dosage: 0.36 mg/kg) followed by biweekly 25% dose adjustments based on individual weight loss, produced 1-2% weekly weight loss and total weight loss of 18.8% in 12 weeks at a final mean dosage of 0.41 mg/kg (range: 0.15-0.60); a dosage range of 0.10-0.34 mg/kg stabilized body weight. Body weight changes for placebo-treated dogs were -0.8% to +0.9% weekly; total weight gain during the weight loss phase was 10.6%. No apparent change in food intake, percentage of fecal fat, and serum cholesterol was observed in the placebo group. Food intake and body weight increased when dirlotapide was discontinued. Dirlotapide produced weight loss by both reducing appetite (about 90% of the weight loss activity) and by increasing fecal fat excretion (about 10% of the weight loss activity).

The neurokinin-1 antagonist activity of maropitant, an antiemetic drug for dogs, in a gerbil model.

Maropitant is a novel synthetic nonpeptide neurokinin type 1 (NK1) selective receptor antagonist, recently developed for use in the dog as an antiemetic. The in vivo functional activity of maropitant was investigated in the gerbil foot-tapping model, to determine the ability of maropitant to penetrate the central nervous system and inhibit foot-tapping induced by the selective NK1 agonist GR73632. In comparison with CP-122,721, a previously characterized NK1 receptor antagonist, maropitant (1 mg/kg by s.c. injection) was found to inhibit foot-tapping for significantly longer (P < 0.01). Inhibition of foot-tapping by maropitant was 100% at 2 h and approximately 50% at 8 h postdosing. The mean brain:plasma concentration ratio at 8 h post-treatment was 3.59. These data demonstrate the central functional action of maropitant as a selective and potent NK1 receptor antagonist and help to support and explain its clinical potential as a broad-spectrum antiemetic agent.

Chronic ingestion of high concentrations of cholecalciferol in cats.

OBJECTIVE: To determine whether ingestion of 63 times the recommended amount of vitamin D3 (cholecalciferol) results in renal calcification or damage in cats. ANIMALS: 20 four-month-old kittens, 17 queens, and 20 kittens born to these queens. PROCEDURE: 4-month-old kittens and queens were given a purified diet with 846 microg of cholecalciferol/kg of diet (high vitamin D3 diet) or 118 microg of cholecalciferol/kg of diet (control diet) for 18 months. Kittens born to queens were weaned onto the same diet given to dams. RESULTS: There were no apparent adverse effects of the high vitamin D3 diet. Plasma cholecalciferol and 25-hydroxycholecalciferol (25-OHD3) concentrations of queens and 4-month-old kittens given the high vitamin D3 diet significantly increased with time. At 6 months, plasma cholecalciferol concentrations in these kittens and queens were 140.0+/-7.3 nmol/L and 423.6+/-26.6 nmol/L, respectively (10 times initial values). Corresponding 25-OHD3 concentration in queens was 587.5+/-59.4 nmol/L (2.5-fold increase over initial values). At 3 months of age, kittens born to queens given the high vitamin D3 diet had an increase in serum BUN and calcium concentrations and a decrease in RBC and serum total protein, albumin, and hemoglobin concentrations. By 18 months, these kittens had an increase in plasma cholecalciferol (276.0+/-22.2 nmol/L) and 25-OHD3 (1,071.9+/-115.3 nmol/L) concentrations. However, all indices of renal function and the appearance of renal tissue on histologic evaluation were normal. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicate that cats are resistant to cholecalciferol toxicosis when the diet is otherwise complete and balanced.

Maternal expression of functional lipoprotein lipase and effects on body fat mass and body condition scores of mature cats with lipoprotein lipase deficiency.

OBJECTIVE: To assess effects of deficiency of lipoprotein lipase (LPL) on body condition scores and lean and fat body masses of adult cats. ANIMALS: 12 cats without LPL mutations and 23 cats that were heterozygous or homozygous carriers of the Gly412Arg LPL mutation. PROCEDURE: Lean and fat body masses were estimated by use of body condition scores and change in enrichment of serum after IV administration of deuterium oxide. Mass spectroscopy and infrared absorbance methods were used to determine deuterium enrichment. RESULTS: Fat body mass (mean +/- SD; 0.2 +/- 0.1 kg) and percentage body fat (6.2 +/- 1.4%) of homozygotes were significantly less than those of clinically normal cats and heterozygotes (0.7 +/- 0.1 kg, 18.2 +/- 1.6% and 0.5 +/- 0.1 kg, 15.6 +/- 1.7%, respectively). Homozygous offspring of homozygous dams had significantly less fat body mass (0.1 +/- 0.1 kg) and percentage body fat (2.1 +/- 1.0%) than homozygous offspring of heterozygous dams (0.3 +/- 0.1 kg and 9.2 +/- 1.7%, respectively). Lean body mass did not differ significantly among groups. For all groups, percentage body fat was significantly correlated with body condition score (r= 0.65), and body condition scores supported findings for fat body mass. CONCLUSIONS AND CLINICAL RELEVANCE: Deficiency of LPL activity in cats diminishes stores of body fat. This is consistent with a low rate of de novo synthesis of fat. The effect of dam on body masses in mature LPL-deficient cats indicates nutrient programming of adipose formation during gestation or lactation.

Kinetics of taurine depletion and repletion in plasma, serum, whole blood and skeletal muscle in cats.

The relationship between taurine concentrations of plasma, whole blood, serum and skeletal muscle during taurine depletion and repletion was investigated in cats, to identify the most useful indicators of taurine status. Sixteen cats were fed a purified diet containing either 0 or 0.15 g/kg taurine for 5 months. Treatments were then reversed and the taurine concentration was measured during repletion and depletion phases. Plasma taurine exhibited the fastest rate (slow component) of depletion (t 1/2 = 4.8 wk), followed by serum (5.3 wk), whole blood (6.2 wk), and skeletal muscle (11.2 wk). Whole blood taurine was the first to replete at a rate of 0.74 wk to 1/2 maximal repletion, followed by serum (2.1 wk), skeletal muscle (3.5 wk), and plasma (3.5 wk). Whole blood more closely reflected skeletal muscle taurine concentrations than plasma during depletion, while plasma taurine concentrations appear to be the most valuable predictor of skeletal muscle taurine concentrations during repletion. This study suggests that the best clinical method to evaluate the taurine status of the cat is the determination and interpretation of both plasma and whole blood taurine concentrations.

Preparturient hypocalcemia in four cats.

Preparturient hypocalcemia was identified in 4 cats in a specific pathogen-free colony between 1995 and 1996. All cats had an acute onset of clinical signs, 3 to 17 days prior to parturition. Signs of depression, weakness, tachypnea, and mild muscle tremors were the most common clinical signs, following by vomiting and anorexia. Additional abnormalities included hypothermia, third eyelid prolapse, dehydration, pallor, lethargy, flaccid paralysis, and hyperexcitability. Hematologic abnormalities included leukocytosis with neutrophilia and lymphopenia. Hypocalcemia was documented in each queen. Common serum biochemical abnormalities included high aspartate aminotransferase and creatine kinase activities. All cats responded to IV or SC administration of 10% calcium gluconate. Queens were then given calcium orally prior to and following parturition. The queens did not have additional complications for the duration of the gestational or lactational periods.

Increased intracellular calcium in rat anterior piriform cortex in response to threonine after threonine deprivation.

The anterior piriform cortex (APC) may serve as the chemosensor for amino acid (AA) deficiency in rats. To investigate the mechanism by which the APC recognizes a limiting indispensable AA (IAA), we examined changes in intracellular calcium ([Ca2+]i) in APC slices after culture in medium with or without threonine (Thr) or lysine (Lys). The addition of 1 or 10 mM Thr to slices previously incubated in Thr-devoid medium resulted in a significant and sustained increase in [Ca2+]i compared to control slices; an effect not seen when isoleucine, another IAA, was added. Similar results were seen when lysine, but not threonine, was added to slices incubated in lysine-devoid medium. The rise in [Ca2+]i resulting from the addition of the limiting IAA to deficient slices may be linked to enhanced activity of the appropriate AA transporter. This is suggested by preliminary findings that serine, a small neutral AA that uses the same transporter as threonine, gave rise to an enhanced response in the Thr-deficient slice.

Interventional nutrition for gastrointestinal disease.

Nutritional intervention plays a key role in the successful management of gastrointestinal disease. This article focuses on several novel areas of nutritional intervention that are becoming increasingly important in gastrointestinal disease, including short-chain fatty acids, omega-3 polyunsaturated fatty acids and glutamine. Short-chain fatty acids are the principal end-products of bacterial fermentation of dietary fibers and have profound effects on normal intestinal cell metabolism and proliferation. Short-chain fatty acids have the potential to improve overall intestinal health, stimulate intestinal healing, and decrease intestinal inflammation. Omega-3 fatty acids, from dietary sources or supplements, may also be useful in decreasing intestinal inflammation and in preventing intestinal cancer. Finally, glutamine also may play an important role in the nutritional management of gastrointestinal disease.

Transfection of avian LMH-2A hepatoma cells with cationic lipids.

LMH-2A is an estrogen-responsive avian hepatoma cell line whose susceptibility to cationic-lipid-mediated transfection is poorly described. 3 beta[N-N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DCC) requires a one-step synthesis, and can be used to formulate transfection-grade liposomes when combined with dioleoylphosphatidyl-ethanolamine (DOPE) 1/1 (wt/wt). Luciferase activities in LMH-2A cells were 8.5-fold and 87.5-fold greater than those in HepG2 and FTO2B cells, respectively, following DCC-liposome-mediated transfection with a reporter consisting of the human cytomegalovirus immediate-early promoter (CMV), joined to Photinus pyralis luciferase (L) cDNA, designated pCMVL. Using pCMVL, N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propana minimun bromide) (BMOP)/DOPE 1/1 (wt/wt), at a 7.5:1 ratio with DNA, produced luciferase activities that were 2.9-fold higher than those of DCC-liposomes, at an optimal 10:1 lipid:DNA ratio. At optimal lipid:DNA ratios, commercially available liposomes, Transfectam, Lipofectamine, and Lipofectin, produced luciferase activities that were 1.39, 1.03, and 0.47-fold those of DCC-liposomes. The effect of 0, 10, 100, or 500 nM/L 17 beta-estradiol on the expression of pCMVL and a second luciferase reporter containing the -593/+48 promoter region of the estrogen-responsive avian apo VLDL-II gene, designated pApoL, was tested in cells cultured in the presence or absence of 10% chicken serum. The CMV promoter supported a high level of expression in LMH-2A cells that was unaffected by serum alone, but was weakly responsive to estrogen. Estrogen responses of both reporters reached a plateau at 10 nM/L. Estrogen increased the expression of pApoL 24-fold and 79-fold in the absence and presence of serum, respectively. The -593/+48 region of the apo VLDL-II promoter may not contain previously reported negative insulin response elements, but chicken serum contains factors that enhance estrogen responsiveness of this region.

Incorporation of tributyrin enhances the expression of a reporter gene in primary and immortalized cell lines.

Advancing the efficiency of foreign gene transfection can be accomplished by improving its DNA delivery or expression or varying the composition of the DNA construct. A novel method of enhancing gene expression by incorporating triacylglycerol into a transfection protocol is described. The triacylglycerol form of butyric acid was chosen because the addition of the free fatty acid to cells increases gene transcription. The effects of this lipid moiety on transfected gene expression were determined for a range of promoters and cell types. In several cases, inclusion of tributyrin resulted in a greater increase in the production of the gene marker product luciferin than that found for the inclusion of free butyric acid. Overall, the relative effects of tributyrin and free butyric acid differ quite markedly for different cell types and promoters. Thus, tributyrin may be a viable alternative to butyric acid in transfection systems where the free fatty acid has little effect.

Gene expression following direct injection of DNA into liver.

The liver is an attractive target tissue for gene therapy. Current approaches for hepatic gene delivery include retroviral and adenoviral vectors, liposome/DNA, and peptide/DNA complexes. This study describes a technique for direct injection of DNA into liver that led to significant gene expression. Gene expression was characterized in both rats and cats following injection of plasmid DNA encoding several different proteins. Luciferase activity was measured after injection of plasmid DNA encoding the luciferase gene (pCMVL), beta-galactosidase (beta-Gal) activity was evaluated in situ using plasmid DNA encoding Lac Z (pCMV beta), and serum concentration of secreted human alpha-1-antitrypsin was measured following injection of plasmid DNA encoding this protein (pRC/CMV-sHAT). Several variables, including injection technique, DNA dose, and DNA diluent, were investigated. Direct injection of pCMVL resulted in maximal luciferase expression at 24-48 hr. beta-Gal staining demonstrated that the majority of transfected hepatocytes were located near the injection site. Significant concentrations of human alpha-1-antitrypsin were detected in the serum of animals injected with pRC/CMV-sHAT. These findings demonstrate the general principle that direct injection of plasmid DNA into liver can lead to significant gene expression.

Dexamethasone enhancement of gene expression after direct hepatic DNA injection.

The critical physiological functions of the liver make hepatocytes important targets for therapeutic gene delivery. This study reports significant gene expression following direct injection of plasmid DNA into the livers of rats and cats. Transfection was characterized using luciferase and Lac Z expression from plasmids with the cytomegalovirus immediate early promoter/enhancer (CMV IE) or the Rous sarcoma virus long terminal repeat (RSV LTR). Dexamethasone treatment enhanced and prolonged transfected gene expression, possibly by activating gene expression. Southern analysis of total DNA extracted from liver at various times following injection detected persistent unintegrated plasmid DNA which maintained a prokaryotic methylation pattern. This study demonstrates the feasibility of direct DNA injection in the experimental analysis of hepatic gene expression in vivo.

An epizootic of feline herpesvirus, type 1 in a large specific pathogen-free cat colony and attempts to eradicate the infection by identification and culling of carriers.

This study describes the clinical course of an inadvertent feline herpesvirus, type 1 (FHV-1) outbreak in 2 specific pathogen-free (SPF) research and breeding colonies housing 690 cats and assesses a programme that was designed to eradicate the virus from the colonies. The clinical signs observed in these cats were milder, with more eye involvement than those previously described for FHV-1 infection and did not include abortion. FHV-1 eradication was based on the detection and elimination of both active and latent viral carriers. Carrier cats were detected by virus isolation from oral swabs before and after corticosteroid-induced reactivation of FHV-1 excretion. Four per cent of recovered cats were actively shedding virus prior to corticosteroid treatment; 21% of the virus negative cats shed virus after one corticosteroid injection, and 12% of remaining culture negative cats tested positive upon a second corticosteroid treatment 6 weeks later. The colony remained virus free for 8 months after all detectable virus carriers were culled and there was no seroconversion among new kittens. A second epizootic of FHV-1 then occurred among susceptible animals. At this time, all breeding cats that had tested negative after 2 injections of corticosteroids were treated a third time; 23% of them now tested positive for FHV-1. This study demonstrates that corticosteroid treatment can be useful in improving the rate of detection, essential as a basis for decreasing the incidence of enzootic disease, but it is unlikely to detect all possible FHV-1 carriers in large populations of cats.

Dietary cysteic acid serves as a precursor of taurine for cats.

The ability of cats to use dietary cysteic acid as a precursor for taurine was examined. For 39 wk, six groups of four specific-pathogen-free cats each were fed purified diets that provided either 1.25 g taurine or 0, 1.0, 2.0, 10 or 20 g cysteic acid/kg diet. Concentrations of taurine in plasma and whole blood were measured weekly for 13 wk and monthly until 39 wk. Muscle concentrations of taurine at 12 wk were measured on a biopsy sample of the semitendinosus muscle. Fecal and urinary excretions of taurine and cysteic acid at 17 wk were measured. Concentrations of taurine in plasma and whole blood were linearly and positively correlated with cysteic acid in the diet (r2 = 0.88). Urinary taurine, a major route of excess taurine excretion, was positively correlated with the cysteic acid concentration of the diet (r2 = 0.94). Muscle and whole-blood taurine concentrations were also positively correlated (r2 = 0.96) with dietary cysteic acid concentration. Gross and histopathological examinations were performed on cats fed diets containing 20 g cysteic acid/kg diet. No adverse gross clinical signs were observed in cats fed any diets containing cysteic acid; minor histopathologic changes of the pancreas and thyroid were found in three of four cats fed 20 g cysteic acid/kg diet. Results suggest that cats are able to use dietary cysteic acid as a precursor for taurine biosynthesis.

Detection of active and latent feline herpesvirus 1 infections using the polymerase chain reaction.

A polymerase chain reaction (PCR) assay was developed to detect the thymidine kinase gene of feline herpesvirus 1 (FHV-1) and to study the active and latent carrier state in a group of naturally FHV-1 infected specific pathogen free (SPF) cats. The detection limit of PCR products on ethidium bromide stained gels was 390 fg or about 3 x 10(3) copies of the FHV-1 genome. The PCR was 25% more sensitive than conventional cell culture based virus isolation techniques in detecting FHV-1 in oral/ocular swabs and 100 times more sensitive in detecting virus in cell culture supernatants. Sites of FHV-1 latency in FHV-1 carriers as determined by PCR were mainly tissues of the head, especially the trigeminal ganglia, optic nerves, olfactory bulbs and corneas. Oral fauces, salivary glands, lacrimal glands, cerebellum and conjunctiva were less consistently positive. The cerebral cortex, thymus, trachea, lung, liver, spleen, kidney, and peripheral blood mononuclear cells were consistently negative for FHV-1 genome. The distribution of FHV-1 DNA in the tissues of the head was similar whether or not corticosteroid-induced virus shedding was occurring at the time the tissues were collected. Infectious virus was never recovered from tissue homogenates regardless of the PCR status of the tissues.

Dietary protein source (soybean vs. casein) and taurine status affect kinetics of the enterohepatic circulation of taurocholic acid in cats.

The effect of dietary protein source (soybean vs. casein) and taurine status on kinetics of [24-14C] and [taurine-2-3H]taurocholic acid was determined by isotope dilution in 10 adult male cats (six taurine-replete and four taurine-depleted). Taurine-replete cats were fed 1500 mg taurine/kg purified diets containing either 435 g/kg casein (1500 Cas) or soybean protein (1500 Soy) in a crossover design. Taurine-depleted cats were fed the soybean protein diet with no taurine (0 Soy). Specific activity of [14C]- and [3H]taurocholic acid in bile was determined for 6 d following a pulse dose of dual-labeled taurocholic acid. Taurocholic acid pool size was significantly greater in cats when fed the 1500 Soy diet than when fed the 1500 Cas or than in cats fed the 0 Soy diet. Total entry rate, irreversible loss rate and recycling rate of [taurine-2-3H]taurocholic acid and the irreversible loss rate of [24-14C]taurocholic acid tended to be greater in cats when fed the 1500 Soy than the 1500 Cas diet. Irreversible loss rates of taurocholic acid in taurine-replete cats fed the 1500 Soy diet were significantly greater than in taurine-depleted cats, 356 vs. 120 mumol/d [24-14C]taurocholic acid and 445 vs. 56 mumol/d [taurine-2-3H]taurocholic acid. The fraction of taurocholic acid was greater, and the fraction of taurochenodeoxycholic and taurodeoxycholic acids lower in cats when fed the 1500 Soy than when fed the 1500 Cas diet. Taurine-depleted cats had less taurocholic, taurochenodeoxycholic, and taurodeoxycholic acids and greater glycocholic and cholic acids than taurine-replete cats fed the 1500 Soy diet. This study demonstrates that both dietary protein source and taurine status affect taurocholic acid kinetics and bile acid composition in cats.

Taurine balance is different in cats fed purified and commercial diets.

Ileal fluxes, urinary losses and taurine balance were determined in six taurine-replete and four taurine-depleted cats. Digesta samples collected at the terminal ileum were used to assess ileal flux of taurine. Four diets were tested: a commercial diet in two forms (heat-processed and frozen) and two purified diets containing either 1225 or 0 mg taurine/kg diet. Five-day balance trials were performed on d 3-7 with measurement of food intake and taurine in urine and ileal digesta. Substantially greater quantities of total taurine (free + bound) were found in ileal digesta from cats fed the heat-processed rather than the frozen preserved diet (205 vs. 101% of the average daily taurine intake, respectively), with calculated taurine balances of -609 vs. -212 mumol/d, respectively. The quantity of taurine in ileal digesta from taurine-replete cats fed the 1225 or 0 mg taurine/kg purified diets was not significantly different, indicating that taurine found at the terminal ileum is mostly of endogenous origin. Taurine-depleted cats had significantly lower amounts of taurine in ileal digesta, with a taurine balance of -77 mumol/d. These results demonstrate that a heat-processed diet causes substantially greater losses of taurine from the intestine than does a frozen diet. This phenomenon may explain the inability of some heat-processed diets to maintain normal plasma taurine concentrations in cats.