Protracted release of the LHRH agonist avorelin (MF 6001) from two depot formulations in dogs and men

Summary Avorelin is a new superagonist of natural luteinizing-hormone-releasing-hormone. Avorelin has been formulated in high molecular weight polylactic glycolic acid to afford protracted and continuous release of the peptide from subcutaneous implants. Two different formulations (10 and 15 mg) were tested first in dogs and then in men during a clinical phase II trial. Chemical castration was maintained for at least 6 months in dogs with both formulations. A similar duration of activity (approximately 6 months) was observed in men.     

Radioimmunoassay of [D-Trp6]-luteinizing hormone-releasing hormone: its application to animal pharmacokinetic studies after single injection and long-acting formulation administration

A sensitive radioimmunoassay (RIA) for [D-Trp6]-luteinizing hormone-releasing hormone (LHRH) has been developed. This assay allowed measurement of the LHRH analog in unextracted plasma with a minimum detectable concentration of 10 pg/ml. Validation of plasma assays was performed through Sep-Pak and HPLC purification. The in vivo fate of the peptide was investigated in dogs after subcutaneous or intravenous injections. In both cases, the LHRH analog showed longer plasma half-life than native LHRH with an elimination half-life superior to 80 min. Long-acting formulations were tested in dogs and rats: the day following administration, [D-Trp6]-LHRH plasma level rose to 2.9-4.6 ng/ml in dogs and 0.8-3.8 ng/ml in rats. From day 4 to day 30, [D-Trp6]-LHRH plasma level followed a plateau with concentrations of 0.3-0.8 ng/ml in dogs and 0.2-0.4 ng/ml in rats. In parallel, testosterone plasma concentration was reduced to castrate level between day 4 and day 7 in dogs and was significantly lowered in rats. This sensitive [D-Trp6]-LHRH RIA will be particularly useful for the evaluation of long-acting formulations in patients with advanced prostate cancer.     

A long-lasting topical deltamethrin treatment to protect dogs against visceral leishmaniasis

To develop long‐lasting, topical pour‐on insecticides for dogs to control zoonotic visceral leishmaniasis, two deltamethrin‐based formulations (emulsifiable concentrate [EC] and suspension concentrate [SC]) were tested for their efficacy against the phlebotomine sandfly Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae), vector of Leishmania infantum Nicolle (Kinetoplastida: Trypanosomatidae). The entomological outcomes tested were anti‐feeding effect (proportion of female sandflies unfed), lethal effect (24‐h female sandfly mortality) and these two effects combined, and the insecticide persistence time at 50% (residual activity, RA₅₀) and 80% (RA₈₀) efficacy. On initial application, the proportions of female flies that demonstrated anti‐feeding activity or were killed were similar for both formulations, at 0.93 (95% confidence interval [CI] 0.856–0.977) vs. 0.81 (95% CI 0.763–0.858) (anti‐feeding) and 0.86 (95% CI 0.787–0.920) vs. 0.76 (95% CI 0.698–0.817) (24‐h mortality) for EC and SC, respectively. The RA₅₀ rates for anti‐feeding and mortality caused by the EC formulation were 4.7 months (95% CI 4.18–5.84) and 2.5 months (95% CI 2.25–2.90), respectively, compared with 1.1 months (95% CI 0.96–1.15) and 0.6 months (95% CI 0.50–0.61), respectively, for the SC formulation. The RA₅₀ for the combined anti‐feeding and mortality effects of EC was 5.2 months (95% CI 4.73–5.96), compared with only 0.9 months (95% CI 0.85–1.00) for the SC formulation. The four‐ to six‐fold superior residual activity of the EC formulation was attributed to the addition of a solvent‐soluble resin in the formulation which improved fur adhesion and acted as a reservoir for the slow release of the active ingredient. These results identify the potential of such a low‐cost formulation to reduce the inter‐intervention interval to 5–6 months, similar to that recommended for deltamethrin‐impregnated dog collars or for re‐impregnation of conventional bednets, both of which are currently used to combat Leishmania transmission. Finally, a novel bioassay was developed in which sandflies were exposed to fur from treated dogs, revealing no detectable tolerance (24‐h mortality) in wild‐caught sandflies to the insecticide formulations up to 8 months after the initiation of communitywide application of the insecticides to dogs.     

Reversible inhibition of testicular androgen secretion by 3-, 5- and 6-month controlled-release microsphere formulations of the LH-RH agonist [D-Trp6, des-Gly-NH10(2)] LH-RH ethylamide in the dog

This study examines the effect of treatment with controlled-release poly(DL-lactide-coglycolide) microsphere formulations of the LH-RH agonist [D-Trp6, des-Gly-NH10(2)]-LH-RH ethylamide (LH-RH-A) designed to release about 100 or 200 micrograms of the peptide per day for 3, 5 or 6 months in male dogs. Plasma levels of testosterone and LH-RH-A were measured at 2-day intervals. After the first injection of the 100-micrograms/day formulation, plasma testosterone increased from 1.6 +/- 0.2 to 3.5 +/- 0.6 ng/ml for 5-7 days before decreasing and remaining at 0.05 +/- 0.008 ng/ml for approximately 150 days (5 months). After two months of recovery, microspheres designed to release 100 micrograms for 6 months of LH-RH agonist per day were then injected. Plasma testosterone levels showed an elevation from 1.5 +/- 0.5 to 4.7 +/- 2.0 ng/ml during the first few days before gradually decreasing to castration levels for 200 days (6 months). One month later, plasma testosterone had returned to normal levels. When microspheres designed to deliver an average of 200 micrograms per day of the peptide for 3 months were injected in another series of animals, castration levels of plasma testosterone were maintained for 95 days with a progressive increase to normal values at later time intervals. The animals of the first series of experiments were then sacrificed after 4 months of recovery following maintenance of plasma testosterone at castration levels for a total period of 11 months. The testes, prostate and pituitary gland were kept for histological examination which was completely normal in all tissues. The efficacy and excellent tolerance of the controlled-release form of LH-RH-A as inhibitor of the pituitary-gonadal axis strongly support the use of such long-term controlled-release formulations of LH-RH agonists for the treatment of sex steroid sensitive diseases.     

Positive Impact of Nutritional Interventions on Serum Symmetric Dimethylarginine and Creatinine Concentrations in Client-Owned Geriatric Dogs

A prospective study was conducted in client-owned geriatric dogs to evaluate the short-term effects of a test food on serum symmetric dimethylarginine (SDMA) and creatinine (Cr) concentrations. Test food contained functional lipids (fish oil), antioxidants (lipoic acid, vitamins C and E), L-carnitine, botanicals (fruits and vegetables), controlled sodium concentration, and high quality protein sources (high bioavailability and an ideal amino acid composition). Dogs (n = 210) were fed either test food or owner’s-choice foods (non-nutritionally controlled cohort). Dogs were included based on age and body weight: small (6.8 to 11.4 kg) and medium dogs (11.5 to 22.7 kg) were ≥ 9 years, whereas dogs >22.7 kg were ≥ 7 years at baseline. At baseline, all dogs had to have serum Cr concentrations within the reference interval and be free of chronic disease. Renal function biomarkers and urinalysis results at baseline, and after consuming test food or owner’s-choice foods for 3 and 6 months, were evaluated. Only dogs consuming test food showed significant decreases in serum SDMA and Cr concentrations (both P ≤ 0.05) across time. At baseline or during the 6-month feeding trial, 18 dogs (8.6%) had increased serum SDMA, but normal serum Cr, consistent with IRIS Stage 1 chronic kidney disease. This included 9 dogs fed test food and 9 dogs fed owner’s-choice foods. Compared with baseline, after feeding 9 dogs test food for 6 months, serum SDMA decreased in 8 dogs and increased in 1 dog. After feeding 9 dogs owner’s-choice foods for 6 months, serum SDMA decreased in 4 dogs and increased in 4 dogs (remained stable in 1 dog). The decreases in serum SDMA and Cr concentrations were significant (both P = 0.03) only for dogs fed test food. These results suggest that nonazotemic dogs with elevated serum SDMA (early renal insufficiency) when fed a test food designed to promote healthy aging are more likely to demonstrate improved renal function compared with dogs fed owner’s-choice foods.     

Renal norepinephrine content decreases after chronic flow probe implantation.

Electromagnetic flow probes were chronically implanted around the left renal artery in 6 female beagle dogs. 3-10 months after implantation, renal cortex norepinephrine contents of right and left kidneys were compared (liquid chromatography). In 4 out of the 6 dogs investigated 3-6 months after implantation, the norepinephrine content of the left kidney was reduced by 27-72%. In 2 dogs investigated 7 and 10 months after implantation, the difference between left and right kidney was not significant. These findings should be taken into consideration when interpreting results from chronically instrumented kidneys.     

Storage stability of Anagrapha falcifera nucleopolyhedrovirus in spray-dried formulations

A multiply embedded nucleopolyhedrovirus isolated from Anagrapha falcifera (Kirby) (AfMNPV) can lose insecticidal activity during months of dry storage in ambient room conditions. We tested the spray-dried AfMNPV formulations after storage for up to 1 year at room temperatures for insecticidal activity against neonate Trichoplusia ni (Hübner). Experimental formulations were made using combinations of corn flours, lignin, and sucrose, and were selected based on previous work which demonstrated that these formulations resisted solar degradation in field experiments. Twelve experimental formulations (organized in three groups of four formulations) compared the effect of (1) the ratio of formulation ingredients (lignin and corn flour) to virus concentration, (2) different sources of lignin, or (3) different corn flours and sugar. Based on a single-dose plant assay with these 12 formulations, none of the formulations lost significant activity due to the drying process, when compared with the unformulated wet AfMNPV. Samples of the 12 dried formulations were stored at room (22+/-3 degrees C) and refrigerated (4 degrees C) temperatures. Insecticidal activity (LC(50)) was determined with a dosage-response assay for neonates fed on treated cotton-leaf disks. After 6 (or 9) and 12 months storage, refrigerated samples maintained insecticidal activity better than corresponding samples stored at room temperatures with LC(50)s that averaged 2.0 x 10(6) polyhedral inclusion bodies per milliliter (pibs/ml) for refrigerated samples and 5.4 x 10(6) pibs/ml for samples stored at room temperatures. Compared with unformulated stock virus stored frozen, six formulations stored at room temperature and 10 formulations stored in the refrigerator did not lose significant insecticidal activity after 1 year based on overlapping 90% confidence intervals. Changing the ratio of virus to formulation ingredients did not provide a clear trend over the range of concentrations tested, and may be less important for shelf-life of virus activity compared with formulations made with different ingredients. Two of the four formulations made with different lignins were about 15 times less active after 1 year at room temperature compared with refrigerated samples, indicating that specific formulation ingredients can affect storage stability. Formulations that contained sugar generally maintained activity during storage better than formulations without sugar. Unformulated virus stock maintained insecticidal activity (ranged from 0.20 to 2.5 x 10(6) pibs/ml) better during storage than dried formulations with LC(50)s that ranged from 0.39 to 27 x 10(6) pibs/ml. Unformulated virus stock, which is essentially a suspension of virus occlusion bodies in homogenized insect cadavers, did not lose activity when stored at refrigerated or room temperature. We believe that stability of AfMNPV insecticidal activity during storage as dry formulations is related to the general composition of the formulation and that sugar may play a critical role in maintaining insecticidal activity.     

Oral KCl dietary supplement extends survivability of dogs with Heidenhain pouch gastric fistulas

Supplementing the food of Heidenhain pouch gastric fistula dogs with a KC1 product greatly extends the survivability of these dogs. Since there is a continuous discharge of gastric juice from the pouch to the exterior each time a meal is consumed, clinical signs such as dehydration, anorexia, rough hair coat and lethargy usually occur within a few months after gastric pouch surgery and, unless extensive supportive measures are taken, most dogs will die shortly thereafter. The five dogs which did not receive KC1 supplementation died within 6 months after surgery with a mean survival time of 2.4 +/- 0.9 months. Seven dogs that received a daily oral supplement of 1.5 g KC1 (20 mEq) in their food have, on average, survived more than ten times longer than dogs which received no KC1 supplementation, with a mean survival time of 25.1 +/- 4.4 months. All KC1 supplemented dogs survived for more than 15 months with three dogs currently surviving for 36-40 months. When two dogs experienced decreased serum potassium, sodium and/or chloride levels and showed clinical signs of electrolyte imbalance despite receiving daily oral KC1 supplementation, intervention with intravenous (i.v.) lactated Ringer's solution and increased amounts of oral KC1 supplement reversed these symptoms within 1-2 weeks. Dogs that received only i.v. Ringer's therapy died with 1 week of the onset of clinical signs. Daily oral KC1 supplementation, careful observation of behavior and eating patterns, and routine physical examinations and serum electrolyte measurements can greatly extend the life expectancy of dogs with Heidenhain pouch gastric fistulas.     

Behavior and cortisol levels of dogs in a public animal shelter, and an exploration of the ability of these measures to predict problem behavior after adoption

Behavior and plasma cortisol levels were examined in puppies and juvenile/adult dogs admitted to a public animal shelter. A behavioral test was developed to assess the responses of the dogs to novel or threatening conditions. Factor analysis of the behavioral responses of 166 dogs on day 3 in the shelter yielded six factors (locomotor activity, flight, sociability, timidity, solicitation, and wariness) that accounted for 68% of the total variance. Among those dogs remaining in the shelter for 9 days, plasma cortisol levels declined from day 2 to 9. Cortisol levels were weakly related to factor scores. In order to explore the relation of measures in the shelter to later behavior, questionnaires assessing problem behaviors were mailed to new owners of dogs 2 weeks and 6 months following adoption. Among puppies, wariness scores were negatively correlated with behavior problems at 2 weeks and cortisol levels were negatively correlated with behavior problems at 6 months. These results suggest how measures of behavior and endocrine activity obtained in shelters might prove useful for screening dogs for adoption or targeting dogs for behavioral intervention.     

Prevalence of species of Toxocara in dogs, cats and red foxes from the Poznan region, Poland

The prevalence of toxocariasis was evaluated for 445 dogs, 105 cats and 92 foxes from the Poznan region during 1997-1998. Forty one cats were infected (39%), 140 dogs (32%) and 15 red foxes (16%). Toxocara canis was found most frequently in puppies up to 3 months old (58%) and T. cati in kittens 4-6 months old (64%). Toxocariasis was much more prevalent amongst adult foxes (14%) than adult dogs (3%). In contrast to cats, female dogs and foxes were less infected than males. The present study suggests that cats may constitute an underestimated risk of transmission of Toxocara spp. to humans and the progressive synatropization of red foxes may also increase the sources of environmental contamination with Toxocara eggs.     

Gender and post-ischemic recovery of hypertrophied rat hearts

Background

Cardiac mortality in Duchenne muscular dystrophy (DMD) has recently become important, because risk of respiratory failure has been reduced due to widespread use of the respirator. The cardiac involvement is characterized by distinctive electrocardiographic abnormalities or dilated cardiomyopathy, but the pathogenesis has remained obscure. In research on DMD, Golden retriever-based muscular dystrophy (GRMD) has attracted much attention as an animal model because it resembles DMD, but GRMD is very difficult to maintain because of their severe phenotypes. We therefore established a line of dogs with Beagle-based canine X-linked muscular dystrophy in Japan (CXMD_J) and examined the cardiac involvement.

Methods

The cardiac phenotypes of eight CXMD_J and four normal male dogs 2 to 21 months of age were evaluated using electrocardiography, echocardiography, and histopathological examinations.

Results

Increases in the heart rate and decreases in PQ interval compared to a normal littermate were detected in two littermate CXMD_J dogs at 15 months of age or older. Distinct deep Q-waves and increase in Q/R ratios in leads II, III, and aVF were detected by 6–7 months of age in all CXMD_J dogs. In the echocardiogram, one of eight of CXMD_J dogs showed a hyperechoic lesion in the left ventricular posterior wall at 5 months of age, but the rest had not by 6–7 months of age. The left ventricular function in the echocardiogram indicated no abnormality in all CXMD_J dogs by 6–7 months of age. Histopathology revealed myocardial fibrosis, especially in the left ventricular posterobasal wall, in three of eight CXMD_J dogs by 21 months of age.

Conclusion

Cardiac involvement in CXMD_J dogs is milder and has slower progression than that described in GRMD dogs. The distinct deep Q-waves have been ascribed to myocardial fibrosis in the posterobasal region of the left ventricle, but our data showed that they precede the lesion on echocardiogram and histopathology. These findings imply that studies of CXMD_J may reveal not only another causative mechanism of the deep Q-waves but also more information on the pathogenesis in the dystrophin-deficient heart.     

The Use of a 4% (w/w) Deltamethrin Collar (Scalibor® ProtectorBand) in the Extended Control of Ticks on Dogs

Deltamethrin (4%, w/w) formulated in a polymer matrix system (Scalibor ProtectorBand, Intervet International BV) was evaluated for the prevention of ticks on dogs. Controlled laboratory trials were conducted, wherein dogs wearing anti-tick collars were compared with control dogs and artificially infested by adult Ixodes ricinus and/or Rhipicephalus sanguineus ticks. The activity of the collars increased from 24 to 48 h post-treatment (pt) wherein tick numbers were reduced to 78.7% for I. ricinus and to 77% for R. sanguineus. The proportion of ticks controlled after the second infestation on D7 pt increased to 99% for I. ricinus and 99.5% for R. sanguineus. For I. ricinus adult ticks, an efficacy of 95.4% was reached over a period of 5.5 months and approximated 90% for R. sanguineus. Hence, the efficacy of the deltamethrin-impregnated dog collar was >90% for 6 months against both tick species. There was no significant difference between the efficacy of the collar against either R. sanguineus or I. ricinus. A subsequent field study wherein 82 dogs were wearing the collar and monitored for tick infestation indicated that the duration of efficacy was between 5 and 6 months.     

Carrier-based Preparations of Plant Growth-promoting Bacterial Inoculants Suitable for use in Cooler Regions

Summary Carrier-based preparations of two plant growth-promoting rhizobacteria (PGPR) viz. Bacillus subtilis and Pseudomonas corrugata, developed in five formulations were evaluated for their growth promotion, rhizosphere colonization, and viability under storage. The effect of these formulations as fresh preparations, and after 6 months of storage at 4 °C and room temperature, was also determined. The bacterial inoculants in all the formulations were found to enhance the growth parameters of the test plant species; best results were obtained in case of alginate-based formulations. Maximum numbers of inoculated bacteria were recovered from the rhizosphere of alginate-based formulation-treated plants after 6 weeks of growth. Viability of bacterial inoculants was maximal in alginate beads, and alginate beads supplemented with skim milk formulations, after 180 days of storage at 4 °C.     

Evaluation of different formulations of potential biocontrol yeast isolates efficacy on apple blue mold at storage condition

Post-harvest pathogens cause major losses in apple production. Biological control by using epiphytic yeasts against Penicillium expansum has been considered as an alternative method for controlling the post-harvest decays. The yeast isolates Rhodotorula mucilaginosa, Pichia guilliermondii, which showed high biocontrol efficacy against P. expansum, were selected for formulation tests. Formulation is an important step in developing a biocontrol product. The successful delivery of biocontrol agents, shelf life, stability and effectiveness in commercial conditions depend on the formulation. In the formulation, the carrier is the primary material used to allow a bioproduct to be dispersed effectively. Yeast isolates were grown in a cane molasses-based medium. Harvested yeast cells were combined with inorganic (talk, kaolin) and organic (Rice bran, wheat bran) carriers. Viability of the yeast cells in formulations stored at 4°C and 24°C was determined each month during 6 months storage. After 6 months storage to evaluate efficacy of formulations, all formulations were tested on apple to control blue mold in storage condition. High stability of antagonistic yeasts was achieved by using organic and inorganic carriers. Rice bran and wheat bran stimulated the germination of the yeasts cells during storage period. Both of the yeasts had significantly highest viable yeast cell content over 6 months in formulation containing wheat bran as a carrier. P.guillermondii in all formulations had significantly higher shelf life and was effective than R. mucilaginosa.     

Taurocholic acid synthesis in dogs maintained on a high cholesterol diet

Labeled 35S-taurocholic acid synthesis was studied in dogs with gall bladder fistulae, fed high cholesterol diet. During the first period (1--2 months) dietary cholesterol caused a significant increase in the 35S-taurocholic acid synthesis. In 5--6 months, at the period of development of the pathological process in the liver, there was a decrease of synthesis and secretion of bile acids, and of cholesterol sediment in the bile. The level of plasma cholesterol in these dogs increased only slightly.     

Antibiotic control of tissue reactions in dogs vaccinated with viable cells of Coccidioides immitis

Castleberry, Merida W. (U.S. Army Biological Laboratories, Fort Detrick, Frederick, Md.), John L. Converse, and Peter J. Soto, Jr. Antibiotic control of tissue reactions in dogs vaccinated with viable cells of Coccidioides immitis. J. Bacteriol. 87:1216-1220. 1964.-A total of 12 dogs (15 to 25 lb each), vaccinated with viable Coccidioides immitis (subcutaneous injection of 260 viable arthrospores in the medial surface of the hind leg), resisted a respiratory challenge (aerosol) with the same organism (13,000 viable arthrospores) administered (aerosol) 2 months after vaccination. Oral amphotericin B therapy (150 mg of Fungizone per day for 21 days) of 6 of the 12 dogs, initiated immediately after vaccination, eliminated the undesirable side reactions of the viable vaccine (ulcerated vaccination site and inguinal lymphadenopathy exhibited by the 6 untreated dogs) without affecting the immunogenicity of the vaccine. Clinical observation (blood-urea nitrogen levels) during and after therapy and histological examination approximately 3 months after respiratory challenge failed to disclose any evidence of nephrotoxicity or renal damage due to the oral antibiotic therapy (total doses of more than 3 g of amphotericin B).     

The effect of particle size on bioavailability in cyclosporine preparations based on submicron dispersions

The effect of particle size on bioavailability of 9 different formulations with cyclosporine A was studied. A common feature of all the formulations was the ability to form submicron dispersions under dilution. The composition of individual formulations was chosen in such a way that they were based on same or similar excipients. For each formulation, pharmacokinetic study was carried out in beagle dogs. On groups of 10 dogs, the average AUC was evaluated. Particle size of formulations under dilution in water was measured by laser scattering method. According to the results of particle size measurement, the formulations were sorted out into groups of similar particle size distribution by use of two methods of multivariate statistical analysis. The average AUC within groups and between-groups was compared, and the effect of particle size on bioavailability was evaluated.     

Preparation and characterization of Eudragit Retard nanosuspensions for the ocular delivery of cloricromene

The purpose of this study was to improve the stability of cloricromene (AD6) in ophthalmic formulations and its drug availability at the ocular level. To this end, AD6-loaded polymeric nanoparticle suspensions were made using inert polymer resins (Eudragit RS100 and RL100). We modified the quasi-emulsion solvent diffusion technique by varying some formulation parameters (the drug-to-polymer ratio, the total drug and polymer amount, and the stirring speed). The chemical stability of AD6 in the nanosuspensions was assessed by preparing some formulations using (unbuffered) isotonic saline or a pH 7 phosphate buffer solution as the dispersing medium. The formulations were stored at 4°C, and the rate of degradation of AD6 was followed by high performance liquid chromatography (HPLC). The obtained nanosuspensions showed mean sizes and a positive surface charge (ζ-potential) that make them suitable for an ophthalmic application; these properties were maintained upon storage at 4°C for several months. In vitro dissolution tests confirmed a modified release of the drug from the polymer matrixes. Nanosuspensions prepared with saline solution and no or lower amounts of surfactant (Tween 80) showed an enhanced stability of the ester drug for several months, with respect to an AD6 aqueous solution. Based on the tecnological results, AD6-loaded Eudragit Retard nanoparticle suspensions appear to, offer promise as a means to improving the shelf life and bioavailability of this drug after ophthalmic application. Published: March 24, 2006