Long term breeding of the Lmna G609G progeric mouse: Characterization of homozygous and heterozygous models.

The transgenic LmnaG609G progeric mouse represents an outstanding animal model for studying the human Hutchinson-Gilford Progeria Syndrome (HGPS) caused by a mutation in the LMNA gene, coding for the nuclear envelope protein Lamin A/C, and, as an important, more general scope, for studying the complex process governing physiological aging in humans. Here we give a comprehensive description of the peculiarities related to the breeding of LmnaG609G mice over a prolonged period of time, and of many features observed in a large colony for a 2-years period. We describe the breeding and housing conditions underlining the possible interference of the genetic background on the phenotype expression. This information represents a useful tool when planning and interpreting studies on the LmnaG609G mouse model, complementing any specific data already reported in the literature about this model since its production. It is also particularly relevant for the heterozygous mouse, which mirrors the genotype of the human pathology however requires an extended time to manifest symptoms and to be carefully studied.

A quick and simple method for the determination of ivermectin in dog plasma by LC-MS/MS.

Ivermectin is an endectocide belonging to the macrocyclic lactone class, commonly used in dogs as a heartworm preventative and for the treatment of several external and internal parasite infections. Among the analytical methods for ivermectin determination in plasma available in literature, many require a laborious clean-up step on SPE cartridge, and use fluorescence detection instead of the more reliable mass spectrometry. In the context of a project aimed at its pharmacokinetic evaluation in this species, a liquid chromatography-tandem mass spectrometry method for the determination of ivermectin in dog plasma was developed and validated, using blank plasma provided by a local canine blood transfusion service. Samples underwent a quick liquid-liquid extraction before analysis in the LC-MS/MS system, operating in selected reaction monitoring (SRM) mode. The method provided satisfactory linearity (R2 >0.99), accuracy (bias <3%) and precision (CV <10%) over the 0.5-20.0 ng/mL range. •This is to our knowledge the first validated LC-MS/MS method for ivermectin determination in dog plasma.•Sample preparation is simple, rapid and inexpensive, without compromising sensitivity.•The modest amount of plasma required makes the proposed technique suitable for pharmacokinetic studies also in small dogs.

A fully-virulent retargeted oncolytic HSV armed with IL-12 elicits local immunity and vaccine therapy towards distant tumors.

Oncolytic herpes simplex viruses (oHSVs) showed efficacy in clinical trials and practice. Most of them gain cancer-specificity from deletions/mutations in genes that counteract the host response, and grow selectively in cancer cells defective in anti-viral response. Because of the deletions/mutations, they are frequently attenuated or over-attenuated. We developed next-generation oHSVs, which carry no deletion/mutation, gain cancer-specificity from specific retargeting to tumor cell receptors-e.g. HER2 (human epidermal growth factor receptor 2)-hence are fully-virulent in the targeted cancer cells. The type of immunotherapy they elicit was not predictable, since non-attenuated HSVs induce and then dampen the innate response, whereas deleted/attenuated viruses fail to contrast it, and since the retargeted oHSVs replicate efficiently in tumor cells, but spare other cells in the tumor. We report on the first efficacy study of HER2-retargeted, fully-virulent oHSVs in immunocompetent mice. Their safety profile was very high. Both the unarmed R-LM113 and the IL-12-armed R-115 inhibited the growth of the primary HER2-Lewis lung carcinoma-1 (HER2-LLC1) tumor, R-115 being constantly more efficacious. All the mice that did not die because of the primary treated tumors, were protected from the growth of contralateral untreated tumors. The long-term survivors were protected from a second contralateral tumor, providing additional evidence for an abscopal immunotherapeutic effect. Analysis of the local response highlighted that particularly R-115 unleashed the immunosuppressive tumor microenvironment, i.e. induced immunomodulatory cytokines, including IFNγ, T-bet which promoted Th1 polarization. Some of the tumor infiltrating cells, e.g. CD4+, CD335+ cells were increased in the tumors of all responders mice, irrespective of which virus was employed, whereas CD8+, Foxp3+, CD141+ were increased and CD11b+ cells were decreased preferentially in R-115-treated mice. The durable response included a breakage of tolerance towards both HER2 and the wt tumor cells, and underscored a systemic immunotherapeutic vaccine response.

Tolerability and pharmacokinetics of two antimony products after subcutaneous administration in dogs.

INTRODUCTION: Pentavalent antimony is the first choice drug for leishmaniasis in dog. Leishmaniasis has a complex pathogenesis and it manifests various clinical signs, some of which are often similar to those associated with the toxicity induced by antimonial treatment. Among the reasons for this toxicity, also a general problem of drug's quality has been reported. METHODOLOGY: The general and local tolerability of two commercially available meglumine antimoniate based veterinary products was evaluated in 12 healthy dogs, 6 receiving Antimania (Fatro, Italy) and 6 receiving Glucantime (Merial, Spain), following repeated subcutaneous administrations of therapeutic doses for 14 days. RESULTS: Individual and mean values of haematological and biochemical parameters in both groups remained in the physiological range, with no considerable differences within the two groups. The general tolerability of the drugs was also supported by clinical observations and physical examination of the dogs throughout the whole study period. Only slight local reactions at the injection sites, that spontaneously disappeared, were observed for both products starting from 12-84 hours after the administration. The pharmacokinetic parameters indicated no antimony accumulation. CONCLUSIONS: These results suggest that meglumine antimoniate administered at the recommended dosage regimen is well tolerated by healthy dogs, and that there is no significant difference between the two tested products.

Pharmacokinetics of buprenorphine following constant rate infusion for postoperative analgesia in dogs undergoing ovariectomy.

OBJECTIVE: To investigate the pharmacokinetics of buprenorphine and its main active metabolite, norbuprenorphine, after administration of an intravenous loading dose followed by constant rate infusion (CRI) in dogs. STUDY DESIGN: Prospective, clinical study. ANIMALS: A total of seven healthy dogs undergoing elective ovariectomy. METHODS: Buprenorphine was administered as a loading dose (intravenous bolus of 15 µg kg-1) followed by CRI (2.5 µg kg-1 hour-1 for 6 hours). Moreover, intraoperative analgesia was supplemented by an intramuscular carprofen (4 mg kg-1) injection, administered prior to surgery, and by lidocaine, administrated through subcutaneous infiltration and through a splash on the ovarian vascular pedicle during surgery. Pain and sedation were scored for all animals throughout the 24-hour study period and rescue analgesia was administered when a visual analogue scale score was > 40 mm. Blood samples were collected from a jugular catheter at regular intervals, and plasma concentrations of buprenorphine and norbuprenorphine were determined by a validated liquid chromatography-tandem mass spectrometry method. RESULTS: Buprenorphine showed a two-compartment kinetic profile. Maximum concentration was 23.92 ± 8.64 ng mL-1 at 1 minute (maximum time); elimination half-life was 41.87 ± 17.35 minutes; area under the curve was 486.68 ± 125.66 minutes ng-1 mL-1; clearance was 33.61 ± 13.01 mL minute-1 kg-1, and volume of distribution at steady state was 1.77 ± 0.50 L kg-1. In no case was rescue analgesia required. Norbuprenorphine resulted below the lower limit of quantification in almost all samples. CONCLUSIONS AND CLINICAL RELEVANCE: The results suggest that a buprenorphine CRI can be a useful tool for providing analgesia in postoperative patients, considering its minor side effects and the advantages of a CRI compared to frequent boluses. The negligible contribution of norbuprenorphine to the therapeutic effect was confirmed.