Methods to evaluate and improve the injection site tolerability of intravenous formulations prior to first-in-human testing.

INTRODUCTION: Evaluation of infusion site tolerability is required for the development of intravenous formulations of New Molecular Entities and is of particular importance for investigational drugs that have the potential to precipitate on contact with the blood stream. Based on a comprehensive set of in vitro and in vivo studies conducted with JNJ-X, a development stage small molecule investigational drug, with a pH-dependent solubility that showed potential to cause infusion site irritation at high concentrations, we have developed a systematic approach for evaluating and selecting suitable intravenous formulations for compounds that show potential to precipitate at the infusion site. METHODS: Aqueous formulations containing a range of concentrations of JNJ-X with different excipients, and buffering agents at different pHs (3.9-7.4) were evaluated in an in vitro solubility assay, a modified hen's egg test-chorioallantoic membrane assay (HET-CAM(VT)) and in vivo in rabbit, rat, and dog intravenous infusion toxicity studies. RESULTS: The data obtained with JNJ-X in the different in vitro and in vivo studies were compared and used to support the development of an in silico model and to create a systematic approach to screen and identify candidate intravenous formulations with improved tolerability. DISCUSSION/CONCLUSION: This approach provides a framework that can be used to assess the risk for infusion site irritation and identify better tolerated formulations with a reduced need for in vivo testing.

Impact of gavage dosing procedure and gastric content on adverse respiratory effects and mortality in rat toxicity studies.

Unscheduled mortality preceded by adverse respiratory clinical signs in rats dosed by oral gavage may not only be caused by technical gavage error or systemic toxicity but may also be caused by gastro-esophageal reflux and subsequent aspiration of high concentrations of drug formulation. In a 3 week oral gavage rat toxicity study for an early drug development compound, preterminal deaths (approximately 20% of animals) at high doses (≥1000 mg kg(-1) ) and concentrations (≥60 mg ml(-1) ) were preceded by recurrent dyspnea, rales or excessive salivation, without evidence of accidental intrapulmonary gavage error. Histological evaluation revealed extensive necrosis and inflammatory changes in the upper respiratory tract, especially in the nasal turbinates and/or nasopharynx. The presence of food particles in inflammatory exudates suggested a retrograde aspiration of stomach content with test formulation via the nasopharyngeal duct into the posterior region of the nose. In contrast, no mortality or adverse respiratory effects were observed in rats following 2 week intravenous administration at comparable exposures or oral gavage administration at lower concentrations (≤20 mg ml(-1) ). In a pharmacology study, the compound caused a dose-dependent increase in gastric content (partly due to inhibition of gastric emptying), providing a pharmacological basis for the suspected gavage-mediated gastroesophageal reflux. Reducing the dose volume and dosing fasted animals substantially reduced or eliminated the respiratory effects and mortality at the high test article concentrations, demonstrating that the adverse effects are related to the gavage method.

Vaccination of calves against Ostertagia ostertagi with cysteine proteinase enriched protein fractions.

Cysteine proteinase enriched fractions obtained by thiol-sepharose chromatography of Ostertagia ostertagi membrane-bound protein extract (S3-thiol) or total adult excretory-secretory (ES-thiol) products were tested in a vaccination experiment to evaluate their protective efficacy against O. ostertagi in cattle. Calves were vaccinated three times and subsequently challenged with a trickled infection of 25,000 infective larvae in total over 25 days (1000 L3/day, 5 days/week). Geometric mean cumulative egg counts in the ES-thiol group were reduced by 60% during the 2-month period between the first challenge infection and necropsy, compared to the control group (P < 0.002). No reduction in egg output was observed in the S3-thiol group. At necropsy, calves immunized with ES-thiol had a significantly higher percentage of inhibited L4 larvae (9.8%) and had in total 18% less worms than the control calves, but this reduction was not statistically significant. Both the female and male adult worms were significantly smaller in the ES-thiol group than in the control group. Although no significant difference was observed in the number of eggs per female worm between the groups, there was a trend to less eggs per female worm in the ES-thiol group. Number of worms, size of adult worms and number of eggs per female worm were not significantly different between the S3-thiol group and the control group. Systemic immunization with QuilA as adjuvant induced a significant rise in Ostertagia-specific antibody levels in the abomasal mucosa. Ostertagia-specific local antibody levels showed a significant negative correlation with the size of the adult worms, the number of eggs per female worm and the cumulative faecal egg counts. However, these correlations were quite weak and did not appear to be isotype-specific.

Cellular immunological responses of pheasant during endogenous development of Eimeria colchici.

We examined the time course and histological localisation of the developmental stages of Eimeria colchici. The prepatent period in the caeca of pheasants was 6 days. The patent period began on day 7 post-infection (p.i.) and ended on day 11 p.i. with peak production of oocysts on days 8-9. The peripheral blood lymphocytes of pheasant chicks showed a significant increase in proliferation to E. colchici antigen from day 5 p.i., with peak on day 14 p.i. The metabolic activity (respiratory burst) of heterophils increased on days 3, 4 and 14 p.i. The total number of peripheral blood leukocytes and lymphocytes in the infected pheasant chicks had increased by day 2 p.i. and reached a maximum on day 4 of the experiment. Days 5 and 6 p.i. were characterised by a drop in the number of these cells.