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.

Cosmetics Europe multi-laboratory pre-validation of the SkinEthic™ reconstituted human corneal epithelium test method for the prediction of eye irritation.

Cosmetics Europe, The Personal Care Association, known as Colipa before 2012, conducted a program of technology transfer and assessment of Within/Between Laboratory (WLV/BLV) reproducibility of the SkinEthic™ Reconstituted Human Corneal Epithelium (HCE) as one of two human reconstructed tissue eye irritation test methods. The SkinEthic™ HCE test method involves two exposure time treatment procedures - one for short time exposure (10 min - SE) and the other for long time exposure (60 min - LE) of tissues to test substance. This paper describes pre-validation studies of the SkinEthic™ HCE test method (SE and LE protocols) as well as the Eye Peptide Reactivity Assay (EPRA). In the SE WLV study, 30 substances were evaluated. A consistent outcome with respect to viability measurement across all runs was observed with all substances showing an SD of less than 18%. In the LE WLV study, 44 out of 45 substances were consistently classified. These data demonstrated a high level of reproducibility within laboratory for both the SE and LE treatment procedures. For the LE BLV, 19 out of 20 substances were consistently classified between the three laboratories, again demonstrating a high level of reproducibility between laboratories. The results for EPRA WLV and BLV studies demonstrated that all substances analysed were categorised similarly and that the method is reproducible. The SkinEthic™ HCE test method entered into the experimental phase of a formal ECVAM validation program in 2010.

Cosmetics Europe multi-laboratory pre-validation of the EpiOcular™ reconstituted human tissue test method for the prediction of eye irritation.

Cosmetics Europe, The Personal Care Association (known as Colipa before 2012), conducted a program of technology transfer and within/between laboratory reproducibility of MatTek Corporation's EpiOcular™ Eye Irritation Test (EIT) as one of the two human reconstructed tissue test methods. This EIT EpiOcular™ used a single exposure period for each chemical and a prediction model based on a cut-off in relative survival [ ≤60%=irritant (I) (GHS categories 2 and 1); >60%=no classification (NC)]. Test substance single exposure time was 30 min with a 2-h post-exposure incubation for liquids and 90 min with an 18-h post-exposure incubation for solids. Tissue viability was determined by tetrazolium dye (MTT) reduction. Combinations of 20 coded chemicals were tested in 7 laboratories. Standardized laboratory documentation was used by all laboratories. Twenty liquids (11 NC/9 I) plus 5 solids (3 NC/2 I) were selected so that both exposure regimens could be assessed. Concurrent positive (methyl acetate) and negative (water) controls were tested in each trial. In all, 298 independent trials were performed and demonstrated 99.7% agreement in prediction (NC/I) across the laboratories. Coefficients of variation for the% survival for tissues from each treatment group across laboratories were generally low. This protocol has entered in 2010 the experimental phase of a formal ECVAM validation program.

Prevalidation of a new in vitro reconstituted human cornea model to assess the eye irritating potential of chemicals.

This multicentre study aimed at evaluating the reliability (reproducibility) and relevance (predictivity) of a new commercially available human corneal epithelial (HCE) model (SkinEthic Laboratories, Nice, France) to assess acute ocular irritation. A prevalidation approach (protocol optimisation, transfer and performance) was followed and at each of the four participating laboratories, 20 coded reference chemicals, covering the whole range of irritancy, were tested. The compounds were applied topically to the HCE cultures and the level of cytotoxicity (tissue viability and histological analysis) was determined. Once a standardised protocol was established, a high level of reproducibility between the laboratories was observed. In order to assess the capability of the HCE model to discriminate between irritants (I) and non-irritants (NI), a classification prediction model (PM) was defined based on a viability cut-off value of 60%. The obtained in vitro classifications were compared with different in vivo classifications (e.g. Globally Harmonised System) which were calculated from individual rabbit data described in the ECETOC data bank. Although an overall concordance of 80% was obtained (sensitivity = 100% and specificity = 56%), the predictivity of the HCE model substantially increased when other sources of in vivo and in vitro data were taken into account.

The mucosal toxicity of different benzalkonium chloride analogues evaluated with an alternative test using slugs.

PURPOSE: The objective of this study was to evaluate the mucosal toxicity of different benzalkonium chloride (BAC) analogues using slugs as the alternative test organism. METHODS: The effect of different BAC analogues on the mucosal tissue of slugs was determined from the protein, lactate dehydrogenase, and alkaline phosphatase released from the foot mucosa after treatment. Additionally, mucus production and reduction in body weight of the slugs were measured. The eye irritation potency of the molecules was evaluated with the Bovine Corneal Opacity and Permeability (BCOP) assay. The antimicrobial activity of the different BAC analogues was also assessed. RESULTS: All BAC analogues induced severe damage to the mucosal epithelium of the slugs, and the irritation increased with decreasing alkyl chain length: BAC-C16 < BAC-C14 < BAC-C12 approximately BAC-mix. A similar ranking was obtained with the BCOP assay for eye irritation. The relative order of activities among the three BAC analogues was the same, i.e., BAC-C14 > or = BAC-C16 > BAC-C12. The BAC-C14 exhibited higher activity than the BAC-mix. CONCLUSIONS: The toxicity and activity of BAC analogues depend on the alkyl chain length. The use of BAC-C14 as a conservative agent in pharmaceutical preparations instead of the BAC-mix should be considered.

Comparative evaluation of the in vitro micronucleus test and the alkaline single cell gel electrophoresis assay for the detection of DNA damaging agents: genotoxic effects of cobalt powder, tungsten carbide and cobalt-tungsten carbide.

Although it is well known that micronuclei may arise from either DNA breakage leading to acentric chromosome fragments or from chromosome/chromatid lagging in anaphase, the ratio between the amount of DNA breakage induced and the frequency of micronuclei expressed in the following interphase is unclear. With the development of the alkaline single cell gel electrophoresis assay, which measures single strand and/or double strand breaks in a cell by cell approach, it is new possible to address this question at the cellular level. We therefore compared the genotoxic potential of pure cobalt powder (Co) and a cobalt-containing alloy, cobalt-tungsten carbide (WC-Co), involved in specific lung disorders, in parallel with the alkaline single cell gel electrophoresis (SCGE) assay (comet assay) and the cytokinesis-blocked micronucleus (MN) test, both carried out in vitro on isolated human leukocytes. The comet assay indicated that the WC-Co mixture produced a higher level of DNA damage than Co alone; WC alone was not able to induce a dose-dependent DNA breakage effect as was seen for Co and WC-Co. Results from the MN test confirmed these observations. It was clear that the clastogenic property of Co-containing dust is significantly enhanced when the Co metal is mixed with WC and suggested that their physicochemical characteristics may act as one of the important parameters responsible for the increased incidence of lung cancers observed in the population of hard metal workers. In agreement with data obtained in the same laboratory on liposoluble chemicals (PCBs and chlorinated aliphatic hydrocarbons) and from the literature, the results indicate that both the comet assay and the micronucleus test were able to detect differences in the genotoxic potential of the compounds studied. Although the micronucleus test seemed to be less sensitive to assess a synergistic DNA damaging potential of the mixture involved, it detects chromosomal aberrations (chromosome/genome mutations) and not just repairable DNA breakage or alkali-labile sites. Combination of the comet assay and the in vitro MN test might therefore be recommended for genotoxins to understand the mechanisms underlying mutagenicity and to assess the lowest efficient dose.

Identification of clastogenic and/or aneugenic events during the preneoplastic stages of experimental rat hepatocarcinogenesis by fluorescence in situ hybridization.

A growing body of evidence from human and animal cancer cytogenetics studies indicates that aneuploidy is an important chromosome change in carcinogenesis. To understand the role of this genetic phenomenon during the first steps of an experimental cancer model, molecular and cellular techniques were combined. A sequential cytogenetic study of a modified Solt-Farber liver cancer model in the rat was performed to identify the importance of chromosome versus genome mutations. Male Wistar rats were initiated with diethylnitrosamine (DENA), followed by a 2-acetylaminofluorene exposure to select resistant hepatocytes. Chronic phenobarbital (PB) treatment was used to induce promotion. Cell proliferation was induced by a necrogenic dose of CCl4, administered during the selection period (Gerlans protocol) or 3 days before hepatocyte isolation (experimental protocol). In order to discriminate between genetic events causing chromosome breakage (clastogenic) and those that induce chromosome loss (aneugenic), isolated micronucleated hepatocytes (MNH) were analysed for the presence of a centromere in the micronucleus (MN). Non-radioactive in situ hybridization with a rat centromere satellite 1 DNA probe was applied. Our results show that the majority of the observed genetic changes, expressed as MN during different preneoplastic stages, were of clastogenic origin. However, the number of induced aneugenic hepatocytes increased markedly during the promotion period of the Gerlans protocol (approximately 7-fold above control) and during PB exposure in the experimental protocol (approximately 4-fold above control). Additionally, these stages were also characterized by an increased level of MN expression (20.3 < % MNH < 32.8), in comparison with the initiation stage after DENA exposure (13.5 < % MNH < 17.1). Although it is not yet clear if these genetic alterations have a causative nature in neoplastic liver transformation, the use of interphase cytogenetics certainly might lead to a better understanding of the genomic changes which occur during experimental hepatocarcinogenesis.

Frequency and DNA content of micronuclei in rat parenchymal liver cells during experimental hepatocarcinogenesis.

Liver carcinogenesis is considered to be a good experimental model to study the sequential changes leading to cancer and was applied here for the analysis of chromosome/genome mutations. Since the micronucleus test was shown to be an adequate method to detect and analyse chromosome changes in dividing cells, the frequency of micronuclei (MN) together with their relative DNA content (DNA content of the MN divided by the DNA content of the corresponding nucleus) were analysed in hepatocytes isolated from rats at different stages of experimentally induced hepatocarcinogenesis. The protocol used for the induction of liver cancer was based on the triphasic 'Gerlans protocol', a Solt-Farber procedure supplemented with a phenobarbital (PB) promotion step. Male Wistar rats were initiated by a single i.p. dose of diethylnitrosamine (DENA), followed by selection of the resistant hepatocytes by 2-acetylaminofluorene (2-AAF). Subsequent promotion was accomplished by chronic exposure to phenobarbital. For each group of rats a mitotic stimulator (CCl4) is necessary at the end of their treatment period to express the clastogenic and/or aneugenic lesions which may lead to micronuclei. The results of these experiments do confirm that genetic alterations are occurring at the chromosome level (MN expression) during the different steps of experimental rat liver carcinogenesis. DNA measurements seem to be a good genetic parameter to detect eventual differences between the chromosomal content (whole chromosome or chromosome fragments) of MN populations appearing in different stages of the carcinogenic process. Moreover, a comparison between the mono- and bi-nucleated cell population showed that the frequency of micronuclei is higher in mononuclear parenchymal liver cells.

Clinical experience with fetal continuous monitoring by pH electrode.

Experiences about continuous measurement with the pH electrode are reported. Difficulties with the use of the electrode noticed when sterilizing, calibrating, and fixing it on the fetal head are discussed separately.