Jean-Jacques Legrand

Systemic exposure to parabens: Pharmacokinetics, tissue distribution, excretion balance and plasma metabolites of [14C]-methyl-, propyl- and butylparaben in rats after oral, topical or subcutaneous administration

Parabens (PB) are preservatives used in food, drugs and personal care products preventing microbial and fungal contamination. We investigated ADME profiles of [14C]-methyl-, propyl- or butylparaben (MP, PP, BP) following single oral, dermal or subcutaneous (BP) doses at 100 mg/kg to Sprague-Dawley rats. Plasma Cmax and AUC values after oral or subcutaneous doses were 4- to 10-fold higher relative to respective values after dermal administration. tmax ranged from 0.5, 2 or 8 h after oral, subcutaneous or dermal administration, respectively. MP produced higher blood Cmax and AUC levels relative to those after PP or BP. Following oral or subcutaneous administration, urinary excretion was predominant (>70%, mainly during the first 24 h), less than 4% were eliminated in the feces, 2% were retained in the tissues and carcasses. Following dermal application, >50% of the dose was unabsorbed, 14-27% or <2% were respectively excreted in the urine or feces, respectively. Overall, parabens were well absorbed after oral and subcutaneous, and partially absorbed after dermal administration. All administration routes produced a single peak in the plasma, corresponding to that of para-hydroxybenzoic acid (PHBA) suggesting that PB produce no significant systemic exposure of mammalian organisms after oral, topical or subcutaneous administration.

Immunotoxic effects of cyclophosphamide and cyclosporine in the dog

Limited non-clinical immunotoxicity data are available in the dog, although this is a major non-rodent species in regulatory safety studies. The present study aimed to test whether widely accepted immunotoxicity endpoints including lymphocyte subset immunophenotyping, the anti-KLH TDAR assay, and histological examination of the main lymphoid organs were reliable to detect immunosuppression induced by cyclosporine and cyclophosphamide in dogs and could, therefore, be used for non-clinical immunotoxicity evaluation in this species. Male and female Beagle dogs were treated orally from Day 1 for 4 weeks with 25 mg/kg cyclosporine daily, or with 2 mg/kg cyclophosphamide on 4 consecutive days each week, or the same volume of drinking water daily. Blood samples were withdrawn pre-test and on Days 11, 18, and 23 to measure standard hematology parameters and analyze lymphocyte subsets. All animals received an intramuscular injection of 5 mg KLH on Day 11. Sandwich ELISA assays were used to quantify anti-KLH IgM and anti-KLH IgG levels in blood samples taken pre-test, on Days 18 and 23, and pre-test, on Days 23 and 28, respectively. At the end of the treatment period, all animals were submitted to histological examination of lymphoid organs, liver, and kidneys. No signs of marked toxicity were observed. No changes in lymphocyte subsets, but markedly decreased primary anti-KLH IgM and IgG responses, and a slightly-to-markedly increased cortex/medulla ratio in the thymus were observed in cyclosporine-treated dogs. Lower total WBC counts correlating with lower total and B-lymphocyte subset and decreased germinal center development in mesenteric lymph nodes, but no changes in primary anti-KLH IgM and IgG responses were observed in cyclophosphamide-treated dogs. These results demonstrate that widely accepted immunotoxicity endpoints can adequately detect the effects of known immunosuppressive drugs in the dog and support the conclusion that it is a relevant animal species for immunotoxicity evaluation.

Recording of the full‐field electroretinogram in minipigs

PURPOSE To test a simple electroretinographic protocol on a representative sample of minipigs. ANIMAL STUDIED Minipig. PROCEDURES Electroretinogram recordings were conducted on 162 healthy minipigs (81 males and 81 females) aged 4-6 months. After a 1.5-h light-adaptation period, the animals were anesthetized with general anesthesia. First, binocular full-field photopic electroretinogram recordings were conducted under photopic conditions. Subsequently, scotopic electroretinogram recordings were conducted during dark-adaptation periods every 4 min for a 20-min period. At the end of this period, the maximal combined rod-cone response was recorded by measuring the retinal response to a single high-intensity flash. We used sclerocorneal clip electrodes as active electrodes and needle electrodes as reference and ground electrodes. RESULTS The a-wave and b-wave peak times and amplitudes have been measured and statistically analyzed. For each of the statistical comparisons, normality and homogeneity of variances were evaluated. No significant gender differences were observed, with the exception of a higher b-wave amplitude for the photopic ERG recordings observed in females when compared to males (48.14 ± 12.909 μV vs. 42.88 ± 10.666 μV; P = 0.005). The process of dark adaptation was evaluated, and the maximal combined rod-cone response was measured (a- and b-waves amplitude and peak time). CONCLUSIONS We conducted photopic and scotopic electroretinogram recordings from a protocol based on light adaptation followed by dark adaptation using sclerocorneal clip electrodes, which allows quick assembly and examination.

Development of a Delayed-Type Hypersensitivity (DTH) Model in the Cynomolgus Monkey.

Although a T-dependent antibody response (TDAR) assay is generally recommended as the first-line immune function assay in nonclinical immunotoxicity evaluation, second-line assays such as delayed-type hypersensitivity (DTH) to measure cell-mediated responses can provide helpful additional information. In this study, male Cynomolgus monkeys were injected intramuscularly either once or twice with 1 mg Keyhole Limpet Hemocyanin (KLH) or twice with a commercially available tetanus vaccine (40 IU tetanus toxoid + 0.06 mg aluminum hydroxide). All animals were subsequently challenged by intradermal injections of the same antigen or aluminum hydroxide after 4, 6 and 8 weeks. Clinical reactions at the injection sites were scored 24, 48 and 72 h post challenge. Skin biopsies were taken on completion of the observation period after each challenge for standard histological examination and immunolabeling using CD3 (T lymphocytes), CD19 (B lymphocytes) and CD68 (macrophages) antibodies. Tetanus toxoid induced stronger clinical reactions than KLH, whereas aluminum hydroxide induced no clinical reaction. Perivascular mononuclear cell infiltrates, a histopathological finding consistent with a DTH reaction, were seen after all challenges with tetanus toxoid or KLH, but not with aluminum hydroxide. Immunohistochemistry evidenced the presence of T lymphocytes and macrophages within these infiltrates. These results suggest that tetanus toxoid adjuvanted with aluminum hydroxide can induce a consistent DTH response for use as a model of cell-mediated response in Cynomolgus monkeys.

Lymphocyte subset analysis by flow cytometry in Beagle dogs

Materials and methods Lymphocyte subset analysis The analysis of blood lymphocyte subpopulations with a Navios 10-color flow cytometer was validated for linearity and precision, and absence of non-specific binding, using the PE-LSM 11.425 antibody for B cells, APC-LSM 8.358 for total T cells, PE-LSM 12.125 for CD4+ T cells, and FITC-LSM 1.140 for CD8+ T cells (BD Pharmingen). CD4+ T cells were also analyzed with the RPE-YKIX302.9 antibody, CD8+ T cells with the AF-647-YCATE55.9 antibody and CD3+ T cells with the FITCCA.17.2A12 antibody (AbD Serotec). Treatment Groups of 3 male and 3 female Beagle dogs were treated orally for 4 weeks with 2 mg/kg cyclophosphamide on 4 consecutive days each week, or 25 mg/kg cyclosporine daily, or the same volume of drinking water daily. Lymphocyte subsets were analyzed twice at a 7-day interval before the start of treatment, and then on days +11, +18 and +28 using the validated assay with the BD Pharmingen antibody combinations.

Compared immunotoxic effects of rituximab and cyclosporine in the Cynomolgus monkey

Introduction The immunotoxic potential of the anti-CD20 monoclonal antibody rituximab (RTX) compared to the reference immunosuppressive agent cyclosporine (CSP) has been investigated in the Cynomolgus monkey. The aim of this study was to evaluate the effects of RTX and CSP on anti-KLH IgM and IgG levels, lymphocyte subsets in the peripheral blood and cell suspensions from selected lymph nodes, and histological and immunohistochemical examination of the main lymphoid tissues.

Tolerance of two saline solutions by intravitreal route in the rabbit and minipig

Injections were performed with the animal under general anesthesia, xylazine (RompunTM) and ketamine (ImalgeneTM) in rabbits and medetomidine (DomitorTM) and a mixture of tiletamine and zolazepam (ZoletilTM) in minipigs. Local analgesia was achieved by corneal instillation of tetracaine 1% and mydriasis was obtained by topical instillation of the antimuscarinic agent tropicamide (MydriaticumTM). The eyes were fl ushed with povidone-iodine (BetadineTM 2%) for prophylactic local antisepsy.

Safety assessment of auditory function in rat: Ototoxicity and measurements of Auditory Brainstem Response

Evaluation of the auditory function is part of the functional assessment of the Central Nervous System in the follow-up studies mentioned in ICH S7A guideline for Safety Pharmacology Studies for Human Pharmaceuticals. The Auditory Brainstem Response globally tests functional integrity of the inner ear and auditory nerve, and their ability to properly transmit a sound signal along the whole pathway to the brainstem. The purpose of the present study was to validate the Auditory Brainstem Response (ABR) as a general evaluation of auditory loss in rats. Cisplatin was used to induce hearing function impairment as it is a well documented ototoxic compound which induces early outer hair cell death through free radical generations beginning at the basal turn of the cochlea.

Use of a functional observational battery in the assessment of safety pharmacology endpoints in general toxicology studies in conscious cynomolgus monkey

Assessment of a drug effect on the Central Nervous System (CNS) during the preclinical development of a new chemical entity is part of the ICH S7A guideline on Safety Pharmacology studies for human pharmaceuticals. This is commonly performed in dedicated safety pharmacology studies involving mainly rodents. However, for biotechnology derived products, integrated safety pharmacology assessment in regulatory toxicology studies are required (ICH S6, S9 and M3(R2)). These integrated safety pharmacology studies are generally focused on cardiovascular and respiratory functions, but also on the central nervous system in appropriate animal models. The non-human primate is one of the large animal models commonly used for the development of biologics. Therefore, the aim of this study was fi rst to validate a functional observational battery (FOB) as a neurobehavioral screening method that could be easily integrated in a 4-week toxicity study or greater. The second objective of the study was to defi ne and validate a tool to help in the interpretation of the results obtained from these tests. The validation of this model was performed using two reference compounds: D-amphetamine hemisulfate salt, a sympathomimetic molecule known to increase the CNS activity, and ketamine hydrochloride, an NMDA receptor antagonist known to induce a state referred to as “dissociative anaesthesia”.