Investigation of maternal and fetal exposure to an IgG2 monoclonal antibody following biweekly intravaginal administration to cynomolgus monkeys throughout pregnancy

To assess the potential for male-mediated drug transfer to their female partner and/or developing conceptus, vaginal uptake of a monoclonal antibody (mAb) biotherapeutic was assessed in cynomolgus monkeys. A human IgG2 mAb (IgG2X; bound human and cynomolgus monkey neonatal Fc-receptor, FcRn, with similar high affinity) was administered intravaginally (IvG; 100 mg/dose) to 5 pregnant cynomolgus monkeys biweekly from gestation day (gd) 21 to gd133. In all maternal samples collected before gd119, IgG2X plasma concentrations were below the limit of quantification (BLQ; <25 ng/mL). After dosing on gd119 and 133, maternal IgG2X plasma concentrations remained BLQ in 3/5 monkeys and were very low in 2/5 (up to 116 ng/mL; ∼0.01% of the IvG dose). IgG2X was BLQ in all fetal plasma samples. These data indicate that male-mediated mAb drug transfer via seminal fluid does not present a health risk to the female partner and is not bioavailable to the developing conceptus.     

Assessment of placental transfer and the effect on embryo-fetal development of a humanized monoclonal antibody targeting lymphotoxin-alpha in non-human primates

An enhanced embryo-fetal development study was conducted in cynomolgus monkeys using pateclizumab, a humanized IgG1 monoclonal antibody (mAb) targeting lymphotoxin-alpha. Pateclizumab administration between gestation days (GD) 20 and 132 did not induce maternal or developmental toxicities. The ratio of fetal-to-maternal serum concentration of pateclizumab was 0.73% on GD 50 and 61% by GD 139. Decreased fetal inguinal lymph node-to-body weight ratio was present in the high-dose group without microscopic abnormalities, a change attributable to inhibition of lymphocyte recruitment, which is a pharmacologic effect of pateclizumab during late lymph node development. The effect was observed in inguinal but not submandibular or mesenteric lymph nodes; this was attributed to differential susceptibility related to sequential lymph node development. Placental transfer of therapeutic IgG1 antibodies; thus, begins during the first trimester in non-human primates. Depending on the potency and dose levels administered, antibody levels in the fetus may be pharmacologically or toxicologically relevant.     

Application of high-frequency rheology measurements for analyzing protein-protein interactions in high protein concentration solutions using a model monoclonal antibody (IgG2)

The purpose of this work was to explore the utilization of high-frequency rheology analysis for assessing protein-protein interactions in high protein concentration solutions. Rheology analysis of a model monoclonal immunoglobulin G2 solutions was conducted on indigenously developed ultrasonic shear rheometer at frequency of 10 MHz. Solutions at pH 9.0 behaved as most viscous and viscoelastic whereas those at pH 4.0 and 5.4 exhibited lower viscosity and viscoelasticity, respectively. Intrinsic viscosity, hydrophobicity, and conformational analysis could not account for the rheological behavior of IgG2 solutions. Zeta potential and light scattering measurements showed the significance of electroviscous and specific protein-protein interactions in governing rheology of IgG2 solutions. Specific protein-protein interactions resulted in formation of reversible higher order species of monomer. Solution storage modulus (G'), and not loss modulus or complex viscosity, was the more reliable parameter for predicting protein-protein interactions. Predictions about the nature of protein-protein interactions made on the basis of solution G' were found to be consistent with observed effect of pH and ionic strength on zeta potential and scattered intensity of IgG2 solutions. Results demonstrated the potential of high-frequency storage modulus measurements for understanding behavior of proteins in solutions and predicting the nature of protein-protein interactions.     

Effect of in utero exposure to di-(2-ethylhexyl)phthalate: Distribution in the rat fetus and testosterone production by rat fetal testis in culture

DEHP is known to cause reproductive toxicity in rats, particularly during the neonatal period. Pregnant and brood rats were treated by gavage with 750 mg/kg b.w./day DEHP starting on GD14 within PND4. Two hours after ¹⁴C-DEHP administration on GD15, GD18, GD21 and PND4, the radioactivity content was measured in the dams blood and in the liver, gonads and carcass of the offspring. The radioactivity concentration recovered in the fetuses was one or two order of magnitude lower than the concentration found in the dam plasma. A low proportion of radioactivity was present in fetal gonads, ca. 2%, 5% and 3.6% on GD18, GD21 and PND4, respectively. The effect on testosterone production of DEHP and its metabolites (MEHP, metabolites VI and IX) was assessed in fetal testis cultures using a dose-range which included the maximal exposure observed in vivo. None of the compounds affected testosterone production. Thus, DEHP and/or its metabolites appear to cross the placental barrier, reach the fetal gonads. In vitro, neither DEHP nor its main metabolites decreased the testosterone production.     

Radiolabelled monoclonal antibodies (McAb): An alternate approach to the conventional methods for the assessment of cardiomyocyte damage in an experimental brain-death pig model

The present study was carried out to determine the possible use of cTn-I in the cardiac myofibrillar architecture, as a potential target forin vivo radioimmunodetection of cardiac damage in a brain death pig model. Radioiodiantion of the anti-cTn-I 5F4 McAb was carried out by lactoperoxidase method. The percentage iodine incorporation achieved was 70∼75%. The radioiodinated McAbs were purified on Sephadex G-25 column and characterised by Paper chromatography, Phast Gel electrophoresis and electroimmunoblotting. Radioiodinated anti-cTn-I 5F4 McAbs were employed alongside Pyrophosphate (Tc^99m-PPi) and Thallium²⁰¹ chloride (Tl²⁰¹) in 24 landrace pigs (brain-dead=18 & sham-operated=6). The percentage cardiac uptake of the radiolabelled antibody injected dose was significantly higher in the brain dead animals (0.196%) as compared to that of sham-operated animals (0.11%). Specificin vivo localization of radiolabelled McAbs in the infarcted cardiac tissue was confirmed by computer-aided reconstruction of 3-D images of the isolated heart. The preliminary results of the study revealed preferential uptake of radiolabelled antibody at the site of myocyte damage resulting from artificially induced brain death.     

Percutaneous absorption and distribution of organophosphates (chlorpyrifos and dichlorvos) following dermal exposure and decontamination scenarios using in vitro human skin model

To date, there has been little research investigating low-level human exposure to chemicals, and so the aim of this study was to examine the percutaneous penetration of organophosphates (dichlorvos and chlorpyrifos) using low-level exposure scenarios in vitro. Dermal absorption of chlorpyrifos applied in different vehicles was measured at 0, 4, 8 and 24 h, after dose application for 4 and 24 h exposure (finite dose, 500 ng/cm²) in isopropanol (IPA), isopropyl myristate (IPM) and propylene glycol (PG). Dichlorvos was applied to the skin for 24 h (infinite dose, 1 mg/cm² and 10 mg/cm²; finite dose, 5 μg/cm²) using the same vehicles. Human skin was mounted in flow through diffusion cells with minimum essential medium eagle pH 7.4 (supplemented with 2% BSA) as receptor fluid. Following exposure, the skin surface dose was removed by tissue swabbing, the stratum corneum removed by sequential tape stripping, and the skin digested prior to scintillation counting (chlorpyrifos), or GC/MS analysis (dichlorvos).     

Antitumor effect of212bismuth-conjugated anti-IL-2R monoclonal antibody(2E4) on a IL-2 receptor positive tumor EL4J3.4

The antitumor effects of the 2E4 and anti-Tac, monoclonal antibodies directed to IL-2 receptor (IL-2R), conjugated with α-particle emitting radionuclide bismuth-212, were compared. The²¹²Bi-2E4 demonstrated specific cytotoxicity to EL4J3.4, a IL-2R⁺ cell line, than to EL4J, a IL-2R⁻ cell line in thymidine incorporation assay.²¹²Bi-2E4 exerted the maximal antitumor effect in that % T/C in C57BL/6 mice implanted with EL4J3.4 ascitic tumor was 331% at the concentration of 50 μCi, while that of²¹²Bi-anti-Tac was 258% at 100 μCi.     

Assessment of herbicide effects on algal production in the Kokai River (Japan) using a model stream and Selenastrum bioassay

The effects of herbicides on algal production in the Kokai River were assessed using a Selenastrum growth inhibition test and by an investigation of the periphyton formed in a channel carrying river water. Herbicides in the water were analysed at the same intervals as the algal growth test (three times per week) from April to August 1992. Growth of Selenastrum in the river water samples was inhibited severely in mid-May, mostly by the joint effects of butachlor and pretilachlor and from early to late June mostly by simetryn alone. The influence of six other kinds of herbicide was low or not detectable. Selenastrum growth ratios (SGR) in the river water samples were correlated with fluctuations in SG-IP (Selenastrum growth inhibition potential), the sum of the growth inhibition ratios by the respective herbicides at the concentrations detected in the river. However, the expected growth inhibition (SG-IP) was usually larger than the observed value (SGR); the reasons for this are discussed.The rate of accumulation of periphyton, which formed on a substrate in the channel, decreased from early May and stayed at relatively low levels until early June. The low accumulation rate from mid-June to mid-July, when growth of Selenastrum had almost recovered, can be attributed to grazing by invertebrates. These results suggested that algal production in the Kokai River is affected by the single or joint action of herbicides, at least in the period from early May to early June.     

Effects of vascular endothelial growth factor (VEGF)A and VEGFB gene transfer on vascular reserve in a conscious rabbit hindlimb ischaemia model

1. As a result of the ageing population, there are increasing numbers of patients with severe peripheral vascular occlusive disease associated with intermittent claudication (pain on walking) and decreased exercise tolerance. There is a great clinical need for pharmacological treatments that may stimulate collateral blood vessel growth, increase vascularity and improve skeletal muscle function. 2. Therapeutic angiogenesis using growth factors such as vascular endothelial growth factor (VEGF) has been used to improve collateral artery development in myocardial or skeletal muscle ischaemia. The broad aims of the work briefly summarized here were to compare the effects of VEGFA165 and VEGFB167 (500 micro g, i.m., gene transfer) on calf blood pressure ratio and reactive hyperaemia in a chronic rabbit preparation with unilateral limb ischaemia. 3. Unilateral femoral artery ligation caused an immediate deficit (compared with the contralateral limb) of 72% in calf systolic blood pressure. There were improvements 14 days after ligation with VEGFA and VEGFB treatments compared with the vehicle control plasmid treatment, but a deficit remained of some 32%. 4. Reactive hyperaemic responses were significantly attenuated 7 days after ligation in the vehicle and VEGFA treatment groups. On day 14, this loss of vascular reserve was restored in the VEGFA group, but remained in the vehicle group (-30%). In VEGFB-treated animals, there was no deficit in reserve 7-14 days post-ligation. 5. In conclusion, there is considerable value in the serial measurements of calf blood pressure ratio and reactive hyperaemia in the rabbit unilateral hindlimb ischaemia model. Gene transfer of either VEGFA or VEGFB allowed significant improvements in these indices compared with vehicle but, at 14 days post-ligation, large deficits still remained. Studies extending this experimental period are in progress.     

The high sensitivity of the rabbit to the teratogenic effects of 13-cis-retinoic acid (isotretinoin) is a consequence of prolonged exposure of the embryo to 13-cis-retinoic acid and 13-cis-4-oxo-retinoic acid,and not of isomerization to all-trans-retinoic acid

Previous studies suggested that the rabbit is much more susceptible to the teratogenic action of 13-cis-retinoic acid (13-cis-RA) than the mouse or the rat, while the teratogenicity of all-trans-RA was comparable in these species. In the present study we investigated if pharmacokinetics can explain these species- and structure-related differences. The embryotoxic and teratogenic potential of all-trans-retinoid acid (all-trans-RA) and 13-cis-RA were evaluated in the Swiss hare rabbit after oral administration of daily doses of the two drugs throughout organogenesis, from gestation day (GD) 6 to 18 (plug day=GD 0). All-trans-RA was given at dose levels of 0.7, 2 or 6 mg/kg body weight per day and 13-cis-RA at 3, 7.5 or 10 mg/kg per day. The doses needed to elicit a minimum teratogenic response were found to be 6 mg/kg per day for all-trans-RA and 10 mg/kg per day for 13-cis-RA. Using these doses, transplacental pharmacokinetics of all-trans- and 13-cis-RA were performed. Pregnant rabbits were treated once daily from GD 7 to 12 and plasma and embryo samples were collected for HPLC analysis at various time intervals after the final dose. The main plasma metabolites of all-trans-and 13-cis-RA were all-trans-β-glucuronide (all-trans-RAG) and 13-cis-4-oxo-RA, respectively. The elimination of 13-cis-RA and its metabolites from maternal plasma were much slower than of all-trans-RA resulting in accumulation of the 13-cis-isomers in plasma. Marked differences in the placental transfer of the two drugs and their metabolites were observed. All-trans-RA and all-trans-4-oxo-RA were efficiently transferred to the rabbit embryo, reaching concentrations similar to the plasma levels. On the contrary, the 13-cis-isomers reached the embryo to a lesser extent. Despite its limited placental transfer, a considerable embryonic exposure to 13-cis-RA and 13-cis-4-oxo-RA was noticed after treatment with isotretinoin, as indicated by their area-under-the-concentration-time-curve (AUC) values in the embryo, which were in the same range as the corresponding AUC value of all-trans-RA after treatment with the all-trans-isomer. Our results suggest that the high sensitivity of the rabbit to the teratogenic effects of 13-cis-RA can be attributed mainly to the 13-cis-isomers and not to isomerization to all-trans-RA. The significant exposure of the rabbit embryo to 13-cis-RA and its 4-oxo metabolite is a result of their very slow excretion rates from the maternal organism. Furthermore, this study supports the view that embryonic AUC values should be considered as the most suitable pharmacokinetic correlate to retinoid induced teratogenesis. Results of the pharmacokinetic study have been presented in part at the 20th Conference of the European Teratology Society, August 31–September 3, 1992, Würzburg, Germany, and have appeared in a short abstract (Tzimas et al. 1992) Supported by a grant from the Deutscher Akademischer Austauschdienst