Aggregation of biopharmaceuticals in human plasma and human serum

Analytical methods based on light microscopy, 90° light-scattering and surface plasmon resonance (SPR) allowed the characterization of aggregation that can occur when antibodies are mixed with human plasma. Light microscopy showed that aggregates formed when human plasma was mixed with 5% dextrose solutions of Herceptin^® (trastuzumab) or Avastin^® (bevacizumab) but not Remicade^® (infliximab). The aggregates in the plasma-Herceptin^®-5% dextrose solution were globular, size range 0.5–9 μm, with a mean diameter of 4 μm. The aggregates in the plasma-Avastin^®-5% dextrose samples had a mean size of 2 μm. No aggregation was observed when 0.9% NaCl solutions of Herceptin^®, Avastin^® and Remicade^® were mixed with human plasma. 90° light-scattering measurements showed that aggregates were still present 2.5 h after mixing Herceptin^® or Avastin^® with 5% dextrose-plasma solution. A SPR method was utilized to qualitatively describe the extent of interactions of surface-bound antibodies with undiluted human serum. Increased binding was observed in the case of Erbitux^® (cetuximab), whereas no binding was measured for Humira^® (adalimumab). The binding of sera components to 13 monoclonal antibodies was measured and correlated with known serum binding properties of the antibodies. The data presented in this paper provide analytical methods to study the intrinsic and buffer-dependent aggregation tendencies of therapeutic proteins when mixed with human plasma and serum.     

A jejunal pouch technique in the rhesus monkey and related clinical observations.

A simple, well-tolerated surgical technique is described for development of a jejunal pouch in Macaca mukitta as a model for the delivery of antiparkinsonian drugs at a site distal to the stomach.     

A novel nuclear protein in rat ventral prostate androgen-dependent and age-related change

A novel protein was found in the nuclei of rat ventral prostate. This protein has a molecular weight of about 21 kDa as measured by SDS-polyacrylamide gel electrophoresis. It showed a characteristic change between 3 and 84 weeks after birth in close association with the level of testosterone in the blood. After castration, the level of the 21-kDa protein decreased to $\text{1}\text{60}$ of normal in 7 days, but on daily injection of testosterone the level was restored to normal in 8 days and to twice the normal level in 14 days. Unlike H₁ and H₁⁰ histone and high mobility group proteins, the 21-kDa protein was not extracted with 5% HClO₄, but was partially extracted with 0.35 M NaCl. The 21-kDa protein was not found in kidney, liver, or brain, suggesting that it is specific to the ventral prostate.     

Antinociceptive, prolactin releasing and intestinal motility inhibiting activities of dermorphin and analogues after subcutaneous administration in the rat

A series of analogues and shorter homologues of dermorphin (DM), a frog skin heptapeptide with potent morphine-like activity, have been assayed in the rat after subcutaneous (SC) administration at the screening dose of 4 mg/kg. The effects taken into account are: analgesia (tail-pinch test), stimulation of prolactin (PRL) secretion, and inhibition of gastro-intestinal (GI) motility (charcoal meal transit). Effective doses were calculated for the most active compounds. The potency of DM (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) in the different tests was: tail-pinch: ED50 = 0.83 mg/kg; PRL release: ED100 = 0.3 mg/kg; inhibition of GI motility: ED30 = 1.8 mg/kg.     

Safety evaluation of formulations containing carboxymethyl derivatives of starch and chitosan in albino rats

Two composite formulations, based on carboxymethyl derivatives of starch (formulation I) and chitosan (formulation II), used in the preparation of coating formulations to enhance post harvest shelf-life of fruits and vegetables, were evaluated for safety by single dose dietary (formulation I, coating on feed pellet-1.3% w/w and formulation II, coating on feed pellet-1% w/w) and oral (1 ml, 2% aqueous solution) administration to albino rats. Experiment was carried out for 4 weeks. No significant changes were observed in gain in weekly body weight, weight of vital organs and in parameters of haematology and histopathology among experimental groups, thus indicating safety (and non-toxicity) of the coating formulations.     

Formation of 5α-dihydrotestosterone from 5α-androstane-3α,17β-diol in prostate cancer LAPC-4 cells – Identifying inhibitors of non-classical pathways producing the most potent androgen

5α-Dihydrotestosterone (5α-DHT) possesses a great affinity for the androgen receptor (AR), and its binding to AR promotes the proliferation of prostate cancer (PC) cells in androgen-dependent PC. Primarily synthesized from testosterone (T) in testis, 5α-DHT could also be produced from 5α-androstane-3α,17β-diol (3α-diol), an almost inactive androgen, following non-classical pathways. We reported the chemical synthesis of non-commercially available [4-¹⁴C]-3α-diol from [4-¹⁴C]-T, and the development of a biological assay to identify inhibitors of the 5α-DHT formation from radiolabeled 3α-diol in LAPC-4 cell PC model. We measured the inhibitory potency of 5α-androstane derivatives against the formation of 5α-DHT, and inhibition curves were obtained for the most potent compounds (IC₅₀ = 1.2–14.1 μM). The most potent inhibitor 25 (IC₅₀ = 1.2 μM) possesses a 4-(4-CF₃-3-CH₃O-benzyl)piperazinyl methyl side chain at C3β and 17β-OH/17α-CCH functionalities at C17 of a 5α-androstane core.     

Pharmacological intervention of biosynthesized Nigella sativa silver nanoparticles against hexavalent chromium induced toxicity in male albino mice

Hexavalent chromium, toxic heavy metal, among the top-rated environmental contaminants, is declared a potent endocrine disruptor in humans and animals. The present study was planned to find harmful effects on the reproductive system caused by Cr (VI) and the ameliorative effect of Nigella sativa and Nigella sativa-mediated AgNP on male mice (Mus musculus). In the present study, known infertility medicine, clomiphene citrate is also used as a positive control. The main objective of the present study was to assess the ameliorative potential of oral administration of a dose of 50 mg/kg BW clomiphene citrate (control), AgNP via chemical synthesis, Nigella sativa seed extract, and Nigella sativa-mediated AgNP against the Cr (VI) at the dose of 1.5 mg/kg BW from K₂Cr₂O₇ orally induced toxicity over eight weeks on the reproductive performance of male albino mice. Nigella sativa mediated AgNPs were characterized by UV, SEM, FTIR, and XRD. The histological analysis, smear study, antioxidant capacity test, and hormone analysis were conducted by blood samples of albino mice. Cr exposed groups showed a significant decrease in sperm head breadth (5.29 ± 0.54 µ) and length (19.54 ± 1.18 µ), middle piece length, tail length, LH (1.65 ± 0.15 ng/mL), testosterone (2.63 ± 0.29 ng/mL), SOD (61.40 ± 2.48 mmol/mL), CAT (87.40 ± 6.01 mmol/mL), GSH (1.54 ± 0.09 µmol/mL), and no of spermatogonia (1.22 ± 0.25), and spermatocytes (2.33 ± 0.943). However, FSH level (160.00 ± 4.98 ng/mL), seminiferous tubule CSA (1094.69 ± 49.76 mm²), size of spermatogonia (41.30 ± 1.24 µ), and spermatocytes (26.07 ± 1.34 µ) were significantly increased. Administration of Nigella sativa and Nigella sativa-mediated AgNPs reduced the toxicity.     

The effects of the environmental antiandrogen vinclozolin on the induction of granulosa cell apoptosis during follicular atresia in pigs

The aim of this study was to investigate whether the androgens testosterone and dihydrotestosterone (DHT) and the antiandrogenic fungicide vinclozolin (Vnz) exert proapoptotic effects on porcine granulosa cells (GCs), and to examine the roles of these compounds in follicular atresia. Granulosa cells isolated from pig follicles were cultured for 24 hours, and then exposed to 0.1 μM testosterone, 0.1 μM DHT, 14 μM Vnz, or the equivalent concentrations of testosterone and Vnz or DHT and Vnz for a further 24 hours. Apoptosis and necrosis of the GCs were determined via Hoechst staining and flow cytometry analyses of annexin V-stained cells. Whole porcine follicles were also exposed to the same compounds and combinations of compounds for 24 hours. The sections were stained with hematoxylin and eosin for morphologic assessments, and a Terminal deoxynucleotidyl Transferase Biotyn-dUTP Nick-End Labeling (TUNEL) assay was performed to determine the number of apoptotic cells. The progesterone and estradiol concentrations secreted into the culture media by isolated GCs and follicles were also measured. Exposure to the androgens resulted in an increased number of apoptotic GCs both in vitro and in the organotypic model. Vinclozolin exposure increased and decreased the number of necrotic and apoptotic GCs, respectively. Furthermore, compared with control follicles, those exposed to testosterone, DHT, or Vnz displayed enhanced atresia, and coadministration of Vnz attenuated the promotive effect of these androgens on atresia. Estradiol secretion was stimulated by the combination of testosterone and Vnz, whereas exposure to Vnz alone reduced it. Progesterone production declined after the combined addition of androgens and the antiandrogen. In summary, Vnz caused massive necrosis of GCs in vitro and induced apoptosis of GCs in whole follicles. The androgens testosterone and DHT enhanced these effects. The results presented here suggest that selective destruction of porcine follicles is a serious consequence of exposure to Vnz, and may lead to premature ovarian failure in affected animals.