Discovery of potent BET bromodomain 1 stereoselective inhibitors using DNA-encoded chemical library selections.

BRDT, BRD2, BRD3, and BRD4 comprise the bromodomain and extraterminal (BET) subfamily which contain two similar tandem bromodomains (BD1 and BD2). Selective BD1 inhibition phenocopies effects of tandem BET BD inhibition both in cancer models and, as we and others have reported of BRDT, in the testes. To find novel BET BD1 binders, we screened >4.5 billion molecules from our DNA-encoded chemical libraries with BRDT-BD1 or BRDT-BD2 proteins in parallel. A compound series enriched only by BRDT-BD1 was resynthesized off-DNA, uncovering a potent chiral compound, CDD-724, with >2,000-fold selectivity for inhibiting BRDT-BD1 over BRDT-BD2. CDD-724 stereoisomers exhibited remarkable differences in inhibiting BRDT-BD1, with the R-enantiomer (CDD-787) being 50-fold more potent than the S-enantiomer (CDD-786). From structure­activity relationship studies, we produced CDD-956, which maintained picomolar BET BD1 binding potency and high selectivity over BET BD2 proteins and had improved stability in human liver microsomes over CDD-787. BROMOscan profiling confirmed the excellent pan-BET BD1 affinity and selectivity of CDD-787 and CDD-956 on BD1 versus BD2 and all other BD-containing proteins. A cocrystal structure of BRDT-BD1 bound with CDD-956 was determined at 1.82 Å and revealed BRDT-BD1­specific contacts with the αZ and αC helices that explain the high affinity and selectivity for BET BD1 versus BD2. CDD-787 and CDD-956 maintain cellular BD1-selectivity in NanoBRET assays and show potent antileukemic activity in acute myeloid leukemia cell lines. These BET BD1-specific and highly potent compounds are structurally unique and provide insight into the importance of chirality to achieve BET specificity.

Sperm impairing microbial factor: potential candidate for male contraception.

BACKGROUND: Despite significant advances in contraceptive options for women, vasectomy and condoms are the only options available for male contraception. Due to this limitation, the burden of contraception resides on the shoulders of females only. Therefore, there is an urgent need to develop a safe, effective and reversible method of contraception for men. Amongst the alternative approaches, microbial derived products are gaining attention of the scientific world to combat unintended pregnancies. Earlier in our laboratory, sperm impairing microbial factor (Sperm immobilization factor) isolated from Staphylococcus aureus has shown excellent contraceptive efficacy in female mice. Keeping this in mind, the present study was carried out to exploit the sperm immobilization factor (SIF) as potential male contraceptive using vas deferens for administration in mouse model. METHODS: SIF (10, 50, 100 or 200 µg) was inoculated in the lumen of right vas deferens whereas the left vas deferens served as control. The mice were sacrificed at Day 3, 7, 14, 21, 30, 45, 60 and 90 after inoculation and the results in terms of change in body weight, seminal parameters, Tissue somatic indices (TSI), haematological parameters, serum level of testosterone, lipid peroxidation and histology were studied. In order to ratify the SIF induced azoospermia SIF (200 µg) was administered with different doses viz. 100, 200, 300, 400 or 500 µg of SIF binding receptor extracted from mouse spermatozoa. RESULTS: The weight profile studies of all the experimental groups showed no significant change in the initial and final body weight. In case of seminal parameters, the results revealed that right vas deferens treated with SIF showed azoospermia and with 200 µg of SIF it persisted up to 90 days. TSI of reproductive organs and non-reproductive organs showed no significant change in all the experimental groups. The haematological indices were found to be unaltered throughout the course of investigation however significant decrease in testosterone level was observed in the treated mice. The treatment also affected the oxidative status of the testis. Further, histological studies revealed hypospermatogenesis and late maturation arrest on treated side whereas the left side which served as control showed normal tissue histology. SIF induced azoospermia was ameliorated when administered with 400 µg of SIF binding receptor from mouse spermatozoa. CONCLUSION: SIF, when administered via intra vas deferens route, could lead to complete azoospermia. Therefore, it could be considered as a potential male contraceptive.

A biochemical and histological approach to study antifertility effects of methanol leaf extract of Asplenium dalhousiae Hook. in adult male rats.

The study was designed to investigate the antifertility properties of methanol leaf extract of Asplenium dalhousiae in adult male rats. Forty adult male Sprague Dawley rats (150 ± 10 g) divided into four groups (n = 10 animals/group) were administered with different doses (0, 50, 100, 150 mg/kg) of plant extract for 28 days. On day 29th, rats were decapitated, trunk blood and reproductive tissues were collected, and blood plasma was separated and stored until use for measuring reproductive hormones, while epididymis and testis were used for assessment of sperm parameters, oxidative stress status and morphometric analysis. Sperm motility, viability and sperm production rates were lowered in high dose treatment groups. Levels of catalase (CAT), sodium dismutase (SOD) and peroxidase (POD) decreased while stress biomarkers including reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) were increased among all treatment groups. Concentrations of plasma testosterone and follicle stimulating hormone (FSH) were decreased while levels of luteinizing hormone (LH) increased in high extract treated groups. Histological examination of testis showed disorganisation of seminiferous tubule and reduced spermatocytes number. The findings of current study revealed that methanol leaf extract of A. dalhousiae might induce antifertility effects via oxidative stress and interfering with testicular architecture leading to spermatogenic arrest.

Bioactive compounds in Pinang Yaki (Areca vestiaria) fruit as potential source of antifertility agent.

Fruits of Pinang Yaki (Areca vestiaria) are used by the people around Bogani Nani Wartabone as contraseption for men. Extracts from the fruit contain tannin, triterpenoid, flavonoid and saponin which are potential as bioactive compounds. This research aimed at exploring the fractions or bioactive compounds contained in the fruit. The extract was prepared by fractionation using hexane. The fractions were separated and analysed by gas chromatography mass spectrometry (GC-MS) technique. The fractions revealed the presence of five compounds. These compounds were identified by interpretation of mass spectra and comparing their retention time and covate indexes with those from literature. The five compounds are pentadecane, methyl-dodecanate, methyl-tetradecanoate, hexadecanoic acid and methyl-octadecanate.

Characterization of EPPIN's semenogelin I binding site: a contraceptive drug target.

Epididymal protease inhibitor (EPPIN) is found on the surface of spermatozoa and works as a central hub for a sperm surface protein complex (EPPIN protein complex [EPC]) that inhibits sperm motility on the binding of semenogelin I (SEMG1) during ejaculation. Here, we identify EPPIN's amino acids involved in the interactions within the EPC and demonstrate that EPPIN's sequence C102-P133 contains the major binding site for SEMG1. Within the same region, the sequence F117-P133 binds the EPC-associated protein lactotransferrin (LTF). We show that residues Cys102, Tyr107, and Phe117 in the EPPIN C-terminus are required for SEMG1 binding. Additionally, residues Tyr107 and Phe117 are critically involved in the interaction between EPPIN and LTF. Our findings demonstrate that EPPIN is a key player in the protein-protein interactions within the EPC. Target identification is an important step toward the development of a novel male contraceptive, and the functionality of EPPIN's residues Cys102, Tyr107, and Phe117 offers novel opportunities for contraceptive compounds that inhibit sperm motility by targeting this region of the molecule.

Update on Novel Hormonal and Nonhormonal Male Contraceptive Development.

BACKGROUND: The advent of new methods of male contraception would increase contraceptive options for men and women and advance male contraceptive agency. Pharmaceutical R&D for male contraception has been dormant since the 1990s. The Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) has supported a contraceptive development program since 1969 and supports most ongoing hormonal male contraceptive development. Nonhormonal methods are in earlier stages of development. CONTENT: Several hormonal male contraceptive agents have entered clinical trials. Novel single agent products being evaluated include dimethandrolone undecanoate, 11ß-methyl-nortestosterone dodecylcarbonate, and 7α-methyl-19-nortestosterone. A contraceptive efficacy trial of Nestorone®/testosterone gel is underway. Potential nonhormonal methods are at preclinical stages of development. Many nonhormonal male contraceptive targets that affect sperm production, sperm function, or sperm transport have been identified. SUMMARY: NICHD supports development of reversible male contraceptive agents. Other organizations such as the World Health Organization, the Population Council, and the Male Contraception Initiative are pursuing male contraceptive development, but industry involvement remains limited.

Influence of Echeveria gibbiflora DC aqueous crude extract on mouse sperm energy metabolism and calcium-dependent channels.

ETHNOPHARMACOLOGY RELEVANCE: In traditional Mexican medicine, Echeveria gibbiflora DC has been used as a vaginal post-coital rinse to prevent pregnancy. The aqueous crude extract (OBACE) induces sperm immobilization/agglutination and a hypotonic-like effect, likely attributed to the high concentration of calcium bis-(hydrogen-1-malate) hexahydrate [Ca2+ (C4H5O5)2•6H2O]. Likewise, OBACE impedes the increase of [Ca2+]i during capacitation. AIM OF THE STUDY: Evaluate the effect of OBACE on sperm energy metabolism and the underlying mechanism of action on sperm-specific channel. MATERIAL AND METHODS: In vitro, we quantified the mouse sperm immobilization effect and the antifertility potential of OBACE. The energetic metabolism status was also evaluated by assessing the ATP levels, general mitochondrial activity, mitochondrial membrane potential, and enzymatic activity of three key enzymes of energy metabolism. Furthermore, the effect of the ion efflux of Cl- and K+, as well as the pHi, were investigated in order to elucidate which channel is suitable to perform an in silico study. RESULTS: Total and progressive motility notably decreased, as did fertility rates. ATP levels, mitochondrial activity and membrane potential were reduced. Furthermore, the activities of the three enzymes decreased. Neither Cl- or K+ channels activities were affected at low concentrations of OBACE; nevertheless, pHi did not alkalinize. Finally, an in silico analysis was performed between the Catsper channel and calcium bis-(hydrogen-1-malate) hexahydrate, which showed a possible blockade of this sperm cation channel. CONCLUSION: The results were useful to elucidate the effect of OBACE and to propose it as a future male contraceptive.

Evaluation of Sperm Impairing Factor from Serratia marcescens as Male Contraceptive in Mouse Model.

The present study was carried out to assess the contraceptive efficacy of sperm agglutinating factor (SAF) isolated from Serratia marcescens, in male Balb/c mice. Mice were administered via an intratesticular route with different concentrations of SAF, viz., 10, 50, 100, 200, or 400 µg, in the right testis only which served as a test while the left side served as control except otherwise stated. Mice were sacrificed on day 3, 7, 14, 21, 30, 45, 60, and 90 after administration, and results in terms of change in body weight, seminal parameters, tissue somatic indices (TSI), hematological parameters, serum level of testosterone, lipid peroxidation, and histology were studied. The body weight and TSI remained unaffected in all the experimental groups. In case of seminal parameters, the right testis treated with 10 µg, 50 µg, 100 µg, 200 µg, or 400 µg of SAF showed azoospermia up to day 7, 14, 21, 45, and 90, respectively. The hematological indices, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were found to be unaltered when the group receiving SAF (test) was compared with the groups receiving phosphate buffer saline (control) in the right testis; however, the treatment had a negative effect on the serum level of testosterone. It also affected the oxidative status of the right testis. Furthermore, histological studies revealed hypospermatogenesis and alterations in the seminiferous tubules which included intraepithelial vacuolation and exfoliation in the right side as compared to the left side. Thus, the results suggest that SAF (400 µg) causes suppression of spermatogenesis, without causing apparent toxic effects.

Antifertility effects of gossypol and its impurities on male hamsters.

Recent papers reporting on the male antifertility effects of gossypol have been criticized due to the possible influence of impurities on efficacy and toxicity. Our laboratory has prepared purified gossypol and a fraction containing the total impurities. The effects of these two substances and impure gossypol on the fertility of male hamsters was evaluated. Only pure gossypol produced a decrease in fertility after six weeks of dosing. No effects on male fertility were observed in the other treatment groups. A full recovery of fertility in the pure gossypol group was observed four weeks after cessation of treatment. These data support the claim that gossypol is indeed an active antifertility agent for males. It is also clear that the impurities may have the ability to block or decrease the efficacy of gossypol.

PIP: Recent papers reporting on the male infertility effects of gossypol have been criticized due to the possible influence of impurities on efficacy and toxicity. Our laboratory has prepared purified gossypol and a fraction containing the total impurities. The effects of these 2 substances and impure gossypol on the fertility of male hamsters was evaluated. Only pure gossypol produced a decrease of fertility after 6 weeks of dosing. No effects on male fertility were observed in the other treatment groups. A full recovery of fertility in the pure gossypol group was observed 4 weeks after cessation of treatment. These data support the claim that gossypol is indeed an active antifertility agent for males. It is also clear that the impurities may have the ability to block or decrease the efficacy of gossypol.

A search for immunocontraceptive agent from marine sources--role of antispermatheca globulin of Telescopium telescopium on fertility regulation in male rat.

Polyclonal antisera were developed in rabbits against 'spermatheca', the reproductive gland of T. telescopium, a marine mollusc. The gland contains spermatozoa. Antisera and its IgG fraction (ASTG) indicated common antigenic determinants by immunodiffusion and had titer values 81920 and 1280 against crude antigen extract. Cycling female rats when exposed to passively immunized male rats with different doses of ASTG, had reduction in implantation sites and litter size. Females had pseudopregnancy when exposed to higher doses of immunized males and had normal cycle after 20 days in average. ASTG in male rats caused decrease in weight of the reproductive glands, alteration in sperm concentration, motility and morphology, formation of multinucleated giant cells and vacuoles leading to arrest of spermatogenesis and reduction in seminiferous tubular diameter. The effects were dose dependent with reversible infertility. The results indicate presence of a common antigenic determinants which cross-react with vertebrates and existence of common relation through phylogenetic evolution and their immune responses.

Antifertility investigations on the crude chloroform extract of Carica papaya Linn. seeds in male albino rats.

Crude chloroform extract of C. papaya seeds (5 mg/animal/day, po, for 20, 40 and 60 days) was investigated for contraceptive efficacy and related side effects in male albino rats. The crude extract reduced fertility to zero per cent by 40 to 60 days of treatment. Suppression of cauda epididymal sperm motility was the most pronounced effect of the drug administration. Scanning electron microscopic observations revealed treatment induced abnormalities in sperms. Cauda epididymal and testicular sperm counts decreased following treatment. Clinical parameters did not show any alterations. Results suggest that the contraceptive effects of chloroform extract of papaya seeds are mainly post-testicular in nature without influencing toxicological profile and libido of the animals.

Antifertility effect of Tinospora cordifolia (Willd.) stem extract in male rats.

Oral administration of 70% methanolic extract of T. cordifolia stem to male rats at the dose level of 100 mg/rat/day for 60 days did not cause body weight loss but decreased the weight of testes, epididymis, seminal vesicle and ventral prostate in a significant manner. Sperm motility as well as sperm density were reduced significantly which resulted in reduction of male fertility by 100%. The stem extract brought about an interference with spermatogenesis. The round spermatids were decreased by 73.12%. However, the population of preleptotene and pachytene spermatocytes were decreased by 47.60% and 52.85% respectively, followed by secondary spermatocytes (48.10%). Leydig cell nuclear area and mature Leydig cell numbers were significantly reduced when compared with controls. Serum testosterone levels showed significant reduction after Tinospora extract feeding. Seminiferous tubule diameter, Leydig cell nuclear area as well as cross sectional surface area of Sertoli cells were reduced significantly when compared to controls. Biochemical parameters i.e. protein, sialic acid, glycogen contents of testes decreased significantly. Seminal vesicular fructose also depleted whereas, testicular cholesterol was elevated significantly followed by a reduction in testosterone levels. These results suggested antifertility effects of the stem extract of T. cordifolia in male rats.

[16-Hydroxytriptolide, a new active diterpene isolated from Tripterygium wilford II].

A new diterpene triepoxide, 16-hydroxytriptolide was isolated from the root and leaves of Tripterygium wilfordii Hook.f. 16-Hydroxytriptolide was obtained as white cluster crystal, mp 232-233.5 degrees C. Its molecular formula is C20H24O7. The structure and stereochemistry of 16-hydroxytriptolide was established as L2 on the basis of spectral data (IR, MS, UV, 1H-NMR, 13C-NMR, 2d-NMR, NOE) and X-ray crystallographic analysis. In the pharmacologic screening, 16-hydroxytriptolide showed definite antiinflammatory actions and strong immunosuppressive and antifertile activities. In antiinflammatory action, its half effective dose (ED50) was 0.12 mg/kg with the model of croton oil induced ear swelling of mice. In immunosuppressive action, its ED50 was 0.05 mg/kg with the model of the formation of haemolysinantibody of mice. Its lowest effective dose (po) was 0.027mg/kg x 33d in antifertile action.

[Screening of active anti-inflammatory, immunosuppressive and antifertility components from Tripterygium wilfordii. IV. A comparison of the male antifertility activities of 7 diterpene lactone epoxide compounds].

The male antifertility activities under equal toxic dosages 1/10-1/60 LD50 and hemolysin antibody formation parameters under effective threshold dosages respectively of 7 diterpene lactone epoxide compounds extracted from Tripterygium wilfordii (TW) were assayed in mice. The results indicated that among the 7 compounds, 6 (T10, T4, T7, T8, T9 and L2) showed antifertility activities, the effective thresholds of T7, T8, T10 and L2 were below 1/40 LD50, those of T4 and T9 were below 1/60 LD50, while the other one (T11) did not. The above results reconfirm that the antifertility and immunosuppressive activities of all 6 active compounds are inseparable, but the formation of hemolysin antibodies was not inhibited under the total dosages which resulted in antifertility. The lowest dosages which were positive in terms of antifertility activity were 5-28 times lower than those for antiinflammatory activity and 5-12 times lower than those for immunosuppressive activity. And the total dosages necessary for effective immunosuppression were 0.9-1.8 times than the total dosages needed for effective antifertility. Finally, the value for the use of antifertility activity of these compounds, the influence of antifertility on antiinflammatory and immunosuppressive therapy, and the use of mouse models for screening of male antifertility activity are discussed.

Natural male contraceptive: phytochemical investigation and anti-spermatogenic activity of Pistia stratiotes Linn.

This work is an attempt to explore the anti-spermatogenic activity of Pistia stratiotes and to investigate it as a male contraceptive. The prepared extracts were screened for the presence of alkaloids, glycosides, steroids, flavonoids, saponin and phenolic compounds. To assess the anti-spermatogenic activity, mice were orally administered with the various extracts of P. stratiotes (dose: 100 and 200 mg/kg body weight/day, for 45 days) and the most active, ethanolic extract was subjected to the isolation of phytoconstituent responsible for the activity. Diethyl ether fraction of ethanolic extract was taken to isolate a saponin, sitosterol-3-O-[2,4-di-O-acetyl-6-O-stearyl-ß-D-glucopyranoside]. Anti-spermatogenic activity of the isolated saponin was evaluated at a dose of 50 mg/kg body weight/day, for 45 days. The treatment caused significant decrease (P < 0.01) in the weight of reproductive organs (testis, epididymis and seminal vesicle). The sperm count, sperm viability and serum testosterone levels were significantly lowered compared with that of the control group.

Male contraceptive efficacy of Ricinus communis L. extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Ricinus communis L. (Rc), of Euphorbiaceae family is a widespread plant in tropical regions and it is used in traditional medicines as an antifertility agent in India and different parts of the world. AIM OF THE STUDY: The aim of the present study is to revalidate the ethnobotanical knowledge by evaluating the activity of only crude stem bark extracts of Rc. In this study, effects of extracts on male contraceptive efficacy were experimented in vitro with human sperm sample. The work is based on primordial and contemporary therapeutic uses of this plant. MATERIALS AND METHODS: In this study, dose of petroleum ether extract, ethyl acetate extract, acetone extract and lyophilised aqueous extract of Rc were added to fresh human semen in 1:1 volumetric ratio. As the aqueous extract showed a promising result in 1:1 ratio, therefore, the Hypo-osmotic swelling test (HOS), Nuclear chromatin decondensation test (NCD) and Acrosomal status and function test (AFT) were also carried out with the aqueous extract of Rc. RESULTS: The sperm immobilisation effects of the extract appeared immediately in a dose-dependent manner when the samples were treated with four different extracts of this plant. At a concentration of 100mg/mL, 100% (p<0.001 and p<0.05) sperms lost their progressive motility. At a concentration of 300 mg/mL, 100% (p<0.001 and p<0.05) became immotile when treated with aqueous extract. There was 88% (p<0.001 and p<0.05) morphological deformities in sperm sample due the effect of aqueous extract when they were tested for HOS and 91% (p<0.05) sperms behaved against NCD as compared to control group. Also there was a distinct decline (p<0.05) in AFT with increase in dosage concentration. CONCLUSION: The findings of the study revealed that aqueous stem bark extract of the plant showed dose dependent loss of sperm motility by influencing the morphological deformation, blockage in nuclear envelope and distinct declination in acrosomal status of spermatozoa. This research, thus, opens up scope for future exploration of bark of the plant as commercial source of new male contraceptive.

Steroidogenic enzyme histochemistry in the testis of Sprague Dawley rats following the administration the water extracts from Carica papaya seed.

Water extracts from pawpaw seed have been reported to reversibly decrease the testicular weight and to suppress spermatogenesis, and fertility of Wistar rats. The reversible changes become evident, 30 - 45 days after the withdrawal of the extract. The possible effect of this extract on the activities of steroidogenic enzymes of the testis has not been investigated. Water extract of papaya seeds was administered to male Sprague Dawley rats ad libitum for 84 days. Following the discontinuation of the extracts, ten rats each were sacrificed on days 0, 10, 20 and 30 after the withdrawal. Their testes were quickly dissected out and frozen. Cryostat sections, 10µm thick were cut. These sections were used for immunohistochemical stains for side chain cleavage enzyme and aromatase, and for histochemical stains for 17-ß Hydroxysteroid dehydrogenase, 3-ß Hydroxysteroid dehydrogenase. We conclude that the water extract of papaya seed suppresses the activities of steroidogenic enzymes in the testis of Sprague Dawley rats, and that this may contribute to reversible suppression of spermatogenesis, a property that gives a possible male contraceptive potential.

Safety evaluation of long term oral treatment of methanol sub-fraction of the seeds of Carica papaya as a male contraceptive in albino rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The manuscript is one of the series of attempts in authenticating scientific documentation of the seeds of Carica papaya being traditionally used for contraception. AIMS OF THE STUDY: To establish safety of the methanol sub-fraction (MSF) of the seeds of Carica papaya as a male contraceptive following long term oral treatment. MATERIALS AND METHODS: MSF was administered orally to albino rats at multiples of contraceptive dose (CD) at 50 (1x), 100 (2x), 250 (5x) and 500 (10x)mg/kg body weight daily for 52 weeks. Body weight, organs weight, morbidity, mortality, clinical chemistry, sperm analysis, histopathology and serum testosterone were evaluated to assess the safety and contraceptive efficacy. RESULTS: MSF treatment at various dose regimens, daily for 52 weeks did not show significant changes in body weight, organs weight, food and water intake and pre-terminal deaths compared to those of control animals. Sperm count and viability in 50mg/kg body weight treated animals and the weight of epididymis, seminal vesicle and prostate of all the treated animals showed significant reduction compared to control. Cauda epididymal spermatozoa of 50mg/kg body weight treated animals were immotile. Azoospermia was observed in 100, 250 and 500 mg/kg body weight treated animals. Serum clinical parameters, serum testosterone and histopathology of vital organs were comparable to those of control animals. Histology of testis revealed adverse effects on the process of spermatogenesis, while the histology of epididymis, seminal vesicles and ventral prostate showed no changes compared to control. CONCLUSION: The long term daily oral administration of MSF affects sperm parameters without adverse side effects and is clinically safe as a male contraceptive.

Expression of a recombinant human sperm-agglutinating mini-antibody in tobacco (Nicotiana tabacum L.).

The murine monoclonal antibody (mAB) S19 recognizes an N-linked carbohydrate antigen designated sperm agglutination antigen-1 (SAGA1) located on the membrane protein CD52. This antigen is added to the sperm surface during epididymal maturation. Binding of the S19 mAB to SAGA-1 causes the rapid agglutination of sperm and blocks pre-fertilization events. Previous studies indicated that the S19 mAB may be a potential specific spermicidal agent (termed a spermistatic) capable of replacing current spermicidal products that contain harsh detergents with harmful side effects. The nucleotide sequences encoding the heavy (H) and light (L) chains of the S19 antibody were cloned. A chimeric gene was constructed using the nucleotide sequences encoding the variable regions of both the H and L chains, and this gene (scFv1 9) was expressed in transgenic tobacco (Nicotiana tabacum L.) to produce a recombinant anti-sperm antibody (RASA). Highest levels of RASA expression were observed in BY-2 plant cell suspension cultures and regenerated N. tabacum cv. Xanthi plants transformant in which the RASA coding sequences were expressed under the control of the Cauliflower Mosaic Virus 35S promoter containing a double-enhancer sequence (2X CaMV 35S). Subsequent modifications of the transgene including the addition of a 5'-untranslated sequence from the tobacco etch virus (TEV leader sequence), N-terminal fusion of the coding region with an endoplasmic reticulum targeting signal of patatin (pat) and C-terminal fusion with the endoplasmic reticulum retention signal peptide KDEL showed further enhancement of RASA expression. The plant-expressed RASA formed intrachain disulfide bonds and was primarily soluble in the cytoplasmic fraction of the cells. Introduction of a poly-histidine (6xHIS) tag in the recombinant RASA protein allowed for rapid purification of the recombinant protein using Ni-NTA chromatography. Optimization of scale-up production and purification of this plant-derived recombinant protein should provide large quantities of an inexpensive spermistatic plantibody.

Chiral separation of rac-Ornidazole and detection of the impurity of (R)-Ornidazole in (S)-Ornidazole injection and raw material.

(S)-Ornidazole is a subject of research as an antifertility agent in male animals at present. However, there seems to be no relative report on chiral separation for rac-Ornidazole, which has been used as an effective medicine for more than 30 years. In this article, the chiral separation of rac-Ornidazole on a Chiralcel OB-H column based on normal-phase high-performance liquid chromatography (NP-HPLC) is investigated and the methodology for detection of impurity of (R)-Ornidazole in (S)-Ornidazole injection and raw material is established. The novel mobile phase is utilized by mixing n-hexane, methanol and isopropyl alcohol (95:4:1, v/v/v) instead of the typical mobile phase of n-hexane and isopropyl alcohol, although the methanol, which offers a good resolution factor for the enantiomeric separation in this system, is not recommended on the Chiralcel OB-H column according to the instruction supplied by Daicel Chemical Ind., LTD (Japan).