Osmond J. D’Cruz

Gel-microemulsions as vaginal spermicides and intravaginal drug delivery vehicles

There is a need for novel formulations to improve the bioavailability through the vaginal/rectal mucosa of microbicidal drug substances against sexually transmitted diseases. In addition, there is a need for more effective and less toxic vaginal spermicides. Here we review our recent discovery of novel gel-microemulsions (GM) as nontoxic, dual-function intravaginal spermicides, which can be used as delivery vehicles for lipophilic drug substances targeting sexually transmitted pathogens. We describe the formulation and biologic properties of 2 novel, submicron-particle-size GMs, GM-4 and GM-144, which were prepared from commonly available pharmaceutical excipients. These GMs comprising oil-in-water microemulsion and polymeric hydrogels were designed to solubilize lipophilic antiviral/antimicrobial agents and exhibited rapid spermicidal activity in human semen. Preclinical studies comparing the in vivo contraceptive efficacy of GM-4 and GM-144 versus nonoxynol-9-based detergent spermicide (Gynol II) in the rigorous rabbit model confirmed the potent contraceptive activity of these GMs. Unlike nonoxynol-9, repeated intravaginal applications of GM-4 and GM-144 in the rabbit vaginal irritation test were not associated with local inflammation or damage of the vaginal mucosa or epithelium. Furthermore, in short-term toxicity studies performed in mice, repetitive intravaginal application of spermicidal GM-4 and GM-144 for up to 13 weeks was not associated with any local, systemic, or reproductive toxicity. Spermicidal GMs have unprecedented potential as dual function microbicidal contraceptives to improve vaginal bioavailability of poorly soluble antimicrobial agents without causing significant vaginal damage.

Comparison of the indirect immunobead, radiolabeled, and immunofluorescence assays for immunoglobulin G serum antibodies to human sperm.

The relative sensitivities of the indirect immunobead test, the indirect flo cytometric immunofluorescence assay, and an indirect radiolabeled antiglobulin assay were compared. Eighteen immunobead test positive sera and 18 negative sera were used as the standard for the other two assays. Of the 18 positive sera, 14 (77%) and 5 (27%) were positive in the immunofluorescence assay and the radiolabeled antiglobulin assay, respectively. Four (22%) of the low titer immunobead test positive sera were negative by both the immunofluorescence assay and the radiolabeled antiglobulin assay. However, there was a significant positive correlation between the results of the immunofluorescence assay and the radiolabeled antiglobulin assay (r = 0.73) and between the results of the radiolabeled antiglobulin assay and the titer of the immunobead test (r = 0.82). The use of an unselected sperm population in the radiolabeled antiglobulin assay and the classical indirect immunofluorescence method using methanol-fixed sperm gave false-positive results in the radiolabeled antiglobulin assay and the immunofluorescence assay. These results suggested that immunoglobulin G antisperm antibody positive sera may be reactive both to sperm surface and internalized sperm antigens.

Antibodies to carbonic anhydrase in endometriosis: prevalence, specificity, and relationship to clinical and laboratory parameters *

OBJECTIVE To investigate the presence and clinical association of serum autoantibodies to carbonic anhydrase (CA) in women with and without endometriosis. DESIGN Sera were tested in an ELISA against human and bovine CAI and/or CAII isoenzymes and by Western immunoblotting of trypsin-digested fragments of human CAII as antigens. The ELISA positivity was defined as mean + 2 SD of 100 control sera. Positive sera also were tested for the presence of antiendometrial antibodies and antinuclear antibodies (ANA) by indirect immunofluorescence assays (IFA) on endometrial (ECC) and HEp-2 cells, antibodies to single-stranded (ss) and double-stranded (ds) DNA by the Farr-type RIA and Crithidia IFA, and extractable nuclear antigens (Sm, nRNP, Ro, and La) by an ELISA. PATIENTS Sera from 319 patients with laparoscopic diagnosed pelvic endometriosis (100 stage I, 95 stage II, 67 stage III, and 57 stage IV), 100 with other gynecologic disorders, and 100 control women were used. RESULTS In the ELISA, 113 of 319 (35.4%) endometriosis sera had elevated immunoglobulin G antibodies against nondenatured CA isoenzymes. The reactivity of sera from the endometriosis group was significantly higher (35%) in all four subgroups of patients than each of the nonendometriosis sera (< 12% and < 6%, respectively). No stage-dependent variation of an autoantibody pattern was evident. However, anti-CA autoantibodies were present in 66.3% of women with endometriosis-associated infertility. The frequency of anti-CA autoantibodies was significantly higher (by 51.7%) in women with antiendometrial antibodies detectable by IFA. In addition, in sera positive for anti-CA antibodies, the frequency of ANA also was increased (20/113 [17.6%]) with titers of 1:40 to 1:1,080. The ANA-positive sera were negative for anti-ssDNA, anti-dsDNA, anti-Sm, anti-nRNP, and anti-La. However, three sera were positive for anti-Ro antibodies. Immunoblotting study of autoantibody reactivity with trypsin-digested subfragments of human CAII revealed consistent immunoreactivity with 14 to 6.2-kd range CAII peptides. CONCLUSIONS [1] A subgroup of patients with endometriosis have autoantibodies directed to native and linear epitopes of the CA protein. [2] Prevalence of anti-CA antibodies was associated with antiendometrial antibodies and ANA. [3] Anti-CA antibodies were associated with a higher predictive value of the disease when all patient subgroups were considered together.

The effect of fixatives and/or air-drying on the plasma and acrosomal membranes of human sperm

It has been hypothesized that some fixatives and conditions of slide preparation expose internal sperm antigens and thus are not suitable for demonstrating surface-specific antigens by immunocytochemical assays. This study examined the ability of five fixatives (glutaraldehyde, acetone, methanol, paraformaldehyde, and periodate-lysine-paraformaldehyde [PLP]) and two conditions of slide preparation (air-drying or maintaining sperm in a liquid phase) to maintain the integrity of human spermatozoal membranes at the ultrastructural level as monitored by transmission electron microscopy. Regardless of the fixative employed, air-drying was detrimental to plasma and acrosomal membrane integrity. Acetone and methanol completely disrupted the plasma and acrosomal membranes over the entire sperm surface whether air-drying or liquid phase conditions were employed. Fixation with glutaraldehyde and paraformaldehyde (to a lesser degree) maintained the morphologic integrity of acrosomal and plasma membranes provided the sperm were maintained in a liquid phase. However, glutaraldehyde and paraformaldehyde fixation increased the postfixation binding of immunoglobulins to the sperm surface. We concluded that immunocytochemistry at the light microscopy level may imply erroneous interpretations regarding antigen/antibody interactions on the sperm plasma membrane surface unless care is taken to verify that the antibodies of interest are binding to the antigenically intact plasma or acrosomal membrane.

Flow cytometric quantitation of the expression of membrane cofactor protein as a marker for the human sperm acrosome reaction

A mAb (TRA-2-10, IgG1) to an embryonal carcinoma cell line (2102ep) that recognizes an antigen termed membrane cofactor protein (CD 46, TLX antigen) binds to human sperm after chemical induction of acrosomal loss by Ca2+ ionophore. An indirect immunofluorescence assay was developed in which sperm membrane cofactor protein was detected by flow cytometry on acrosome-reacted living human sperm. The expression of the membrane cofactor protein antigen on acrosome-reacted sperm may represent a marker that can be used in a rapid, quantitative, and reproducible flow cytometric assay for the evaluation of human sperm acrosomal loss.

Pokeweed antiviral protein: a potential nonspermicidal prophylactic antiviral agent.

OBJECTIVE To investigate the effects of pokeweed antiviral protein (PAP), a 29-kDa anti-human immunodeficiency virus (HIV) protein purified from the leaves of Phytolacca americana, on human sperm function. DESIGN Prospective, controlled study. SETTING Reproductive biology department. PATIENT(S) Seven sperm donors. INTERVENTION(S) Human sperm and female genital tract epithelial cells were exposed to PAP ranging in concentration from 1 to 1,000 microg/mL. MAIN OUTCOME MEASURES Effect of PAP on sperm motility, kinematics, and sperm penetration through bovine mucus, as well as binding, penetration, and fusion of zona-free hamster eggs. RESULTS Exposing human sperm to PAP (IC(50) p24 = 14 +/- 2 nM) did not affect sperm motility and kinematics over a dose range of 1 to 1,000 microg/mL. Treating sperm with either 100 or 1,000 microg/mL of PAP had no effect on cervical mucus penetrability, nor did it affect sperm binding, penetration, and fusion of zona-free hamster eggs. PAP was noncytotoxic to genital-tract epithelial cells. CONCLUSIONS The broad-spectrum antiviral agent PAP was nontoxic to human sperm and female genital tract epithelial cells even at a concentration 2,000 times higher than its IC(50) value against HIV-1. PAP has particular clinical usefulness both as a nonspermicidal intravaginal microbicide and as a prophylactic antiviral agent that can inactivate infective viruses and virus-infected cells in semen before assisted reproductive technology procedures are undertaken.

Preclinical Evaluation of a Dual-Acting Microbicidal Prodrug WHI-07 in Combination with Vanadocene Dithiocarbamate in the Female Reproductive Tract of Rabbit, Pig, and Cat

The mucosal safety of the combination antiretroviral spermicide, WHI-07 [5-bromo-6-methoxy-5,6-dihydro-3′-azidothymidine-5′-(p-bromophenyl)-methoxy alaninyl phosphate] and vanadocene dithiocarbamate (VDDTC), was evaluated in 3 different animal models. Twenty-seven NZW rabbits in four subgroups were exposed intravaginally to a gel-microemulsion (GM) with and without three dose levels of WHI-07 plus VD-DTC (0.5 + 0.06%, 1.0 + 0.12% and 2.0 + 0.25%) or 4% nonoxynol-9 (N-9; Conceptrol®) for 14 consecutive days. Ten nonestrus gilts (Duroc) in three subgroups received either a single or daily intravaginal application of GM with and without 2.0% WHI-07 plus 0.25% VDDTC or 2.0% benzalkonium chloride (BZK)-containing gel for 6 and 4 consecutive days, respectively. Five cats received a single intravaginal application of GM incorporating 2.0% WHI-07 plus 0.25% VDDTC. Genital tract histopathology was performed in the pig and rabbit at the end of dosing period but after 18 weeks post-dosing in the cat. Porcine cervicovaginal lavage (CVL) fluid was obtained for up to 72 hours after a single exposure and changes in the levels of inflammatory cytokines (IL-1β, IL-8, IFN-γ, and TNF-α) were quantitated by a multiplexed chemiluminescence-based immunoassay. Rabbit vaginal tissues were evaluated for localized cellular inflammation and in situ apoptosis by immunohistochemical staining for CD45, nuclear factor (NF)-κB, and terminal deoxynucleotidyl transferase-mediated FITC-deoxyuridine triphosphate nick-end labeling (TUNEL) using confocal laser scanning microscopy (CLSM), respectively. Vanadium content in selected organs and body fluids from rabbits and pigs was determined by atomic absorption spectroscopy. When compared with 4% N-9 (total irritation score 13–14 out of a possible 16), none of the rabbits given WHI-07 plus VDDTC intravaginally, developed histological alterations such as epithelial erosion, edema, leukocyte influx or vascular congestion characteristic of inflammation (total irritation score 4–6). CD45 and NF-κB immunoreactivity was limited to cells within the vascular lumen of both control and WHI-07 plus VDDTC-treated vaginal tissues. TUNEL assay revealed lack of increased apoptotic cells in vaginal mucosa exposed to increasing concentrations of WHI-07 plus VDDTC. Basal levels of proinflammatory cytokines (IL-1β, IL-8, IFN-κ and TNF-α) in porcine CVL were unaffected by intravaginal exposure to WHI-07 plus VDDTC when compared with BZK used as a positive control. Endpoint histology of the reproductive tract from cats and pigs after a single or repeated intravaginal exposure to WHI-07 plus VDDTC, respectively, revealed lack of irritation/inflammation in the epithelium, subepithelium/lamina propria, vessels/perivascular tissues, and underlying/surrounding muscles. Vanadium was not preferentially incorporated into rabbit or porcine tissues and body fluids at levels above 1 μg/g. Based on comparative histologic data and surrogate markers for inflammation, repeated intravaginal administration of WHI-07 plus VDDTC via a gel-microemulsion did not result in vaginal irritation, mucosal toxicity, or systemic absorption of vanadium. Therefore, the combined use of WHI-07 and VDDTC via gel-microemulsion appears safe for topical use as a prophylactic anti-HIV microbicide.

Members of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway are present and active in human sperm

OBJECTIVE To investigate whether components of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway are present and active in human sperm. DESIGN Comparative study. SETTING Reproductive biology department. PATIENT(S) Nine sperm donors. INTERVENTION(S) Sperm were exposed to interferon-alpha (IFN-alpha), IFN-gamma, interleukin-12 (IL-12), Ca2+ ionophore (A23187), or progesterone under capacitating conditions. MAIN OUTCOME MEASURE(S) Cell lysates prepared from sperm and Jurkat T-cell line were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the expression of JAKs (1-3 and TYK 2) and STATs (1-6) was examined by Western blot analysis. Effect of IFN-alpha, IFN-gamma, IL-12, A23187, or progesterone on sperm STAT 1 or STAT 4 phosphorylation was determined by phospho-STAT 1 antibody or antiphosphotyrosine (APT) Western blot analysis. Indirect immunofluorescence and confocal laser scanning microscopy was used to confirm the specific staining of anti-TYK 2, anti-STAT 1, and anti-STAT 4 antibodies. RESULT(S) By Western blot analysis, only antibodies to TYK 2 of the JAK family, and antibodies to STAT 1 and STAT 4 members of the STAT family specifically recognized protein bands corresponding to TYK 2, STAT 1, and STAT 4 described in other cell types. By confocal microscopy, antibodies to TYK 2 reacted with the sperm tail as well as the apical region of sperm head, whereas antibodies to STAT 1 and STAT 4 reacted with the apical region of the sperm head. Tyrosine phosphorylation of STAT 1 in capacitated sperm was enhanced by IFN-alpha and IFN-gamma, and that of STAT 4 was enhanced by IL-12. Both A23187 and progesterone markedly inhibited tyrosine phosphorylation of sperm STAT 4. CONCLUSION(S) Members of the JAK/STAT proteins, TYK 2, STAT 1, and STAT 4 are present and active in human sperm. The localization of STAT 1 and STAT 4 proteins to the apical region of the sperm head and their activation by IFN-alpha, IFN-gamma, or IL-12 implicate a role for sperm STAT proteins in fertilization. We hypothesize that sperm-derived phosphorylated STAT 1 and STAT 4 could contribute to the pool of transcription factors during sperm-oocyte fusion as well as transmit signal to the oocyte nucleus. Therefore, defects in sperm TYK 2 and STAT 1- or STAT 4-mediated signaling pathway may have relevance to male factor infertility.

GM-144, a novel lipophilic vaginal contraceptive gel-microemulsion

In a systematic effort to develop a dual-function intravaginal spermicide as well as a drug delivery vehicle against sexually transmitted pathogens, a submicron particle size (30–80 nm), lipophilic and spermicidal gel-microemulsion (viz GM-144) containing the pharmaceutical excipients propylene glycol, Captex 300, Cremophor EL, Phospholipon 90G, Rhodigel, Pluronic F-68, and sodium benzoate was formulated. GM-144 completely immobilized sperm in human or rabbit semen in less than 30 seconds. Therefore, thein vivo contraceptive potency of intravaginally applied GM-144 was compared in the standard rabbit model to those of the detergent spermicide, nonoxynol-9 (N-9)-containing formulation. Eighty-four ovulated New Zealand White rabbits in subgroups of 28 were artificially inseminated with and without intravaginal administration of GM-144 or 2% N-9 (Gynol II) formulation and allowed to complete term pregnancy. GM-144 showed remarkable contraceptive activity in the rigorous rabbit model. When compared with control, intravaginal administration of GM-144 and Gynol II resulted in 75% and 70.8% inhibition of fertility (P<.0001 versus control, Fisher’s exact test), respectively. Thus, GM-144 as a vaginal contraceptive was as effective as the commercially available N-9 gel. In the rabbit vaginal irritation test, none of the 6 rabbits given daily intravaginal application of spermicidal GM-144 for 10 days developed epithelial ulceration, edema, leukocyte influx, or vascular congestion characteristic of inflammation (total score = 5). Therefore, GM-144 has the potential to become a clinically useful safe vaginal contraceptive and a vehicle for formulating lipophilic drugs used in reducing the risk of heterosexual transmission of sexually tranmitted diseases.

Targeting Spleen Tyrosine Kinase (SYK) for Treatment of Human Disease

Targeting Spleen Tyrosine Kinase (Syk) for Treatment of Human Disease Spleen tyrosine kinase (SYK) plays a crucial role in the coordination of immune recognition receptors and orchestrates multiple downstream signaling pathways in various hematopoietic cells. In addition to its well-known function in transducing Fcγ receptor- and B cell receptor-mediated events, SYK signals downstream of a growing list of immunoreceptor pathways that modulate the innate and adaptive responses.