There is an urgent need to expand the range of interventions to prevent HIV transmission and acquisition, especially those that can be controlled by women. Microbicides, defined as antimicrobial products that can be applied topically for the prevention of HIV and other sexually transmitted infections, may offer one of the most promising preventive interventions, because they could be inexpensive, readily available, and widely acceptable. The first microbial product to be clinically evaluated contained Nonoxynol-9 (nonylpenoxypolyethoxyethanol [N-9]), a nonionic surfactant, as the active agent. This article presents a review of the in vitro, ex vivo, and animal model data on the safety of N-9 and a critical analysis of their predictive power based on the results of multiple safety and efficacy trials.

In vitro and in vivo: the story of nonoxynol 9.

Incomplete coverage and short duration of action limit the effectiveness of vaginally administered drugs, including microbicides, for preventing sexually transmitted infections. We investigated vaginal distribution, retention, and safety of nanoparticles with surfaces modified to enhance transport through mucus. We show that mucus-penetrating particles (MPPs) provide uniform distribution over the vaginal epithelium, whereas conventional nanoparticles (CPs) that are mucoadhesive are aggregated by mouse vaginal mucus, leading to poor distribution. Moreover, when delivered hypotonically, MPPs were transported advectively (versus diffusively) through mucus deep into vaginal folds (rugae) within minutes. By penetrating into the deepest mucus layers, more MPPs were retained in the vaginal tract after 6 hours compared to CPs. After 24 hours, when delivered in a conventional vaginal gel, patches of a model drug remained on the vaginal epithelium, whereas the epithelium was coated with drug delivered by MPPs. We then developed MPPs composed of acyclovir monophosphate (ACVp). When administered before vaginal herpes simplex virus 2 challenge, ACVp-MPPs protected 53% of mice compared to only 16% protected by soluble drug. Overall, MPPs improved vaginal drug distribution and retention, provided more effective protection against vaginal viral challenge than soluble drug, and were nontoxic when administered daily for 1 week.

Mucus-Penetrating Nanoparticles for Vaginal Drug Delivery Protect Against Herpes Simplex Virus

Tests of human sperm function and fertilization in vitro

Acrosin (ACR), a serine proteinase located in the acrosome of the sperm, has been presumed to be involved in the recognition and binding of the sperm to the zona pellucida of the ovum and the sperm penetration through the zona pellucida. To examine the function of acrosin in vivo, we have generated mice carrying a mutation at the acrosin locus (Acr) through targeted disruption in embryonic stem (ES) cells. One chimeric male and female transmitted the targeted gene through their germ line. Homozygous Acr-/- mice are fertile and yield litters comparable in number and size to those of Acr+/+ mice. These data show that sperm of the homozygous Acr-/- mice are able to penetrate the zona pellucida, fertilize the ovum, and produce viable offspring. However, spermatozoa lacking acrosin protein show a delayed fertilization. One chimeric male which contained the targeted gene in 20% of its sperm transmitted only the Acr+ allele to its progeny. Furthermore, in vitro fertilization with equally mixed sperm cells of Acr+/+ and Acr-/- mice resulted in fertilization only with the Acr+ sperm cells. Incubation of oocytes with Acr+ or Acr- sperm show that the Acr+ sperm are faster to fertilize the oocytes than the Acr- sperm cells. These results suggest that Acr- sperm have a selective disadvantage when they are in competition with Acr+ sperm.

Spermatozoa lacking acrosin protein show delayed fertilization.

Effects of Smoking on Testicular Function, Semen Quality and Sperm Fertilizing Capacity

Acrosin, a sperm-specific acrosomal proteinase, has an essential role in the fertilization process. Low levels of acrosin appear to be associated with subfertility and infertility, and the acrosin activity of spermatozoa may potentially be a useful indicator of semen quality. The standard acrosin tests employed by research laboratories are too complicated and/or time consuming for clinical use; therefore, a simple assay has been developed to assess total acrosin activity (acrosin and activatable proacrosin). To perform the test, liquefied semen is centrifuged over Ficoll, the washed sperm pellet is suspended in a detergent (Triton X-100)-substrate (N-alpha-benzoyl-DL-arginine p-nitroanilide) buffer, pH. 8.0, and the amidase activity is determined spectrophotometrically after a 3-hour incubation period. Amidase activity can be inhibited with benzamidine, indicating that the activity is primarily or entirely due to acrosin. The absence of detergent in the incubation medium results in greatly reduced activity. The assay is repeatable, linear with increasing sperm concentration, sensitive to a lower limit of 2 x 10(6) spermatozoa, and the results correspond to those obtained with a standard acrosin extraction and assay technique. Storage of ejaculates at 3 to 6 C or at 22 to 24 C for 24 hours does not affect the acrosin activity significantly but much higher temperatures can cause a loss of activity. Freezing ejaculates results in a large decrease in sperm acrosin activity. Leukocytes show minimal activity in the assay. Sperm populations prepared by a swim-up procedure average approximately a 2-fold higher acrosin activity than the original ejaculates. Preliminary experiments indicate that the average sperm acrosin activity of ejaculates whose spermatozoa successfully fertilize human eggs in vitro is significantly higher than those that do not fertilize eggs.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.1989.tb00092.x

A simple, clinical assay to evaluate the acrosin activity of human spermatozoa.

Inhibition of human sperm penetration into zona-free hamster oocytes by proteinase inhibitors *

Vaginal contraceptive activity of aryl 4-guanidinobenzoates (acrosin inhibitors) in rabbits☆

Certain aryl 4-guanidinobenzoates (AGs; inhibitors of proteinases, including the sperm enzyme acrosin) have been shown to be more potent vaginal contraceptives in rabbits and less toxic than nonoxynol-9, the active ingredient of most marketed vaginal contraceptive formulations. To determine if these AGs can contact sperm and inhibit acrosin when mixed with the entire human ejaculate for a short period of time (roughly imitating clinical conditions), the inhibitors were added to semen at various concentrations for 2 min, after which the seminal plasma and unbound inhibitor were removed from the sperm by Ficoll centrifugation. Subsequently, the total arginine amidolytic activity of the spermatozoa was determined spectrophotometrically after a combined treatment that resulted in extraction, proacrosin activation, and reaction with substrate. Dose-response curves were prepared. All AGs studied were effective inhibitors of the amidolytic activity under these conditions, with ED50 values (the dose levels at which half of the acrosin associated with 10(6) sperm is inhibited) ranging from 10(-5) to 10(-7) M. To determine the effect on the proteolytic activity of individual spermatozoa, the experiment was repeated with 4'-acetamidophenyl 4-guanidinobenzoate (AGB), and the protease released from the sperm was measured by the gelatin-plate assay. The inhibition results were similar to those obtained by extraction of the spermatozoa and measurement of amidolytic activity. Thus, when mixed with the human ejaculate, AGs interact rapidly with spermatozoa to inhibit both their arginine amidolytic and proteolytic activity (probably due primarily or only to inhibition of acrosin) and remain bound even after removal of the seminal plasma. These data encourage further study of the compounds for contraceptive purposes.

Effect of aryl 4-guanidinobenzoates on the acrosin activity of human spermatozoa.

In view of the need for improved vaginal formulations that are contraceptive, that may prevent transmission of sexually transmitted infections, or both, a new delivery system (base formulation; called Long Acting, Sustained Release of Spermicide, or LASRS) was developed that contains bioadhesive and other ingredients with a long history of safety, and was designed to provide long-lasting vaginal retention of the formulation and to minimize possible vaginal irritation caused by incorporated active ingredients. Nonoxynol-9 (N-9) was added as an active ingredient to study the vaginal irritating properties of the formulation and to assess its long-term effectiveness by postcoital spermicidal tests. In the first series of experiments, in vitro studies showed that the formulation spreads rapidly over a cellulose membrane, forming a bioadhesive layer that remained for at least 12 hours. The second series of experiments addressed the safety of the LASRS suppository in rabbits and primates. Even with a very high concentration of N-9 (20.5%), LASRS caused only mild/moderate but acceptable irritation in the rabbit. No vaginal irritation occurred in the primate at an even higher concentration (22.5%). During the third series of experiments, the long-lasting vaginal retention properties were evaluated by postcoital spermicidal tests in the primate. LASRS with N-9 was highly spermicidal even when mating was delayed for 12 hours after placement of the formulation. Spermicidal activity was also observed when 1) mating was delayed for 24 hours after insertion of the formulation, and 2) if the females were mated 2 or even 3 times without reinsertion of the suppository before collection of the vaginal contents. In the final series of tests, the postcoital spermicidal properties of menfegol, another cytotoxic spermicide, were evaluated as were several modifications in the base formulation. Menfegol produced essentially the same results as N-9. Altering the base formulation proved to be nonbeneficial because a decrease in the long-term spermicidal effectiveness was obtained. These results suggest that the LASRS suppository has good vehicle properties for the delivery of active ingredients to the vagina.

Joanne M. Kaminski

Papers

A simple, clinical assay to evaluate the acrosin activity of human spermatozoa.

Inhibition of human sperm penetration into zona-free hamster oocytes by proteinase inhibitors *

Vaginal contraceptive activity of aryl 4-guanidinobenzoates (acrosin inhibitors) in rabbits☆

Effect of aryl 4-guanidinobenzoates on the acrosin activity of human spermatozoa.

Properties of a new, long-lasting vaginal delivery system (LASRS) for contraceptive and antimicrobial agents.