4
Contraceptive Technology and the State of the Science: New Horizons

Beyond those contraceptive technologies that are likely to be developed within the next decade (Chapter 3), several lines of scientific research offer the promise of new and innovative approaches to contraception in the longer term. This chapter looks at these approaches within three broad categories: (1) female methods, (2) male methods, and (3) immunocontraception, areas that offer promise for both males and females. Together, these sections constitute a summary of the longer, fully referenced authored papers that were specifically commissioned for this study and appear in this report as Appendixes A, B, C, and D. Each section of this chapter is also linked to a table which summarizes the mechanisms that this committee considers important candidates for contraceptive research and the development of new methods.

A Strategy for Basic Research on Contraception

Identification of Targets Through Molecular Biology

In all organisms a small number of germ cells are set aside from somatic cells in early embryogenesis. Usually they remain in an undifferentiated quiescent state while somatic cells are dividing and forming recognizable tissues and organs. Germ cells begin to divide rather late in fetal life after they have settled in the germinal ridge.

This gonial stage is followed by cessation of mitotic cell division, differentiation, maturation, and meiosis. Every step of gamete formation is unique. In addition there are support organs that are partly or entirely devoted to conception



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--> 4 Contraceptive Technology and the State of the Science: New Horizons Beyond those contraceptive technologies that are likely to be developed within the next decade (Chapter 3), several lines of scientific research offer the promise of new and innovative approaches to contraception in the longer term. This chapter looks at these approaches within three broad categories: (1) female methods, (2) male methods, and (3) immunocontraception, areas that offer promise for both males and females. Together, these sections constitute a summary of the longer, fully referenced authored papers that were specifically commissioned for this study and appear in this report as Appendixes A, B, C, and D. Each section of this chapter is also linked to a table which summarizes the mechanisms that this committee considers important candidates for contraceptive research and the development of new methods. A Strategy for Basic Research on Contraception Identification of Targets Through Molecular Biology In all organisms a small number of germ cells are set aside from somatic cells in early embryogenesis. Usually they remain in an undifferentiated quiescent state while somatic cells are dividing and forming recognizable tissues and organs. Germ cells begin to divide rather late in fetal life after they have settled in the germinal ridge. This gonial stage is followed by cessation of mitotic cell division, differentiation, maturation, and meiosis. Every step of gamete formation is unique. In addition there are support organs that are partly or entirely devoted to conception

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--> (testis, ovary, epididymis, prostate, fallopian tubes, uterus, sperm duct, penis, vagina) and subsequently to the protection and nourishment of the embryo (uterus). There cannot be another bodily function that has so much unique and essential paraphernalia devoted to it. It is this fact that provides the opportunities for selectively interfering with reproduction without affecting any other biological function. Drosophila geneticists estimate that about 100 genes—or 1 percent of the genome—can cause male and female sterility, respectively. While humans may dedicate less of the genome to conception than do fruit flies, the number of tissues, organs, hormones, and the like devoted to conception suggests that there must be many genes with no other function as well. Look, for example, at the number of sterile humans who are otherwise perfectly healthy. It is this fact that provides abundant opportunities for selectively interfering with reproduction—in both males and females—without affecting any other biological function. The basic knowledge of reproduction that is needed to conduct rational research on contraception can be summarized by two questions. First, what are the gene products that are expressed specifically in the various cells, tissues, and organs involved in reproduction? Second, which of these products is required for conception? If we were trying to devise contraceptives for Drosophila, there would be little doubt how to proceed. Both questions would be asked simultaneously by carrying out saturation mutagenesis and isolating all sterile males and females. Saturation mutagenesis is the mutation of all genes within a species. This is accomplished by mutating each gene in one individual until all the genes have been mutated in a separate individual of Drosophila. Then a genetic screen would be used to select for the mutated individuals that are sterile to determine which genes are needed for reproduction. Each of the mutant genes would be cloned and sequenced so that some idea of their function could be predicted from similarities to genes known in the published data base. We would then have in hand a list of many genes that affect conception. We would select for further study only those genes that, when mutated, do not affect any other biological process but conception. Genes that meet these two criteria are candidates for contraceptive agents. Stated another way, the products of these genes are candidates for interference. Next we would determine in what tissue and at what time each candidate gene is expressed. From its sequence we could predict whether the gene's product is a secreted extracellular product, a protein bound to cell membranes, a transcription factor, a component of the extracellular matrix, a growth factor, or perhaps a key hormone in the feedback loop that is required for reproduction. Having gathered this information, the basic research phase of Drosophila contraceptive development would be concluded, because we would have found our targets for contraception. We would have identified a large number of genes and their products that deal specifically with conception. Furthermore, we would

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--> know enough about what many of their products do to consider the rational design of contraceptive agents and to select a few that seem promising. To what extent can we apply this strategy to human contraception? What tools are available to accomplish in humans (or the closest model organism) the same results? The only higher vertebrate that currently can be mutated to saturation is the zebrafish. There are powerful genetic tools available in the mouse such as transgenesis; the introduction of a gene; "knockouts," that is, the inactivation of an existing gene; and even a large number of mutants that have been identified and maintained over the years. Nonetheless, saturation mutagenesis in the mouse is not now feasible. However, an important truth that has emerged through decades of research is that gene sequences and functions have been remarkably preserved throughout evolution. This makes it logical to assume that genes affecting conception that are identified in a zebrafish screen could be shown to play a similar role in the mouse by selectively "knocking out" the homologous mouse gene and observing the results. If the gene turns out to play a similar role in mouse reproduction and also has a human homologue with an expression pattern resembling that of the mouse and the zebrafish—then a potential human contraceptive target has also been identified. It is by no means far-fetched to assume that Drosophila genes that are involved in conception may have homologues with the same function in humans. A purely molecular biological method for identifying gene products that are specific for conception would use the principle of subtractive hybridization or the differential display of DNA copies of the messenger RNAs in a particular cell or tissue. Suppose, for example, that we wish to identify genes that are expressed solely in the epididymis. We would collect mRNA from epididymis tissue as well as from many other tissues and organs and then compare the populations of mRNA in order to identify genes that are expressed exclusively in the epididymis. Several genome companies are now sequencing all mRNAs (cDNAs) in many different tissues and then finding differences by computer search. Alternatively, it might be desirable to identify just those epididymis-specific genes that are regulated by androgen. Then the mRNA from control and hormone-stimulated epididymides would be compared for differentially expressed genes. One can imagine a number of important collections of tissue-specific or hormone-induced genes from which one could then choose possible targets for interfering with conception. These include cell-specific proteins of eggs, sperm, prostate, epididymis, and uterus. We would want to identify the battery of genes that is regulated by androgen, estrogen, progesterone, gonadotropin-releasing hormone (GnRH, sometimes referred to as LHRH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in their respective target reproductive tissues. Once we have identified genes that are specific for the target tissues and have cloned and sequenced their full-length cDNA, it is time to assay the gene for function because this method which, unlike a genetic screen, does not reveal

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--> whether the gene is required for conception. We only know that a given gene is tissue-specific and/or regulated by a hormone. The gene sequence is critical because it gives the best clue to the gene's function. The gene can then be knocked out in the mouse and antibodies made against the gene's product to see if they neutralize its activity. There are a number of methods to test whether the gene really is essential for conception but, in each case, the best functional assay can be chosen only when the gene's product has been identified. In summary, the fact that successful conception relies on many different, highly specific biological events and organs presents a rich array of targets for contraceptive research. What modern methods provide are new and powerful ways of identifying the molecules required for conception. In this field the basic research need not be far in advance of the practical applications. This proposed strategy will provide an efficient means of identifying new and relevant targets. Some of these targets will be more amenable to attack than others, either because of accessibility for delivery of inhibitory agents or because of the length of the window of biological need for normal function. Thus, selection among such targets will have to be made using criteria related to successful product development and not solely scientific merit. Identification of Targets Through Research in Reproductive Biology In addition to any new targets identified by the strategy outlined above, there are also already available for exploitation various exciting new targets that have been identified by ongoing basic research in reproductive biology and other related fields. Figure 4-1 displays the areas of the male and female reproductive structures where today's science points to potential targets for potentially significant advances in making more contraceptive choices available to more individuals. In both men and women, the hypothalymus secretes the gonatropin-releasing hormone (GnRH) which, in turn, stimulates the pituitary to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH), both of which are needed for steroid hormone production as well as sperm and egg development. Since the primary structure of GnRH was identified two decades ago, researchers have been designing, synthesizing, and testing agents that may be able to block the hormone's action. Some of these show promise for the development of new contraceptive products, some of which could conceivably appear by the end of the next decade. In addition to the neuron itself, there are targets throughout the GnRH neuronal network that might be activated or inhibited, providing a wealth of possible contraceptive agents. In fact, several peptidic agonists and antagonists of GnRH have already been identified. Each of these two classes has a different mode of action: While antagonists work by blocking a receptor, agonists operate by stimulating it so much that it is ultimately inhibited.

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--> Figure 4-1 Potential targets for development of new contraceptive technologies. Source: Prepared for this report by Neena Schwartz. The GnRH agonists available today have proved useful in the treatment of certain conditions, including prostate cancer, endometriosis, polycystic ovarian disease, and the induction of ovulation for in vitro fertilization. So far, however, they have not been potent enough to be used as contraceptives. The best hope in this area lies in the development of nonpeptide GnRH antagonists which, because they are orally active, make better drug candidates than do peptides. While all GnRH antagonists reported to date are, in fact, peptides, new approaches that have permitted development of a nonpeptidic growth hormone analogue makes the possibility of a nonpeptidic GnRH analogue more likely. Several GnRH agonists have also been developed. Current clinical applications of these compounds include the treatment of precocious puberty and restoration of fertility in GnRH-deficient men and women, the treatment of prostate cancer in men, and the treatment of uterine fibroids, endometriosis, and polycystic ovarian disease in women. Higher doses of the agonists could be used to suppress spermatogenesis in males. While the reversibility and absence of toxicity of these analogues have been established, there are some potential problems. The most important is the need to replace testosterone, whose production is also stopped by blocking GnRH, in order to maintain libido. Also promising is current research that focuses on suppressing the production or action of FSH and LH, attractive targets because of the important role these hormones play in gametogenesis in both sexes. In males, researchers believe that FSH analogues will be able to selectively block spermatogenesis without affecting testosterone secretion. In females, the degree to which FSH affects estrogen production remains unclear. If an FSH inhibitor did decrease estrogen, the hormone would have to be

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--> replaced. Another approach would be to target agents such as inhibin and activin, growth factors which also affect FSH secretion. Several industrial and academic programs are working to develop FSH agonists or antagonists and are taking advantage of the new availability of cloned human FSH receptors. They are also profiting from the new techniques of combinatorial chemistry, a chemistry-based technology platform that generates large arrays of screenable compounds for rapid drug discovery that are, in effect, libraries of small organic molecules, peptides, or oligonucleotides. Nonsteroidal agents in general offer good opportunities for innovative companies, particularly those specializing in design of small molecules, drug delivery systems for proteins or large molecules, and in combinatorial chemistry. Approaches to New Contraceptives for Females* Recent advances in molecular and cellular biology have provided a better understanding of female reproductive processes—including oogenesis, follicular maturation, ovulation, fertilization, and implantation—than ever before. This knowledge will have major impact on the development of new contraceptives for women. The challenge now is to select from the many potential targets those that can be manipulated to achieve contraception with minimal or no impact on other organ systems; the more specific and more limited the systemic effects of a contraceptive are, the less likely they may be to produce the sorts of side effects that are troublesome in greater or lesser degree to many women. Table 4-1 summarizes those potential target areas the study committee identified as being particularly promising. The cascade of events involving GnRH, LH, and FSH described above and depicted in Figure 4-1 is what regulates successful follicular development and ovulation. Recently, the GnRH receptors were cloned, an advance that will permit a wider range of possibilities for intervening in these processes. One possibility would be to suppress the receptor, either through agonists or antagonists, which would certainly halt follicular maturation and ovulation. However, this approach would also require steroid replacement to avoid unwanted effects elsewhere in the body. Another possibility would be to modify the intermittent or pulse-like rhythm of GnRH secretion in such a way as to exert a disproportionate effect on follicular development relative to steroid synthesis. Another option would be to target FSH from the start. It is now considered likely that the growth factor activin plays a key role in maintaining FSH expression. Two activin inhibitors, inhibin and follistatin, have now been identified and shown to suppress both FSH production and follicular development following *   Please refer to Appendix A for the full text of the authored and fully referenced paper on which this section is based.

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--> systemic administration in animals. This interference was specific to activin's role in the reproductive system (DePaolo et al. 1991; Vale et al. 1994, 1990). Once FSH reaches the ovary, there are still many ways to interfere with follicular maturation. A unique organ, the ovary contains hundreds of thousands of follicles—the sacs containing developing ova—that die naturally during a female's lifetime. Of the 400,000 follicles found in human females at puberty, for example, only about 400 will ever make it to ovulation, a process of attrition that is depicted in Figure 4-1 (see Appendix A). A better understanding of this process could lead to new contraceptive approaches. Most ovarian cell death is caused by apoptosis, or programmed cell death. Researchers have now identified several substances in the ovary that affect apoptosis by acting either as follicular survival factors or as mediators of cell death. These substances include gonadotropins, steroid hormones, interleukins, and cytokines such as IGF- 1. Researchers have also have identified a number of transcription factors—factors controlling gene expression—that regulate apoptosis (Artini et al. 1994; Erickson and Danforth 1995; Hsueh et al. 1994). Meiotic cell division, which allows for the combination of maternal and paternal DNA when two gametes meet, occurs only in male and female gonads. The process therefore provides a good target for contraception. One particularly promising point of intervention occurs when the ovary's primary oocytes are released from prophase I and allowed to progress to metaphase II (Grigorescu et al. 1994), an event regulated by a factor called maturation promoting factor (MPF). Further study could provide clues to new drugs that might interfere with follicular maturation (Dorce 1990). Greater research into and understanding of the molecular and cellular events involved in oocyte maturation have provided some new and some unexpected targets. Among the unexpected are a class of ''orphan receptors," some of which may have endogenous ligands yet to be discovered, while others appear not to be ligand-dependent and rather respond to other metabolic influences or synthetic molecules. Many of these "orphans" are nuclear receptors active in gametogenesis and further study may provide important new reproductive leads (Becker-Andre et al. 1994; Chen et al. 1994; Heyman et al. 1992; Ikeda et al. 1994; Lala et al. 1992; Luo et al. 1994; O'Malley 1991; Shen et al. 1994). Specific Targets Contraceptive Targets Between Oocyte Development and Ovulation The final stage of follicular development, follicular rupture, presents yet another promising contraceptive target. One factor essential to the process, the PGS-2 gene, could provide an ideal way to inhibit this one specific event while otherwise leaving the reproductive system alone (Morris and Richards 1993, 1995).

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--> TABLE 4-1 New Horizons for Contraceptive Research and the Development of New Female Methods Mechanism Description I. Mechanisms underlying the pulsatile release of GnRH (gonadotropin releasing hormone) and the differential regulation of FSH and LH synthesis and secretion. GnRH   Long-acting agonist analogues Initial stimulation and eventual desensitization of the receptor and attenuation of receptor signal transduction. These agents inhibit fertility but also reduce steroid production and induce postmenopausal symptoms, and they therefore would require steroid replacement therapy. Receptor antagonists Immediate suppression of gonadotropin secretion, although higher doses are required than of the agonists. Potential for further optimization using high throughput GnRH receptor assays. Antagonists of SF-1 SF-1 is a transcription factor that controls the development of the gonadotrope. Antagonists to it could suppress the pituitary-gonadal axis; however, unless it were highly selective for oogenesis/ovulation, replacement therapy would still be required. FSH (follicle-stimulating hormone) Activin inhibitors Inhibin Follistatin Activin is probably the key tropic factor maintaining expression of FSH. Inhibin provides a negative feedback signal that shuts off FSH secretion; follistatin serves to limit all effects of activin. Small molecules could interfere with these functions. Inhibin suppresses only a subset of activin effects, so that the drugs could be relatively specific to the suppression of reproduction. II. Specific molecular events associated with maintenance of oocytes in prophase I and release of this block by the ovulatory stimulus only in mature follicles; determination of the suitability of these targets for pharmaceutical intervention. Meiosis One potential point of intervention is the regulation of progression of primary oocytes from prophase I to metaphase II. Synthetic analogues may be efficient as agonists, as well as antagonists, for pharmacologic manipulation of the onset of meiosis. MPF has been identified as an intracellular factor that regulates this transition. MPF (maturation promoting factor) OMI (oocyte maturation inhibitor) GVB (germinal vesicle breakdown)  

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--> Mechanism Description III. Apoptosis research, to include the developing follicle as a target. Apoptosis research in diverse areas, including this one, could be mutually informative. An important research objective is better understanding of the mechanism by which the dominant follicle progresses while other developing follicles undergo atresia. FSH blockers Blocking the continuing maturation of preovulatory follicles or causing their premature demise using apoptotic factors is one potential contraceptive target. Gonadotropins, estrogens, growth hormone, growth factors, a cytokine, and nitric oxide act to ensure preovulatory follicle survival, while androgens, interleukin-6, and gonadal GnRH-like peptides are apoptotic factors. FSH, a gonadotropin, acts as a survival factor preventing the demise of early antral follicles, one of which is selected for final maturation and ovulation. Blocking FSH using an antagonist or a neutralizing protein would act in an ovary-specific manner, preventing ovulation. This method would therefore require physiologic replacement therapy. Deglycosylated FSH of FSH receptors Extracellular fragment antagonists IV. Examination of molecular and cellular aspects of follicular development and definition of the key players, their specific targets, and identification of endogenous and synthetic ligands should become major research objectives. Melatonin A hormone intimately involved in reproduction, its transcriptional effects are manifest through the orphan receptor RXR. A combination of melatonin and a synthetic progestin has been tested as a novel type of oral contraceptive preparation (see Chapter 3). Orphan receptors These receptors can be regulated by a synthetic pharmaceutical in a manner that impacts on relevant biological processes without knowing whether or not a given receptor has an endogenous ligand. GCNF (germ cell nuclear factor) A nuclear hormone restricted in expression to developing gametes and detectable in all stages of oocyte development. SF-1 (steroidogenic factor 1) May also play a key regulatory role in gametogenesis, in addition to being a positive transcriptional regulator of continued on next page

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--> Mechanism Description V. Elucidation of the factors that control follicular rupture: Inhibition of this process would be an ideal way to prevent fertilization and simulate a normal nonconception cycle with unaltered steroid patterns and levels and cycle length. PGS-2 (COX II) The LH surge induces the expression of the prostaglandin synthase 2 gene (PGS-2) that codes for an enzyme whose activity is essential for follicular rupture. If this enzyme were selectively inhibited, ovulation would be eliminated without the blocking of luteinization and the synthesis of steroid hormones. VI. A reexamination of established targets, for example, the steroid hormone receptors, would seem merited, given the recent observation that tissue-selective compounds can be developed to control specific subsets of genes that are regulated by the natural hormone. Estradiol and tamoxifen Both are ligands for the estrogen receptor but induce distinct conformational changes within the receptor with distinct biological consequences in vivo. VII. Expeditious examination of combinations of antiprogestins and other hormonal or antihormonal drugs should be undertaken, toward a method of emergency or once-a-month contraception. A more effective combination could be developed in a relatively short time frame. Oxytocic agents Drugs that stimulate uterine contractility alone or in combination with drugs that accelerate tubal transport and cause expulsion of the embryo from the uterus. Delivery Vaginal, instead of oral, administration of hormonal formulations based on single or combined steroids may increase efficacy and reduce gastrointestinal side effects of emergency contraception. Mifepristone An antiprogestin that can delay ovulation owing to temporary arrest of the growth of the dominant follicle, can offset the positive feedback of estrogen on the discharge of gonadotropin from the pituitary gland, and can disrupt the required secretory changes of the endometrium. This, in turn, could prevent either fertilization or implantation, depending on the stage of the menstrual cycle at which it is taken. Taken in combination with estrogen, it may be more effective and have fewer side effects. In addition, given in the time frame between ovulation and implantation, mifepristone prevents fertility.

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--> Mechanism Description Epostane and Azastene These enzyme inhibitors act to prevent progesterone synthesis. Epostane has been shown to terminate pregnancy in about 80 percent of women up to the eighth week of pregnancy; however, nausea was a common side effect. This approach may affect synthesis of adrenal steroids and is therefore problematic. Combinations Combinations of progesterone synthesis inhibitors and progesterone receptor blockers (mifepristone) might be more effective than either alone. A combination of an anti-estrogen and an antiprogestin might also be effective, given the objective of putting endometrial development out of synchrony with or making it hostile to the embryo. VIII. Studies should be carried out in nonhuman primates to develop the concept, and test the safety, of immunization against progesterone as a simple and easily reversible contraceptive method. Progesterone antibodies Active immunization against progesterone would result in exposure of the endometrium to unopposed estrogen. However, through administration of a synthetic progestin that does not cross-react with the antibody, during the last quarter of the cycle withdrawal bleeding would occur. IX. The most promising of the adhesion molecules should be studied to determine how essential they are for initial blastocyst attachment to the endometrial epithelium. Integrins Collagen, laminin, fibronectin, vitronectin integrin α4ß1 Changes in the expression of several integrins may define the putative period of uterine receptivity for blastocyst attachment. Blockade or disruption of this expression could provide a specific means of preventing implantation. High-molecular-weight (MW) glycoprotein A high MW glycoprotein involving N-acetyl-galactosamine and other determinants and secreted from the endometrial glands during the period of uterine sensitivity for implantation in humans, it is believed to be involved in the initial adhesion phase of implantation. Muc-1 (episialin) A member of the family of mucin glycoproteins and found in the endometrial epithelial cells of mouse uterus. Ways of decreasing it prematurely or delaying its down-regulation, necessary for implantation, could be the basis for a contraceptive approach.

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--> sperm-surface domains: the tail, midpiece, head, and perhaps even the inner-acrosomal membrane, which forms a major part of the head's anterior surface after the acrosome reaction. While inner-acrosomal antigens alone would offer the advantage of a highly specific, precisely timed target, they would also have to stimulate antibodies at a high enough level to affect the antigens within a narrow window of time. For that reason, focusing on surface antigens may make more sense at present. Because no single antigen may provoke a strong enough immune response, researchers working on different proteins may ultimately combine their efforts to produce one formulation containing several sperm immunogens. Multideterminant Immunogens In research less advanced than in the case of hCG, the reproductive hormone gonadotrophin-releasing hormone (GnRH) has shown promise as the base of contraceptive immunogens for both men and women. In research with male rats, these formulations resulted in a significant suppression of spermatogenesis that proved to be reversible. One drawback in the male is that it also affects libido and secondary sexual characteristics and would therefore have to be supplemented with testosterone (Ladd et al. 1988 and 1989). In females, these formulations have been tested in primates, with mixed results (Talwar et al. 1993), and phase I human trials are currently under way in India. More promising as a male contraceptive are immunogens based on the hormone FSH. In trials with male monkeys, FSH immunizations caused significant oligospermia (70 percent reduction in sperm count), which resulted in virtually zero fertility (Moudgal et al. 1988a, 1988b, 1992; Moudgal and Aravindan 1993). This effect was reversible, and toxicity studies in both monkeys and rats have shown no complications. A clear advantage of the FSH immunogen is that, unlike GnRH, it does not require androgen supplements to maintain libido and secondary sexual characteristics. In immunocontraceptive research to date, the best efficacy results have been at the level of 80 percent (for the hCG-based immunogen) and 75 percent (for the LDH-C4-based formulation). Because both these percentages are well below the 95 percent efficacy of today's oral contraceptives, no formulation so far has provided a significant improvement over current contraceptive methodologies. These results have led many researchers to believe that the ultimate contraceptive immunogen may turn out to be a combination of different immunogens, possibly hCG and one of the sperm proteins. The chances of developing such a multideterminant formulation could be enhanced by changes in current FDA requirements to test individually for safety and efficacy each component of any new drug containing several different compounds. Several recent areas of research should contribute significantly to the development of an effective immunogen. In the area of antigen delivery, for example,

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--> researchers have inserted the sperm antigen SP-10 into live, attenuated salmonella bacteria. When administered through the gut to female mice, this novel formulation induced an effective gamete-specific immune response (Curtiss 1990; Srinivasan et al. 1995). Another interesting possibility comes from recent research on the direct injection of DNA rather than of an antigen. While not yet attempted with DNA encoding reproductive molecules, this work on so-called ''naked DNA immunization" has spawned a paradigm shift in the broader field of vaccine research that may yet have a major impact on the field of immunocontraception (Wolff et al. 1990). Mucosal Immunity* In females, the vaginal and cervical mucosa are the first line of defense against invasion by foreign molecules. Given proper reinforcements, this natural barrier could be used to block the successful entry of both microbial pathogens and sperm. The mucosal membranes of the body include the respiratory tract, conjunctiva (inner surface of the eyelids), intestinal tract, mammary glands, and the urogenital tracts of the male and female. The surface area of this complex system is nearly 400 square meters and, within it, nearly 80 percent of all immunoglobulin-producing plasma cells reside in the intestinal mucosa, making this organ the largest reservoir of immune cells in the body. Because stimulation of the mucosal immune system at one site could elicit an immune response at a distant site, researchers believe that oral administration of immunogens could lead to effective localized immunity in the urogenital tract (Haneberg et al. 1994; Lehner et al. 1992 and 1993; Ogra and Karzon 1969; Ogra and Ogra 1973). The most promising achievement would be a vaccine that produces both a local as well as a systemic immune response that would in turn supplement the mucosal immunity. An example of where early work is going on at this interface is Reprogen's attempt to define the molecular mechanisms involved in fertility and the mucosal immune function of the urogenital tract, toward development of interventions that are both immunocontraceptive and anti-infective. One foundation concept is that, in about five percent of infertile couples, at least one member of the couple produces an antibody that binds to the male's sperm cells and blocks activity; antibodies are found in both the cervical and male urogenital mucosa. Thus the developmental effort is toward formulating sperm head and tail proteins that can induce both a strong IgA secretory and a systemic antibody response, for use in contraception. The other piece of the foundation is identification of antigens on *   Please refer to Appendix D for the full text of the authored and fully referenced paper on which this section is based.

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--> gonorrhea and chlamydia organisms that will generate both IgA and serum antibodies to block bacterial attachment and infection (Robbins-Roth 1994). Three general requirements exist for successful oral immunization: (1) a formulation that achieves survival in the mucosal environment, (2) an adequate delivery of antigen to inductive sites, and (3) stimulation of an appropriate protective immune response. Researchers face several challenges to developing an oral immunization (e.g., Challacombe and Tomasi 1980; Mowat 1987). These include low absolute absorption of antigen via oral administration (meaning that a large initial dose is required) and the possibility that the systemic immune system may not respond to oral immunization. Nevertheless, because orally administered immunogens could have fewer adverse side effects than those delivered systemically, research in this area is considered important. Isolation of appropriate immunogens is a critical step in the overall process but, as with vaginal formulations, the delivery system can play a key role in the effectiveness of a mucosal vaccine, just as it does in the case of vaginal formulations. Mucosally active adjuvants, or "helpers," administered with immunogen, increase the immune response to the immunogen and decrease the quantity needed (Grun and Maurer 1989). The candidate most likely to succeed will be some combination of adjuvants or a "hybrid" mucosal immunogen. The site of immunization may also play an important role in efficacy. Vaginal immunization has a relatively low success rate when compared with injection into the pelvic presacral space, via the intraperitoneal route, or via rectal administration. The vaginal epithelium provides a significant barrier to antigen uptake. Relatively little is known about the male urogenital mucosal system, but it would appear that rectal immunization could be effective in inducing certain mucosal immune reactions in the male as well. Because the lower female urogenital tract already has a strong defense system in place, it makes sense to take advantage of this natural characteristic to block the entry of both sperm and pathogens. Primary oral immunization followed by repeated local boosting (vaginal or rectal) may be particularly effective. By combining the body's general natural defense mechanisms with the specificity possible through immunology, such an approach offers promise for development of a highly effective, user controlled, and reversible contraceptive as well as an anti-STD agent. Concluding Comment This chapter was prepared as a summary of the individually authored technical papers that constitute the appendixes to this report, to which the reader is referred. It is intended as a bridge among what the committee believes to be the pressing public health and individual needs for new contraceptive technologies; the market that will or will not respond to those needs; and the regulatory, legal,

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--> Notes 1.   During the first 14 days of its development, the product of the fertilized egg is variously termed the conceptus, the zygote, the blastocyst, or the "preembryo." It is after implantation that what is properly termed the embryo forms in the center of the conceptus; the remaining 99 percent of the tissue gives rise to the placenta and other extraembryonic membranes. The embryo develops into a fetus at about 8 weeks after fertilization or 10 weeks after the last menstrual period (Cook 1989). 2.   The first laboratory and clinical data on RU 486 were presented by Herrman et al. in 1982.