Title: Compositions and Methods for Enhancing Ovulation Inducing Agents FIELD OF THE INVENTION

The invention relates generally to compositions, conjugates, and methods comprising an ovulation inducing agent and an ovulatory function enhancing agent, and uses thereof. BACKGROUND OF THE INVENTION

The likelihood that a couple presenting with delayed conception has unexplained infertility is approximately 15% [I]. Ovarian stimulation with intrauterine insemination (IUI) is a commonly prescribed therapeutic option before resorting to the more sophisticated in vitro fertilization (IVF) if a pregnancy does not occur after three to six cycles of treatment [2]. Although the absolute treatment effect is small, given the low cost and ease of administration, clomiphene citrate (CC) appears to be a rational first choice treatment for women with unexplained infertility [3].

Three or four cycles of CC administration, IUI, followed by three or four cycles of controlled ovarian hyperstimulation using gonadotropins (COH) with IUI, prior to IVF are common treatment regimens for unexplained infertility in the United States [4]. Variable success rates have been reported after CC/IUI. Clomiphene therapy is successful in inducing ovulation in 50-75% of the cases, [5], however much lower pregnancy rates were reported.

Recently, it has been reported that pregnancy rates in COH/TUI cycles are significantly reduced after four unsuccessful CC-IUI cycles [6]. This discrepancy has been attributed to a negative effect of CC on the endometrium, i.e., to its prolonged antiestrogenic effects on endometrial receptivity and cervical mucus [6,7]. Moreover, Hsu and colleagues demonstrated that CC affects uterine blood flow, which was lower in the early luteal phase and in the perimplantation phase, compared with that of untreated women [8]. Recently Unfer and colleagues suggested that a high dose of phytoestrogens can reverse the deleterious effects of clomiphene citrate on endometrial thickness and could contribute to higher pregnancy rates [9]. SUMMARY OF THE INVENTION Broadly stated, the invention relates to compositions, conjugates, and methods forthe prevention and/or treatment of a condition disclosed herein comprising therapeutically effective amounts of an ovulation inducing agent and an ovulatory function enhancing agent. The combination of an ovulation inducing agent and an ovulatory function enhancing agent may provide beneficial effects in the prevention and/or treatment of conditions for which an ovulation inducing agent has been demonstrated to have a therapeutic effect, including but not limited to infertility, unexplained infertility, polycystic ovary syndrome (PCOS), anovulatory infertility associated with polycystic ovary syndrome, and related conditions, diseases and disorders.

Combinations of an ovulation inducing agent and an ovulatory function enhancing agent may be selected to provide unexpectedly additive effects or greater than additive effects i.e., synergistic effects. Further, a composition, conjugate, or method of the invention may provide sustained beneficial effects following treatment or termination of treatment.

A composition, conjugate, or method comprising an ovulation inducing agent and an ovulatory function enhancing agent may employ different mechanisms to achieve maximum therapeutic efficacy, and may improve tolerance to the therapy with a reduced risk of side effects that may result from higher doses or longer term monotherapies. A composition, conjugate, or method of the invention may permit the use of lower doses of one or both compounds with reduced adverse toxic effects of each compound. A suboptimal dosage may provide an increased margin of safety, and may also reduce the cost of a drug necessary to achieve therapeutic results. In certain aspects of the invention, the increased convenience of a single combination dosage unit may result in enhanced compliance. Other advantages of a composition, conjugate, or combination therapy may include higher stability towards degradation and metabolism, longer duration of action, and/or longer duration of action or effectiveness at particularly low doses.

In aspects of the invention, a composition, conjugate, or combination therapy of the invention may result in a decreased risk of multifollicular development and spontaneous abortion, and/or a reduced risk of developing complications such as multiple pregnancies and ovarian hyperstimulation syndrome.

In an aspect, the invention contemplates a composition, preferably a pharmaceutical composition, comprising therapeutically effective amounts of an ovulation inducing agent and an ovulatory function enhancing agent. In another aspect the invention provides a pharmaceutical composition comprising therapeutically effective amounts of an ovulation inducing agent and an ovulatory function enhancing agent that provide beneficial effects relative to an ovulation inducing agent alone following treatment. In particular aspects, the beneficial effects comprise an increased ovulation rate. In other particular aspects the beneficial effects comprise an increased preganacy rate. A pharmaceutical composition of the invention may optionally comprise a pharmaceutically acceptable carrier, excipient, or vehicle.

In a further aspect, the invention relates to a two component pharmaceutical composition for treating a condition disclosed herein (e.g., infertility), in particular unexplained infertility, in a subject comprising one or more daily doses of an ovulation inducing agent together with a pharmaceutically acceptable carrier, in combination with one or more daily doses of an ovulatory function enhancing agent together with a pharmaceutically acceptable carrier. The amount of ovulation inducing agent used in combination with an ovulatory function enhancing agent may be substantially less than in methods or preparations where the ovulation inducing agent is used alone.

The invention also contemplates a pharmaceutical composition in separate containers and intended for simultaneous or sequential administration to prevent or treat a condition disclosed herein (e.g., unexplained infertility), and in particular to provide beneficial effects, more particularly sustained beneficial effects in treating unexplained infertility, comprising an ovulation inducing agent and an ovulatory function enhancing agent, both optionally together with pharmaceutically acceptable carriers, excipients, or vehicles.

The invention further contemplates a conjugate comprising an ovulation inducing agent interacting with or linked to an ovulatory function enhancing agent, in particular to provide beneficial effects, more particularly

sustained beneficial effects. The invention further contemplates a single combination dosage unit comprising an ovulation inducing agent and an ovulatory function enhancing agent.

The invention still further contemplates methods for preparing compositions and conjugates of the invention that result in compositions and conjugates to prevent or treat a condition disclosed herein (e.g. infertility), in particular to provide beneficial effects, more particularly sustained beneficial effects.

In an aspect of the invention, a method is provided for preparing a pharmaceutical composition of an ovulation inducing agent and an ovulatory function enhancing agent adapted in particular to provide beneficial effects, more particularly sustained beneficial effects, following treatment, comprising preparing a composition comprising the ovulation inducing agent, an ovulatory function enhancing agent, and a pharmaceutically acceptable carrier, excipient, or vehicle.

In another aspect of the invention, a method is provided for preparing a stable pharmaceutical composition of an ovulation inducing agent comprising mixing an ovulation inducing agent, an ovulatory function enhancing agent, and a pharmaceutically acceptable carrier, excipient, or vehicle effective to physically stabilize the ovulation inducing agent. Such a stable pharmaceutical compositions may be adapted to provide beneficial effects, in particular sustained beneficial effects.

The invention relates to a combination treatment for preventing or treating a condition disclosed herein (e.g., unexplained infertility) in a subject comprising administering to the subject a therapeutically effective amount of at least one ovulation inducing agent and at least one ovulatory function enhancing agent, or a composition, conjugate or dosage unit of the invention, in particular to provide beneficial effects. In an aspect the invention provides a combination treatment and/or intervention which provides sustained beneficial effects following treatment.

In an aspect, the invention provides a method for the prevention and/or intervention of a condition disclosed herein (e.g., unexplained infertility) in a subject comprising administration of at least one ovulation inducing agent and at least one ovulatory function enhancing agent, or a dosage unit, composition or conjugate of the invention.

In an aspect, the invention provides a method for the prevention and/or intervention of a condition disclosed herein (e.g., unexplained infertility) in a subject comprising administration of at least one ovulation inducing agent and at least one ovulatory function enhancing agent to a subject in need thereof to provide beneficial effects. In another aspect, the invention provides a method for the prevention and/or intervention of a condition disclosed herein (e.g., unexplained infertility) in a subject comprising co-administering at least one ovulation inducing agent and at least one ovulatory function enhancing agent to a subject in need thereof.

In aspects of the invention, methods are provided for inducing or augmenting ovulation in females with anovulatory infertility or unexplained infertility and other types of ovulatory infertility comprising administering therapeutically effective amounts of at least one ovulation inducing agent and at least one ovulatory function enhancing agent.

The invention provides a method of inducing or augmenting ovulation in a subject suffering from unexplained infertility comprising administering to the subject daily doses of an ovulation inducing agent and an ovulatory function enhancing agent early in one or more menstrual cycles.

In aspects of the invention, a method is provided for treating a subject with unexplained infertility that is undergoing intrauterine insemination comprising administering to the subject therapeutically effective amounts of at least one ovulation inducing agent and at least one ovulatory function enhancing agent.

The invention further provides a method for decreasing the risk of multifollicular development, spontaneous abortion, developing multiple pregnancies and/or developing ovarian hyperstimulation in a subject to be treated with intrauterine insemination comprising administering to the subject therapeutically effective amounts of at least one ovulation inducing agent and at least one ovulatory function enhancing agent prior to intrauterine insemination.

In an aspect the invention provides a method of substantially reducing dosage levels of an ovulation inducing agent, in particular a clomiphene agent (e.g. clomiphene citrate), for administration to a female which comprises administering a combination of at least one ovulatory function enhancing agent with the ovulation inducing agent. In a particular aspect the invention provides a method of substantially reducing dosage levels of an ovulation inducing agent, in particular a clomiphene agent (e.g., clomiphene citrate), for administration to a female, with comprises administering a combination of one or more daily doses of at least one ovulatory function enhancing agent with one or more daily doses of the ovulation inducing agent.

In another aspect the invention provides a method of increasing response to an ovulation inducing agent, in particular a clomiphene agent (e.g., clomiphene citrate), in a subject who is a poor responder to the ovulation inducing agent, which comprises administering an ovulatory function enhancing agent in combination with the ovulation inducing agent. In a particular aspect the invention provides a method of increasing response to an ovulation inducing agent, in particular a clomiphene, in a subject who is a poor responder to the ovulation inducing agent, which comprises administering a combination of one or more daily doses of at least one ovulatory function enhancing agent in combination with one or more daily doses of at least one ovulation inducing agent.

The invention relates to the prevention and/or treatment, in a subject, of conditions using an ovulation inducing agent and an ovulatory function enhancing agent, or a composition, or conjugate of the invention. In a particular aspect the invention provides for the use of one or more daily doses of an ovulation inducing agent for treating infertility, each dose comprising a therapeutically effective amount of an ovulatory function enhancing agent for inducing or augmenting ovulation.

In aspects of the methods and compositions of the invention, the ovulation inducing agent is an agent that increases viscosity of cervical mucosa, in particular a clomiphene agent, more particularly clomiphene or clomiphene citrate.

In aspects of the methods and compositions of the invention, the ovulatory function enhancing agent is a mucolytic agent, an anti-oxidant, or a thiol-based compound, in particular N-acetylcysteine (NAC), dithiothreitol, pepsin, pilocarpine, glyceryl guaiacolate, terpin hydrate, ambroxo, or tyloxapol, more particularly NAC.

In an embodiment, a method is provided for treating in a subject unexplained infertility comprising administering clomiphene citrate and N-acetylcysteine in one or more treatment cycles followed by intrauterine insemination.

In an embodiment, a method is provided for treating in a subject unexplained infertility comprising administering clomiphene citrate and N-acetylcysteine in one, two or three treatment cycles each followed by intrauterine insemination.

The invention further relates to the use of one or more daily doses of an ovulatory function enhancing agent in combination with one or more daily doses of an ovulation inducing agent for treating infertility (e.g., unexplained infertility) in a female wherein the amount of ovulation inducing agent is substantially reduced as compared with the use of the ovulation inducing agent on its own.

The invention further provides for the use of one or more daily doses of an ovulatory function enhancing agent in combination with one or more daily doses of an ovulation inducing agent for treating a female who is a poor responder to the ovulation inducing agent.

The invention also contemplates the use of a composition comprising a combination of at least one ovulation inducing agent and at least one ovulatory function enhancing agent for the preparation of one or more medicament for preventing or treating a condition disclosed herein. The invention further contemplates use of an ovulation inducing agent in combination with an ovulatory function enhancing agent for the manufacture of a medicament for the treatment of a condition disclosed herein. Still further the invention provides use of an ovulation inducing agent for the manufacture of a medicament for the treatment of a condition disclosed herein used in combination with an ovulatory function enhancing agent.

In an aspect, the invention relates to the use of synergistically effective amounts of at least one ovulation inducing agent and at least one ovulatory function enhancing agent for the preparation of a medicament for preventing or treating a condition (e.g. infertility, in particular unexplained infertility). In another aspect, the invention relates to the use of an ovulation inducing agent and an ovulatory function enhancing agent for the preparation of a medicament which has a protracted profile of action. The invention additionally provides uses of a pharmaceutical composition and a conjugate of the invention in the preparation of medicaments for the prevention and/or treatment of conditions such as infertility, in particular unexplained infertility. The medicaments may provide beneficial effects, in particular sustained beneficial effects following treatment.

Since the present invention relates to a method of prevention and/or treatment comprising a combination of active agents which may be administered separately or as conjugates, the invention also provides a kit comprising an ovulation inducing agent and an ovulatory function enhancing agent, and a pharmaceutical composition, or conjugate of the invention in kit form.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various

changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Glossary

The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about." Further, it is to be understood that "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "a compound" includes a mixture of two or more compounds. The term "about" means plus or minus 0.1 to 50%, 5-50%, or 10-40%, preferably 10-20%, more preferably 10% or 15%, of the number to which reference is being made.

The terms "administering" and "administration" refer to the process by which a therapeutically effective amount of compounds or a composition contemplated herein are delivered to a subject for prevention and/or treatment purposes. Compositions are administered in accordance with good medical practices taking into account the subject's clinical condition, the site and method of administration, dosage, patient age, sex, body weight, and other factors known to physicians.

Compounds/agents described herein may contain one or more asymmetric centers and may give rise to enantiomers, diasteriomers, and other stereoisomeric forms which may be defined in terms of absolute stereochemistry as (R)- or (S)-. Therefore, the compounds and agents used in the present invention include all possible diasteriomers and enantiomers as well as their racemic and optically pure forms. Optically active (R)- and (S)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds/agents described herein contain centers of geometric asymmetry it is intended that the compounds include both E and A geometric isomers. All tautomeric forms are intended to be included within the scope of the invention.

The terms "subject" and "patient" are used interchangeably herein and refer to an animal including a warm-blooded animal such as a mammal, which is afflicted with or suspected of having or being pre-disposed to a condition disclosed herein. Preferably, the terms refer to a human. The terms also include domestic animals bred for food, sport, or as pets, including horses, cows, sheep, poultry, fish, pigs, cats, dogs, and zoo animals. The methods herein for use on subjects and patients contemplate prophylactic as well as curative use. Typical subjects for treatment include persons susceptible to, suffering from or that have suffered a condition disclosed herein. In aspects of the invention, a subject is an infertile female, in particular a female with unexplained infertility, more particularly a female with physiological indications of normal estrogen levels.

The term "pharmaceutically acceptable carrier, excipient, or vehicle" refers to a medium which does not interfere with the effectiveness or activity of an active ingredient and which is not toxic to the hosts to which it is administered. A carrier, excipient, or vehicle includes diluents, binders, adhesives, lubricants, disintegrates, bulking agents, wetting or emulsifying agents, pH buffering agents, and miscellaneous materials such as absorbants that may be needed in order to prepare a particular composition. The use of such media and agents for

an active substance is well known in the art. In certain aspects of the invention, a carrier, excipient, or vehicle is selected to stabilize an ovulation inducing agent and/or an ovulatory function enhancing agent.

"Pharmaceutically acceptable salt(s)," means a salt that is pharmaceutically acceptable and has the desired pharmacological properties. By pharmaceutically acceptable salts is meant those salts which are suitable for use in contact with the tissues of a subject or patient without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are described for example, in S. M. Berge, et al, J. Pharmaceutical Sciences, 1977, 66:1. Suitable salts include salts that may be formed where acidic protons in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, e.g. sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g. ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Suitable salts also include acid addition salts formed with inorganic acids (e.g. hydrochloric and hydrobromic acids) and organic acids (e.g. acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benezenesulfonic acid). When there are two acidic groups present, a pharmaceutically acceptable salt may be a mono-acid-mono-salt or a di-salt; and similarly where there are more than two acidic groups present, some or all of such groups can be salifϊed.

The terms "preventing and/or treating", "prevention and/or treatment" and "prevention and/or intervention" refer to the administration to a subject of biologically active agents, conjugates or compositions either before or after onset of a condition. An objective of prevention and/or treatment is to combat the condition and includes administration of the active compounds, conjugates or compositions to prevent or delay the onset of the symptoms or complications, or alleviating the symptoms or complications, or eliminating or partially eliminating the condition. In particular, "prevention" or "preventing" includes the management and care of a subject at risk of developing a condition disclosed herein prior to the clinical onset of the condition. "Preventing" or "prevention" also refers to preventing the recurrence of a condition or of one or more symptoms associated with such condition. "Treatment" or "treating" refers to the management and care of a subject at diagnosis or later. A treatment may be either performed in an acute or chronic way.

A "beneficial effect" refers to an effect of a combination of an ovulation inducing agent and an ovulatory function enhancing agent, or dosage unit, composition or conjugate thereof, that is greater than the effect of an ovulation inducing agent alone. A beneficial effect includes favorable pharmacological and/or therapeutic effects, and improved pharmacokinetic properties and biological activity. A beneficial effect maybe an additive effect or synergistic effect. In aspects of the invention, beneficial effects include but are not limited to the following: increased ovulation, increased pregnancy rate, pregnancy before 4, 5, or 6 cycles of treatment, higher number of follicles greater than 15 mm, decreased risk of multifollicular development and spontaneous abortion, and/or a reduced risk of developing complications such as multiple pregnancies and ovarian hyperstimulation syndrome. In a particular aspect of the invention, the beneficial effect is a "sustained beneficial effect" where the beneficial effect (e.g., increased ovulation) is sustained for a prolonged period of time after termination of

treatment. In an embodiment, one or more of the aforementioned effects are sustained for a prolonged period of time after termination of treatment. A beneficial effect may be sustained for at least about 1 to 5 days, 2 to 7 days, 1 to 2 weeks, 1 to 4 weeks, and 1 to 6 weeks, 2 to 16 weeks, 2 weeks to 6 months or periodically following treatment. The period of time a beneficial effect is sustained may correlate with the duration and timing of the treatment. A subject may be treated continuously for about 1 to 3 days, 1 to 5 days, 1 to 7 days, 2 to 7 days, 1 to 2 weeks, 1 to 4 weeks, and 1 to 6 weeks, 2 to 16 weeks, 2 weeks to 6 months or periodically.

The beneficial effect may be a statistically significant effect in terms of statistical analysis of an effect of the two agents versus the effects of an ovulation inducing agent alone or ovulatory function enhancing agent alone. "Statistically significant" or "significantly different" effects or levels with two agents compared with each compound alone, in particular an ovulation inducing agent alone, may represent levels that are higher or lower than a standard. In embodiments of the invention, the difference may be 1.5, 2, 3, 4, or 5 times higher or lower compared with the effect obtained with each compound alone.

An "additive effect" of an ovulation inducing agent and an ovulatory function enhancing agent refers to an effect that is equal to the sum of the effects of the two individual agents. A "synergistic effect" of an ovulation inducing agent and an ovulatory function enhancing agent refers to an effect that is greater than the additive effect which results from the sum of the effects of the two individual agents.

"Combination treatment", "combination therapy", and "in combination" are used interchangeably herein and mean that the active ingredients are administered concurrently to a patient being treated. When administered in combination each component may be administered at the same time, or sequentially in any order at different points in time. Each component may be administered separately, but sufficiently close in time to provide a desired effect, in particular a beneficial, additive, or synergistic effect. The first agent may be administered in a regimen which additionally comprises treatment with the second agent. In certain embodiments, the term refers to administration of an ovulation inducing agent and an ovulatory function enhancing agent to a patient daily, within one week, one month, 6 months, or one year, including separate administration of two medicaments each containing one of the agents as well as simultaneous administration whether or not the two agents are combined in one formulation or whether they are two separate formulations.

A "medicament" refers to a pharmaceutical composition suitable for administration of a pharmaceutically active compound(s) (e.g. an ovulation inducing agent and an ovulatory function enhancing agent) to a patient.

"Therapeutically effective amount" relates to the amount or dose of active agents (i.e., ovulation inducing agent and ovulatory function enhancing agent), compositions or conjugates of the invention that will lead to one or more desired thereapeutic effect or beneficial effect, in particular one or more sustained beneficial effects. A "therapeutically effective amount" can provide a dosage which is sufficient in order for prevention and/or treatment of a subject to be effective compared with no treatment or an ovulation inducing agent alone.

"Synergistically effective amount" relates to the amount of dose of active agents [i.e., ovulation inducing agent and ovulation function enhancing agent (e.g., an acetylcysteine compound], compositions or conjugates of the invention that will provide a synergistic effect, in particular a synergistic beneficial effect.

"Suboptimal dose" or suboptimal dosage" refers to a dose or dosage of an active agent which is less than the optimal dose or dosage for that agent when used in monotherapy.

The terms "associated", "linked", "interact", "interaction", or "interacting" refer to any physical association between molecules. The terms more particularly refer to a stable association between two molecules due to, for example, electrostatic, hydrophobic, ionic, hydrogen-bond interactions, or covalent interactions. Certain interacting or associated molecules interact only after one or more of them have been activated. In the present context, an "ovulation inducing agent" is understood to refer to any compound, which fully or partially induces ovulation. Examples of ovulation inducing agents include without limitation a clomiphene agent, in particular clomiphene (2-[4-(2-chloro-l,2-diphenylethenyl)phenoxy]-N,N- diethylethanamine) or clomiphene citrate, tamoxifen (TAM), ((Z)-2-[4-(l,2-diphenyl-l-butenyl)phenoxy]-N,N- dimethylethanamine, a gonadotrophin (HCG, HMG, LH, uFSH, or rFSH), an insulin sensitizing agent including metformin (e.g., GLycon®, Glucophage XR®, and Glucophage®) or an agent of the thiazolidinedione group of antidiabetic drugs such as rosiglitazone (Avandia®) and troglitazone (e.g., Rezulin®), gonadotrophin releasing hormone (GnRH), a bromocriphinemesylate, an aromatase inhibitor, or derivatives, analogs, and prodrugs thereof.

In aspects of the invention an ovulation inducing agent is selected which increases the viscosity of the cervical mucosa. In particular aspects the ovulation inducing agent is a clomiphene agent. Commercially available preparations of clomiphene agents include without limitation Clomid® (Aventis), Serophene® (Serono), Dyneric®, Ikaclomin®, Phemilon®, Milophene® (Milex), Clomiphene Citrate (Par Pharm), and generic equivalents.

In other aspects of the invention gonadotrophins can be employed as ovulation inducing agents either alone or with a clomiphene agent. For example, GnRH [e.g. Gonadorelin (Lutrepulse®, Ferring, Factrel®, Relisorm, Serono) and Leuprolide (Lupron®)], GnRH agonist (Synarel®, Pfizer), hCG (Pregnyl®, Organon; Profasi®, Pharmascience and Serono, Chorex®, Choron®, Ovidrel®, and Novarel®), HMG (Pergonal®, Serono; Humegon®, Organon, Repronex®), recombinant FSH [Follitropin alfa (Gonal-F®, Serono) and follitropin beta (Follistim®, Puregon®, Organon)], human FSH (urofollitropin such as Fertinex®, MetrodinMP®, and Neo- Fertinom®); and bromocriphinemesylate (Parlodel®, Sandoz).

An "ovulatory function enhancing agent" refers to any compound, which fully or partially improves the function or activity of, or response to, an ovulation inducing agent. In aspects of the invention an ovulatory function enhancing agent provides a beneficial effect. In particular aspects the agent provides an additive effect. In other aspects the agent provides a synergistic effect. Examples of ovulatory function enhancing agents include without limitation mucolytic agents, antioxidants, and thiol-based compounds. Certain ovulatory function

enhancing agents may fit into more than one category of compounds. For example an agent may be a mucolytic agent and an antioxidant.

Examples of mucolytic agents include N-acetylcysteine, dithiothreitol, pepsin, pilocarpine, glyceryl guaiacolate, terpin hydrate, ambroxo, and tyloxapol, and derivatives, analogs, and prodrugs thereof. Examples of antioxidants include without limitation vitamins A, C, and E, β-carotene, metabisulfϊte, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, butylated hydroxytoluene, glutathione, cysteine, methionine, 2-mercaptoethanol, trolox, citrate salt, sodium metabisulphate, thiourea, and trisodium citrate.

Examples of thiol-based compounds include without limitation sodium thiosulfate, N-acetylcysteine, glutathione ethyl ester, D-methionine, S-adenosyl-methionine, cysteine, N,N'-diacetylcysterine, cystathione, glutathione, glutathione ethyl ester, glutathione diethyl ester, S-(l ,2-dicarboxyethyl) glutathione triester, cysteamine, cysteine isopropylester, thiol amifostine, tyloxapol, and combinations thereof. In certain aspects, the thiol-based compound is sodium thiosulfate, N-acetylcysteine, derivatives, analogs or prodrugs thereof, or combinations thereof.

In particular aspects of the invention the ovulatory function enhancing agent is N-acetylcysteine, optionally encompassing salts, prodrugs, modifications, and metabolites thereof. For example, the hydrophilicity of an N-acetyl-L-cysteine can be reduced by chemical modification of the carboxylic group and/or the thiol group. Several pro-drugs of N-acetyl-L-cysteine are described in Anne H. Kahns et al., "Prodrugs as drug delivery systems. The synthesis and chemical and enzymatic hydrolysis kinetics of various mono- and diester prodrugs ofN-Acetyl-cysteine" in Intemat. J. Pharma, 1990;62:193-205. "Condition(s)" refers to one or more pathological symptoms or syndromes for which an ovulation inducing agent provides a beneficial or therapeutic effect, including without limitation infertility and polycystic ovary syndrome (PCOS). "Infertility" refers to a reduction in the ability to reproduce or achieve conception. "Primary infertility" refers to infertility occurring in subjects who have never conceived while "secondary infertility" refers to infertility occurring in patients who have previously conceived. "Anovulatory infertility" refers to infertility occurring in subjects who do not ovulate. Anovulatory infertility is frequently associated with polycystic ovary syndrome (PCOS). Infertility may arise in an anovulatory subject or an ovulatory subject suffering from unexplained infertility or other types of ovulatory infertility. Alternatively, the subject may be a poor responder to an ovulation inducing agent. In particular aspects of the invention, the condition is unexplained infertility. In other particular aspects, the condition is one that involves a risk of multifollicular development, spontaneous abortion, or developing complications such as multiple pregnancies or ovarian hyperstimulation syndrome. DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The invention is related to compositions, conjugates, and methods that utilize an ovulation inducing agent and an ovulatory function enhancing agent. In particular, the invention relates to compositions, conjugates, and methods for the prevention, intervention and/or treatment of a condition discussed herein comprising an ovulation inducing agent and an ovulatory function enhancing agent. In aspects of the invention, the

compositions, conjugates and methods of the invention provide beneficial effects, in particular sustained beneficial effects relative to an ovulation inducing agent alone. The beneficial effects may be additive or synergistic effects.

In aspects of the invention, where the condition is infertility, in particular unexplained infertility, beneficial effects, in particular sustained beneficial effects of a composition, combination treatment, or conjugate of the invention may manifest as one or more of the following: a) An increase in ovulation rates relative to the levels for an ovulation inducing agent alone. Preferably the compounds together provide at least about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%, 45%, or 50% increase in ovulation rates. In aspects of the invention the ovulation rate is at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90%. b) An increase in pregnancy rates relative to the levels for an ovulation inducting agent alone. Preferably the compounds together provide at least about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%, 45%, or 50% increase in pregnancy rates. In aspects of the invention the pregnancy rate is at least about 20%, 30%, 35%, 40%, or 50%. c) Pregnancy before three, four, five or six cycles of treatment, in particular pregnancy before four, five, or six cycles of clomiphene or clomiphene citrate, NAC, and IUI. d) An increase in follicles greater than 15 mm. The increase may represent at least about a 0.05%,

0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%, 45%, or 50% increase in follicles greater than 15 mm. e) A decreased risk of multifollicular development and spontaneous abortion. f) A reduced risk of developing complications such as multiple pregnancies and ovarian hyperstimulation syndrome.

Beneficial effects in respect to unexplained infertility may be evidenced by one or more of the beneficial effects described herein, in particular (a); (b); (a) and (b); (a), (b) and (c); (a), (b), (c), and (d); (a), (b), (c), (d) and (e); or (a) through (f).

An ovulatory function enhancing agent may be selected for particular compositions, methods and kits of the present invention to provide one or more specific beneficial effect(s) described herein based on characteristics including the ability to augment the activity of an ovulation inducing agent, and/or increase the physical or chemical stability of an ovulation inducing agent. In certain aspects of the invention, pharmaceutically acceptable salts of an ovulation inducing agent and/or pharmaceutically acceptable salts of an ovulatory function enhancing agent are utilized.

In aspects of the methods, conjugates, uses and compositions of the invention, the ovulation inducing agent is an agent that increases viscosity of cervical mucosa, in particular a clomiphene agent, more particularly clomiphene or clomiphene citrate. In aspects of the invention, an ovulatory function enhancing agent used in the methods, compositions, uses and conjugates of the invention is a mucolytic agent, in particular a thiol-based compound. In an

embodiment of the invention, an ovulatory function enhancing agent is acetylcysteine, and optionally analogs, derivatives, prodrugs and metabolites thereof.

In other aspects of the invention a pharmaceutical composition with beneficial effects, in particular statistically significant beneficial effects or sustained beneficial effects, is provided comprising an ovulation inducing agent which increases the viscosity of the cervical mucosa and a mucolytic agent. In a particular aspect, a pharmaceutical composition with statistically significant beneficial effects or sustained beneficial effects is provided comprising a clomiphene or clomiphene citrate and acetylcysteine.

The invention in particular aspects provides a pharmaceutical composition which has been adapted for administration to a subject to provide sustained beneficial effects to treat a condition (e.g.,infertility). In an embodiment for the prevention and/or treatment of unexplained infertility the composition is in a form such that administration to a subject results in increased ovulation rates. In another embodiment for the prevention and/or treatment of unexplained infertility the composition is in a form such that administration to a subject results in increased pregnancy rates.

This invention provides a conjugate comprising an ovulation inducing agent linked to or interacting with an ovulatory function enhancing agent. The invention also relates to isolated covalent conjugates of the invention. An ovulation inducing agent may be conjugated to an ovulatory function enhancing agent via an ester bond. Conjugates of an ovulation inducing agent and an ovulatory function enhancing agent may be conjugated with an intermediate spacer or linker. A suitable spacer or linker may be a mono- or disaccharide, an amino acid, a sulfate, a succinate, an acetate, or an oligomeric polymeric spacer or linker comprising one or more of such moieties.

The invention also provides methods that result in conjugates with improved pharmacokinetic properties, biological activity and/or beneficial effects. The methods comprise incubating the ovulation inducing agent with an ovulatory function enhancing agent under conditions that allow formation of a covalent linkage between the two compounds. The invention therefore contemplates a process for preparing a covalent conjugate comprising an ovulation inducing agent covalently bonded or linked to an ovulatory function enhancing agent, the process comprising: incubating the ovulation inducing agent with an ovulatory function enhancing agent under conditions and at a pH and for a time sufficient for formation of a covalent bond or linkage between the ovulation inducing agent and ovulatory function enhancing agent; and isolating the covalent conjugate. The above process for preparing a conjugate comprising an ovulation inducing agent and an ovulatory function enhancing agent may provide a conjugate with a substantial amount of an ovulation inducing agent covalently linked to the ovulation inducing agent.

The invention also relates to a conjugate prepared by a process described herein. The invention also relates to pharmaceutical formulation or composition comprising conjugates of the invention and a pharmaceutically acceptable carrier, excipient, or vehicle. In an aspect, the invention relates to a pharmaceutical formulation or composition of substantially pure covalent conjugates comprising an ovulation inducing agent covalently linked to an ovulatory function enhancing agent which in particular provides beneficial effects, more

particularly sustained beneficial effects, compared to the ovulation inducing agent alone. In an embodiment, a pharmaceutical formulation is provided consisting essentially of covalent conjugates comprising an ovulation inducing agent covalently linked without an intermediate spacer or linker to an ovulatory function enhancing agent. In another embodiment, a pharmaceutical formulation is provided consisting essentially of a covalent conjugate comprising an ovulation inducing agent covalently linked with an intermediate spacer or linker to an ovulatory function enhancing agent

The invention provides methods for the prevention, treatment and/or intervention of a condition (e.g., infertility) in a subject comprising administering an ovulatory function enhancing agent and an ovulation inducing agent or a pharmaceutical composition or conjugate of the invention in particular to provide a beneficial effect, more particularly a sustained beneficial effect.

In methods of the invention providing beneficial effects, in particular statistically significant beneficial effects or sustained beneficial effects, an ovulation inducing agent is selected that increases the viscosity of the cervical mucosa, and an ovulatory function enhancing agent is selected that is a mucolytic agent, an anti-oxidant or a thiol-based compound. In an aspect, the invention provides a method for the prevention and/or intervention of a condition discussed herein (e.g. infertility) in a subject comprising administering in combination at least one ovulation inducing agent and at least one ovulatory function enhancing agent. The invention also provides a combination treatment for preventing or treating a condition discussed herein (e.g. infertility) in a subject comprising administering to the subject a therapeutically effective amount of at least one ovulation inducing agent and an ovulatory function enhancing agent to provide beneficial effects. In an aspect the invention provides a combination treatment or intervention which provides sustained beneficial effects following treatment.

The invention also relates to a method of treatment comprising administering a therapeutically effective amount of at least one ovulation inducing agent in combination with the administration of at least one ovulatory function enhancing agent which upon administration to a subject with symptoms of or diagnosed infertility produces beneficial effects, preferably sustained beneficial effects, manifested as increased ovulation rate and/or increased pregnancy rate.

In an aspect of the invention therapeutically effective amounts of an ovulation inducing agent and an ovulatory function enhancing agent are combined prior to administration to a subject. In an embodiment, therapeutically effective amounts of an ovulation inducing agent and an ovulatory function enhancing agent are mixed at a physiologically acceptable pH. In an embodiment, therapeutically effective amounts of an ovulation inducing agent and an ovulatory function enhancing agent form a single combination dosage unit.

In a particular aspect, the invention provides methods for treating infertility in a patient in need thereof by administering a composition comprising an ovulatory function enhancing agent and an ovulation inducing agent in an amount sufficient to increase ovulation. The invention also contemplates the use of a composition comprising a combination of at least one ovulation inducing agent and at least one ovulatory function enhancing agent for the preparation of a medicament.

In an aspect, the invention relates to the use of a therapeutically effective amount of at least one ovulation inducing agent, and at least one ovulatory function enhancing agent for preparation of a medicament for providing beneficial effects, preferably sustained beneficial effects, in treating infertility, in particular unexplained infertility. In another aspect the invention provides the use of an ovulation inducing agent and an ovulatory function enhancing agent for the preparation of a medicament for increasing (preferably sustained increasing of) ovulation in a subject after treatment.

The compounds, compositions, medicaments, and conjugates of the present invention can be administered by any means that produce contact of the active agent(s) with the agent's sites of action in the body of a subject or patient. The compounds, compositions, medicaments, and conjugates of the present invention in the described dosages are administered by conventional methods including without limitation orally, intranasally, by inhalation, intraperitoneally, subcutaneously, intramuscularly, transdermally, sublingually or intravenously.

The active ingredients can be administered simultaneously or sequentially, and in any order at different points in time, to provide the desired beneficial effects. The compounds, conjugates and compositions can be formulated for sustained release, for delivery locally or systemically. It lies within the capability of a skilled physician or veterinarian to select a form and route of administration that optimizes the effects of the compositions, conjugates, and treatments of the present invention.

The compositions may be administered in oral dosage forms such as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. In an aspect, the invention provides an oral dosage form, in particular a tablet, comprising therapeutically effective amounts of an ovulation inducing agent and an ovulatory function enhancing agent. They may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular forms all utilizing dosage forms well known to those of ordinary skill in the pharmaceutical arts. The compositions of the invention may be administered by intranasal route via topical use of suitable intranasal vehicles, or via a transdermal route, for example, using conventional transdermal skin patches. A dosage protocol for administration using a transdermal delivery system may be continuous rather than intermittent throughout the dosage regimen.

The present invention includes combination treatments providing additive or synergistic activity, delivering an additive or synergistically effective amount, or an amount to provide a therapeutically effective amount of an ovulation inducing agent and an ovulatory function enhancing agent, or a conjugate or composition of the invention. Therefore, pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in a synergistically effective amount or a therapeutically effective amount.

The dosage regimen of the invention will vary depending upon known factors such as the pharmacodynamic characteristics of the agents and their mode and route of administration; the species, age, sex, health, medical condition, and weight of the patient, the nature and extent of the symptoms, the kind of concurrent treatment, the frequency of treatment, the route of administration, the renal and hepatic function of the

patient, and the desired effect. The effective amount of a drug required to prevent, counter, or arrest progression of a condition can be readily determined by an ordinarily skilled physician or veterinarian.

A composition, medicament, or treatment of the invention may comprise a unit dosage of at least one ovulation inducing agent and a unit dosage of at least one ovulatory function enhancing agent. In an aspect, a composition, medicament, or treatment of the invention may comprise a single unit dosage of at least one ovulation inducing agent and at least one ovulatory function enhancing agent. A "unit dosage" refers to a unitary i.e. a single dose which is capable of being administered to a patient, and which may be readily handled and packed, remaining as a physically and chemically stable unit dose comprising either the active agents as such or a mixture with one or more solid or liquid pharmaceutical excipients, carriers, or vehicles. In an aspect, a pharmaceutical composition is provided comprising a therapeutically effective suboptimal dosage of an ovulation inducing agent and an ovulatory function enhancing agent that are more effective at increasing ovulation following treatment, preferably for a sustained period, compared with a dosage of an ovulation inducing agent alone.

In another aspect, an improved pharmaceutical composition is provided comprising therapeutically effective suboptimal amounts of an ovulation inducing agent and an ovulatory function enhancing agent in a form for chronic or acute therapy of a condition (e.g. infertility). In a particular aspect, the composition comprises an ovulation inducing agent in doses that are equal to or at least 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold lower than the doses of the agent alone required to treat a condition (e.g. infertility).

In another particular aspect a pharmaceutical composition is provided comprising about 0.5 to 1000, 0.5 to 750, 0.5 to 500, 1 to 500, 1 to 400, 1 to 300, 1 to 250, 10 to 200, 10 to 150, 10 to 125, 10 to 150, 25 to 200, 25 to 150, 25 to 125, 25 to 100, or 50 to 100 micrograms of an ovulation inducing agent per single unit and 0.5 to

2000, 1 to 2000, 100-2000, 500 to 2000, 500-1500, 750-1500 and 1000-1500 micrograms ovulatory function enhancing agent compound per single unit.

In another aspect the invention provides a pharmaceutical composition comprising between 0.1 to 200, 0.1 to 150, 0.1 to 125, 0.1 to 100, 0.1 to 50, 1 to 200, 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 to 5, or 1 to 2 micrograms/kg/day of an ovulation inducing agent and 5 to 2000, 5 to 1000, 5 to 500, 5 to 250, 5 to 50, 5 to 30,

10 to 25, or 10 to 20 micrograms/kg/day ovulatory function enhancing agent.

In an embodiment of the invention a pharmaceutical composition comprises clomiphene or clomiphene citrate in a formulation and dose suitable to administer 250-500 mg/month to a patient, preferably given in divided dosages of 50-200, 50-150, 50-100, 75-150, 75-1 10, or 75-100 mg/day on the 3rd through the 8th days or

3 ^rd through the 7 ^th days after menstruation.

In an embodiment of the invention a pharmaceutical composition comprises N-acetylcysteine in a formulation and dose suitable to administer 0.5 to 2gm/day, 1 to 2gm/day, 1.2 to 1 ,5gm/day, or 1 to 1.5gm/day on the 3rd through the 8th days or 3 ^rd through the 7 ^th days after menstruation. A composition or formulation of the invention may be administered to a subject continuously for 4, 5, 6 or 7 days, or periodically.

In an embodiment, the ratio of an ovulation inducing agent to ovulatory function enhancing agent in a composition of the invention is selected to augment the activity of the ovulation inducing agent to provide beneficial effects, preferably sustained beneficial effects.

An ovulation inducing agent and an ovulatory function enhancing agent may be in a ratio selected to augment the activity of one or both compounds to produce a beneficial effect(s), in particular a sustained beneficial effect(s), and/or to produce an additive or synergistic effect. In embodiments, the ratio of an ovulation inducing agent to an ovulatory function enhancing agent may be from 1 : 1 to 1 : 120, 1 : 1 to 1 : 110, 1 : 1 to 1 : 100, 1:1 to 1:75, 1:1 to 1:50, 1 :1 to 1:25, 1 :1 to 1 :15, 1:1 to 1:10, l :10 to 1:110, 1 :20 to 1:110, 1:30 to 1:110, 1:40 to 1 : 110, 1 :50 to 1 : 100, in particular 1 : 10 to 1 : 15, more particularly 1 : 15. In other particular embodiments, the ratio of an ovulatory function enhancing agent to an ovulation inducing agent may be from 1 : 1 to 1 :120, 1 :1 to 1 :110, 1 : 1 to 1 :100, 1 : 1 to 1 :75, 1 :1 to 1 :50, 1 : 1 to 1 :25, 1 :1 to 1 :15, 1 :1 to 1 :10, and 1: 1 to 1 :5.

An ovulation inducing agent may be used in combination with an ovulatory function enhancing agent at therapeutically effective weight ratios of between about 1 : 1 to 1 : 100, 1 :1 to 1 :50, 1 :10 to 1 :100, 1 :50 to 1 :100, in particular 1: 1 to 1 :25, more particularly 1:1 to 1:15 or 1 :1 to 1 :10. The compositions of the present invention or fractions thereof typically comprise suitable pharmaceutical diluents, excipients, vehicles, or carriers selected based on the intended form of administration, and consistent with conventional pharmaceutical practices. The carriers, vehicles etc. may be adapted to provide an additive, synergistically effective or therapeutically effective amount of the active compounds.

Suitable pharmaceutical diluents, excipients, vehicles, and carriers are described in the standard text, Remington: The Science and Practice of Pharmacy, [(formerly called Remington's Pharmaceutical Sciences), Editor: University of the Sciences in Philadelphia (USIP), 21 ^st Edition, May, 2005]. By way of example, for oral administration in the form of a capsule or tablet, the active components can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, methyl cellulose, magnesium stearate, glucose, calcium, sulfate, dicalcium phosphate, mannitol, sorbital, and the like. For oral administration in a liquid form, the agents may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Suitable binders (e.g. gelatin, starch, corn sweeteners, natural sugars including glucose; natural and synthetic gums, and waxes), lubricants (e.g. sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride), disintegrating agents (e.g. starch, methyl cellulose, agar, bentonite, and xanthan gum), flavoring agents, and coloring agents may also be combined in the compositions or components thereof.

In an aspect of the invention a pharmaceutical composition has a pH from about 7 to 10. Formulations for parenteral administration of a composition of the invention may include aqueous solutions, syrups, aqueous or oil suspensions and emulsions with edible oil such as cottonseed oil, coconut oil or peanut oil. Dispersing or suspending agents that can be used for aqueous suspensions include synthetic or natural gums, such as tragacanth, alginate, acacia, dextran, sodium carboxymethylcellulose, gelatin, methylcellulose, and polyvinylpyrrolidone.

Compositions for parenteral administration may include sterile aqueous or non-aqueous solvents, such as water, isotonic saline, isotonic glucose solution, buffer solution, or other solvents conveniently used for parenteral administration of therapeutically active agents. A composition intended for parenteral administration may also include conventional additives such as stabilizers, buffers, or preservatives, e.g. methylhydroxybenzoate or similar additives.

In an embodiment, a solid form pharmaceutical composition is provided (e.g. tablets, capsules, powdered, or pulverized form) comprising a crystalline or amorphous ovulation inducing agent and a crystalline or amorphous ovulatory function inducing agent.

In another embodiment, the invention relates to a liquid drug formulation comprising an ovulation inducing agent and an ovulatory function enhancing agent or pharmaceutically acceptable salts thereof, and to lyophilized drug formulations that can be reconstituted to provide suspensions that are stable and suitable for parenteral administration.

Agents, compositions, conjugates, and dosage units may be sterilized by, for example, filtration through a bacteria retaining filter, addition of sterilizing agents to the composition, irradiation of the composition, or heating the composition. Alternatively, the compounds, conjugates, and compositions of the present invention may be provided as sterile solid preparations e.g. lyophilized powder, which are readily dissolved in sterile solvent immediately prior to use.

After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labelled for treatment of an indicated condition. For administration of a composition of the invention, such labelling would include amount, frequency, and method of administration.

The invention provides kits. In an aspect, a kit comprises or consists essentially of agents or compositions described herein. The kit is a package that houses a container which contains agents (an ovulation inducing agent and an ovulatory function enhancing agent), a composition or conjugate of the invention, and also houses instructions for administering the agents, conjugate or composition to a subject. In an aspect of the invention, a pharmaceutical pack or kit is provided comprising one or more containers filled with one or more of the ingredients of a composition of the invention. Associated with such container(s) can be various written materials such as instructions for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration. As there may be advantages to mixing a component of a composition of the invention and a pharmaceutically acceptable carrier, excipient or vehicle near the time of use, the invention encompasses kits in which components of the compositions are packaged separately. For example, the kit can contain an agent in a powdered or other dry form in, for example, a sterile vial or ampule and, in a separate container within the kit, a carrier, excipient, or vehicle, or a component of a carrier, excipient, or vehicle (in liquid or dry form). In an aspect, the kit can contain a component (e.g. an ovulation inducing agent) in a dry form, typically as a powder, often in a lyophilized form in, for example, a sterile vial or ampule and, in a separate container within the kit, a

carrier, excipient, or vehicle, or a component of a carrier, excipient, or vehicle. Alternatively, the kit may contain a component (e.g. an ovulation inducing agent) in the form of a concentrated solution that is diluted prior to administration. Any of the components described herein, any of the carriers, excipients or vehicles described herein, and any combination of components and carriers, excipients or vehicles can be included in a kit. A kit can include two or more of the agents e.g., an ovulation inducing agent and an ovulatory function enhancing agent (in any sufficiently stable form). The components can be combined with a carrier, excipient, or vehicle or packaged separately. For example, a kit can contain an ovulation inducing agent, and, in a separate container, an ovulatory function enhancing agent.

Optionally, a kit may also contain instructions for preparation or use (e.g., written instructions printed on the outer container or on a leaflet placed therein) and one or more devices to aid the preparation of the solution and/or its administration to a patient [e.g., one or a plurality of syringes, needles, filters, tape, tubing (e.g. , tubing to facilitate intravenous administration) alcohol swabs and/or Band-Aids®]. Compositions that are more concentrated than those administered to a subject can be prepared. Accordingly, such compositions can be included in the kits of the invention with, optionally, suitable materials (e.g., water, saline, or other physiologically acceptable solutions) for dilution. Instructions included with the kit can include, where appropriate, instructions for dilution.

In other embodiments, the kits of the invention can include pre-mixed compositions and instructions for solubilizing any precipitate that may have formed during shipping or storage. Kits containing solutions of one or more ovulation inducing agent and ovulatory function enhancing agent and one or more carriers, excipients or vehicles may also contain any of the materials mentioned above (e.g., any device to aid in preparing the composition for administration or in the administration per se). The instructions in these kits may describe suitable indications (e.g., a description of patients amenable to treatment) and instructions for administering the solution to a patient.

The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. Example 1 MATERIALS AND METHODS Patients and ovarian stimulation

Patients with unexplained infertility (n=40) referred for IUI were included from July, 2003 to May, 2004. Patients in this group received N-acetylcysteine (NAC )1.2 gm/day with clomiphene citrate (CC) 100 mg/day for 5 days starting at day 3 of the cycle. The study group was compared to 2 historical control groups of similar diagnosis who underwent IUI. The first control group received CC 100 mg/day for 5 days starting at day 3 of the cycle (n=32) and the second control group was natural cycle IUI (n=47). Patients were included in the study if they had a history of at least 2 years of primary or secondary infertility with the current partner and had

completed a basic evaluation that included medical history, physical examination and at least 2 semen analyses. All women had at least unilateral tubal patency, as demonstrated by hysterosalpingogram, chromopertubation at laparoscopy or both. Women with suspected female factor infertility due to pelvic disease (endometriosis or adhesions) were evaluated via laparoscopy to rule out any female factors. The ethical committee of Assiut University approved this study. Sperm Preparation:

Semen was collected by masturbation after 2 to 3 days of sexual abstinence. SelSelect (Scandinavia IVF Science, Gothenburg, Sweden), was used to prepare a density gradient for centrifugation. Aliquots (3 mL) of liquefied semen were placed on the upper (47%) layer of a 2-layered density gradient (47% and 90%) in a sterile conical centrifuge tube. Specimens were centrifuged for 20 minutes at 60Og. The supernatant was removed and the pellet was resuspended in 2mL of media. The specimen was then centrifuged for 7 minutes at 60Og and the supernatant was removed again. The final pellet was resuspended in a volume of 0.5mL media. Insemination procedure:

Insemination was performed 36 hours after hCG administration under sterile conditions using a flexible plastic Wallace catheter (Cooper Surgical, Shelton, Connecticut) with the patient in the lithotomy position. The processed sperm sample was collected into the insemination catheter connected to a 1-mL tuberculin syringe. After an examination of the cervix, the catheter was passed through the cervix and internal os into the uterine cavity in an aseptic technique. The sperm sample was then gently deposited into the uterus. Only clinical pregnancies, defined as a visible gestational sac on vaginal ultrasonography, were recorded. Statistical analysis:

Comparisons were performed using χ ² analysis. PO.05 was considered statistically significant. Results:

All 3 groups were comparable regarding age, body mass index, and duration of infertility. Patients who received NAC+CC had significantly higher number of follicles >15 mm at the day of HCG administration as compared to the other 2 groups (Table 1 ). The same level of significance was maintained for follicles <12mm and 12-15mm. Pregnancy rates in the 3 groups were 35%, 21.8% and 8.5% respectively. The NAC+CC group had significantly higher pregnancy rate as compared to natural cycle/IUI (P=0.002). On the other side, although pregnancy rate in group I (NAC/CC) is higher than that of group II (CC) it was not statistically significant. No ovarian hyperstimulation was reported in any treatment cycle. Discussion:

Clomiphene citrate is frequently used for ovulation induction in women undergoing IUI. Although the antiestrogenic effects of clomiphene on the cervix may be by-passed by the insemination procedure the same effect on the endometrium is probably persistent and repetitive in subsequent cycles. This consistent phenomenon is not responsive to the addition of supplemental estrogen [H]. Moreover, CC therapy may be associated with progressive thinning of the endometrium at midcycle leading to low pregnancy rates. For women who experience this adverse effect of clomiphene, the next step is induction of ovulation with gonadotropins, which significantly

increases both the cost of and risk associated with treatment. In this study, NAC was added to CC for induction of ovulation. NAC is a safe, well tolerated, cheap mucolytic agent. The peak plasma level of NAC is attained one hour after an oral dose and it disappears from the plasma after twelve hours. The biological activity of NAC is attributed to its sulfhydryl group which enhances glutathione - S - transferase activity aiding in the protection of all cells and membranes against free oxygen radical induced damage. NAC was also found to improve ovulation and pregnancy rate in CC-resistant PCOS patients [10]. This was probably due to the multiple biological effects of NAC. First and most important its proved antioxidant effects [12]. This is in line with the finding reported by Wang and associates that showed patients with idiopathic infertility had oxidative stress in the peritoneal fluid. [13]. In addition, NAC protected against focal cerebral ischemia in a rat model [14]. Moreover, NAC inhibited phospholipid metabolism, proinflammatory cytokine release, protease activity, and nuclear factor-kappa B deoxyribonucleic acid-binding activity in human fetal membranes in vitro [15]. Lastly, it significantly decreased the apoptosis rate in the glomerular cells of an experimental diabetes model [16] and it had anti-insulin effects [17]. Given these diverse biological activities, NAC may exert the same effects at the ovarian level and thus improving the ovulatory functions. Increasing the number of follicle > 15mm and improving pregnancy rate in this study indicates that NAC is a potent adjuvant to CC in ovulation induction. These results suggest that CC/NAC/IUI could be used before resorting to the more expensive gonadotrophins/IUI or the more sophisticated IVF. This overall positive effect may be due to positive effects of NAC at the ovarian and endometrial levels. In conclusion, NAC is a novel adjuvant treatment for unexplained infertility patients undergoing IUI. It is a simple, well-tolerated and inexpensive agent. It could be used as an alternative step before resorting to the gonadotrophins. A new ovulation induction drug under the name clomocysteine combining CC and NAC in one tablet, may be highly effective in ovulation induction. Example 2

Polycystic ovary syndrome (PCOS) affects up to 10% of women of reproductive age, in which hyperandrogenism, enlarged cystic ovaries, and chronic anovulation often coexist with obesity, hyperinsulinemia, and insulin resistance [17, 18], Obesity in women with PCOS is rather high, ranging from 30% to 60% [19], whereas hyperinsulinemia is present in more than 50% of patients with PCOS.

Clomiphene citrate therapy has variable success rates in anovulatory women; however it is the lowest in women with PCOS particularly those with insulin resistance. Currently there is increasing evidence that insulin sensitizers are particularly effective in inducing ovulation in PCOS patients [20]. However, not all cases respond to insulin sensitizers [21]. Exploring other mechanisms to induce or augment ovulation in CC resistant patients is a desirable goal in reproductive medicine.

The study described in this example was performed to evaluate the effect of NAC administration as an adjuvant to CC on ovulation and pregnancy rates as compared to placebo in patients with CC resistant PCOS. The study was a placebo-controlled, double blind randomized trial conducted at a University based hospital and private infertility practice.

MATERIALS AND METHODS

The study was conducted in a university based hospital and private infertility practice. 150 women affected by PCOS, aged 18-39 years were studied. As described elsewhere [Acbay, O. and, Gundogdu, S. Fertil Steril, 1996; 65, 946-9], PCOS was diagnosed by a finding of bilaterally normal or enlarged ovaries (ovarian volume >12 cm ³ ) with the presence of at least 7-10 peripheral cysts per ovary. No patient showed hyperprolactinemia or clinical evidence of hypercorticism or thyroid dysfunction. AU patients had to have at least one patent fallopian tube observed at hysterosalpingography or laparoscopy. The patients' male partners underwent a semen analysis and the results were determined to be adequate according to the latest WHO guidelines. Eligible patients could not have been receiving any hormonal medications except progesterone for withdrawal bleeding for 2 months before the study. No patient had taken any medication known to affect carbohydrate metabolism for at least 3 months before the study. The body mass index (BMI) was calculated according to the following formula: body weight in kilograms/height in meters squared and obesity was defined as BMI > 30 kg/m ² . Informed consent was obtained from each patient before the entry into the study. The study was approved by Benha School of Medicine Institutional Review Board.

Patients who met the inclusion criteria were found to have CC resistance, which was defined as lack of ovulation after treatment with CC, 100 mg, for 5 days in 3 consecutive cycles [Coelingh Bennink, H. J., et al., European Puregon Collaborative Anovulation Study Group. Fertil Steril, 1998; 69, 19-25]. Experimental Protocol Amenorrheic patients began treatment with induction of menses using progesterone in oil (100 mg).

On day 3, each patient underwent a baseline ultrasonographic examination. Clomiphene citrate, 100 mg, was given from day 3 until day 7. In addition to the CC, each patient was selected randomly to receive either NAC (Sedico, Cairo, ARE), in a dose of 1.2 g/day orally, or a placebo (sugar) of the same volume twice daily from day 3 until day 7. Monitoring of the cycle included transvaginal determination of the mean follicular diameter and measurement of serum E ₂ levels. Monitoring intervals were determined by patient response. Human chorionic gonadotropin was administered when at least one follicle measured 18 mm and the E ₂ level had raised.

Timed intercourse was advised 24-36 hours after hCG injection. A serum progesterone level was checked on cycle day 21-22. A serum hCG level was determined 14 days after hCG injection if menses had not yet occurred. Pregnancy was defined as a rise in the serum hCG level on serial determinations at least 2 days apart. Randomization and Blinding

In both groups, patients were randomized to receive CC and either NAC or placebo using sealed envelopes. Each participant had only one treatment cycle. Allocation was done by a third party (nurse). The NAC and placebo were supplied in identical sachets. The patients and the physician monitoring the cycles were blinded to the identity of each medication.

Outcome measures

The primary outcome was the ovulation rate in the treatment cycle. Secondary outcomes included pregnancy rate, number of follicles of > 18 mm, the serum E2 concentration, serum progesterone, endometrial thickness. The major safety end points were the incidence of ovarian hyperstimulation syndrome (OHSS) and multiple gestations. An on going pregnancy was defined as a viable pregnancy at least 12 weeks after hCG administration. Hormonal Assay:

Estradiol was measured with a radioimmunoassay (RIA) using direct double-antibody kits (Pantex, Santa Monica, CA). The assay sensitivity was 10 pg/mL. The interassay and intraassay precision of low, middle, and high controls were 14.2% and 16%, 10.6% and 7.9%, and 11.4% and 4.2%, respectively. Follicle-stimulating hormone and Luteinizing hormone were measured with the Flourimetric Enzyme Immunoassay kits (Baxter Diagnostics Inc., Miami, FL). The assay sensitivity of both assays was 0.3 mlU/mL. The interassay and Intraassay precision of low, middle, and high controls were 1.5% and 4.3%, 2.95% and 2.1%, and 3.15% and 3%, respectively, for FSH. For LH, the values were 6.35% and 8.1%, 2.9% and 1.9%, and 2.8% and 2.5%, respectively. Progesterone was measured with an RIA using the antibody coated-rube method (Coat-A-Count; Diagnostic Products Corporation, Los Angeles, CA). The sensitivity of this assay was 0.02 ng/mL. The interassay precision of low, middle, and high controls for the assay was 8.8%, 3.6%, and 3.9% respectively. Insulin was measured with the AxSym™ insulin assay (Abbott Laboratories, Abbott Park, USA). The sensitivity of the assay was 6-24 mIU/ml. The interassay and intraassay precision of low, middle and high controls were (6-10) (32-48) and (96-144), respectively. Statistical Analysis

The proportion of pregnancies that occurred in each group was compared with Fisher's exact test. Comparisons of serum levels between the NAC and placebo groups were analyzed with Student's t-test. A P level of <0.05 was considered significant. RESULTS

A total of 150 patients were randomized to. receive either NAC or a placebo (NAC: n = 75; placebo: n = 75) in a total of 150 CC cycles. As shown in Table 2, there was no difference in age, infertility duration, BMI, WVH, FSH/LH the cycles in which NAC or placebo was given. All participants had BMI more than 25 kg/m ² and the mean BMI in both groups was more than 30 kg/m ² (obese). The mean E ₂ level and the number of follicles >18 mm at the time of hCG administration in the NAC were significantly higher than the placebo group. Similarly, significantly higher ovulation rates as well as pregnancy rates were noted in the NAC group.

There were five cases of multiple pregnancies in the NAC group. No cases of ovarian hyperstimulation syndrome were reported. There were two cases of miscarriage (12.5%) (one singletone and one multiple pregnancy). On performing subgroup analysis in the NAC group, it was found that at insulin level >20 μ/mL there were 8 pregnancies (one twin) out of 35 (22.8%) while at insulin < 20 μ/mL, 8

pregnancies (three multiple pregnancies) out of 40 (20%) were observed (odds Ratio = 1.14 95%, confidence interval [CI] = 0.38 to 3.36).

In summary, a combination of CC and NAC significantly increased both ovulation rate and pregnancy rate in women with CC resistant PCOS (49.3% vs 1.3 and 21.3% vs 0% respectively). No cases of ovarian hyperstimulation syndrome were reported in the NAC group. There were two cases of miscairriage (12.5%). DISCUSSION

In this study, NAC was well tolerated by all the patients and no adverse effects were observed. A significant increase of both ovulation and pregnancy rates were obtained in the NAC group. All participants in the study had only one cycle and this facilitated completion of the study. In addition, the study participants were on oral medications of well-known tolerability and compliance. These two factors made this study achieve a high level of compliance and completion. Furthermore, no manifestations of OHSS were reported.

In conclusion, NAC is a novel adjuvant treatment for PCOS patients. It is a simple, well tolerated and inexpensive agent. It can be used as an alternative to other insulin sensitizing agents like metformin or troglitazone.

The present invention is not to be limited in scope by the specific embodiments described herein, since such embodiments are intended as but single illustrations of one aspect of the invention and any functionally equivalent embodiments are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

All publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. The citation of any reference herein is not an admission that such reference is available as prior art to the instant invention.

Table 1

Cycle characteristics of the 3 groups:

P value P value

Variable Group I Group II Group III I vs II I vs IH

No of patients 40 32 47

No of cycles 40 32 47

Age 34.58±1.18 32.52±1.17 32.0±1.32 NS NS

Wt (Kg) 74.3 ±12.4 73.2±12.3 74.5±11.5 NS NS

Height (cm) 164.1 ±5.31 162.5±5.7 163.2±4.9 NS NS

BMI (kg/m ² 27.62± 2.6 27.8 ± 3.1 28.08 ± 2.8 NS NS

Infertility duration (ys) 3.23±0.66 3.21±0.7 3.4±0.59 NS NS

Follicles <12mm 2.75±0.36 0 0 <0.0001 <0.0001

Follicles 12-15mm 1.97±0.30 0 0 0.0001 O.OOOl

Follicles > 15mm 1.80±0.18 1.04±0.18 l±0.2 O.0001 O.OOOl

Clinical pregnancy (%) 14/40 (35%) 7/32 (21.8%) 4/47(8.5%) NS 0.002

Table 2

Comparison of the baseline features and clinical outcomes of the 2 treatment groups

*only one follicle was shown to be more than 18 mm in one patient.

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