Topical peptide formulations for the treatment of sexual dysfunction

Abstract

Aspects of the present disclosure relate to topical pharmaceutical formulations comprising a nitric oxide synthase inducer (NOS) synthetic peptide and a permeation-enhancing excipient mixture. In some embodiments, the formulations provide enhanced absorption and penetration of the peptide through skin and mucous membranes over previously described formulations. In some aspects, the present disclosure provides methods for preparing such formulations. In some embodiments, the present disclosure provides methods for treating sexual dysfunction in humans using the formulations.

Description

[Technical Field]

[0001] The present disclosure relates to topical pharmaceutical formulations and methods for treating sexual dysfunction in a subject. [Background technology]

[0002] Sexual dysfunction is a common condition in which men and women experience difficulty during some phase of normal sexual activity, which can have a significant impact on an individual's quality of life and can cause high levels of personal and relationship distress. Summary of the Invention

[0003] PnPP-19 is a nitric oxide synthase (NOS) enhancer and increases the level of nitric oxide (NO), a potent vasodilator. PnPP-19 is an unstable peptide. Furthermore, PnPP-19 induces vasodilation in the corpus cavernosum (CC), and to be useful for the treatment of sexual dysfunction, it must reach tissue targets (e.g., the penile foreskin and the smooth muscle layers of the penis and vagina). However, if PnPP-19 reaches systemic blood vessels, it may cause vasodilation in undesirable tissues.

[0004] Aspects of the present disclosure relate to compositions and methods for treating sexual dysfunction. The disclosure is based, in part, on topical formulations comprising a therapeutic synthetic peptide, PnPP-19, set forth in SEQ ID NO: 1, and other pharmaceutically acceptable excipients, such as a penetration enhancer, stabilizer(s), and surfactant(s). Surprisingly, it has been discovered that not only are the formulations described by the present disclosure useful for treating sexual dysfunction in subjects (e.g., male and female subjects), but also that such formulations possess several improved properties compared to previously described formulations, such as improved stability (e.g., shelf life), as well as penetration into target tissues, rapid onset of action, comfort upon application, and reduced irritation to the skin and / or mucous membranes. In some embodiments, these improved properties are important factors for increasing patient compliance when using the formulations described herein.

[0005] Unexpectedly, it has been found that formulating therapeutic peptides (e.g., PnPP-19) derived from the venom of the black widow spider (Phoneutria nigriventer) with other excipients, such as propylene glycol and surfactants, in certain ratios can substantially increase the absorption and bioavailability of the peptide in the skin / mucosa of a subject.

[0006] The methods and formulations provided herein exhibit several attractive features and desirable properties, including comfort and ease of use, that make them suitable for use to treat a variety of conditions associated with sexual dysfunction.

[0007] Accordingly, in some aspects, the present disclosure provides a topical pharmaceutical formulation comprising a synthetic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1, present at a concentration of between 0.1% and 0.7% (weight / volume) w / v, a surfactant, propylene glycol, and a buffer having a pH between 4.0 and 6.5.

[0008] In some aspects, the present disclosure provides a topical pharmaceutical formulation comprising between 0.1-0.7% w / v of a synthetic peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, a surfactant in a concentration range of between about 2.5-18.0% w / v, propylene glycol in a concentration range of between about 7.5-30.0% w / v, and a buffer in a concentration range of between about 50.0-73.5% (volume / volume) v / v, wherein the buffer has a pH between about 4.0-6.5. In some embodiments, such formulations may be referred to herein as "FAPP20," "FAPP22.1," "FAPP22.2," "FAPP22.3," "FAPP22.4," "FAPP22.5," or "FAPP23.1" formulations.

[0009] In some embodiments, the surfactant comprises or consists of a poloxamer, hi some embodiments, the poloxamer comprises poloxamer 188, poloxamer 407, or a combination thereof.

[0010] In some embodiments, the buffer comprises sodium acetate or potassium phosphate.

[0011] In some embodiments, the formulation further comprises one or more preservatives or antioxidants.

[0012] In some embodiments, the formulation further comprises one or more additional excipients, hi some embodiments, the one or more excipients comprise fillers, lubricants, wetting agents, flavoring agents, fragrances, and / or emulsifiers.

[0013] In some embodiments, the formulation is in the form of a cream, gel, lotion, ointment, or spray.

[0014] In some embodiments, the formulation penetrates the fibrous layer of the tunica albuginea of ​​a subject without exhibiting systemic effects in the subject.

[0015] In some aspects, the present disclosure provides methods of treating a disease that would benefit from modulation of smooth muscle cell (SMC) contractility, comprising locally administering to a subject in need thereof a formulation described herein.

[0016] In some embodiments, the present disclosure provides a method of treating male sexual dysfunction, comprising topically administering to a subject in need thereof a formulation described herein.

[0017] In some embodiments, the male sexual dysfunction is selected from the group consisting of erectile dysfunction (ED), delayed ejaculation, premature ejaculation, and male hypoactive sexual desire disorder.

[0018] In some embodiments, the present disclosure provides a method of treating female sexual dysfunction (FSD), comprising topically administering to a subject in need thereof a formulation described herein.

[0019] In some embodiments, the FSD is selected from the group consisting of female sexual arousal disorder (FSAD), female orgasmic disorder, female hypoactive sexual desire disorder, and genito-pelvic pain / insertion disorder. In some embodiments, the genito-pelvic pain / insertion disorder is dyspareunia or vaginismus.

[0020] In some embodiments, the formulation is administered to the skin, mucosa, or urethra of a subject.

[0021] In some embodiments, the subject is male and the formulation is administered to the subject's penis or urethra.

[0022] In some embodiments, the subject is female and the formulation is administered to the subject's genitals, for example, the labia minora and labia majora and clitoris.

[0023] In some embodiments, the formulation is applied daily, hi some embodiments, the formulation is administered less than once a day, for example, once every 2, 3, 4, 5, 6, 7, or 8 or more days.

[0024] In some embodiments, the formulation is administered prior to sexual activity (eg, between 60 minutes and about 1 minute prior).

[0025] In some aspects, the present disclosure provides a topical pharmaceutical formulation described herein for use in the treatment of a disease that would benefit from modulation of SMC contractility.

[0026] In some aspects, the present disclosure provides a topical pharmaceutical formulation described herein for use in treating sexual dysfunction in a subject.

[0027] In some embodiments, the sexual dysfunction is a male sexual dysfunction selected from the group consisting of ED, delayed ejaculation, premature ejaculation, and male hypoactive sexual desire disorder.

[0028] In some embodiments, the sexual dysfunction is a female sexual disorder (FSD) selected from the group consisting of female sexual arousal disorder (FSAD), female orgasmic disorder, female hypoactive sexual desire disorder, and genito-pelvic pain / insertion disorder. In some embodiments, the genito-pelvic pain / insertion disorder is dyspareunia or vaginismus.

[0029] In some aspects, the disclosure provides for the use of a pharmaceutical formulation described herein for the preparation of a medicament for treating a disease that would benefit from modulation of SMC contractility.

[0030] In some aspects, the present disclosure provides for the use of a pharmaceutical formulation described herein for the preparation of a medicament for the treatment of sexual dysfunction in a subject.

[0031] In some embodiments, the sexual dysfunction is a male sexual dysfunction selected from the group consisting of ED, delayed ejaculation, premature ejaculation, and male hypoactive sexual desire disorder.

[0032] In some embodiments, the female sexual dysfunction is an FSD selected from the group consisting of FSAD, female orgasmic disorder, female hypoactive sexual desire disorder, and genito-pelvic pain / insertion disorder. In some embodiments, the genito-pelvic pain / insertion disorder is dyspareunia or vaginismus.

[0033] In some aspects, the disclosure provides methods of making a pharmaceutical formulation described herein, comprising introducing a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 into a pharmaceutically acceptable excipient, wherein the peptide is present in an amount of between about 0.1% and 0.7% w / v. In some embodiments, the pharmaceutical compositions described herein have a rapid onset of activity (e.g., within about 5 to 30 minutes, e.g., within 15 minutes) after administration to a subject.

[0034] Additional embodiments and advantages of the disclosure will be set forth in part in the description that follows and in part may be learned from the description or learned by the practice of the disclosure. The embodiments and advantages of the disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. [Brief explanation of the drawings]

[0035] [Figure 1] 1 is a graph showing representative data illustrating the release profile (%) of the amount of PnPP-19 in a FAPP20 formulation placed in the donor compartment. [Figure 2] 1 is a graph showing representative data showing the permeation profile (%) of PnPP-19 in FAPP20 formulations from the amount added to the donor medium. [Figure 3] Figure 1 shows representative data for the photodegradation of a representative FAPP20 formulation with and without UV protection. Formulations were incubated in a UVA / UVB irradiation chamber for 96 hours, and PnPP-19 degradation was calculated based on a previously validated HPLC-DAD analytical method. (*) With UV protection. [Figure 4]1 is a graph showing representative data on the kinetics of peptide PnPP-19 in target tissues. When applied in a FAPP20 formulation, the peptide was significantly detected in target tissues (e.g., foreskin and smooth muscle layer) during the first analysis period (15 minutes), demonstrating rapid release. In the smooth muscle layer, the peptide remained stable for 30 minutes, while detection in the skin decreased and detection increased in the foreskin. Within 60 minutes, small amounts were detected in the tissues. Peptide distribution was determined by radioactivity in each tissue relative to the administered dose per gram of target tissue. [Figure 5] 1 shows representative data on the biodistribution of peptide PnPP-19 15 minutes after application of the FAPP20 formulation, demonstrating rapid release. The data shown are the results of normalizing the percentage of applied dose per gram of tissue. In this calculation, the total applied dose (the sum of the quantifications in all analyzed tissues) was considered to be 100% of the applied dose, and the percentage of the applied dose in each tissue was calculated. Thus, for the FAPP20 formulation, of the 100% applied dose per gram of the animal's penis, approximately 42% was found in the skin, 18% in CC tissue (e.g., target tissues containing SMCs), and 40% in the foreskin (e.g., target tissues containing the dorsal nerve). The peptide was not detected in other organs or systemically. [Figure 6] Graph showing representative data demonstrating the enhancement of erectile function by locally administered PnPP-19 peptide in the FAPP20 formulation in healthy rats. Penile erection was induced by stimulation of the pelvic ganglion. ICP: intracavernosal pressure. MAP: mean arterial pressure, ratio (ICP / MAP). N=8 per group. *=p<0.05, **=p<0.01, ***=p<0.001. (One-way ANOVA followed by Bonferroni). DETAILED DESCRIPTION OF THE INVENTION

[0036] Aspects of the present disclosure relate to compositions and methods for treating sexual function in a subject in need thereof. The present disclosure is based, in part, on pharmaceutical compositions (also referred to herein as formulations) comprising peptides derived from the toxin PnTx2-6. In some embodiments, the pharmaceutical compositions described by the present disclosure are safe and effective in treating certain signs and symptoms associated with sexual dysfunction, such as erectile dysfunction.

[0037] U.S. Patent No. 9,279,004 (the entire contents of which are incorporated herein by reference) discloses a peptide consisting of 19 amino acids (PnTx(19)) and having a molecular weight of 2,485.85 Da, which is composed of the toxin PnTx2-6. The naturally occurring toxin PnTx2-6 causes priapism in male patients bitten by the black widow spider. Similarly, the peptide PnTx(19), also referred to as PnPP-19, is a non-naturally occurring molecule engineered from a non-contiguous domain of the naturally occurring toxin PnTx2-6. In some embodiments, PnPP-19 can enhance erectile function, as demonstrated by improved relaxation of isolated strips of mouse corpus cavernosum ex vivo. However, challenges exist in creating formulations that can deliver effective amounts of the peptide PnPP-19 to target tissues without simultaneously causing systemic exposure of the peptide PnPP-19.

[0038] The traditional therapeutic route involving protein or peptide drugs is usually parenteral administration (i.e., by injection). This is primarily because such drugs based on active peptides are not absorbed through the gastrointestinal tract. However, injections are painful and sometimes more difficult to administer than other dosage forms. Furthermore, parenterally injected peptides often result in systemic exposure in subjects, thus increasing the risk of side effects. Oral delivery is preferable to injections in terms of patient compliance because it is less painful and more convenient for patients. However, delivery of therapeutic (poly)peptides via the gastrointestinal tract has several challenges, including low stomach pH, ​​proteolysis of peptide-based drugs in the small intestine, poor absorption across the intestinal membrane, and limited stability of such formulations, especially as aqueous solutions, all of which are potential barriers to the absorption of (poly)peptides after oral administration. Furthermore, oral delivery cannot avoid systemic exposure of peptides.

[0039] Topical administration appears to be a good option for transdermal delivery of peptide-based drugs through the skin or mucosa. However, peptide-containing formulations have several major drawbacks, which often prevent their practical application. In particular, the administration of peptide-based formulations can be very difficult due to the lack of stability of these formulations, degradation of these molecules, and loss of function, especially during long-term storage and transportation.

[0040] Furthermore, some auxiliary components, such as poloxamers, "trap" the peptide inside the formulation. This feature is important for parenteral delivery because such auxiliary components protect the peptide from thermal and enzymatic degradation, delay the release of the peptide into the target tissue, and improve the bioavailability of the peptide. On the other hand, in transdermal delivery, this feature delays the onset of action and increases the loss of peptides that do not penetrate and remain trapped in the skin.

[0041] Another challenge in local administration is the penetration through the skin or mucous membrane to achieve effective peptide delivery to target tissues. Particularly in the case of transdermal delivery through the skin of the penis to CC, the peptide must pass through a very strong fibrous layer called the tunica albuginea, which makes it very difficult for the peptide to penetrate. In addition, once the peptide reaches the well-vascularized target tissue, the peptide can be easily applied systemically, which is an undesirable feature for a drug intended to act only locally.

[0042] Finally, topical formulations must have a pleasant feel to the subject to facilitate patient acceptance and must not cause irritation or sensitivity of the skin or mucous membranes.

[0043] It is desirable that all of the aforementioned characteristics be present simultaneously in a topical formulation.

[0044] Aspects of the present disclosure relate to peptide formulations (e.g., peptide formulations for treating sexual dysfunction in a subject) that can be delivered to target tissues (e.g., reproductive organs such as the penis or vagina) by a more desirable route (e.g., topical administration) without causing undesirable systemic side effects. In some embodiments, the compositions and methods described herein address challenges associated with delivering agents to treat sexual dysfunction, such as skin penetration, peptide "trapping," and maintaining the stability and function of the active ingredient (e.g., PnPP-19). In some embodiments, the compositions enhance peptide stability; do not "trap" the peptide, allowing the peptide to have a rapid onset of action; have optimal delivery—enabling the peptide to reach tissue targets in effective amounts without systemic exposure (e.g., cross the tunica albuginea); have good sensory properties; and do not cause skin / mucosal irritation or sensitivity. For the first time, active topical formulations comprising the synthetic peptide PnPP-19 are provided that are useful for treating sexual dysfunction.

[0045] Thus, in some aspects, the present disclosure provides a topical pharmaceutical formulation comprising a synthetic peptide having the sequence set forth in SEQ ID NO: 1 (e.g., in a concentration range of 0.1-0.7% w / v), a surfactant (e.g., in a concentration range of about 2.5-18.0% w / v), propylene glycol (e.g., in a concentration range of about 7.5-30.0% w / v), and a buffer (e.g., in a concentration range of about 50.0-73.5% v / v). In some embodiments, the buffer has a pH of about 4.0-6.5.

[0046] definition Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Those skilled in the art may refer specifically to "Current Protocols in Molecular Biology" for definitions and terms of the art (AUSUBEL, FM, BRENT, R., KINGSTON, RE, MOORE, DD, SEIDMAN, G., SMITH, JA, STRUHL, K. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., Media Pa. 2015).

[0047] As used herein, the term "peptide" refers to a compound formed by a chain of amino acids linked by peptide bonds. Peptides can vary in length. In some embodiments, peptides are less than 50 amino acids in length. In some embodiments, peptides are in the range of about 5 to about 25 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acids in length. In some embodiments, peptides are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. Peptides can be naturally occurring peptides or recombinantly or synthetically produced peptides. In some embodiments, peptides are recombinantly produced. Amino acid residue abbreviations are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids. In some embodiments, the peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:1.

[0048] The singular terms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0049] In the context of this specification, the term "comprising" means "including primarily, but not necessarily solely." Furthermore, variations of the word "comprising," such as "comprise" and "comprises," have correspondingly varied meanings.

[0050] As used herein, the terms "treat," "treating," and "treatment" refer to any objective or subjective parameter, e.g., any indication of successful treatment or amelioration of an injury, disease, or condition, including the alleviation, remission, or reduction of symptoms, or making the injury, disease, or condition more tolerable to the patient; a slowing of the rate of degeneration or decline; or an improvement in the patient's physical or mental well-being. Treatment or amelioration of symptoms may be based on objective or subjective parameters, including the results of a physical examination, neuropsychiatric examination, or psychiatric evaluation.

[0051] An "effective amount" or "therapeutically effective amount" of a drug or pharmacologically active agent refers to a nontoxic but sufficient amount of the drug or agent to produce a desired effect. The amount that is "effective" will vary from subject to subject, depending on the individual's age and general condition, the specific active agent(s), and the like. Thus, it is not always possible to specify an exact "effective amount." However, an appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation. In some embodiments, an "effective amount" of a peptide (e.g., PnPP-19, SEQ ID NO: 1) ranges from about 0.5 mg / ml to about 8.0 mg / ml. In some embodiments, an effective amount of a peptide ranges from about 1 mg / ml to about 7 mg / ml. In some embodiments, an effective amount is about 1, 2, 3, 4, 5, 6, or 7 mg / ml. In some embodiments, an "effective amount" of a peptide (e.g., PnPP-19, SEQ ID NO: 1) in a formulation ranges from 0.1 to 0.7% w / v.

[0052] The terms "excipient," "additive," or "other ingredient or component" refer to a substance that is a non-therapeutic agent and is intended for use as a carrier or vehicle for delivery of a therapeutic agent or for addition to a pharmaceutical composition comprising a therapeutic agent. In some embodiments, an excipient enhances handling and storage characteristics or allows or facilitates the formation of a unit dose of a composition (e.g., a composition comprising a PnPP-19 peptide).

[0053] The term "topical administration" refers to administration of a substance for absorption through the skin, mucous membranes, and tissues. Non-limiting examples of formulations for topical administration include creams, gels, lotions, emulsions, aqueous suspensions, ointments, or sprays. In some embodiments, the formulation for topical administration is not a foam.

[0054] The term "sexual dysfunction," as used herein, refers to any sexual dysfunction in a woman or a man, including, but not limited to, deficits in sexual desire, arousal, orgasm, or satisfaction, or premature ejaculation, any or all of which may be attributable to, for example, psychogenic, biological (including vasculogenic, endocrine-related, menopausal, and neurological) or medication-induced mechanisms, excluding pain or paraphilia. The sexual dysfunction may be, for example, a disorder selected from the group consisting of FSD, including FSAD, female orgasmic disorder, female hypoactive sexual desire disorder, and genito-pelvic pain / insertion disorders (such as dyspareunia and vaginismus), and male sexual dysfunction, including ED, delayed ejaculation, premature ejaculation, and male hypoactive sexual desire disorder.

[0055] As used herein, the term "about" means ±10% of a particular value, unless otherwise indicated.

[0056] The term "at least" before a number or series of numbers is understood to include the number following the term "at least," and all subsequent numbers or integers that can be logically included as is clear from the context. When at least precedes a series or range of numbers, it is understood that "at least" can modify each of the numbers in the series or range.

[0057] Pharmaceutical preparations Aspects of the present disclosure relate to pharmaceutical compositions comprising a PnPP-19 peptide (e.g., a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1). PnPP-19 is a non-toxic synthetic peptide composed of 19 amino acid residues derived from the PnTx2-6 toxin isolated from the venom of the Brazilian armadeira spider (Ardeira nigra). In some embodiments, the isolated peptide (e.g., PnPP-19) exists as a lyophilized white powder (e.g., a solid physical form). In some embodiments, PnPP-19 is an amphipathic peptide, with approximately 26% cationic amino acid residues. In some embodiments, PnPP-19 is positively charged at pH 7. In some embodiments, the PnPP-19 peptide has a pI of 10.68 and a pKa of 12.37.

[0058] In some embodiments, the pharmaceutical formulations of the present disclosure comprise an effective amount of a peptide having the amino acid sequence set forth in SEQ ID NO: 1 in a pharmaceutically acceptable medium. In some embodiments, the peptide is acetylated at the N-terminus and / or amidated at the C-terminus. In some embodiments, the peptide is in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts of peptides that may be used in formulating the compositions include, but are not limited to, acetate, chloride, bromide, carbonate, phosphate, palmitate, caproate, histidine, or trifluoroacetate. In some embodiments, the PnPP-19 peptide is acetylated, e.g., by incorporating an acetyl group at the N-terminus of the peptide (e.g., glycine (G) of the peptide). In some embodiments, the PnPP-19 peptide is amidated, e.g., by incorporating an amide group at the C-terminus of the peptide (e.g., lysine (K) of the peptide). In some embodiments, the PnPP-19 peptide is both acetylated and amidated.

[0059] The peptides described herein can be prepared by any methodology known to those skilled in the art, including recombinant and non-recombinant methods. Synthetic routes (non-recombinant) include, but are not limited to, solid-phase chemical peptide synthesis, liquid-phase chemical peptide synthesis, and biocatalytic synthesis. In some embodiments, peptides are obtained by liquid-phase or solid-phase chemical synthesis using manual, automated, or semi-automated systems. Alternative embodiments and methods for peptide synthesis are described, for example, in U.S. Patent Application Publication No. 2021 / 0060125, the entire contents of which are incorporated herein by reference.

[0060] The amount of therapeutic peptide (e.g., the PnPP-19 peptide set forth in SEQ ID NO: 1) present in the formulation can vary. In some embodiments, the amount of peptide in the formulation ranges from about 0.5 mg / ml to about 8.0 mg / ml. In some embodiments, the amount of peptide in the formulation ranges from about 1 mg / ml to about 7 mg / ml. In some embodiments, the amount of peptide in the formulation is about 1, 2, 3, 4, 5, 6, or 7 mg / ml, depending on various factors, such as the dosage of peptide to be delivered, the indication being treated, the individual patient, and the like. In some embodiments, the concentration of therapeutic peptide present in the formulation ranges from about 0.1 to 0.7% w / v (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7% w / v). In some embodiments, the amount of PnPP-19 peptide is a therapeutically effective amount, i.e., an amount that produces a therapeutic effect.

[0061] An embodiment of the present disclosure relates to a pharmaceutical formulation comprising a PnPP-19 peptide (e.g., as set forth in SEQ ID NO: 1) and a surfactant. The surfactant used in the formulations of the present invention can be a conventional surfactant in the art. An exemplary surfactant that can be used with the formulations of the present invention is a poloxamer or a mixture thereof. Topical formulations containing poloxamers have been difficult to develop. For example, Akash et al. (2014) Polymer Reviews, 54:4, 573-597 disclose that topical administration of a formulation containing PF127 poloxamer resulted in incomplete protein release and skin irritation. In another example, WO 2009 / 090558 discloses that topical foam compositions containing poloxamers lack stability and disintegrate upon administration to a delivery site. The present disclosure is based, in part, on the surprising discovery that combining a PnPP-19 peptide with one or more poloxamers in a pharmaceutical formulation results in increased stability compared to previously described topical pharmaceutical formulations. In some embodiments, the surfactant comprises (or consists of) poloxamer 188 or poloxamer 407, or a mixture thereof. The concentration range of surfactants present in the formulation can vary. In some embodiments, the concentration of surfactant (e.g., poloxamer 188, poloxamer 407, or a combination thereof) ranges from about 2.5% to about 18.0% when measured by weight / volume (w / v). In some embodiments, the concentration of poloxamer 188 in a formulation (e.g., a formulation comprising a PnPP-19 peptide) ranges from about 2.5% to about 18.0% when measured by weight / volume (w / v). In some embodiments, the concentration of poloxamer 407 in a formulation (e.g., a formulation comprising a PnPP-19 peptide) ranges from about 2.5% to about 18.0% when measured by weight / volume (w / v). In some embodiments, the total concentration of poloxamer 188 and poloxamer 407 in a formulation (e.g., a formulation comprising a PnPP-19 peptide) ranges from about 2.5% to about 18.0%, as measured by weight / volume (w / v).In some embodiments, the pharmaceutical formulation comprises about 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, or 18% surfactant (e.g., poloxamer 188, poloxamer 407, or a combination thereof), measured by weight / volume (w / v).

[0062] In some embodiments, the pharmaceutical formulation comprises a buffer (e.g., an aqueous solution comprising one or more weak acids or bases, such as acetate or phosphate, and one or more cognate salts, such as sodium or potassium). In some embodiments, the buffer comprises sodium acetate and / or potassium phosphate. In some embodiments, the buffer comprises sodium acetate. In some embodiments, the buffer comprises potassium phosphate. The concentration range of the buffer in a formulation (e.g., a formulation comprising a PnPP-19 peptide) can vary. In some embodiments, the concentration of the buffer in the pharmaceutical formulation ranges from about 50.0% to about 73.5%, measured by volume / volume (v / v). In some embodiments, the concentration of the buffer in the pharmaceutical formulation is about 50%, 55%, 60%, 65%, 70%, or about 75%, measured by volume / volume (v / v). In some embodiments, the concentration of the buffer in the pharmaceutical formulation is about 60%, measured by volume / volume (v / v).

[0063] Aspects of the present disclosure relate to pharmaceutical formulations comprising a PnPP-19 peptide (e.g., as set forth in SEQ ID NO: 1) and propylene glycol. Topical formulations comprising propylene glycol have been difficult to develop. For example, U.S. Patent No. 6,019,988 discloses that formulations comprising propylene glycol and peptides exhibited poor stability upon topical administration. The present disclosure is based, in part, on the surprising discovery that combining a PnPP-19 peptide with propylene glycol in a pharmaceutical formulation results in increased stability compared to previously described topical pharmaceutical formulations. In some embodiments, the pharmaceutical formulation comprises propylene glycol. In some embodiments, the concentration of propylene glycol in the formulation, measured by weight / volume (w / v), ranges from about 7.5% to about 30.0%. In some embodiments, the concentration of propylene glycol in the formulation, measured by weight / volume (w / v), is about 7.5%, 8%, 10%, 15%, 20%, 25%, or 30%.

[0064] In some embodiments, the pharmaceutical formulation has a pH ranging from about 4.0 to about 6.5. In some embodiments, the pH of the pharmaceutical formulation is about 4.0, 4.5, 5.0, 5.5, 5.8, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In some embodiments, the pH of the pharmaceutical formulation is 5.0.

[0065] In addition to the pharmaceutical ingredients described above, the pharmaceutical formulations of the present disclosure may further include additives or stabilizers to maintain the stability and functionality of the peptide in the formulation. In some embodiments, the stabilizer is selected from one or more preservatives and one or more antioxidants. In some embodiments, the concentration of each stabilizer in the formulation ranges from about 0.01 to about 2.0% m / m. Examples of preservatives include, but are not limited to, Microcare® PEHG e Euxyl® PE9010 (phenoxyethanol and ethylhexylglycerin), Microcare® SB (sodium benzoate and potassium sorbate), SymOcide® PC (phenoxyethanol and caprylyl glycol), and Nipaguard SCE (sorbitan caprylate, 1,3-propenodiol, and benzoic acid). Examples of antioxidants include, but are not limited to, vitamin E (tocopheryl acetate), Tinogard® TS (octadecyl di-t-butyl-4-hydroxyhydrocinnamate), Tinogard® TT (pentaerythrityl tetra-di-t-butyl hydroxyhydrocinnamate), BHT (butylated hydroxytoluene), and ferulic acid.

[0066] In some embodiments, the pharmaceutical formulation comprises one or more additional excipients, such as fillers, lubricants, wetting agents, flavoring agents, perfumes, and emulsifiers. The amount of the additional excipients contained in the pharmaceutical formulation of the present invention is not particularly limited and can be appropriately adjusted within the range of the amount used in conventional formulations.

[0067] The pharmaceutical formulations of the present disclosure may be found in, for example, British, European, and United States Pharmacopoeias (British pharmacopoeia. Vol. 1, London: Medicines and Healthcare products Regulatory Agency, 2018; European pharmacopoeia. 9th Edition, Strassbourg: Council of Europe: 2018; United States Pharmacopoeia, 42, National Formulary 37, 2018), Remington's Pharmaceutical Sciences (REMINGTON, JP and GENNARO, AR Remington's Pharmaceutical Sciences. Mack Publishing Co., 18th Edition, 1990), Martindale: The Extra Pharmacopoeia (MARTINDALE, W. and REYNOLDS, JEF F Martindale: The Extra Pharmacopoeia. London, The Pharmaceutical Press, 31st Edition, 1996), Harry's Cosmeticology (HARRY, R. and ROSEN, MR Harry's cosmeticology. Leonard Hill Books, 9th Edition, 2015), and Prista's Pharmaceutical technology (PRISTA, LVN, ALVES, AC, MORGADO, RMR Tecnica Farmaceutica e Farmacia Galenica. 4th Edition, Fundacao Calouste Gulbenkian. Servico de Educacao e Bolsas, 1996), each of the foregoing disclosures is incorporated herein by reference in its entirety.

[0068] In some embodiments, the formulation can be prepared by dissolving the peptide (e.g., PnPP-19 peptide), solubility enhancer, and buffer in water, adjusting the pH, sterilizing by filtration if necessary, and aseptically filling into a suitable dispenser.

[0069] The pharmaceutical formulations of the present disclosure may be in any pharmaceutically acceptable form for topical administration to the skin, mucous membranes, or urethra, such as, for example, creams, gels, lotions, ointments, or sprays, and the like. In some embodiments, the pharmaceutical formulation is a gel. Gels may provide a convenient and improved form for handling by the subject and may also allow for preparation to contain a homogeneous mixture of ingredients.

[0070] In some embodiments, the pharmaceutical formulations described herein are formulated for immediate release of the active peptide. In some embodiments, the pharmaceutical formulations described by the present disclosure have a rapid onset of activity, e.g., between about 5 minutes and 30 minutes (e.g., 5, 10, 15, 20, 25, or 30 minutes) after administration to a subject.

[0071] Treatment methods Aspects of the present disclosure relate to methods of treating sexual dysfunction in a subject in need thereof. The subject may be a mammal. In some embodiments, the subject is a human, such as a male human or a female human. In some embodiments, the method comprises topically administering to the subject a pharmaceutical formulation according to the present disclosure. In some embodiments, the pharmaceutical formulation is self-administered by the subject.

[0072] Previous topical formulations for the treatment of sexual dysfunction have faced challenges. For example, U.S. Patent Nos. 6,284,735 and 5,576,290 disclose the local (e.g., intravaginal, transmucosal, etc.) administration of melanocyte-stimulating hormone-like (MSH-like) peptides for the treatment of sexual dysfunction. However, MSH-like peptides have been found to have systemic effects (e.g., acting on neurons in the subject's CNS) and are therefore less desirable than locally acting therapeutic agents. The present disclosure is based, in part, on the surprising discovery that the topical formulations comprising PnPP-19 described herein act locally with fewer systemic effects than previously described formulations for the treatment of sexual dysfunction.

[0073] The amount of peptide (e.g., PnPP-19) in a pharmaceutical formulation administered to a subject can vary. In some embodiments, the amount of peptide in the formulation ranges from about 0.5 mg / ml to about 8.0 mg / ml. In some embodiments, the amount of peptide in the formulation ranges from about 1 mg / ml to about 7 mg / ml. In some embodiments, the amount of peptide in the formulation is about 2, 3, 4, 5, 6, or 7 mg / ml.

[0074] The present disclosure is based, in part, on the use of the pharmaceutical formulations described herein to treat certain disorders that benefit from modulation of SMC contractility, including, but not limited to, male and female sexual dysfunction.

[0075] In some aspects, the sexual dysfunction is a disorder selected from the group consisting of FSD, including but not limited to FSAD, female orgasmic disorder, female hypoactive sexual desire disorder, and genito-pelvic pain / insertion disorders (e.g., dyspareunia and vaginismus), and male sexual dysfunction, including ED, delayed ejaculation, premature ejaculation, and male hypoactive sexual desire disorder.

[0076] In some embodiments, the pharmaceutical formulations described herein are applied topically to relax SMC tissue in the genital area of ​​a human. In some embodiments, administration of the pharmaceutical formulations described herein relaxes the SMC tissue of a subject (e.g., SMC tissue in the genital area).

[0077] In some embodiments, the pharmaceutical formulations of the present disclosure are administered topically to the subject's genital mucosa, such as the penis, vulva, or other suitable skin site.

[0078] Surprisingly, it has been observed that the pharmaceutical formulations disclosed herein, in some embodiments, facilitate sufficient delivery of a therapeutically effective amount of a peptide (e.g., a PnPP-19 peptide) to its site of action in SMC tissue of the penis. In some embodiments, administration of the pharmaceutical composition to the penis of a subject causes the peptide to cross the tunica albuginea of ​​the penis of the subject. In some embodiments, the peptide (e.g., PnPP-19) formulations in the pharmaceutical compositions described herein result in stabilization of the peptide and a rapid onset of action following administration to a subject (e.g., within about 5 to 30 minutes, e.g., within 15 minutes, of administration to a subject).

[0079] In some embodiments, the frequency of administration to a subject varies depending on the needs of each subject and the severity of the disease being treated, and administration is recommended daily or whenever convenient for the subject. In some embodiments, a subject is administered a pharmaceutical formulation described herein once a day. In some embodiments, a subject is administered a pharmaceutical formulation described herein before engaging in sexual activity (e.g., between 5 minutes before engaging in sexual activity and about 1 day before engaging in sexual activity).

[0080] In some embodiments, the formulation is applied daily, hi some embodiments, the formulation is administered less than once a day, for example, once every 2, 3, 4, 5, 6, 7 or more days.

[0081] In some embodiments, the formulation is administered before sexual activity (e.g., between 60 minutes and about 1 minute before), hi some embodiments, the formulation is administered about 60 minutes, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute before sexual activity.

[0082] Method of preparation The present disclosure also provides methods of making the pharmaceutical formulations described herein. In some embodiments, the methods include incorporating a peptide (e.g., a PnPP-19 peptide) into a pharmaceutically acceptable excipient, wherein the peptide is present in an amount sufficient to treat sexual dysfunction in a subject. In some embodiments, the amount of peptide is in the range of about 0.5 mg / ml to about 8 mg / ml (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, or 8 mg / ml).

[0083] Preferably, the method further comprises the step of processing the mixture into a formulation suitable for administration as a topical application.

[0084] Preferably, the method of making a pharmaceutical formulation of the present invention provides a therapeutic formulation in the form of a gel.

[0085] The present disclosure is based, in part, on the surprising discovery that storing a formulation comprising a PnPP-19 peptide in a light-blocking container improves the stability and preserves the activity of the PnPP-19 peptide. In some embodiments, the disclosure relates to a container containing a pharmaceutical formulation (e.g., a formulation comprising a PnPP-19 peptide). In some embodiments, the container is configured to block the passage of light (e.g., UV light, visible light, infrared light, etc.) through the container. Examples of containers that block the passage of light include, but are not limited to, containers made of metal (e.g., aluminum) or coated with metal, dark glass containers, and UV-protective plastic containers. Any suitable container can be used, e.g., a vial, jar, tube, bottle, box, etc.

[0086] The headings provided herein are not limitations on the various aspects of the disclosure, which aspects may be defined by reference to the specification as a whole. It is also understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting, since the scope of the disclosure will be limited only by the appended claims. [Example]

[0087] The following examples are intended to provide one of ordinary skill in the art with a general description of how to make and use the present invention, and are not intended to limit the scope of the invention in any way, nor are they intended to imply that the experiments below are all experiments that have been performed or that they are the only experiments that may be performed.

[0088] Sexual dysfunction has a multifactorial etiology, and the causes of sexual dysfunction can stem from a variety of conditions, such as biological, organic, or psychological conditions, and / or social factors.

[0089] The prevalence often increases with age and can affect, for example, penile erection, libido, sexual desire, physical pleasure, sexual pain, sexual arousal, and / or orgasmic capacity in humans.

[0090] In terms of physiology, penile erection and clitoral enlargement are neurovascular events that depend on the integrity of the vascular, muscular, and neural infrastructure that comprises the penis and clitoris, for example. Sexual stimulation results in the release of neurotransmitters at nerve endings in the penis and vagina, which increases blood flow, resulting in erection and clitoral enlargement.

[0091] Nitric oxide (NO) is the primary neurotransmitter involved in penile erection and clitoral enlargement and is synthesized by the enzyme nitric oxide synthase (NOS). This enzyme has three major isoforms: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). eNOS and nNOS are said to be constitutive because they synthesize NO under physiological conditions. iNOS is an inducible isoform that is expressed only in the presence of stimuli. All isoforms of this enzyme are detected in the penis, particularly in the corpus cavernosum (CC). Expression of these isoforms has been demonstrated in the endothelial nerve endings and neuronal cell bodies of the CC, primarily in parasympathetic nonadrenergic, noncholinergic (NANC) neurons, and in several cellular structures.

[0092] The ensuing signaling cascade leads to relaxation of the SMCs in the corpora cavernosa, increased blood flow to these structures, and ultimately to erection and clitoral enlargement. Failure of this mechanism can result in erectile dysfunction insufficient for sexual activity, a hallmark of ED; and lack of female sexual arousal, a hallmark of FSAD.

[0093] There are two sets of nerves that innervate the penis and vagina: the cavernous nerves, which are autonomic, and the dorsal nerves, which are somatic. The cavernous nerves are primarily autonomic and therefore controlled by the sympathetic / parasympathetic nervous system. When stimulated, the cavernous nerves release acetylcholine and nNOS at their terminals. Acetylcholine stimulates the production of eNOS, which produces NO. nNOS produces NO directly at the nerve terminals.

[0094] The dorsal nerve, a primarily somatic nerve, is more associated with sensitivity and dramatically enhances erections, although it itself cannot produce them. The dorsal nerve tract has nNOS-positive branches, which means that their terminals produce and release NO, promoting increased blood flow and, consequently, promoting erections in men and clitoral enlargement in women.

[0095] Once produced, NO diffuses into SMCs adjacent to CCs and initiates a chain reaction through the binding and activation of soluble guanylate cyclase (GC), which then catalyzes the breakdown of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). cGMP activates the enzyme cGMP kinase, which phosphorylates certain proteins and ion channels, resulting in the opening and hyperpolarization of potassium channels, the sequestration of intracellular calcium, the inhibition of calcium channels, and the blockade of calcium influx. This results in a decrease in calcium levels, which leads to relaxation of SMCs and dilation of arteries and arterioles, resulting in increased blood flow to the area. This system is known as NO-GMPc.

[0096] The return to the non-erectile state is primarily initiated by the hydrolysis of cGMP to guanosine monophosphate by phosphodiesterase type 5 (PDE5). In addition, the reduction in tumescence is also due to sympathetic nerve discharge, which promotes vasoconstriction, and inhibits NO release by CC and endothelium. Therefore, when arteries constrict and venous pressure decreases, the penis returns to its normal state.

[0097] The search for active drugs to treat sexual dysfunctions such as ED, to increase libido, sexual satisfaction, and to improve overall sex life in female or male subjects has grown substantially over the years.

[0098] The first-line treatment for ED is the oral PDE5 inhibitors (PDE5i), such as sildenafil (Viagra®), vardenafil (Levitra®), tadalafil (Cialis®), and avanafil (Spedra®). However, up to 36% of ED patients do not respond to this treatment, are intolerant to PDE5i drugs, or have a contraindication for their use.

[0099] The mechanism of action of this class of drugs is that the effect of NO is enhanced by inhibiting the PDE5-mediated hydrolysis of cGMP.Therefore, PDE5i does not induce erection.In the absence of sexual stimulation, these drugs do not have any effect on erectile function.PDE5i can only enhance erection when NO production is complete.Therefore, the failure of PDE5i is particularly caused by the defect of NO production.

[0100] Endothelial dysfunction is the common denominator of many vascular risk factors that can lead to ED in men or FSAD in women due to defective eNOS production, which leads to reduced NO production. Atherosclerosis, hypertension (SAH), hyperlipidemia, smoking, diabetes mellitus (DM), and pelvic irradiation are some of the diseases associated with endothelial dysfunction. Furthermore, patients who have undergone radical pelvic surgery (e.g., radical prostatectomy (RP)) are at particular risk for nerve damage and subsequent parasympathetic nervous system defects, which lead to reduced NO production.

[0101] Regarding the effects of RP on erectile function, it was previously thought that the decrease in penile size after RP was caused by hypoxia. It was thought that common arterial insufficiency resulting from intraoperative damage to the arteries supplying the CC resulted in hypoxemia. This theory lost much of its momentum when it was discovered that the primary damage was intracellular and reduced oxygen levels could not be shown as evidence to support this hypothesis, and the theory of nerve damage gained ground.

[0102] RP can affect the cavernous nerves that pass through the posterolateral aspect of the prostate either by direct injury (neurotomy) during surgery when "nerve-sparing" techniques are not applicable, or by transient conduction disturbances, a condition in which the nerve is intact but rendered "non-functional" by surgical manipulation. In both cases, a significant loss of NO production occurs.

[0103] In the absence of neural NO production (e.g., no nNOS activity) and no stimulation for production by blood vessels or injury to blood vessels (e.g., no eNOS activity), the only means of producing sufficient NO for erection is via iNOS. iNOS can produce NO in much greater quantities than nNOS and eNOS, and even within the target cells themselves. The challenge is that this enzyme is only produced in response to a stimulus such as injury or trauma. Also, in the case of RP, the surgery is performed on the prostate, away from the CC, so the penis is intact.

[0104] At the cellular level, nerve damage and the resulting lack of NO result in an increase in local cytokines and toxic substances with proapoptotic (cell death of SMCs) and profibrotic (increased collagen production) effects. This results in atrophy of the corpus cavernosum tissue, accompanied by a decrease in penile cell mass and its replacement by fibrous tissue. Penile size reduction occurs to varying degrees, clinically resulting in some degree of impairment of erectile function, which may be partial or permanent. In this case, injection of vasoactive substances into the CC of the penis fails to induce erection.

[0105] Certain factors increase the likelihood that a patient will have a sufficient erection in the future for successful penetration: patients under 60 years of age, with good preoperative erectile function, and undergoing "nerve-sparing" techniques are more likely to have their erectile function restored. Other, less significant, risk factors for non-recovery of erectile function are risk factors for atherosclerotic vascular disease: SAH, DM, hypercholesterolemia, and smoking.

[0106] However, recovery is slow. Those who recover require at least 24 months and up to 48 months for improved erectile function. In some cases, a wait of 6 to 48 months for erectile function to return is recommended.

[0107] Due to the lack of NO production, PDE5i are unsuccessful in their ability to restore erectile function in almost all individuals who undergo RP.

[0108] In some embodiments, failure of PDE5i treatment includes the inability to achieve or maintain adequate penile erection during at least four consecutive sexual encounters despite optimal drug administration.All PDE5i are considered to have similar effectiveness.Therefore, if one drug treatment is unsuccessful, the patient will be offered an alternative treatment based on a different class of compound.Furthermore, some patients may be unable to tolerate PDE5i due to adverse events related to vasodilation in non-penile tissues that express PDE5 or adverse events resulting from the inhibition of homologous non-penile isoenzymes (i.e., retinal phosphodiesterase type 6).Finally, in nitrate users, concurrent use of PDE5i is contraindicated because it increases the risk of severe hypotension.

[0109] The combination of oral PDE5i with intracavernosal or intraurethral injection regimens has been shown to rescue as many as 31% of patients who do not respond to these treatments alone, but it is also associated with the occurrence of side effects in 33% of patients, including dizziness in 20% of patients. Patients who do not respond to any medical treatment options may be candidates for penile implant surgery as a third-line treatment.

[0110] Topical treatments, such as nitroglycerin, applied to the skin of the glans penis or shaft of the penis, have been investigated, but none have demonstrated sufficient efficacy for FDA approval in ED, particularly in ED unresponsive to PDE5i drugs. Therefore, a safe, well-tolerated, and effective ED treatment that avoids the side effects discussed above is desirable.

[0111] FSD is a complex and multifaceted problem affecting at least one-third of the female population. Estimates suggest that 40-43% of sexually active women already report sexual dysfunction, 33% of whom are between the ages of 40 and 69.

[0112] FSAD, a type of FSD, is characterized by a repeated inability to achieve or maintain sufficient sexual arousal despite adequate stimulation. The main problem is a decrease in NO production, which prevents the increase in blood flow after genital sexual arousal. The result is a lack of swelling of the labia and vaginal wall, and a lack of clitoral enlargement and prominence. Currently, there is no FDA-approved treatment for FSAD.

[0113] The following non-limiting examples describe pharmaceutical compositions and formulations that have been found to be safe and effective in treating sexual dysfunction.

[0114] Example 1: Peptide synthesis The NO inducer peptides (e.g., PnPP-19) of the present disclosure were chemically synthesized using solid-phase peptide synthesis (SPFS) technology. This technology involves the use of a solid matrix within which the peptide chain is constructed. In this process, amino acids bearing a protecting group, such as 9-fluorenylmethyloxycarbonyl-Fmoc, are used to prevent secondary reactions. Finally, the peptide is cleaved from the solid matrix, and the protecting group is removed to yield the desired peptide. Thus, in the synthesis of PnPP-19, the solid polymer matrix used was Rink amide resin (0.68 mmol / g), which served as a support for coupling amino acid derivatives with the Fmoc group to obtain and protect the peptide sequence of PnPP-19. Furthermore, the peptide was acetylated at the N-terminus and amidated at the C-terminus.

[0115] Example 2: Representative Formulations of FAPP20 Based on the makeup of FAPP20, excipients can be varied to some extent to obtain other preferred formulations.

[0116] These modifications can be made to alter sensory parameters. Table 1 below lists the composition of the FAPP20 formulation and the modifications made to obtain other preferred formulations. In all of these exemplary formulations, the concentration of PnPP-19 varies from 1 mg / mL to 5 mg / mL.

[0117] All of these formulations exhibited peptide contents within the specification range of 80.0–120.0% after storage at room temperature (25°C) for 3 months (Table 1).

[0118] [Table 1]

[0119] Example 3: Preparation of FAPP20 formulations A gel formulation according to the present invention was prepared by mixing the ingredients shown in Table 2 below.

[0120] [Table 2] JPEG2026507422000004.jpg78149

[0121] The above-cited formulations are prepared as follows: Part I 20mM sodium acetate buffer pH 5.0 + peptide (PnPP-19) Calculate the volume of sodium acetate and PnPP-19 solution required to make 60% of the final volume of the formulation. Weigh out enough sodium acetate for a final solution of 20 mM. Then measure the pH and adjust to 5.0 using NaOH or HCl. After this procedure, solubilize the peptide using sodium acetate solution to a concentration of 0.5% (w / v). or 100mM potassium phosphate buffer pH 5.8 + peptide (PnPP-19) Calculate the volume of potassium phosphate buffer and PnPP-19 solution needed to achieve the target percentage of final formulation volume. Weigh out enough KHPO (Solution 1) and KHPO (Solution 2) to make a 1 M solution. To obtain a final concentration of 0.1 M potassium phosphate buffer, Solutions 1 and 2 should be mixed in the ratio of 8.5% Solution 1 to 91.5% Solution 2 using ultrapure water. The pH is then measured and adjusted to 5.8 using Solution 1 or 2 to raise or lower the pH, respectively. After this procedure, the peptide is solubilized using potassium phosphate buffer to a concentration of 0.5% (w / v).

[0122] Part II Propylene glycol Propylene glycol is weighed to obtain the target concentration (w / v) taking into account the final volume of the formulation. Propylene glycol is mixed with the solution containing sodium acetate and peptide until completely solubilized. Poloxamer 407 and / or Poloxamer 188 Weigh out Poloxamer 407 and / or Poloxamer 188 to achieve the target concentration (w / v) taking into account the final volume of the formulation. Mix the poloxamer into the solution containing sodium acetate, peptide, and propylene glycol until completely solubilized. Adjust the final volume of the formulation with distilled water, if necessary.

[0123] Part III Microcare (registered trademark) PEHG In cases where a preservative is added to the formulation, the preservative should be added at the end of the process at a final concentration of 0.8% (w / w).

[0124] Solubilization of the solution The final solution should be stirred until completely solubilized. If necessary, use other methods to facilitate solubilization (stirring and ultrasonic bath - 10-15 minutes). The formulation is then placed in the refrigerator (without stirring) for approximately 24 hours. After this period, the formulation is stored at room temperature.

[0125] Example 3: Determining pH Range The purpose of this experiment was to evaluate the stability of the peptide over a wide range of pH values ​​in order to identify a preferred pH range.

[0126] Seventeen formulations were made at pH values ​​ranging from 4.0 to 8.0. The starting concentration of PnPP-19 in all preparations was 5 mg / mL. All preparations were filled into autoclaved glass vials and stopped using autoclaved stoppers. The vials were then sealed and stored at 5°C, 25°C, and 40°C for 2 and 4 weeks. The preparations were tested by visual assessment (viewing the vials on the bench under ambient light), RP-HPLC, asymmetric field-flow fractionation (AF4-MALLS), and osmolality.

[0127] According to Table 3, formulations with a pH above 6.5 exhibited an unstable chemical profile, defined as a greater than 20% reduction in peptide content when analyzed by RP-HPLC.

[0128] [Table 3]

[0129] In conclusion, to maintain purity and stability, the pH range in PnPP-19 formulations should be between 4 and 6.5.

[0130] Example 4: Determination of buffer type The purpose of this experiment was to assess the stability of the peptide in a wide range of buffers.

[0131] The presence of buffer was essential to stabilize the peptide, as preparations without buffer became turbid with particles and increased viscosity.

[0132] After the buffer is introduced, it is necessary to adjust the pH. If acetic acid is used to adjust the pH, the formulation will have an unpleasant odor, which is an undesirable sensory characteristic, making patient acceptance difficult. If NaOH or HCl are used to adjust the pH, there is no particular odor, and therefore NaOH and HCl are the substances of choice for adjusting the pH of formulations after the buffer is introduced.

[0133] Seventeen formulations were made using different buffer types, including sodium acetate, trisodium citrate dihydrate (citrate), L-histidine, potassium phosphate, and Tris hydrochloride. The starting concentration of PnPP-19 in all preparations was 5 mg / mL. All preparations were filled into autoclaved glass vials and stopped using autoclaved stoppers. The vials were then sealed and placed at 5°C, 25°C, and 40°C for 2 and 4 weeks. The preparations were tested by visual assessment (viewing the vials on the bench under ambient light), RP-HPLC, AF4-MALLS, and osmolality.

[0134] According to Table 4, citrate and Tris do not stabilize PnPP-19, showing a reduction in peptide content of more than 10% when analyzed by RP-HPLC. In F04 with the citrate formulation, the peptide is almost completely lost.

[0135] [Table 4]

[0136] Other preparations with different pH showed that sodium acetate and potassium phosphate increased stability independent of pH, while the effect of L-histidine was small compared to the effect of pH.

[0137] In conclusion, to maintain purity and stability, the buffers of choice for stabilizing PnPP-19 are sodium acetate and potassium phosphate, and any necessary pH adjustments after buffer introduction must be made with NaOH and HCl.

[0138] Example 5: Determination of additional excipients The purpose of this experiment was to evaluate the stability of the peptide in a wide range of additional excipients.

[0139] Seventeen formulations were made with different additional excipients, including hydroxypropyl beta cyclodextrin (HPCD), alpha cyclodextrin, polyethylene glycol (PEG) 400, PEG 6000, poloxamer 407, monoethyl ether (Transcutol P), hydroxyethyl cellulose (HEC), polysorbate 20 (PS20), and microemulsion.

[0140] The starting concentration of PnPP-19 for all preparations was 5 mg / mL. All preparations were filled into autoclaved glass vials and stopped using autoclaved stoppers. The vials were then sealed and placed at 5°C, 25°C, and 40°C for 2 and 4 weeks. Preparations were tested by visual assessment (view of vials on bench under ambient light), RP-HPLC, AF4-MALLS, and osmolality.

[0141] According to Table 5, only alpha cyclodextrin and poloxamer 407 did not increase the chemical degradation of PnPP-19 (less than 10% reduction in peptide content as analyzed by RP-HPLC). Analysis by SE-HPLC also confirmed that formulations with HEC, PEG6000, PEG400, and PS20 reduced physical stability. Microemulsion formulations could not be analyzed by RP-HPLC due to technical limitations.

[0142] [Table 5]

[0143] Example 6: In vivo release and permeation studies using FAPP20 formulations All of the additional excipients selected in Example 5 have the effect of increasing peptide stability, but can also "trap" the peptide, avoiding its release and preventing its penetration through the skin.

[0144] Release and permeation experiments were performed to determine whether PnPP-19 in FAPP20 formulations in microemulsions, formulated with alpha cyclodextrin or Transcutol P, could cross different physical barriers in adequate amounts.

[0145] In the release studies, a cellulose acetate membrane was used to create a physical barrier between the donor compartment, where the formulation was added, and the recipient compartment, where the peptide was measured.

[0146] In the first permeation assay, the physical barrier was whole porcine skin, which mimics the human stratum corneum. In the second permeation study, the physical barrier was a Strat-M membrane, which mimics the human stratum corneum.

[0147] The assay was performed in a 1.767 cm 2 tube with a donor compartment having a volume of 1 mL and a recipient compartment having a volume of 7 mL. 2 The study was performed in a Franz vertical diffusion cell with a peptide diffusion zone of 100 μL. To the recipient compartment, 7 mL of a biorelevant solution was added, in which the medium selected through preliminary testing was 20 mM sodium acetate buffer pH 5.0 + 0.5% Tween® 20. To the donor compartment, 300 μL of FAPP20, microemulsion, alpha cyclodextrin, and Transcutol P formulation were added. The cell was placed in a thermostatic bath at 37°C ± 0.5°C to mimic the surface temperature. The bioreceptor medium was stirred at 350 rpm. At 0.5, 1, 2, 4, 6, and 24 hours, 1 mL of aliquots were withdrawn from the recipient compartment to measure the peptide content.

[0148] In the release study, three controls were used to validate the assay, which demonstrated that PnPP-19 could be quantified in the receptor medium after 6 hours (peak) and up to 24 hours when the peptide was applied in saline or in buffer in different formulations in the donor compartment.

[0149] In the first permeation assay, PnPP-19 was formulated with 6 μM alpha cyclodextrin or 15% Transcutol. However, PnPP-19 was not detected in the receptor medium in these formulations, demonstrating that both formulations "trap" the peptide and prevent its release. In the second permeation assay, PnPP-19 was formulated in FAPP20 or a microemulsion. 0.5% (w / v) PnPP-19 in a microemulsion or FAPP20 formulation was added to the donor compartment.

[0150] The release profile of PnPP-19 in the microemulsions showed a maximum of approximately 11%, which rapidly decreased after 4 hours of assay to 2% after 24 hours.

[0151] The release profile of PnPP-19 in the FAPP20 formulation showed a maximum of 12% after 6 hours of assay, which slowly decreased to 6% after 24 hours (Figure 1).

[0152] Based on the above release results, 20 mM sodium acetate buffer pH 5.0 + 0.5% Tween® 20 was confirmed to be the best recipient medium to use in permeation assays with Strat M membranes.

[0153] A permeation study was performed using one control, which demonstrated that permeation of PnPP-19 when applied in FAPP20 buffer (20 mM sodium acetate buffer solution) was detected in the receptor compartment from the first time point (0.5 h).

[0154] 0.5% w / v PnPP-19 in microemulsion or in the FAPP20 formulation was added to the donor compartment.

[0155] PnPP-19 in the microemulsion formulation was not detected in the receptor medium.

[0156] As shown in Figure 2, PnPP-19 in the FAPP20 formulation was detectable from the first time point (0.5 hours) and the concentration increased up to 24 hours.

[0157] In conclusion, the FAPP20 formulation allowed sustained release and permeation of PnPP-19 through physical barriers over a 24-hour period. PnPP-19 formulated in alpha-cyclodextrin, Transcutol P, or microemulsions "traps" the peptide, preventing its release and permeation.

[0158] Example 7: Analysis of long-term stability of representative formulations Stability analysis of PnPP-19 in formulation FAPP20 and other representative formulations was performed using a long-term protocol (temperature: 30°C ± 2°C and relative humidity (RH) 75% ± 5%) at three different time points: time 0 (T0), one month (T1), and three months (T3). The stability protocol was performed in accordance with Resolution 318 / 2019 of the Brazilian regulatory authority (ANVISA - Agencia Nacional de Vigilancia Sanitaria). A validated high-performance liquid chromatography (HPLC) method was used for content analysis. Additionally, the following criteria were evaluated: physical characteristics, pH, viscosity, weight loss, and microbiological analysis. The results are listed in Table 6 below.

[0159] [Table 6]

[0160] Based on the results shown above, the PnPP-19 peptide remains stable (>80% peptide content) after 3 months of long-term resting conditions.

[0161] For other listed representative formulations (Table 2), long-term stability studies were conducted for up to 3 months at a temperature of 25°C. Each formulation was prepared in triplicate or in six replicates (n = 3-6). The results are shown in Table 7.

[0162] [Table 7]

[0163] Example 8: Photodegradation of PnPP-19 in Representative Formulations and Long-Term Stability in the Dark Photodegradation studies were performed to assess the effect of UVA and UVB irradiation on peptide degradation in representative formulations. The following formulations were tested in this assay: FAPP22.1 and FAPP22.4.

[0164] Therefore, each formulation was evaluated in two ways: I) with UV protection and II) without UV protection. All samples were incubated in a UVA / UVB chamber (UVA source (95%) + UVB (5%)) for 96 hours. After this period, the samples were analyzed by HPLC-DAD using the same previously validated analytical method.

[0165] The results are shown in FIG.

[0166] [Table 8]

[0167] In addition, a long-term stability study was conducted on formulation FAPP20 with and without light protection for 6 months to obtain the peptide content, as shown in Table 9.

[0168] [Table 9]

[0169] Consequently, the formulation should be protected from light to increase the stability of PnPP-19.

[0170] Example 9: Biodistribution and Pharmacokinetics of PnPP-19 in FAPP20 Formulation Following Topical Administration in Rats To assess the in vivo penetration of the peptide (and / or its fragments) in formulation FAPP20, biodistribution studies were performed in healthy rats. The peptide was iodinated using the chloramine T method. 131 I]NaI.

[0171] The study was conducted considering a dose of 5 mg / ml and maximum area of ​​genital application. The final application volume was 100 μL, resulting in a dose of 2 mg / kg. Biodistribution evaluation was performed after administration of a single dose of peptide, which was applied topically to the skin of the penis after dividing the rat's genital area into thirds.

[0172] Regarding biodistribution data, radiolabeled peptide concentrations were higher in skin, foreskin, and SMC tissues at all time points tested. At the initial analysis time of 15 minutes, it was already possible to detect the peptide in the target tissues, the SMC layer and the first layer of skin (referred to herein as the foreskin), where the nerves of the penis are found, indicating that the peptide was rapidly absorbed into the skin. Within 30 minutes, peptide detection decreased at the initial application site (skin) and increased in the foreskin, indicating that the peptide continued to be absorbed and remained stable in the SMC layer. Within 60 minutes, the peptide was practically undetectable in tissues (Figure 4).

[0173] For pharmacokinetic (PK) studies, blood samples were collected from animals at time 0 (pre-dose), 15 minutes, 30 minutes, 60 minutes, 3 hours, 8 hours, and 24 hours. All collected samples were quantified using a Wizard 3 gamma counter (PerkinElmer) for subsequent calculation of the percentage of applied dose per gram of organ. Figure 5 shows another analysis of the biodistribution of peptide PnPP-19 at 15 minutes. While 42% of the applied dose was found in the first layer of skin, up to 18% of the peptide was detected in the SMC layer. Neither the peptide nor its fragments were found systemically (<1%) in selected organs (kidney, testis, and epididymis) nor in blood at all evaluated time points, indicating that the formulation remained at the application site without systemic absorption.

[0174] These data confirm that the FAPP20 formulation allows the peptide to rapidly penetrate the skin, cross the tunica albuginea, and rapidly reach target tissues in high amounts. Additionally, this biodistribution study confirmed the absence of systemic exposure of PnPP-19 or its fragments.

[0175] Example 10: Effect of peptide PnPP-19 in FAPP20 formulation on erectile function in healthy rats In this study, PnPP-19 was produced using either TFA or acetate as a counterion. Typically, peptides with TFA as the counterion are used in the preclinical phase of development. However, for safety reasons, the counterion is changed to acetate during the clinical phase of development. Therefore, this experiment was conducted to evaluate the efficacy of PnPP-19 in the FAPP20 formulation on erectile function in healthy rats. Additionally, to compare whether the exchange of counterions affects the biological effects of PnPP-19, two peptide batches were produced, one with TFA as the counterion and the other with acetate as the counterion. Sildenafil was used as a positive control (subcutaneous administration).

[0176] Erectile function was assessed by the intracavernous pressure / mean arterial pressure (ICP / MAP) ratio at four different time points (0, 15, 30, and 45 min) after application of 8 Hz ganglionic functional electrical stimulation (EFS). Healthy adult male Sprague-Dawley rats were divided into four groups, with eight animals per group: (i) a negative control treated with the formulation of interest (FAPP20) (vehicle) without peptide and saline, applied subcutaneously (SC); (ii) the peptide PnPP-19 formulated in FAPP20 with an acetate counterion; (iii) the peptide PnPP-19 formulated in FAPP20 with a TFA counterion; and (iv) a positive control administered SC with sildenafil.

[0177] Formulated peptides (both with TFA counterions and acetate) were applied topically to the rat penis at a dose of 1000 μg diluted in 200 μL of formulation (4 mg / kg dose), resulting in a concentration of 5 mg / mL or 0.5% w / v in the peptide formulation, 15 minutes prior to ICP / MAP measurements. Sildenafil was applied SC at a dose of 25 mg / kg (1 mL / kg) 30 minutes prior to ICP / MAP measurements.

[0178] Sildenafil showed a trend at 30 minutes, but this was not statistically significant, and sildenafil was not superior to the negative control group (vehicle). Both the peptides with TFA and those with acetate in the FAPP20 formulation were superior to the control, particularly at 15 and 30 minutes, with no statistical difference between the peptides (Figure 6). In conclusion, peptides in the FAPP20 formulation, regardless of counterion, are effective in enhancing erections, with a rapid onset of action.

[0179] Again, no signs of dermal irritation were observed, and there was no difference in MAP between the peptide- and vehicle-treated groups, confirming that the peptide does not cause systemic hypotension in rats when applied topically. Conversely, the sildenafil-treated group had a significantly reduced MAP compared to the vehicle-treated group.

[0180] In conclusion, these data demonstrated that PnPP-19 in the FAPP20 formulation has a rapid onset of action, reaches target tissues in effective amounts, does not cause systemic hypotension, and has no local side effects (no signs of dermal irritation).

[0181] [Table 10]

[0182] Example 11: Subchronic Toxicity and Toxicokinetics of PnPP-19 in FAPP20 Formulation Following Daily Administration to Pigs for 60 Days Subchronic toxicity and toxicokinetic studies were conducted in Brazilian minipigs (male and female) after administration of the peptide in formulation FAPP20. The study was conducted under GLP conditions at the Centro de Estudos Pre-Clinicos of the Instituto Israelita de Ensino e Pesquisa Hospital Albert Einstein.

[0183] This species was chosen based on literature stating that the minipig is the best model for preclinical studies with topical drugs, as the skin of this animal is classified as most similar to human skin, both in terms of structure and pharmacokinetics (Bode, 2010).

[0184] The topical route was chosen because it was the intended route in clinical studies. The application sites, the genital area (penis or vulva, and the entrance to the vaginal canal), were chosen for similar reasons. Regarding dosage, since the investigational drug in question has neither a maximum tolerated dose (MTD) nor a maximum no observed adverse effect dose (NOAEL), it was decided to conduct this study considering the maximum administrable dose (MFD) of the peptide in the FAPP20 formulation (0.7% w / v or 7 mg / mL) and the maximum application volume (the largest possible applicable area within the genital area of ​​the pig, normalized to 1 mL). Therefore, the dose used was 7 mg per pig, which is equivalent to 0.7 mg / Kg.

[0185] A 60-day study period was selected to support a Phase 2 clinical trial of the same duration. The number of pigs per group was selected according to the OECD 409 guide. A recovery group was included to determine the resolution and reversibility of adverse reactions if detected. Thus, the experimental groups were: (1) peptide in FAPP20 formulation (daily application of 7 mg of peptide in 1 mL for 60 days), (2) peptide-recovery in FAPP20 formulation (daily application of 7 mg of peptide in 1 mL formulation for 60 days, followed by an additional 7 days of recovery without drug application), and (3) vehicle (daily application of FAPP20 formulation without peptide for 60 days).

[0186] Each pig was observed at least daily for clinical signs (including skin / hair; respiratory, urinary, and nervous systems; appetite; locomotion, defecation, salivation, vomiting, and others), morbidity, and mortality. Water and feed consumption assessments were qualitative. Body weight was monitored weekly. Blood samples were collected weekly (under general anesthesia) for evaluation of toxicological effects over time (time points 1–10). A baseline sample was collected as an animal control (time point 1) and included in the hematology, biochemistry, coagulogram, and urinalysis analyses. Additionally, for toxicokinetic analysis, blood samples were collected on days 1 and 60 after peptide administration to examine the detection and quantification of the peptide and its fragments. For this analysis, blood was collected at the following time intervals on days 1 and 60: 0 (pre-dose), 15 min, 30 min, 60 min, 3 h, 8 h, and 24 h.

[0187] At the end of the study, pigs were euthanized with a large dose of anesthetic at the time points specified for each group. During necropsy, a gross examination of all organs was performed, and the following organs were collected for histopathological examination: liver with gallbladder, kidneys, adrenal glands, testes, epididymis, ovaries, uterus, thyroid gland (with parathyroid glands), spleen, brain, and heart.

[0188] No mortality or morbidity was observed in either male or female groups throughout the experimental period.

[0189] Regarding clinical changes, these were not considered to be clinically significant because the episodes in males were rare, isolated, resolved spontaneously, and occurred randomly and at different treatment weeks in both groups.

[0190] In females, diarrheal episodes occurred only in the treatment group. Pasty diarrhea began during the third week of treatment and continued through the seventh week of treatment. There were no episodes of chyme during the recovery period. Therefore, chyme was considered clinically relevant, but was reversible and resolved spontaneously.

[0191] Hematology, coagulation, and biochemistry showed only isolated and time-dependent changes in both males and females that were not considered clinically significant.

[0192] Histology revealed no clinically significant differences between the treatment and vehicle groups in either female or male pigs. One treatment male had purulent balanitis, one treatment female had vascular lesions on the mucosal surface, and one vehicle female had apocrine gland inflammation. All of these histological findings had no clinical or laboratory correlation, and because they were isolated, they were not considered clinically significant.

[0193] Finally, with regard to the toxicokinetic analysis, the peptide and its fragments were not detected in the blood of the pigs at any of the time points analyzed.

[0194] Therefore, it was concluded that the peptide PnPP-19 in the FAPP20 formulation of the present invention did not show any toxic effects or local side effects in the dermis in pigs. The only adverse event considered relevant was a more frequent occurrence of episodes of pasty stool in the treated group in females, which was reversible and resolved spontaneously. According to the toxicokinetic analysis, the tested product did not show any systemic exposure in either sex (male or female).

[0195] Example 12: Texture analysis of representative formulations The perception of representative formulations of the present invention was tested in a texture assay.

[0196] Texture assays were performed using a CT310K Texture Analyzer (Brookfield, EUA) in compression mode with a flat baseplate probe TA-DEC. This probe is typically used to determine material hardness values. In this assay, the probe was pressed into each sample to a specified depth (1 mm) at a specified speed (2 mm / s). When an initial force of 2 g was applied, the probe penetrated the sample to a depth of 1 mm. During this time, the force to penetrate into the sample increased. Once a specific penetration distance was reached, the probe was withdrawn from the sample at a post-test speed of 2 mm / s. The maximum force value on the graph is a measure of the sample's hardness at the specified depth. The minimum force value on the graph when the probe was withdrawn is a measure of tackiness.

[0197] The parameter of hardness can provide insight into the spreading behavior of each formulation, as harder formulations spread less and vice versa. Hardness (or stiffness) is the force required until the material exhibits the required deformation. Tack is the amount of work required to overcome the surface attractive forces between the sample and the probe.

[0198] The test was performed in quadruplicate over a temperature range of 28.2°C ± 0.4°C above the gelling temperature of the sample.

[0199] The FAPP22.1 formulation exhibited a more water-like texture (Table 11), indicating that the FAPP22.1 formulation was fluid. The FAPP22.4 formulation had an increased hardness of approximately 600 g and a higher viscosity of approximately 13 mJ. Both formulations are suitable for topical application when some degree of spreadability on the skin and / or mucous membranes is desired. These findings support the results from the rheological studies. The behavior of the FAPP22.4 formulation on the skin follows that of a semisolid gel that remains intact when applied to the skin. By applying a spreading force, the initial tension of the material is released and it begins to flow, followed by a decrease in viscosity and, consequently, easier spreadability. In contrast, the material of the FAPP22.1 formulation begins to flow on the skin immediately after application.

[0200] [Table 11]

[0201] In addition, both FAPP22.1 and FAPP22.4 are translucent, do not have any undesirable odor, and are of uniform appearance, characteristics that are important for facilitating compliance.

[0202] Example 13: Safety and Pharmacokinetics of FAPP20 Formulations in Human Studies The pharmaceutical formulations described by the present disclosure were administered to human subjects during a Phase 1 study entitled "Phase 1 clinical study to evaluate safety and pharmacokinetics of BZ371A in the form of gel applied to the genitalia of healthy men and women." The objectives of the study were to determine the safety and tolerability of BZ371A when applied topically to the male and female genitalia, and to determine the systemic presence of BZ371A and its fragments when BZ371A was applied topically to the male and female genitalia via blood pharmacokinetic evaluation.

[0203] BZ371A was administered at a concentration of 5 mg / mL in formulation FAPP20 and applied topically to the genitals (in men: entire penis including glans; in women: clitoris, labia majora, and labia minora) in a volume of 1.5 mL.

[0204] The study consisted of three visits and was 7 days in duration. Medical and laboratory analyses were performed to assess safety, and blood was collected for pharmacokinetic analysis. Quantitation and detection of BZ371A and its fragments in blood was performed by a suitable analytical method specifically developed and previously validated for this purpose.

[0205] Twelve healthy adult subjects (6 males and 6 females) were included in the study, with no restrictions regarding ethnicity, class, or social group, and all of these subjects were assigned to a group. Only subjects in whom the FAPP20 was properly administered were included in the analysis.

[0206] The following outcomes will be considered in the analysis of the safety and tolerability of BZ371A: (1) reports of adverse events (AEs), (2) local physical examination in the genital area, (3) vital signs, including systemic blood pressure and heart rate; electrocardiogram; and laboratory tests, including complete blood count (including platelets), coagulogram, biochemistry (glucose, sodium, potassium, urea, creatinine, chloride, total calcium, phosphorus, total protein and fractions (albumin and globulin), aspartate transaminase, alanine transaminase, alkaline phosphatase, gamma-glutamyltransferase, total cholesterol, HDL cholesterol, triglycerides, pH; and urinalysis.

[0207] For pharmacokinetic analysis, the following outcomes were considered: detection of BZ371A and its fragments in blood at the following collection times: pre-dose, 15, 30, 60, 180, and 360 minutes. If detected, the pharmacokinetic parameters maximum concentration, half-life, area under the curve, clearance, and volume of distribution were determined.

[0208] FAP20, containing BZ371A at a concentration of 5 mg / mL applied in a volume of 1.5 mL to the genital area (entire penis including glans, and clitoris, labia majora, and labia minora), proved safe and well tolerated. There were no clinically important changes in safety outcomes related to laboratory tests, vital signs, ECG, and physical examination.

[0209] Regarding pharmacokinetics, bioanalytical results demonstrated that plasma concentrations of BZ371A were unquantifiable (lower limit of quantification (LIQ) = 50 ng / mL) and concentrations of BZ371A fragments were undetectable (limit of detection = 40 ng / mL) in all 12 subject recoveries.

[0210] In conclusion, FAPP20 has been proven to be safe and tolerable in humans while avoiding systemic exposure to BZ371A and its fragments.

[0211] [Representative sequences] The amino acid sequence of the peptide PnPP-19 described by the present disclosure (SEQ ID NO: 1) is shown below: GERRQYFWIAWYKLANSKK

[0212] [Related Applications] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 485,866, filed February 17, 2023, entitled "TOPICAL PEPTIDE FORMULATION FOR THE TREATMENT OF SEXUAL DYSFUNCTION," the entire contents of which are incorporated herein by reference.

Claims

1. (i) a synthetic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1, present at a concentration between 0.1% and 0.7% w / v; (ii) a surfactant, (iii) propylene glycol, and (iv) a buffer having a pH between 4.0 and 6.5 1. A topical pharmaceutical formulation comprising:

2. (i) a synthetic peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, present at a concentration of between 0.1 and 0.7% w / v; (ii) a surfactant present in a concentration range of between about 2.5 and 18.0% w / v; (iii) propylene glycol present in a concentration range of between about 7.5 and 30.0% w / v; and (iv) a buffer present in a concentration range between about 50.0 and 73.5% v / v Including, A topical pharmaceutical formulation, wherein the pH of said buffer is between about 4.0 and 6.

5.

3. 3. The topical pharmaceutical formulation of claim 1 or 2, wherein the surfactant is a poloxamer, preferably the poloxamer comprises poloxamer 188, poloxamer 407, or a combination thereof.

4. A topical pharmaceutical formulation according to any one of claims 1 to 3, wherein the buffer comprises sodium acetate or potassium phosphate.

5. A topical pharmaceutical formulation according to any one of claims 1 to 4, further comprising a preservative or antioxidant.

6. 6. The topical pharmaceutical formulation of any one of claims 1 to 5, further comprising one or more additional excipients, optionally wherein said one or more additional excipients comprise fillers, lubricants, humectants, flavoring agents, fragrances, and / or emulsifiers.

7. The topical pharmaceutical formulation of any one of claims 1 to 6, which is a cream, gel, lotion, ointment, or spray.

8. 8. A topical pharmaceutical formulation according to any one of claims 1 to 7, which penetrates the fibrous layer of the tunica albuginea of ​​a subject without exerting systemic effects.

9. 10. A method of treating a disease that would benefit from modulation of smooth muscle cell contractility, comprising the step of locally administering to a subject in need thereof the formulation of any one of claims 1 to 8.

10. 10. The method of claim 9, wherein the disease that would benefit from modulation of smooth muscle cell contractility is male sexual dysfunction or female sexual dysfunction.

11. 10. The method of claim 9, wherein the male sexual dysfunction is selected from the group consisting of erectile dysfunction, delayed ejaculation, premature ejaculation, and male hypoactive sexual desire disorder.

12. 10. The method of claim 9, wherein said female sexual dysfunction is selected from the group consisting of female sexual arousal disorder, female orgasmic disorder, female hypoactive sexual desire disorder, and genito-pelvic pain / insertion disorder, and optionally said genito-pelvic pain / insertion disorder is dyspareunia or vaginismus.

13. 8. A method of treating male sexual dysfunction, comprising topically administering to a subject in need thereof the formulation of any one of claims 1 to 7.

14. 14. The method of claim 13, wherein the male sexual dysfunction is selected from the group consisting of erectile dysfunction, delayed ejaculation, premature ejaculation, and male hypoactive sexual desire disorder.

15. 8. A method of treating female sexual dysfunction, comprising topically administering to a subject in need thereof the formulation of any one of claims 1 to 7.

16. 16. The method of claim 15, wherein said female sexual dysfunction is selected from the group consisting of female sexual arousal disorder, female orgasmic disorder, female hypoactive sexual desire disorder, and genito-pelvic pain / insertion disorder, and optionally said genito-pelvic pain / insertion disorder is dyspareunia or vaginismus.

17. The method of any one of claims 9 to 16, wherein said administering comprises administering to the skin, mucosa, or urethra of said subject.

18. 15. The method of claim 13 or 14, wherein the subject is male and the formulation is administered to the subject's penis or urethra.

19. 17. The method of claim 15 or 16, wherein the subject is a female and the formulation is administered to the subject's genitals, and optionally the formulation is administered to the subject's labia minora and labia majora and / or clitoris.

20. The method of any one of claims 9 to 19, wherein said administering comprises daily application of said formulation to said subject.

21. 9. A method for producing a pharmaceutical formulation according to any one of claims 1 to 8, comprising the step of introducing a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 into a pharmaceutically acceptable excipient, wherein the peptide is present in an amount between about 0.1% and 0.7% w / v.

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