Formulations and methods for treating erectile dysfunction

Abstract

The formulations disclosed herein have beneficial properties that enhance the solubility and permeability (flux) of at least one of vardenafil, sildenafil, and / or related phosphodiesterase inhibitors or other ionizable compounds. In particular, the formulations impart beneficial properties to the compounds as they pass through mucous membranes, resulting in effective plasma drug concentrations. In some embodiments, the formulations include an organic water-soluble solvent that may include at least one of alcohol, polyether, diethylene glycol monoethyl ether, medium chain glycerides, one or more saturated polyglycolized C8-C10 glycerides, and / or organic salts, or any combination thereof. In some embodiments, the formulations have a pH of about 3.5 to about 8.0. Also disclosed herein is the use of the formulations in administering one or more compounds for the treatment of erectile dysfunction or other disorders, where the one or more compounds are administered transmucosally (intranasally or sublingually).

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application is a continuation-in-part of International Application No. PCT / US21 / 34334, filed May 26, 2021.

[0002] There is a need for improved formulations and methods for treating erectile dysfunction. The present technology generally relates to formulations and methods for treating erectile dysfunction using phosphodiesterase inhibitors, but can also be applied to other agents in treating different disease states using transmucosal administration, e.g., sublingual or nasal administration. [Background technology]

[0003] Erectile dysfunction is considered the most common form of sexual dysfunction in men and becomes increasingly common with age. It is estimated that approximately 50% of men aged 40-70 years and 70% of men over 70 years suffer from erectile dysfunction. The condition is not limited to older men, as erectile dysfunction can be caused by one or more of the following factors: neurological, vascular, endocrinological, and psychological. Other risk factors, such as cardiovascular disease, hypertension, diabetes, hypercholesterolemia, and smoking, are strongly associated with an increased prevalence of erectile dysfunction. As a result, there is an increasing need for effective treatment of erectile dysfunction. Summary of the Invention

[0004] For example, there is a need for compositions and methods that can sufficiently solubilize and allow sufficient permeation of phosphodiesterase inhibitors, including vardenafil, sildenafil, and tadalafil. Disclosed herein in some embodiments is an organic-aqueous mixture that can sufficiently solubilize phosphodiesterase inhibitors and is relatively safe or well tolerated by human subjects. In some embodiments, the organic-aqueous mixture is screened and identified based on the solubility of the phosphodiesterase inhibitor. In some embodiments, the phosphodiesterase inhibitor is vardenafil. In some embodiments, the phosphodiesterase inhibitor is sildenafil. In some embodiments, the phosphodiesterase inhibitor is tadalafil. In some embodiments, pH and osmotic effects are determined.

[0005] Described herein, in some embodiments, is a method of identifying a formulation for enhancing the solubility and permeability of one or more phosphodiesterase inhibitors across a mucosa, the method comprising: (a) one or more phosphodiesterase inhibitors; and (b) an organic water-soluble solvent comprising an alcohol, a glycol, diethylene glycol monoethyl ether, a medium chain glyceride, one or more saturated polyglycolized C8-C10 glycerides, or a combination thereof; wherein the formulation has a pH of about 3.5 to about 8.0, and the organic water-soluble solvent enhances the solubility of the one or more phosphodiesterase inhibitors compared to the solubility of the one or more phosphodiesterase inhibitors in water. In some embodiments, the organic water-soluble solvent comprises an alcohol. In some embodiments, the formulations described herein comprise one or more weak salts. Exemplary weak salts include, for example, citric acid, tartaric acid, acetic acid, flumaric acid, lactic acid, ammonium chloride, or similar organic salts. In some embodiments, the formulations described herein comprise N-methylpyrrolidone (NMP), Tween 80, or similar organic compounds. In some embodiments, the formulations described herein are a combination of Tween 80 or similar organic compounds, including citric acid, tartaric acid, acetic acid, flumaric acid, lactic acid, a weak salt such as ammonium chloride or a similar organic salt, or N-methylpyrrolidone (NMP), with one or more alcohols, polyethers, diethylene glycol monoethyl ether, medium chain glycerides, one or more saturated polyglycolized C8-C10 glycerides, or combinations thereof. In some embodiments, the alcohol is ethanol or glycerol. In some embodiments, the ethanol is present at a concentration of 5% to 40%. In some embodiments, the ethanol is present at a concentration of 12%, 25%, or 30%. In some embodiments, the organic water-soluble solvent comprises a polyether. In some embodiments, the polyether is polyethylene glycol. In some embodiments, the polyethylene glycol is PEG 6000 or PEG 400. In some embodiments, the polyethylene glycol is present at a concentration of 1% to 20%.In some embodiments, the polyethylene glycol is present in a concentration of 5%. In some embodiments, the formulation has a pH of about 3.5 to about 8.0. In some embodiments, the phosphodiesterase inhibitor is vardenafil, sildenafil, tadalafil, or a combination thereof. In some embodiments, the phosphodiesterase inhibitor is vardenafil. In some embodiments, the phosphodiesterase inhibitor is sildenafil. In some embodiments, the phosphodiesterase inhibitor is tadalafil.

[0006] In some embodiments described herein, a method of treating erectile dysfunction in a subject in need thereof is provided, comprising contacting a mucosa of the subject with a formulation disclosed herein, thereby treating the erectile dysfunction in the subject. In some embodiments, contacting the mucosa comprises nasal administration. In some embodiments, contacting the mucosa comprises sublingual administration.

[0007] Described herein in some embodiments is a method of preparing a formulation for treating erectile dysfunction in a subject, the method comprising: (a) adding one or more phosphodiesterase inhibitors to an organic water-soluble solvent comprising an alcohol, a polyether, diethylene glycol monoethyl ether, a medium chain glyceride, one or more saturated polyglycolized C8-C10 glycerides, or a combination thereof; (b) adjusting the pH of the organic water-soluble solvent comprising the one or more phosphodiesterase inhibitors to about 3.5 to about 8.0, wherein treating erectile dysfunction comprises contacting the formulation with a mucous membrane of the subject. In some embodiments, the formulation described herein comprises one or more weak salts. Exemplary weak salts include, for example, citric acid, tartaric acid, acetic acid, flumaric acid, lactic acid, ammonium chloride, or similar organic salts. In some embodiments, the formulation described herein comprises N-methylpyrrolidone (NMP), Tween 80, or similar organic compounds. In some embodiments, the formulations described herein are combinations of Tween 80 or similar organic compounds, including citric acid, tartaric acid, acetic acid, flumaric acid, lactic acid, weak salts such as ammonium chloride or similar organic salts, or N-methylpyrrolidone (NMP), with one or more alcohols, polyethers, diethylene glycol monoethyl ether, medium chain glycerides, one or more saturated polyglycolized C8-C10 glycerides, or combinations thereof. In some embodiments, the solubility of one or more phosphodiesterase inhibitors is increased in an organic water-soluble solvent compared to the solubility of one or more phosphodiesterase inhibitors in water. In some embodiments, the permeability of one or more phosphodiesterase inhibitors across mucosa is increased in an organic water-soluble solvent compared to the permeability of one or more phosphodiesterase inhibitors in water. In some embodiments, the permeation of one or more phosphodiesterase inhibitors across an in vitro artificial membrane is increased in an organic water-soluble solvent compared to the permeation of one or more phosphodiesterase inhibitors in water.In some embodiments, the bioavailability of the one or more phosphodiesterase inhibitors is increased in the organic water-soluble solvent compared to the bioavailability of the one or more phosphodiesterase inhibitors in water. In some embodiments, the organic water-soluble solvent comprises an alcohol. In some embodiments, the alcohol is ethanol or glycerol. In some embodiments, the ethanol is present at a concentration of 5% to 40%. In some embodiments, the ethanol is present at a concentration of 12%, 25%, or 30%. In some embodiments, the organic water-soluble solvent comprises a polyether. In some embodiments, the polyether is polyethylene glycol. In some embodiments, the polyethylene glycol is PEG 6000 or PEG 400. In some embodiments, the polyethylene glycol is present at a concentration of 1% to 20%. In some embodiments, the polyethylene glycol is present at a concentration of 5%. In some embodiments, the formulation has a pH of about 3.5 to about 8.0. In some embodiments, the phosphodiesterase inhibitor is vardenafil, sildenafil, tadalafil, or a combination thereof. In some embodiments, the phosphodiesterase inhibitor is vardenafil. In some embodiments, the phosphodiesterase inhibitor is sildenafil.In some embodiments, the phosphodiesterase inhibitor is tadalafil. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows the calibration curve of vardenafil concentration in purified water (y=0.00853x+0.006553, R2=0.9962). [Diagram 2] FIG. 2 shows the stable solubility concentration (mg / ml) of vardenafil hydrochloride trihydrate in water at different pH values ​​using the HPLC method. [Diagram 3] FIG. 3 shows the simultaneous measurement of the solubility of a saturated solution of vardenafil hydrochloride trihydrate (mg / ml) in water, 12% alcohol and 30% alcohol. [Figure 4] FIG. 4 shows the relationship between the apparent permeability coefficient (Papp) of vardenafil at 6 and 12 hours in the PAMPA study. [Diagram 5] FIG. 5 shows a comparison of the effect of pH on Papp (panel a) and Jss (panel b) of various formulations. [Figure 6] Figure 6 shows a comparison of vardenafil Papp values ​​in different solutions using either the PAMPA or Calu-3 cell line model. Open circles represent water solutions. Closed circles represent EtOH (12%), PEG400 (15%), NMP (10%), calcium lactate (5%), EtOH / PEG400 (12% / 15%), EtOH / calcium lactate (12% / 5%). Only the lowest Papp (bottom left) for PEG400 (15%) is significantly lower than in water (p<0.05 t-test). [Figure 7] FIG. 7 shows representative curves of vardenafil concentration in the plasma of subject 11 ("S11") after either intranasal (IN) or oral (PO) administration. [Figure 8] FIG. 8 shows the effect of pH on sildenafil flux (Jss) based on PAMPA studies. [Figure 9] Figure 9 shows a comparison of Papp values ​​in different solutions using either the PAMP or Calu-3 cell line model. Open circles represent aqueous solutions. Closed circles represent acetate / NMP / calcium lactate (5 / 10 / 3.5%), acetate / calcium lactate (5 / 3.5%), acetate / calcium lactate (1 / 3.5%), NMP (10%), calcium lactate (3.5%). [Figure 10] FIG. 10 shows a comparison of vardenafil hydrochloride trihydrate permeation over 24 hours in water (columns 1-5), 12% ethanol in water (columns 6-10), and 30% ethanol-water solution (columns 11-15). Saturation concentrations were used. [Figure 11] FIG. 11 shows a comparison of vardenafil permeation using saturating concentrations in glycerin (glycerol), polyethylene glycol (PEG), and PEG ethanol (EtHO) mixtures. [Figure 12] FIG. 12 shows the calibration curve of vardenafil concentration using purified water (y=0481x+0.0033; R2=0.9994). [Figure 13] FIG. 13 shows the calibration curve of vardenafil in 25% ethanol aqueous mixture (y=0.583x+0.0043; R2=0.9945). [Figure 14] FIG. 14 shows the simultaneous measurement of the saturation solubility of vardenafil drug substance in water, 12% and 30% alcohol (EtOH). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention relates to formulations and methods that optimize the solubility and permeability of phosphodiesterase inhibitors in mucosa.The formulations and methods provided herein can be used, for example, in the treatment of erectile dysfunction.Those skilled in the art will understand that the formulations and methods disclosed herein are applicable to any ionizable compound, including acidic and basic compounds, as well as drugs or other compounds that are not phosphodiesterase inhibitors.Non-limiting examples of ionizable compounds that can be administered into mucosa through the formulations disclosed herein include levodopa, chlorothiazide, furosemide, ibuprofen, levodopa, warfarin, acetazolamide, phenytoin, theophylline, chlorpropamide, bumetanide, diazepam, allopurinol, alprenolol, amphetamine, atropine, codeine, lidocaine, metoprolol, epinephrine, imipramine, methadone, methamphetamine, morphine, nicotine, norepinephrine, and pilocarpine.

[0010] Normal penile erection results from penile blood inflow and smooth muscle relaxation. This process is mediated by spinal reflexes, the L-arginine-nitric oxide-guanylyl cyclase-cyclic guanosine monophosphate (cGMP) pathway, and sensory and psychological stimuli. Nerves and endothelial cells release nitric oxide directly in the penis, where it stimulates guanylyl cyclase to produce cGMP and reduce intracellular calcium concentrations. This induces relaxation of arterial and cavernous smooth muscles, leading to arterial dilation, venous constriction, and erection. The balance of factors stimulating contraction and relaxation determines the tone of the penile vasculature and corpus cavernosum smooth muscle.

[0011] Phosphodiesterase 5 (PDE5) is the predominant phosphodiesterase in the corpus cavernosum. The catalytic site of PDE5 normally degrades cGMP, and PDE5 inhibitors such as sildenafil enhance the endogenous increase in cGMP by inhibiting degradation at the catalytic site. Phosphorylation of PDE5 not only enhances its enzymatic activity but also increases the affinity of its allosteric site (non-catalytic / GAF domain) for cGMP. Binding of cGMP to the allosteric site further stimulates enzymatic activity. Thus, phosphorylation of PDE5 and binding of cGMP to the non-catalytic site mediate negative feedback control of the cGMP pathway.

[0012] Sildenafil, tadalafil, and vardenafil are approved by the FDA for the treatment of erectile dysfunction. All of these drugs act as phosphodiesterase inhibitors. Phosphodiesterase inhibitors are drugs that inhibit one or more of the five subtypes of the enzyme phosphodiesterase (PDE), preventing the inactivation of the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cMP). Phosphodiesterase is involved in the breakdown of cyclic guanosine monophosphate (cGMP), which triggers smooth muscle relaxation and erection during sexual stimulation. Therefore, inhibition of one or more phosphodiesterases by these drugs enhances erectile function by increasing cGMP.

[0013] Sildenafil (Viagra) The effective dose of sildenafil (Viagra) is 25-100 mg once daily as needed. The active ingredient is sildenafil citrate. Its mean peak plasma concentration is approximately 60 minutes (range 30-90 minutes), and its absolute bioavailability is approximately 41%. The drug is metabolized primarily by cytochrome P4503A4 (CYP3A4) and has a half-life of approximately 4 hours (1).

[0014] Tadalafil (Cialis) The effective dose of tadalafil (Cialis) is 5-20 mg once daily as needed. The active ingredient is tardafil. The mean time to peak plasma concentration (Tmax) is approximately 2 hours (range 30 minutes to 6 hours) after a single dose (2). The drug is primarily metabolized by CYP3A4 to catechol metabolites and further to glucuronide conjugates. The mean terminal half-life is approximately 17.5 hours in healthy subjects (2). Absolute bioavailability after oral administration has been reported to not exceed 80% (3).

[0015] Vardenafil (Levitra) The standard recommended dose of vardenafil (Levitra) is 10-20 mg tablets taken once daily as needed. The active ingredient is vardenafil hydrochloride trihydrate. The mean time to peak plasma concentration (Tmax) is approximately 60 minutes (30 minutes to 2 hours), and the absolute bioavailability after oral administration is approximately 15%. The drug is mostly metabolized by CYP3A4, with the M1 metabolite accounting for approximately 7% of the total pharmacological activity. The terminal half-life of vardenafil or the M1 metabolite is approximately 4-5 hours, and the onset of therapeutic effect is approximately 30 minutes (4).

[0016] Each of these three phosphodiesterase inhibitors is approved by the FDA for erectile dysfunction and has a mean time to maximum concentration (Tmax) of approximately 60 minutes or more, with an initial Tmax of 30 minutes. Thus, the onset of action of these drugs is usually after 30 minutes, with a maximum effect after 1 hour. Due to poor water solubility at pH 4.0-7 (close to the physiological pH range in the nasal cavity and sublingual membrane) (5-7), these drugs are not suitable for administration as an aqueous solution when administered sublingually or intranasally for fast action.

[0017] To achieve good permeation and / or absorption at the sublingual or nasal sites, drugs must have a small molecular weight (<1 kD), a good membrane partition coefficient (good log P), and good water solubility (7-9). Nasal and sublingual administration is more rapidly absorbed than oral administration due to the thin membranes of the nose and tongue (6, 7). In addition, the nasal and sublingual routes of administration can bypass initial hepatic metabolism and can result in higher bioavailability than that obtained by oral administration (10-11). However, the aqueous solubility of the three phosphodiesterase inhibitors at pH 4.0-7.0 is low, which is a major obstacle to efficient permeation and / or absorption at the nasal or sublingual sites. To optimize mucosal permeation and / or absorption via the sublingual or intranasal routes of administration, appropriate solvents (e.g., organic-aqueous mixtures) that can improve solubility and achieve permeability similar to or greater than that at the appropriate pH at these sites are needed. However, there is no reliable method to predict the solubility and permeability at the optimal pH in these solvents.

[0018] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the type described herein that will be apparent to those skilled in the art upon reading this disclosure, etc.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In this specification, "about" used when referring to a measurable value such as an amount, a time duration, etc. means to include a variation of ±20%, ±10%, or ±5%, or even ±1% from the specified value, as such variation is appropriate for the disclosed composition or to carry out the disclosed method.

[0020] Definitions of standard chemical terms can also be found in references including the references Carey and Sundberg "ADVNCED ORGANIC CHEMISTRY 4TH ED" Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwise indicated, routine methods of the art of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology and pharmacology are employed. Unless specific definitions are provided, the nomenclature employed in connection with analytical chemistry, organic synthetic chemistry, and medicinal chemistry described herein, as well as the laboratory procedures and techniques, are those known in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Reactions and purification techniques can be performed, for example, using manufacturer's specification kits or as commonly accomplished in the art or as described herein. The aforementioned techniques and procedures can be generally performed according to methods known in the art and as described in the various general and more specific references cited and discussed throughout this specification.

[0021] As used herein, the term "penetration" or "absorption" refers to the "penetration" of a pharmaceutical active compound through a mucous membrane, unless otherwise specified. The terms "penetration" and "absorption" can be used interchangeably.

[0022] As used herein, the terms "mucosal" or "into a mucous membrane" mean, unless otherwise specified, any route of administration through a mucous membrane. Examples include, but are not limited to, sublingual, nasal, vaginal, and rectal administration of a pharmaceutical or pharmaceutical active compound.

[0023] As used herein, the term "phosphodiesterase inhibitor" refers to any agent that blocks one or more subtypes of the enzyme phosphodiesterase (PDE), thereby preventing the inactivation of the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) by each PDE subtype(s).The term "phosphodiesterase inhibitor" can refer to inhibitors of PDE1, PDE2, PDE3, PDE4, PDE5, PDE6, PDE7, PDE8, PDE9, PDE10, PDE11 and / or PDE12.Phosphodiesterase inhibitors include selective inhibitors and non-selective inhibitors.

[0024] As used herein, the term "subject" means animals and humans.

[0025] The term "environment" or "environment of administration" refers to the environment in which the active compound of a pharmaceutical agent is absorbed across a mucous membrane. For example, when administration is sublingual, the environment is the saliva, which contains the drug and "bathes" the sublingual mucosa.

[0026] The embodiment method of providing an environment with a specific pH includes providing an environment with a preferred pH during administration of the medicament, and creating an appropriate formulation of the medicament so that the medicament itself can provide an environment with the desired pH. In some embodiments, the latter is preferred. In this case, a buffering agent is preferably involved in the formulation.

[0027] The embodiments described herein can include calculating an estimated range of the amount of vardenafil that needs to be solubilized and then permeated or absorbed across the mucosa to achieve a therapeutically effective concentration (from the minimum amount of vardenafil API to a two-fold amount that represents the minimum effective amount).

[0028] The embodiments described herein may include various formulations or compositions depending on the dosage form or route of administration. For example, when the formulation or composition containing the pharmaceutical agent is administered sublingually, it may be in the form of a tablet, pill, pellet, powder, liquid or spray. Examples of other suitable formulations or compositions include, but are not limited to, ointments, capsules, solutions, syrups, drops, granules and suppositories. In any formulation or composition, the pharmaceutical agent may include a therapeutically effective amount of an active compound or a pharmaceutically acceptable form thereof, or any entity and a pharmaceutically acceptable carrier. As another example, when the formulation or composition containing the pharmaceutical agent is administered nasally, the formulation or composition may be in liquid form. Liquid forms suitable for nasal administration are, for example, nasal sprays and nasal drops.

[0029] In some embodiments, one or more phosphodiesterase inhibitors are administered sublingually. For sublingual administration of one or more phosphodiesterase inhibitors, the formulation or composition can be in any of the forms described above. Any method of producing a tablet, pill, pellet, powder, liquid or spray for sublingual administration can be used. To produce a tablet, for example, a granulated powder is pressed into small tablets. The tablet disintegrates when mixed with saliva, resulting in solubilization and absorption of the drug. The tablet is formulated, for example, with mixing with saliva in mind, to obtain a desired pH range for drug permeation and / or absorption.

[0030] Alcohol powder can be used to prepare tablets for sublingual administration. As another example, polyethylene glycol (PEG) can be used to prepare tablets for sublingual administration. Both alcohol powder and PEG are miscible with water. Exemplary liquid PEGs that can be used include, but are not limited to, PEG200, PEG400, and PEG600. Exemplary waxy or solid PEGs that can be used include, but are not limited to, PETGs with average molecular weights greater than about 600 g / mol (PEG600), such as PEG3000, PEG3350, PEG4000, PEG6000, and PEG8000.

[0031] In some embodiments, the one or more phosphodiesterase inhibitors are administered intranasally. For intranasal administration of the one or more phosphodiesterase inhibitors, the formulation or composition may be in any of the forms described above, including, for example, a nasal spray or liquid drops. Special devices can be used for intranasal or sublingual administration of a set volume. Exemplary volumes of such devices can range from 10 μl to 1.6 ml, which can be delivered to each of the two nostrils. Further exemplary volumes can range from 25 μl to 1.0 ml, 50 μl to 800 μl, 75 μl to 600 μl, 100 μl to 500 μl, or 200 μl to 300 μl per at least one nostril. Devices for intranasal administration are commercially available, for example, from Aptar.

[0032] Intranasal (IN) drug administration, e.g., by nasal spray, is a convenient route of administration. This route of administration can achieve the following advantages over oral drug administration: (a) it provides a faster effect, (b) it requires a smaller drug exposure to achieve an equivalent effect, and (c) it can be administered without the need for water for swallowing. Such advantages of nasal administration include the leaky epithelium lining the nasal mucosa (compared to the intestinal epithelium), the extensive vascular supply, and the relatively large surface area (approximately 9.6 m2 including microvilli). 2), and avoidance of first-pass metabolism (3-9). The relatively large surface area for drug absorption via the IN route also gives it an advantage over the sublingual route. The sublingual route also allows for rapid onset of action, but the surface area is much smaller (26 cm 2 ), the absorption of drugs such as vardenafil is limited and the full therapeutic effect is only achieved if they are administered multiple times.

[0033] To achieve good penetration and / or absorption at the nasal site, low molecular weight (<1 kD) is preferred, with a good membrane partition coefficient (good logP), good water solubility, and a desirable pKa that can result in ionization and good penetration at the physiological pH of the nose. Since the physiological pH of the nose is 6.4, it is generally recommended to keep the pH of the formulation between pH 3.5-7.5 to avoid irritation of the nasal mucosa (5, 10-11). However, to optimize the solubility and permeability of individual drugs, it may be necessary to deviate the pH of the formulation from the physiological pH. The recommended expected target pH is pH 3.5-7.5 (11).

[0034] The molecular weight of vardenafil HCL trihydrate is 579.1 (12). The molecular weight of vardenafil free base is 488.6, with logP=3.64, pKa=7.15 (mainly basic) and 9.86 (mainly acidic). The water solubility of vardenafil, a basic compound, is pH dependent and has been reported to be less than 2 mg / ml at pH 4-7 (12-13). Although the solubility of vardenafil hydrochloride trihydrate in water is higher than that of vardenafil base, the solubility of vardenafil drug substance still requires further improvement (Figure 2-3). Because the drug must be soluble for rapid nasal absorption before exerting its therapeutic effect, a fundamental step in vardenafil IN formulation is to achieve sufficient solubility and permeability of the desired drug amount. To optimize the IN formulation of vardenafil drug substance, the solubility and permeability at pH 3.5-7.5 must be experimentally determined.

[0035] Formulation Reagents Alcohol In certain embodiments, the formulations and compositions described herein for treating erectile dysfunction, increasing the solubility of one or more phosphodiesterase inhibitors, and / or increasing the permeability of one or more phosphodiesterase inhibitors, comprise at least one alcohol.In some embodiments, the formulations and compositions described herein for treating erectile dysfunction, increasing the solubility of one or more phosphodiesterase inhibitors, and / or increasing the permeability of one or more phosphodiesterase inhibitors, comprise one or more weak salts.Exemplary weak salts include, for example, citric acid, tartaric acid, acetic acid, flumaric acid, lactic acid, ammonium chloride, or similar organic salts.In some embodiments, the formulations described herein comprise N-methylpyrrolidone (NMP), Tween 80, or similar organic compounds. In some embodiments, the formulations described herein are prepared by combining Tween 80 or similar organic compounds, including citric acid, tartaric acid, acetic acid, flumaric acid, lactic acid, weak salts such as ammonium chloride or similar organic salts, or N-methylpyrrolidone (NMP), with one or more alcohols, polyethers, diethylene glycol monoethyl ether, medium chain glycerides, one or more saturated polyglycolized C8-C10 glycerides, or combinations thereof. Alcohols are a family of compounds that contain one or more hydroxyl (-OH) groups attached to a carbon atom of an alkyl group. Alcohols can have any number of carbon atoms in the chain. Alcohols can be primary, secondary, or tertiary alcohols. Monohydric and polyhydric alcohols are known. Exemplary monohydric alcohols include methanol, ethanol, propanol, butanol, pentanol, hexanol, and the like. Exemplary polyhydric alcohols include, for example, ethylene glycol, propylene glycol, glycerol (glycerin), and the like. In some embodiments, the alcohol is ethanol.In some embodiments, the alcohol is present at a concentration of about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, and any number or range therebetween. In some embodiments, ethanol is present at a concentration of about 5% to about 40%. In some embodiments, ethanol is present at a concentration of about 12%, about 25%, or about 30%. In some embodiments, the alcohol is glycerol (glycerin). In some embodiments, glycerol is present at a concentration of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%.

[0036] Polyether The formulations and compositions described herein for treating erectile dysfunction, increasing the solubility of one or more phosphodiesterase inhibitors, and / or increasing the permeability of one or more phosphodiesterase inhibitors may contain a polyether in certain embodiments. A polyether is a polymer that includes one or more ether functional groups. Polyethers include, for example, polyethylene glycol (PEG), polyethylene oxide (PEO), polyoxyethylene (POE), polypropylene glycol (PPG), polytetramethylene glycol (PTMG), polytetramethylene ether glycol (PTMEG), and paraformaldehyde. Aromatic polyethers include, for example, polyphenyl ether (PPE) and poly(p-phenylene oxide) (PPO). In some embodiments, the polyether is polyethylene glycol (PEG). The molecular weight of the polyethylene glycol (PEG) may range from 300 g / mol to 10,000,000 g / mol. In some embodiments, the polyether is PEG6000. In some embodiments, polyethylene glycol (PEG) is present at a concentration of about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, and any number or range therebetween. In some embodiments, polyethylene glycol (PEG) is present at a concentration of about 1% to about 20%. In some embodiments, polyethylene glycol (PEG) is present at a concentration of about 5%.

[0037] Glycerides In certain embodiments, the formulations and compositions described herein for treating erectile dysfunction, increasing the solubility of one or more phosphodiesterase inhibitors, and / or increasing the permeability of one or more phosphodiesterase inhibitors include at least one or more glycerides. Glycerides are esters formed from glycerol and fatty acids. Exemplary glycerides include mono-, di-, and triglycerides. In some embodiments, the formulations and compositions described herein include medium chain glycerides. In some embodiments, the formulations and compositions described herein include polyglycolized C8-C10 glycerides. In some embodiments, the polyglycolized C8-C10 glycerides are saturated polyglycolized C8-C10 glycerides. In some embodiments, the formulations and compositions described herein include a mixture of glycerides. The glycerides in the mixture can be unsaturated or saturated. In some embodiments, the mixture of glycerides includes additional chemicals or compounds. In some embodiments, the glyceride comprises a polyoxylglyceride. In some embodiments, the glyceride comprises caprylocaproyl polyoxyl-8 glyceride or caprylocaproyl macrogol-8 glyceride. In some embodiments, the glyceride comprises caprylic / capric glyceride. In some embodiments, the caprylic / capric glyceride further comprises a polyethylene glycol, such as PEG-8. In some embodiments, the formulations and compositions described herein comprise LABRASOL®.

[0038] Solvent stabilizer / penetration enhancer In some embodiments, the formulations and compositions for treating erectile dysfunction, for increasing the solubility of one or more phosphodiesterase inhibitors, and / or for increasing the permeability of one or more phosphodiesterase inhibitors described herein may contain certain other compounds or chemicals that function as solvents, stabilizers, or penetration enhancers. By way of example, the formulations and compositions described herein may contain diethylene glycol monoethyl ether. Diethylene glycol monoethyl ether is also known as 2-(2-ethoxyethoxy)ethanol and is sold under the trade name TRANSCUTOL. This compound functions as a high-purity solvent and stabilizer and is associated with enhanced skin permeability in topical dosage forms. Other suitable solvents, stabilizers, and penetration enhancers will be known to those skilled in the art. The amount of such compounds may vary depending on the desired formulation, such as in the range of 0.1-99.9% by weight, 1.0-99% by weight, 5-95% by weight, 10-90% by weight, or 20-80% by weight.

[0039] Buffer Buffering agents that can be used in the embodiments described herein will be known to those skilled in the art.See Handbooks Pharmaceutical Excipients (Second Edition), edited by Ainley Wade and Paul JW Weller, The Pharmaceutical Press London, 1994, which is incorporated herein by reference.Exemplary buffering agents include, but are not limited to, phosphates such as sodium phosphate; monobasic phosphates such as sodium dihydrogen phosphate and potassium dihydrogen phosphate; dibasic phosphates such as disodium hydrogen phosphate and dipotassium hydrogen phosphate; citrates such as sodium citrate (anhydrous or dehydrated); bicarbonates such as sodium bicarbonate and potassium bicarbonate.The amount of buffering agent used in the formulations and methods described herein is easily determined by those skilled in the art depending on the preferred pH value. Certain embodiments contemplated herein include about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 8.0, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9 or about 9.0, and any number or range therebetween. In some embodiments, the formulation or composition has a pH of about 3.5 to about 8.0. In some embodiments, the formulation or composition has a pH of about 3.5 to about 6.5. In some embodiments, the formulation or composition has a pH of about 4.0 to about 5.0.

[0040] Career Carriers suitable for use in the embodiments described herein will depend on the particular formulation or composition of the pharmaceutical, and include, but are not limited to, fillers, binders, lubricants, diluents, sweeteners and flavoring agents, preservatives, disintegrants, baking agents, and penetration enhancers. Examples of carriers include starch, gelatin, natural sugars, natural and synthetic gums such as corn, acacia, sodium alginate, methylcellulose, carboxymethylcellulose, polyethylene glycol, waxes, boric acid, sodium benzoate, sodium acetate, sodium chloride, agar, bentonite, agar gum, stearates such as sodium stearate, HPMC, palmitic acid, dimethylsulfoxide, N,N-dimethylacetamide, N,N-dimethylformamide, 2-pyrrolidone, 1-methyl-2-pyrrolidone, 1,5-dimethyl-2-pyrrolidone, 1-ethyl-2-pyrrolidone, 2-pyrrolidone-5-carboxylic acid, N,N-dimethyl-m-toluamide, urea, ethyl acetate, 1-dodecylazacycloheptan-2-one (Azone®), oleic acid, ethylene vinyl acetate copolymers, polyvinyl chloride, polyethylene, polydiethyl phthalate.

[0041] General Description of Various Embodiments Exemplary Formulations for Increasing Solubility / Permeability of One or More Phosphodiesterase Inhibitors Across Mucosa Provided herein, in some embodiments, is a formulation for enhancing the solubility / permeability of one or more phosphodiesterase inhibitors across a mucosa, comprising: (a) one or more phosphodiesterase inhibitors; and (b) an organic water-soluble solvent comprising an alcohol, a glycol, diethylene glycol monoethyl ether, a medium chain glyceride, one or more saturated polyglycolized C8-C10 glycerides, or a combination thereof; wherein the formulation has an pH of about 3.5 to about 8.0, and the organic water-soluble solvent enhances the solubility of the one or more phosphodiesterase inhibitors compared to the solubility of the one or more phosphodiesterase inhibitors in water. In some embodiments, the organic water-soluble solvent comprises an alcohol. In some embodiments, the alcohol is ethanol or glycerol. In some embodiments, the ethanol is present at a concentration of % to 40%. In some embodiments, the ethanol is present at a concentration of 12%, 25%, or 30%. In some embodiments, the organic water-soluble solvent comprises a polyether. In some embodiments, the polyether is polyethylene glycol. In some embodiments, the polyethylene glycol is PEG6000 or PEG400. In some embodiments, the polyethylene glycol is present at a concentration of 1% to 20%. In some embodiments, the polyethylene glycol is present at a concentration of 5%. In some embodiments, the formulations described herein include a weak salt. Non-limiting examples of weak salts include citric acid, tartaric acid, acetic acid, flumaric acid, lactic acid, ammonium chloride, similar organic salts, N-methylpyrrolidone (NMP), Tween 80, or similar organic compounds. In some embodiments, the formulations described herein include one or more alcohols, polyethers, diethylene glycol monoethyl ether, medium chain glycerides, one or more saturated polyglycolized C8-C10 glycerides, or combinations thereof. In some embodiments, the formulations have a pH of about 3.5 to about 8.0. In some embodiments, the formulations have a pH of about 3.5 to about 5.0.In some embodiments, the phosphodiesterase inhibitor is vardenafil, sildenafil, tadalafil, or a combination thereof. In some embodiments, the phosphodiesterase inhibitor is vardenafil. In some embodiments, the phosphodiesterase inhibitor is sildenafil. In some embodiments, the phosphodiesterase inhibitor is tadalafil. In some embodiments, the phosphodiesterase inhibitor is vardenafil in combination with sildenafil and / or tadalafil.

[0042] Exemplary Methods for Treating Erectile Dysfunction In some embodiments described herein, a method of treating erectile dysfunction in a subject in need thereof is provided, comprising contacting a mucosa of the subject with a formulation disclosed herein, thereby treating the erectile dysfunction in the subject. In some embodiments, contacting the mucosa comprises nasal administration. In some embodiments, contacting the mucosa comprises sublingual administration.

[0043] Exemplary methods for preparing formulations for treating erectile dysfunction Described herein in some embodiments is a method of preparing a formulation for treating erectile dysfunction in a subject, the method comprising: (a) adding one or more phosphodiesterase inhibitors to an organic water-soluble solvent comprising an alcohol, a polyether, diethylene glycol monoethyl ether, a medium chain glyceride, one or more saturated polyglycolized C8-C10 glycerides, or a combination thereof; (b) adjusting the pH of the organic water-soluble solvent comprising the one or more phosphodiesterase inhibitors to about 3.5 to about 8.0; wherein treating erectile dysfunction comprises contacting a mucosa of the subject with the formulation. In some embodiments, the solubility of the one or more phosphodiesterase inhibitors is increased in the organic water-soluble solvent compared to the solubility of the one or more phosphodiesterase inhibitors in water. In some embodiments, the permeability of the one or more phosphodiesterase inhibitors in the mucosa is increased in the organic water-soluble solvent compared to the permeability of the one or more phosphodiesterase inhibitors in water. In some embodiments, the bioavailability of the one or more phosphodiesterase inhibitors is increased in the organic water-soluble solvent compared to the bioavailability of the one or more phosphodiesterase inhibitors in water. In some embodiments, the organic water-soluble solvent comprises an alcohol. In some embodiments, the alcohol is ethanol or glycerol. In some embodiments, the ethanol is present at a concentration of 5% to 40%. In some embodiments, the ethanol is present at a concentration of 12%, 25%, or 30%. In some embodiments, the organic water-soluble solvent comprises a polyether. In some embodiments, the polyether is polyethylene glycol. In some embodiments, the polyethylene glycol is PEG6000 or PEG400. In some embodiments, the polyethylene glycol is present at a concentration of 1% to 20%. In some embodiments, the polyethylene glycol is present at a concentration of 5%. In some embodiments, the formulations described herein comprise a weak salt.Non-limiting examples of weak salts include citric acid, tartaric acid, acetic acid, flumaric acid, lactic acid, ammonium chloride, similar organic salts, N-methylpyrrolidone (NMP), Tween 80, or similar organic compounds. In some embodiments, the formulations described herein include one or more alcohols, polyethers, diethylene glycol monoethyl ether, medium chain glycerides, one or more saturated polyglycolized C8-C10 glycerides, or combinations thereof. In some embodiments, the formulations have a pH of about 3.5 to about 8.0. In some embodiments, the formulations have a pH of about 3.5 to about 5.0. In some embodiments, the phosphodiesterase inhibitor is vardenafil, sildenafil, tadalafil, or combinations thereof. In some embodiments, the phosphodiesterase inhibitor is vardenafil. In some embodiments, the phosphodiesterase inhibitor is sildenafil. In some embodiments, the phosphodiesterase inhibitor is tadalafil.

[0044] In some embodiments, the formulations described herein include an organic water-soluble solvent that includes one or more organic solvents or components. Exemplary organic solvents or mixtures of components include, for example, PEG and ethanol in water. In some embodiments, the PEG in the aqueous organic solvent mixture is PEG 400. In some embodiments, the ethanol is present in the aqueous organic solvent mixture at a concentration of about 5% to about 40%. In some embodiments, the ethanol is present in the aqueous organic solvent mixture at a concentration of about 12%. In some embodiments, the PEG is present in the aqueous organic solvent mixture at a concentration of about 1% to about 40%. In some embodiments, the PEG 400 is present in the aqueous organic solvent mixture at a concentration of about 1% to about 40%. In some embodiments, the PEG 400 is present in the aqueous organic solvent mixture at a concentration of about 10%, about 15%, or about 20%. In some embodiments, the formulation comprises 10% PEG 400 in 12% ethanol. In some embodiments, the formulation consists of 15% PEG 400 in 12% ethanol. In some embodiments, the formulation consists of 20% PEG 400 in 12% ethanol. Formulations comprising one or more organic solvents or ingredients in water can be used in any of the methods described herein.

[0045] In some embodiments, when the organic water-soluble solvent comprises one or more organic solvents or components, the second organic component is selected for the purpose of enhancing at least one property selected from the group of solubility, stability, permeability, and safety. In some embodiments, the formulation has a pH of about 3.5 to about 8.0.

[0046] Although many of the examples disclosed herein relate to formulations used to treat erectile dysfunction, it will be understood that the formulations may be combined with any one or more ionizable agents, such that the formulations are contemplated for use in treating any disease, disorder, or condition targeted by one or more ionizable agents.

[0047] Prescription Examples As disclosed herein, novel mucosal formulations (e.g., IN, sublingual formulations) serve to achieve not only (a) a good therapeutic effect or effective drug concentration in the body (e.g., plasma drug concentration), but also (b) a rapid onset of effect or a rapid peak drug concentration in the body (Tmax) compared to that from established oral administration routes, or to achieve similar effects to those from established injection administration routes (e.g., intravenous or subcutaneous routes). In some alternatives, the formulations are optimized for a given ionizable compound using the following methodology:

[0048] Step 1: Estimation of effective drug dose in mucosal formulations The goal of estimating an effective drug dose is to achieve a similar efficacy or area under the concentration-time curve (AUC) as the drug from oral or parenteral routes. In some options, the established oral bioavailability of an oral drug or parenteral bioavailability of an parenteral drug is first considered to calculate the effective mucosal dose. For example, if the oral bioavailability of drug X is 0.5 compared to intravenous administration due to hepatic first-pass metabolism. The IN dose can be estimated to be half of the oral dose because the IN route avoids hepatic first-pass metabolism. Thus, if the oral dose of drug X is 10 mg, the IN dose can be estimated to be approximately 5 mg.

[0049] Step 2: Minimum soluble drug concentration or Sol required for mucosal drug administration min For some mucosal administrations, there is a limit to the amount that can be administered at the site of administration. For example, for IN administration, the optimal volume via nasal spray is typically 0.1-0.15 ml, not exceeding 0.2 ml per nostril, to prevent "dripping". Thus, the volume of solution administered to each nostril is approximately 0.2-0.3 ml. Thus, the Sol required for the IN 5 mg Drug X example above (Step 1) is 0.01 ml. minwould be estimated as 5mg / 0.2ml or 5mg / 0.3ml = 25mg / ml or 17mg / ml, respectively, using 0.1ml or 0.15ml / spray into each of the two nostrils. Similarly, for sublingual sprays, such a Sol min can be calculated similarly.

[0050] Step 3: Reference Flux or JSS (ref) Decision Chemical flux or Jss (ref) is defined as the reference amount of drug per time per area permeating across the mucosa from a drug in aqueous solution administered to the mucosa at a particular pH. It is well known that drug flux or Jss is composed of solubility × apparent permeability (Papp), and the Papp of a drug passing through a biological membrane depends mainly on the unstirred water layer (UWL) and the permeability of the membrane to the drug. The UWL of a mucosa (such as the nasal mucosa) is covered by mucus, a gel-like liquid whose main component is water. From this structure, in this patent, the Jss of a drug in a pure aqueous solution is calculated by multiplying the Jss of a drug in an organic water-soluble solvent (a new formulation intended for mucosal administration). Since Jss = (Papp) (solubility), the Papp can be determined by an artificial membrane PAMPA (both sides of the membrane are in contact with the UWL) (16-19). The Papp from PAMPA is calculated by multiplying the Jss (ref) It is proposed that this is a useful approach for estimating the amount of soluble ...

number

[0051] Step 4: Jss of drug in organic aqueous solvent pHmax Measurement Drug in organic aqueous solvent (ssol)pHmax and pHmax or Papp pHmax The Papp in the drug are screened using the conventional “shake flask” method (solubility determination) (20-22) and PAMPA (permeability determination), respectively. (ssol)pHmax ≧Sol min (as defined in step 2) and the drug complex in a given organic aqueous solvent (or in a specific formulation for mucosal administration) (ssol)pHmax )(Papp pHmax ) or Jss pHmax Jss (ref) If the Tmax is equal to or exceeds the Tmax calculated in step 1, then that particular formulation qualifies as a suitable mucosal formulation for the drug to be administered (e.g., via IN or sublingual administration). For example, IN or sublingual administration can achieve the same therapeutic effect and a shorter Tmax as oral administration as calculated in step 1.

[0052] Step 5: Jss using appropriate cell line models pHmax Conformation of Although the Papp obtained by PAMA is relatively simple and convenient, a more physiological approach to determine the Papp of a drug in aqueous solution or various organic aqueous solvents provides further validation of the selected formulations described in steps 1-4 above. For IN formulations, validation of the appropriate Papp determined using the Calu-3 cell line model can be used (23-24). For sublingual formulations, the HO-1U-1 cell line can be used (25). Organic aqueous formulations that show significantly lower Papp at pHmax compared to formulations in aqueous solution should be excluded.

[0053] Usage example Additional embodiments related to the identification of specific formulations and methods for achieving effective IN or sublingual administration of phosphodiesterase inhibitors are disclosed in further detail. These are not intended to limit the scope of the claims in any way. Furthermore, the identification of specific formulations and methods for achieving effective IN or sublingual administration described herein is applicable to any other ionizable compounds. Specific examples A1, A2, B1, B2, and D1-D4 included below represent successive steps in the method of identifying desirable IN formulations based on a combination of solubility and permeability profiles, and the dosages required for vardenafil and sildenafil to achieve rapid and effective concentrations in the body. Specific examples C1, C2, C3, and D5 are in vivo evidence that confirms the suitability of the formulations and IN administration methods identified by the step-by-step method described above.

[0054] Example A1 Intranasal (IN) Administration and Formulation Embodiments of the minimum IN effective dose requirements of phosphodiesterase inhibitors, e.g., vardenafil / sildenafil, necessary to achieve therapeutic efficacy equivalent to the approved oral doses described herein have been estimated using specific calculations. Such calculations assume that sufficient vardenafil / sildenafil and nasal permeability can be identified to lead to the desired plasma concentrations and bioavailability.

[0055] The desired administration further requires an IN formulation of vardenafil drug substance or sildenafil drug substance, providing an amount of vardenafil hydrochloride trihydrate that achieves effective concentrations similar to those administered via the oral route, but significantly earlier. For example, if an oral dose of 10-20 mg of vardenafil is approved by the FDA, then IN administration with the appropriate formulation should achieve a faster peak time (Tmax) while achieving similar bioavailability as oral administration. Since IN administration is by either nasal spray or nasal drops, the IN dose can be estimated from the amount of vardenafil formulation solution administered nasally and the relative bioavailability adjusted concentration. An example calculation is shown below.

[0056] Since IN dosing typically uses 50-200ul / spray in each nostril (spray volumes greater than 200ul are likely to drip out of the nose), the desired spray volume can be set at 100ul / nostril or 2 x 100ul / 2 nostrils for an IN dose equivalent to a 10mg oral dose of vardenafil. Doubling the amount of nasal spray will give an IN dose equivalent to a 20mg oral dose. IN doses / dosages of other phosphodiesterase inhibitors such as tadalafil and sildenafil can be calculated similarly to be equivalent to oral doses.

[0057] Assuming that the absorption of oral vardenafil in water is related to IN administration of the appropriate formulation, and based on the known bioavailability / pharmacokinetics of oral vardenafil, it can be assumed that intranasal administration will result in a bioavailability equivalent to 0.4-0.8 oral administration, but with a much earlier peak concentration. This is based on the known advantage of intranasal administration, which avoids first-pass metabolism in the liver, and allows rapid penetration through the nasal membrane to be achieved, similar to the previously published bioavailability by pulmonary inhalation (26). For this reason, an IN formulation would require a minimum concentration of 2 mg / 100 ul or a solubility of vardenafil hydrochloride trihydrate of at least 20 mg / ml. Other phosphodiesterase inhibitors such as sildenafil and tadalafil can be calculated in a similar manner. Special devices are used for intranasal administration, allowing administration into each of the two nostrils. Devices for intranasal administration are commercially available, for example from Aptar.

[0058] Example A2 Solubility and stability screening of vardenafil in organic-aqueous mixed solvents The solubility and stability of vardenafil embodiments described herein were measured in various organic-aqueous mixtures.

[0059] The molecular weight of the active pharmaceutical ingredient vardenafil hydrochloride trihydrate is 579.1 g / mol, and the corresponding free base is 488.6 g / mol (12). Vardenafil base has pKa=7.15 and 9.88, and logP=3.64. The solubility of vardenafil is approximately 8.8 g / L at pH 1, 3 g / L at pH 2, 1.6 g / L at pH 3, 0.88 g / L at pH 4, 0.16 g / L at pH 5, and 0.019 g / L at pH 6 (13). The solubility of the active pharmaceutical ingredient (API) vardenafil hydrochloride trihydrate in water is much better (Figure 3). However, such solubility is still low, and the aqueous solubility, which decreases with increasing pH, is a significant obstacle to achieving rapid and sufficient penetration and / or absorption via sublingual and intranasal administration. Thus, despite the excellent membrane partition coefficient of vardenafil base (ρgP=3.64), sublingual or nasal administration of aqueous vardenafil solutions does not provide sufficient bioavailability compared to oral administration when administered close to or at the appropriate physiological pH at the nasal or sublingual sites (see calculations in Example A1).

[0060] However, vardenafil drug substance can achieve improved solubility in certain solvents, such as alcohol (13) or other organic-aqueous mixed solvents. The use of a pure alcohol solution of vardenafil is of concern due to potential membrane irritation and damage. Therefore, an alcohol-aqueous mixed solvent or other organic-aqueous mixed solvent that is relatively safe or well tolerated for human subjects, such as organic compounds (at relatively low concentrations) that belong to the "Generally Regarded as Safe" or "GRAS" category, is desirable. For this reason, the use of a 12% alcohol solvent in nasal sprays has been accepted by the FDA as an acceptable concentration for human subjects (27, 28). Although 12% alcohol can rapidly solubilize vardenafil drug substance, it precipitates within 24 hours. Therefore, a "shake flask" method over a 3-day period was utilized to measure the saturation solubility (20-22).

[0061] As an example, the aqueous solubility of saturated vardenafil at different pHs was first screened, followed by the permeability at different pHs. Such information is used to generate an optimal solubility and permeability combination in aqueous systems (i.e., Jss), which is used to provide initial clues for the desired pH, solubility and permeability of organic aqueous solvents to be used as the desired appropriate IN vardenafil formulation and dose.

[0062] In addition, any mixed solvent must be capable of solubilizing vardenafil for rapid and sufficient absorption when administered sublingually or nasally.

[0063] As an example, vardenafil drug substance was first screened for solubility in aqueous ethanol mixtures, followed by permeability screening at different pH to determine optimal solubility and permeability suitable for sublingual and nasal administration.

[0064] material Vardenafil hydrochloride (CAS number 224785-91-5) was purchased from India Alembic Pharmaceutical Ltd, Gujarat-391450 (Lot number 1704002361).

[0065] Tadalafil (TDA) 5 mg tablets were purchased from Polpharam (Poland).

[0066] Acetonitrile ≧99.5% ACS (CAS No. 75-05-8) was purchased from VWR Chemials BDH®.

[0067] Methanol ("MeOH") was purchased from VWR Chemials BDH®.

[0068] Ethanol 190-Proof (CAS number 64-17-5) was purchased from EDM Millipore (Burlington, MA, USA).

[0069] Syringe filters with pore size w / 0.2 μm cellulose acetate membrane (Cat# 28145-475) were purchased from VWR (Radnor, PA, USA).

[0070] Polyethylene glycol 400 (Lot 52081314) was purchased from EDM Millipore (Burlington, Massachusetts, USA).

[0071] Glycerin or glycerol (Lot 70K0044) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0072] Calcium lactate pentahydrate (Lot SLGB7173) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0073] Glacial acetic acid (Lot B21R026) was purchased from Alfa Aesa (Haverhill, MA, USA).

[0074] NMP (1-methyl-2-pyrrolidinone) (Lot 51K3683) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0075] equipment A Basic pH meter was purchased from Fisher Scientific (Leicestershire, UK).

[0076] An Agilent 1260 Infinity HPLC system consisting of a G1311B 1269 Quat pump, a B7129 1260 vial sampler, and a G1315D 1260 DAD VAL detector was purchased from Agilent (Santa Clara, CA).

[0077] Analytical balances were purchased from Mettler-Toledo, LLC (Columbus, Ohio).

[0078] procedure To screen various organic solvents, we first investigated the solubility of vardenafil drug substance in various % ethanol-water mixed solvents and compared it with the solubility in pure water. As disclosed herein, the solutions were prepared by the "shake flask" method. Briefly, vardenafil drug substance was added to various mixtures until it was saturated. The saturated organic-aqueous mixtures were pH adjusted using a pH meter (pH range 3.5-7.5). The saturated solutions were shaken slowly using a magnetic stirrer at room temperature or rapidly shaken several times a day for 24 hours or more, up to 3 days. The solutions were then filtered through a VWR 0.2 micron filter. The filtrate was used to check for clarity, and the concentration was measured by HPLC. In addition, the saturated solubility of vardenafil in various organic water-soluble solvents, such as glycerin (glycerol), polyethylene glycol 400 (PEG), and combinations of the two organic solvents, was investigated. The solubility of vardenafil in other representative organic water-soluble solvents was also investigated.

[0079] (a) HPLC method for measuring vardenafil concentration For concentration determination from solubility and permeability studies (see Examples B1 and B2), samples were mixed 1:1 with MeOH to prepare for HPLC analysis. Standard curves were prepared in MeOH and mixed with PBS to ensure the same analytical matrix. HPLC analysis was performed using an Agilent 1260 Hypersil BDS-C8, 5 μm, 4×250 mm column (PN79926B8-584, SN USUE000480, LN512010961) with an isocratic flow of acetonitrile:0.02 M sodium phosphate buffer pH 4 (35:65 v / v) at 1 mL / min for 10 min.

[0080] result The validity of the assay was evaluated in accordance with FDA guidance for linearity, sensitivity, repeatability, stability, precision, and accuracy. The calibration curve for vardenafil was linear over the concentration range of 0.2-200ug / ml. The correlation coefficient (r 2) was greater than 0.99 for each of three different runs. For quality control samples of 0.5, 10 and 200ug / ml, the relative standard deviation (RSD) values ​​of precision ranged from 1.8 to 6.1% (interday) and 0.07 to 4.1% (intraday). Precision ranged from -4.2% to 2.2% (interday) and -0.9 to 3.4% (intraday). The lower limit of quantification was 0.2ug / ml.

[0081] Table 1 shows a comparison of the saturation solubilities of vardenafil concentrations in several organic water-soluble solvents at different pH. Table 2 shows the interday precision and accuracy of six different solutions with two different pH values, measured using the standard curves of each solution and compared to the 50% methanol standard curve. Table 3 shows a comparison of the saturation solubilities of vardenafil drug substance at pH 4.0 in different solvents. [Table 1] Comparison of saturation solubilities for each organic water-soluble solvent [Table 2] Comparison of dilution precision and bias in the analysis of indicated organic aqueous solvents.

[0082] Dilution precision was determined by comparing various solutions at 20 mg / ml. The RSD was 3.1%. The stability of quality control samples was tested by reinjecting samples 0, 9, 18 and 24 hours after reconstitution and storing them in the autosampler at 200 °C. The RSD values ​​were 2.8-7.8%. [Table 3] Solubility of Vardenafil Drug Substance at pH 4 in Different Solvents* *Room temperature, average pH4.0 (3.9-4.1), Sat concentration - saturation concentration, SD standard deviation.

[0083] conclusion As disclosed herein, organic aqueous mixtures such as ethanol aqueous mixtures can significantly increase the solubility of vardenafil compared to its solubility in pure aqueous solutions (Figure 3 and Table 3). The solubility of vardenafil drug substance is pH dependent, with the lower the pH, the higher the solubility. Organic aqueous mixtures such as ethanol aqueous mixtures can significantly increase the solubility of vardenafil compared to its solubility in water. The solubility of vardenafil can be further improved by increasing the concentration of organic solvent, for example ethanol. Additionally, the solubility of vardenafil can be improved by combining certain organic solution mixtures. For example, a 15% PEG-12% EtOH-water mixture compared to a PEG15 aqueous solution.

[0084] The results disclosed herein show that the use of a methanol standard curve in the assay allows accurate and precise measurement of vardenafil concentration in different solvents or solvent mixtures and at different pHs. A 100-fold dilution from a 20 mg / mL concentration can also be measured accurately / precisely within ±15%.

[0085] If a minimum solubility of 20 mg / ml at pH 4 of the vardenafil drug substance is desired for an IN formulation, then water, which has an average saturation solubility below 20 mg / ml at pH 4, is not a suitable solvent for a vardenafil IN formulation. However, a number of other GRAS classified organic water-soluble solvents have been identified that have a high solubility of 20 mg / ml at pH 4 (see Table 3), and additional mixtures of these solutions are likely to be suitable for vardenafil IN formulations, as disclosed herein.

[0086] Example B1 Permeability and flux screening using PAMPA for selection of IN vardenafil formulations This example describes the determination of the permeability of vardenafil using a parallel artificial membrane permeability assay (PAMPA).

[0087] As disclosed herein, the permeability of vardenafil drug substance in different solvents at room temperature and atmospheric pressure was screened using in vitro PAMPA. PAMPA predicts the passive absorption of drugs and is suitable for studies with many solutions, including ethanol (up to 30% concentration) (22-24). The unit of measurement is the apparent permeability (Papp) obtained at steady state, expressed in cm / sec. The maximum drug flux (Jss) at a particular pH, calculated from Papp and saturation solubility, is also expressed as the amount of drug crossing a unit area per second.

[0088] As disclosed herein, the effect of aqueous ethanol solution on improving drug permeability / absorption across mucosa was determined. Considering that 12% ethanol-water solution and 30% ethanol-water solution can significantly improve solubility, permeability studies at different pH were further carried out. Prior to this study, the effect of pH on permeation at different aqueous ethanol concentrations was unknown and could not be accurately predicted.

[0089] Materials and Equipment Transport receiver plates (Cat#TRNS50) and multiscreen IP filter plates (Cat#MAIPN4550) were purchased from Millipore (Burlington, MA, USA).

[0090] Vardenafil hydrochloride (CAS number 224785-91-5); India Alembic Pharmaceutical Ltd, Gujarat-391450 (Lot number 1704002361).

[0091] Ethanol 190-proof (CAS number 64-17-5) It was purchased from Millipore (Burlington, Massachusetts, USA).

[0092] Acetonitrile (Cat#BDH83639.400) was Chemicals (Radnor, PA, USA).

[0093] Dodecane (Cat#D221104), sodium phosphate monobasic (Cat#S0751), sodium phosphate dibasic (Cat#S0876) and polyethylene glycol 6000 (Cat#8.07491) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0094] Lecithin purified material (Cat#36486) was purchased from Alfa Aesar (Haverhill, Massachusetts, USA).

[0095] Syringe filters with pore size w / 0.2 μm cellulose acetate membrane (Cat# 28145-475) were purchased from VWR (Radnor, PA, USA).

[0096] Polyethylene glycol 400 (Lot 52081314) was purchased from EMD Millipore (Burlington, Massachusetts, USA).

[0097] procedure (a) Preparation of solutions Saturated solutions of vardenafil hydrochloride trihydrate (5 ml) in different solvents were prepared by increasing amounts of vardenafil and adjusted to the desired pH (range 3.5-6.0 using a pH meter) as described in Example A2 above. The saturated solutions were shaken slowly for 24 hours at room temperature or shaken rapidly several times and kept at room temperature for 24 hours or more. The solutions were then filtered using a 0.2 μm filter. The filtrates were used for permeation studies.

[0098] (b) Permeation test using PAMPA Permeation studies of vardenafil were performed using a Parallel Artificial Membrane Permeation Assay (PAMPA) with receiver plates and MultiScreen-IP filter plates. The PAMPA assay predicts passive absorption of drugs and is suitable for testing with ethanol solvent (22-23). ​​Steady-state permeation assays were performed for 6 or 24 h in triplicates or more to represent steady state. For some solutions, both 6 and 24 h were performed to establish consistency between the two durations. Before transferring 150 μL of desired sample to the donor chamber, the donor chamber was coated with 5 ul of 3% (w / v) lecithin in dodecane. Then, 300 μl of phosphate buffer was transferred to the acceptor well. After 6 or 24 h of permeation, samples were taken from the donor and receiver chambers for concentration analysis using HPLC.

[0099] The apparent permeability coefficient (P app ) was calculated based on the following equation at steady state (24):

number

[0100] Here, the parameters are as follows:

[0101] V D = volume of donor chamber

[0102] V A = volume of the acceptor chamber

[0103] A = effective area of ​​the membrane (PAMPA: 0.3 cm 2 )

[0104] t = time of permeability test

[0105] C A = drug concentration in acceptor wells at the end of the study

[0106] C E = equilibrium concentration in both wells, and

number

[0107] Steady-state flux (Jss) ug / sec / cm 2 Jss, expressed as: was calculated based on the following formula:

number

[0108] Here, the parameters are as follows:

[0109] C D = loading concentration in the donor chamber

[0110] A comparison of the Papp and Jss of vardenafil in different solvents at pH 4 is shown in Table 4. [Table 4] Effect of solvents on permeability and flux* of vardenafil drug substance * Determined by PAMPA at room temperature and 24-hour average pH of 4.0 (3.9-4.1); # Vardenafil organic-aqueous mixture (saturated solubility ≧ 20mg / ml) of Jss is water or Jss (ref) (see below), p<0.05 (-test); SD=standard deviation.

[0111] At pH 3.5-8.0 or higher, the pH-solubility time of vardenafil in aqueous solution is pH-P app The profile is a pH-flux or pH-Jss profile (see profile in Figure 10b). The pH corresponding to the highest Jss is pH max and the corresponding saturated solubility of vardenafil in water is V (ssol)pHmaxTherefore, Jss at pHmax or JsspHmax is Jss or Jss (ref) The reference Sol corresponding to the required minimum solubility is specified as min (The minimum IN dose is 20 mg / ml for nostril, which is 2 mg / ml according to the previous calculation in

[0061] ).

number

[0112] A vardenafil organic aqueous mixture at any pH (within the range of pH 3.5-7.5) having a solubility of greater than 20 mg / ml with a corresponding Jss value ≧Jss (ref) are eligible for IN vardenafil formulations.

[0113] (c) HPLC assay 100 μL of donor or receiving chamber sample was mixed with 100 μL of MeOH and 10 μL of internal standard for 2 min, and then the sample was centrifuged at 10000 rpm for 10 min before HPLC injection. The loading stock solution was diluted with 50% MeOH 50% before HPLC injection. The HPLC assay was performed as described in Example A2.

[0114] result Comparative permeability test results shown in Table 4. Excellent correlation of Papp results between these 6-hour and 24-hour permeability tests was observed, indicating the consistency of the Papp experiments. Additional individual tests using pure aqueous solutions and other ethanol-water mixtures also supported similar trends of the effect of pH on permeability, as shown in Figure 5.

[0115] conclusion As disclosed herein, vardenafil API increases in solubility as the pH decreases. However, the permeability (Papp) of vardenafil is theoretically expected to increase (corresponding to a higher % of combined species) as the pH increases from 3.5 to 5.0 (Figure 5). Drug flux or Jss (Pss) of aqueous vardenafil solutions app , a parameter consisting of times the saturated solubility, is optimal at pH 4.0 (Figure 5b). A similar trend is observed for a representative organic water-soluble vardenafil solvent (EtOH (12%)-water) (Figure 5B). From these data, Jss max , pH 4 is selected.

[0116] Many organic solvents with a solubility of more than 20 mg / ml at pH 4 can be used to determine the aqueous or Jss (ref) Average Jss (8.5E-05ug / s / cm 2 ) and can be considered a suitable formulation for vardenafil IN (Table 4).

[0117] Many organic-aqueous or organic-organic aqueous mixtures can improve the solubility and permeability of vardenafil hydrochloride trihydrate, the current active pharmaceutical ingredient of vardenafil. On the other hand, the combination of two different organic solvents in water can improve the permeability even if the solubility is not improved compared to a single organic-aqueous mixed solvent, such as a 12% EtOH-water solution. Without being limited or bound by any theory, it is believed that the solvents described herein and other suitable organic water-soluble solvents that can improve the solubility of different phosphodiesterase inhibitors in the pH range close to the physiological pH in the nasal or sublingual sites can also improve their permeability and are suitable for nasal and sublingual delivery.

[0118] Example B2 Measurement of vardenafil permeability in selective solutions using the Calu-3 cell line model This example describes the measurement of vardenafil permeability using the Calu-3 cell line model.

[0119] As disclosed herein, the permeability of vardenafil drug substance in different solvents was screened using an in vivo cell line model Calu-3 (non-small cell lung cancer line). The effect of various solvents on the permeability of vardenafil drug substance can be efficiently screened using the PAMPA method, but its reliability can be improved by confirming it using a physiological model such as a cell line similar to the target membrane. Therefore, in this study, the permeability of the PAMPA test results was confirmed using the Calu-3 cell line, which is suitable as a model for IN permeation.

[0120] The Papp of water-soluble drugs determined by the Calu-3 cell line model, when determined at pH 7.4, has been shown to be relevant for IN absorption in animal studies (23-24). As disclosed herein, the Calu-3 cell line model was able to measure the Papp of various organic water-soluble solvents. appwas used to confirm the relative values ​​of β-aminobutyric acid (β-aminobutyric acid) in comparison with water at pH 4.0 (to simulate IN administration of the vardenafil formulation).

[0121] material Glacial acetic acid (purity ≥99%, CAS 64-19-7) was purchased from Alfa Aesar (Haverhill, MA, USA).

[0122] Acetonitrile >= 99.5% ACS (CAS No. 75-05-8) was purchased from VWR Chemicals BDH®.

[0123] Sodium phosphate monobasic monohydrate (phosphate buffer) was purchased from BDH Chemicals (Radnor, PA, USA).

[0124] NaOH (sodium hydroxide) was purchased from Biobasic Canada Inc. (Markham, Ontario, Canada).

[0125] Sodium Phosphate Dibasic, Heptahydrate (Phosphate Buffer) EMD It was purchased from Millipore (Burlington, Massachusetts, USA).

[0126] Vardenafil hydrochloride trihydrate USP was purchased from SMS Pharmaceuticals Ltd. (India).

[0127] Gibco Hank'S Balanced Salt Solution (HBSS), o-phosphoric acid, 85% (HPLC), Transwell® multiwell plates with permeable polycarbonate membrane inserts triethylamine were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

[0128] Syringe filters with 0.2 um cellulose acetate membrane were purchased from Avantor, VWR (Radnor, PA, USA).

[0129] equipment A Basic pH meter was purchased from Fisher Scientific (Leicestershire, UK).

[0130] An Agilent 1260 Infinity HPLC system consisting of a G1311B 1269 Quat Pump, a G7129 1260 Vial Sampler, and a G1315D 1260 DAD VAL Detector was purchased from Agilent (Santa Clara, CA).

[0131] Analytical balances were purchased from Mettler-Toledo, LLC (Columbus, Ohio).

[0132] The water used was obtained from a Nanopure water filtration system (Barnsted Nanopoure Diamond Life Sci UV / UF system (Cat# 119310, purchased from APS Water Servives Corporation, Lake Balboa, CA, USA).

[0133] Transepithelial electrical resistance (TEER, unit: Ω.cm 2 )Measuring device.

[0134] procedure (a) Preparation of solutions Saturated solutions of vardenafil (2 mg / mL) in different solvents were prepared and adjusted to the desired pH (range 3.9-4.1 using a pH meter) as described in Examples A2 and B1 above. Various solutions containing 2 mg / mL vardenafil were prepared by completely dissolving each powder by vortexing and then mixing overnight on a rotating platform. The solutions were then filtered using a 0.2 μm filter. The filtrates were used for the transmission / permeability tests.

[0135] (b) Calu-3 culture and monolayer preparation The study was performed similarly to that described previously (26-27). Calu-3, a human bronchial submucosal adenocarcinoma cell line, was grown in a DMEM:Ham's F-12 (1:1) mixture supplemented with 10% FBS and 1% penicillin / streptomycin solution. Cells were harvested with 0.25% trypsin-EDTA and spread onto polycarbonate filters (pore size: 0.4 μm, growth area: 1.12 cm). 2 , 12 wells / plate, Corning) 5 Cells were seeded at a density of 1000 cells / well. The medium was changed every 2 days during the experimental period. Monolayers were used for in vivo transport studies 9–10 days after seeding.

[0136] (c) In vitro permeability test using Calu-3 cell line model The TEER of the monolayer was measured and found to be approximately 500 Ohm.cm 2 The growth medium was aspirated and the upper and lower chambers were washed with transport medium (TM: Hank's Balanced Salt Solution pH 7.4 supplemented with 15 mM glucose and 10 mM HEPES buffer).

[0137] After 10 min of incubation, the solution in the apical chamber was replaced with the study vardenafil solution at pH 4.0. Under a BSL2 hood, sample aliquots (50 μL) were taken from the basolateral side after 15, 30, 45, 60, 90, and 120 min. TEER measurements were taken periodically, with 50 μL of transport medium being replenished each time.

[0138] (d) Preparation and measurement of HPLC assays 50 μl samples from the receptor and apical chambers were mixed with 50 μl 50% MeOH and 10 μl of internal standard or diluted with 50 μl of medium and internal standard. Supernatants were collected after centrifugation for HPLC analysis. HPLC assays were performed as outlined in Examples A2 and B1.

[0139] (e) Pap calculation P app (Unit: cm / sec) was calculated using the formula from samples analyzed by HPLC.

[0140]

number

[0141] dQdt is the appearance of drug in the receptor chamber (nmol / sec), A is the surface area of ​​the monolayer (1.12 cm 2 ), C is the initial concentration of drug in the apical chamber.

[0142] result Calculated mean P of different vardenafil solutions app are shown in Table 5. Most of the P app Values ​​are the P values ​​of vardenafil in 15% PEG400 solution. app Except for the values, the P value of vardenafil in water app For vardenafil in 15% PEG400 solution, the TEER value was approximately 1200 Ohm.cm above the initial baseline value. 2 After 120 minutes, it was at a level similar to the baseline (approximately 500 Ohm.cm 2 ) The initial TEER was also approximately 1200 Ohm.cm when EtOH / PEG (12% / 15%) was added. 2 The TEER increased but returned more rapidly to baseline levels after 40 minutes. Most other solutions typically reached a TEER of 500 Ohm.cm in less than 20-40 minutes. 2 It rapidly decreased to below baseline levels. [Table 5] Permeability of Vardenafil Drug Substance Using Calu-3 Cell Line Model*

[0143] conclusion In this experiment, the concentration of each solution is 2mg / ml, so the relative Papp values ​​of the solutions also directly reflect the relative Jss values. The Papp values ​​of vardenafil drug substance in different organic water-soluble solvents are almost the same as the Papp value of vardenafil in water in most solutions, except for the 15% PEG400 solution, which is 20 times lower (p<0.001, two-sample t-test). This is because the TEER of the organic water-soluble solvent containing vardenafil increased in the 15% PEG400 solution (the initial TEER of the medium, i.e., 500 Ohm.cm). 2 ) may correspond to increased TEER. An increase in TEER may indicate increased tight junction function in the cell membrane. In support of this hypothesis, when ethanol was added to PEG400 (which is known to increase membrane permeability), the TEER value was significantly decreased. Therefore, using the Calu-3 model to further screen vardenafil formulations initially identified by PAMPA could be a useful step in eliminating undesirable formulations.

[0144] Example C1 Methods for administering phosphodiesterase inhibitors This example illustrates methods for intranasal and sublingual administration of phosphodiesterase inhibitors.

[0145] The phosphodiesterase inhibitor is added to the organic aqueous mixture, and the pH of the mixture is adjusted. Any phosphodiesterase inhibitor can be used, such as vardenafil (Levitra), sildenafil (Viagra), tadalafil (Cialis), etc. The phosphodiesterase inhibitor is added to the organic aqueous solvent at a certain pH, which increases the solubility and permeability of the phosphodiesterase inhibitor. Any organic aqueous mixture or solvent can be used, including any organic aqueous solvent that is relatively safe or well tolerated by human subjects and can sufficiently solubilize the phosphodiesterase inhibitor and increase its permeability.

[0146] Improved solubility of phosphodiesterase inhibitors can result in improved flux (solubility x permeability or Jss) of phosphodiesterase inhibitors in mucosa, such as vardenafil, sildenafil, or tadalafil. The mucosa flux of phosphodiesterase inhibitors in organic aqueous mixtures is favorable in the pH range of about 4.0 to about 8.0. Improved flux in mucosa can increase Cmax and bioavailability and shorten Tmax of phosphodiesterase inhibitors compared to oral administration of the same phosphodiesterase inhibitor. This means that intranasal or sublingual administration results in higher plasma concentration and faster onset of action compared to oral administration of the same dose of phosphodiesterase inhibitor.

[0147] Phosphodiesterase inhibitor can be delivered via nasal or sublingual membrane in any dosage form, but using solution formulation, e.g. spray, should provide the fastest onset of effect compared to other dosage forms.As an example, the amount of phosphodiesterase inhibitor can be added to organic aqueous mixed solvent, and the desired amount of phosphodiesterase inhibitor can be delivered intranasally or sublingually in a volume of 100μl per spray.

[0148] Phosphodiesterase inhibitors having improved solubility and permeability as described above are administered, for example, for the treatment of erectile dysfunction.

[0149] In addition to phosphodiesterase inhibitors, this method is applicable to any ionizable compound.

[0150] Example C2 Comparison of intravenous and oral administration of vardenafil in rats This example describes the measurement of the bioavailability of vardenafil after oral or IN administration and how variations in formulation affect pharmacokinetics.

[0151] IN administration allows the compound to bypass hepatic metabolism. Furthermore, due to the thin layer of the nasal mucosa, IN administration allows for rapid absorption of the drug, higher bioavailability, and faster peak concentration time (Tmax) compared to oral administration, provided the appropriate formulation is administered. Certain formulations have been found to have different Jss or Papp of vardenafil in vivo. Thus, these formulation differences are likely to affect the above pharmacokinetic parameters when administered via the IN route. Prior to the study, a stepwise approach to estimate the appropriate dose / drug concentration and to identify and confirm a specific formulation with the desired solubility and permeability for IN administration to achieve adequate vardenafil plasma concentrations improved compared to oral administration of the dose was unclear.

[0152] procedure Aqueous and organic solutions of vardenafil were prepared using the same protocol described in Example A2. Jugular cannulated Sprague Dawley rats were ordered from Envigo RMA (Indianapolis, IN). Upon arrival, rats were habituated to the Western University Health Sciences animal care room for one week prior to pharmacokinetic studies. Anesthesia and testing procedures were performed similar to previously published studies (29). A total of six formulations were tested via IN administration and one via oral administration (see Table 6 below).

[0153] A subset of rats was administered orally (PO, or orally) with a formulation containing vardenafil (5.6 mg / kg). Another subset of rats was administered intranasally (IN) with a formulation containing vardenafil (2.8 mg / kg). The IN rat group received the formulation via a micropipette at approximately 12.5 ul per nostril. Rats receiving the water and PEG formulations via the IN route received 1.7 mg / kg and 2.0 mg / kg, respectively. Rats were randomized to receive the six formulations (see Table 6).

[0154] After administration of the formulation, 200ul of blood was obtained from the rats at 0, 2, 5, 10, 15, 20, 30, 45, 60, 120, and 180 minutes. One week after administration, the hematocrit of the rats returned to normal values, as confirmed by the plasma of the rats randomly selected in the study. Based on such hematocrit response, general physical activity, and cannula patency, the rats were switched to different formulation treatments with the same blood samples taken one week later. After the end of blood collection, the samples were centrifuged, and the plasma was collected and stored for analysis using liquid chromatography-tandem mass spectrometry (LC / MS / MS, Sciex API4000 and Agilent HPLC1200 systems). For the measurement, rat plasma was used to construct a standard curve, and sildenafil was used as an internal standard (IS). The assay procedure was similar to the human study described below (see Example C3, Measurement of Plasma Concentration of Vardenafil).

[0155] result The standard curve for the measurement of vardenafil concentrations in rat plasma showed excellent correlation coefficients (R 2 = 0.9981). The formulations of vardenafil and the number of rats administered IN and PO are shown in Table 6. The pharmacokinetic parameters of the oral and IN formulations of ethanol / PEG400 (12% / 15%) are shown in Table 7. The pharmacokinetics of formulations 2 to 6 when administered IN are shown in Table 8. [Table 6] A formulation containing vardenafil was administered orally or intravenously to rats. [Table 7] Pharmacokinetic parameters of vardenafil administered intranasally and orally in ethanol / PEG400 (12% / 15%) *Normalized per mg / kg dose [Table 8] Pharmacokinetic parameters of five vardenafil formulations administered IN* F = formulation, *normalized per mg / kg dose. **P<0.01 compared to F6 (water formulation) (t test).

[0156] Observations and Conclusions The relative bioavailability (AUC), Cmax (peak concentration) and Tmax (time to Cmax) results are consistent with those expected from the formulations identified in Examples A2, B2, B2 and C1. Solvents with lower Papp / flux compared to water, such as vardenafil in 15% PEG400 or 15% PEG-10% NMP from the in vivo PAMPA and / or Calu-3 studies, may result in relatively lower bioavailability when administered via the IN route. Compared to the water formulation (F6), the AUC of F5 is significantly lower. The AUC and Cmax of formulation No. 4 are also significantly lower compared to the AUC and Cmax of formulation No. F6, but the values ​​are not statistically significant at p<0.05 due to the small number of rats that completed the study. However, solvents with similar / better Papp / flux compared to water, such as vardenafil in 12% ethanol and in ethanol / PEG400 (12% / 15%), may result in similar / better bioavailability compared to water. Thus, these rat data support the current method of screening and selecting desirable formulations using the stepwise approach demonstrated in Examples A1, A2, B1, and B2.

[0157] Example C3 Comparison of intravenous and oral administration of selective phosphodiesterase inhibitors in humans. This example describes the measurement of the bioavailability of vardenafil after oral or IN administration in humans, and how variations in formulation affect pharmacokinetics.

[0158] As disclosed herein, this study compared an SDS-089 solution (consisting of 20 mg / ml vardenafil dissolved in 12% EtOH / 15% PEG400) administered as a nasal spray (IN administration) with Levitra oral tablets 10 mg (oral, or PO administration). The solubility of vardenafil >20 mg / ml, and the steps for selecting its solution in 12% ethanol-15% PEG400 for nasal drug delivery are described in Examples A1, A2, B1, B2, and C1.

[0159] material Vardenafil nasal solution (SDS-089 nasal solution) was prepared for each human subject participating in the study according to the investigator's prescription.

[0160] The active pharmaceutical ingredient is from Alembic Pharmaceutical Ltd., India (batch 1704002361) conforming to USP specifications.

[0161] The nasal solution was prepared by solubilizing 20 mg / ml vardenafil API in 12% ethanol and 15% PEG400, with a pH of approximately 4.0. The SDS-089 nasal spray solution was filtered (0.22 μm filter) into a small-volume 5 mL amber bottle and fitted with a nasal spray device (Aptar, Pharma, France) that delivered 100 μL per spray. Prior to the pilot clinical trial, it was confirmed that the Aptar nasal spray device could deliver 100 μL per spray. The spray was administered by dissolving 2 mg of vardenafil hydrochloride per spray in a 12% ethanol and 15% PEG400 solution.

[0162] Vardenafil hydrochloride (Levitra) 10 mg oral tablets (NDC: D173-0830-13, lot number: 5930248) manufactured by Bayer Yakuhin were purchased from a pharmacy.

[0163] SFS089 was prepared by a licensed pharmacist under the supervision of a licensed pharmacist at the Medical Center within the Patient Care Center of Western University Health Sciences, Pomona, CA, USA.

[0164] procedure The 12 human subjects recruited for this study were healthy volunteers aged between 21 and 45 years. Each subject received two study treatments: SDS-089 nasal spray (4 mg vardenafil hydrochloride trihydrate) and Levitra oral tablets (10 mg), administered consecutively in a randomized order with a 7±1 day interval between each treatment.

[0165] On the day of the study, subjects had an intravenous catheter inserted. All subjects received either a 10 mg Levitra tablet (oral PO) or a 4 mg SDS-089 solution (2 mg / spray per nostril) nasal spray IN. After dosing, subjects were asked to drink 240 ml of water. One week later, each subject was crossed over to treatment (subjects previously given IN treatment received PO treatment, and subjects previously given PO treatment received IN treatment). Each subject consumed 240 ml of water at the same time as drug administration and was allowed to drink water or clear liquids for 2 hours after the single dose. Food was allowed to be consumed at least 4 hours after dosing.

[0166] A total of 17 blood samples (2 cc each) were collected after administration of the drug formulation. Blood samples were collected at 0 (pre-dose), 2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 60 min, 90 min, 2 h, 3 h, 4 h, 6 h, 8 h, and 10 h. All blood samples were immediately centrifuged at 3000 rpm for 10 min and stored at -80°C until ready for bioanalysis.

[0167] During the study, safety assessments included adverse event monitoring, vital signs, and necessary medical history and physical examinations as per the medical supervisor's discretion.

[0168] (a) Measurement of plasma concentration of vardenafil Analysis of vardenafil concentrations in plasma was performed using liquid chromatography-tandem mass spectrometry (LC / MS / MS, Sciex API4000 and Agilent HPLC1200 systems) through the analytical certification laboratory outsourcing services provided by Dr. Stan Lui, College of Pharmacy, University of Southern California. During the validation experiment, each calibration standard and QC sample was prepared by spiking the specified amount of vardenafil hydrochloride (USP) and sildenafil (LETCOL) as internal standards into blank human plasma. Aliquots of 50ul of analyte were extracted with 850ul of methanol, centrifuged, and the supernatant was dried. The powder was reconstituted in 60ul of 50% methanol, and after filtration, 30ul was injected into the LCMS. The separated analytes were separated using a reversed-phase high-performance Eclipse Plus C18 column (Agilent). The concentration of the analyte in each standard was quantified using a triple quadrupole tandem mass spectrometer operating in the positive mode of electrospray ionization (ESI). Vardenafil and sildenafil were detected using multiple reaction monitoring (MRM) for each analyte. The average measurement precision was 92-110%. R 2 The R ranged from 0.9977 to 0.9998. The precision, defined as the coefficient of variation (CV) = (standard deviation / mean value of measured values ​​× 100%), ranged from 4 to 8%. The lower limit of quantification was 0.05 ng / ml.

[0169] result Representative vardenafil concentration-time curves are shown in Figure 12, and mean comparative pharmacokinetic parameters are shown in Table 9. Area under the concentration-time curve from 0 to infinity (AUC O-inf Based on the time to maximum concentration (T max ) was 58 minutes for oral administration and 10 minutes for nasal SDS089 administration. max The maximum concentration (C) after administration of SDS089 was 6–15 min in 85% of subjects receiving IN administration, compared with 45–60 min in 92% of subjects receiving oral administration. max ) is the C after oral administrationmax These data indicate that the bioavailability of the 4 mg SDS089 nasal spray is close to that of the 10 mg oral dose, but T max was shown to be shorter. [Table 9] Comparison of mean pharmacokinetic parameters of nasal spray and oral tablet

[0170] During the two treatment periods, 47 adverse events (AEs) occurred. Of the 47 AEs, 42 were recorded as adverse drug reactions (ADRs), of which 33 were related to the nasal spray and 9 were related to the oral tablet. Observed adverse events included headache, sneezing, runny nose, watery eyes, nasal irritation, and throat irritation. The SDS-089 nasal spray formulation caused more nasal symptoms, but overall the adverse events were transient and well tolerated by subjects. Reported headaches were classified as mild and moderate headaches, with C max and AUC O-inf When correlated with

[0171] conclusion As disclosed herein, nasal and oral administration of badenafil was compared in 12 healthy volunteers. Side effects were more common with nasal administration, but these effects were transient and tolerable. The overall results of this study were consistent with those in rats, with IN administration resulting in a faster T max These human study results further confirm that a stepwise approach, such as that described in Examples A1, A2, B1 and B2, can be used to identify appropriate formulations and doses. References

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Nielsen PE., Avdeef A. (2004). PAMPA-a drug absorption in vitro model 8. Apparent filter porosity and the unstirred aqueous layer. European J Pharmaceutical Sciences 22:33-41. 20. Birch, H., Redman, AD, Letinski, DJ, Lyon, DY, & Mayer, P (2019) Determining the Water Solubility of Difficult-to-Test Substances: A Tutorial Review Anal Chim Acta, 1086, 16-28 http: / / doi.org / 10.1016 / j.aca.2019.2019.07.034 21. He, Y., Ho, C., Yang, D., Chen, J., & Orton, E. (2017) Measurement and accurate interpretation of pharmaceutical salt solubility. J Pharm Sci, 105(5), 1190-1196 http: / / doi.org / 10.1016 / j.xphs.2017.01.023 22. Bergstrom, C., Avdeef, A., (2019) Perspectives on the measurement and interpretation of solubility Admet & DMPK 7, 88-105 23. Inoue, D., Furubayashi, T., Tanaka, A. et al. (2020) Quantitative evaluation of drug permeation through nasal mucosa using in vitro membrane permeability through Calu-3 cell layer for prediction of in vivo bioavailability after nasal administration in rats. European J. Pharmaceutics and Biopharm., 149, 145-153. 24. Takashi Furubayashi, Daisuke Inoue, Naoki Nishiyama, et al. (2020) Comparison of various cell lines and three-dimensional mucosal tissue model systems to estimate drug permeability and in vitro transport studies to predict intranasal drug absorption in rats. Yakugaku 12, 1-14 25. Wang, Y, Zuo, Z, Chow, MS (2008) HQ-1u-1 model for sublingual drug delivery screening-Effect of pH, osmolality and penetration enhancers. Int J Pharm. 209 Mar 31;370(1-2):68-74. Doi:10.1016 / j.ijpharm.2008.11.010 26.Pharmacokinetics of vardenafil using an inhalation ultrasonic nebulizer compared with a single 10 mg oral tablet. J sex Med.2016 Jul;13(7):1111-8.doi:10.1111 / j.1743-6109.2009.01403.x.PMID:27318021 27. https: / / www.accessdata.fda.gov / drugsatfda_docs / nda / 2012 / 202813Origls000SumR.pdf 28. https: / / www.druginformation.com / RxDrugs / B / Beclomethasone%20Dipropionate%20HFA.html 29.Qian,S,Wong,YC,Zuo,Z(2014),Development,Characterization and application of in situ gel systems for intranasal delivery of tacrine.Int J Pharm 2014 Jul 1;468(1-2):272-82.doi;10.1016 / j.ijpharm.2014.04.015.Epub 2014 Apr 5.pmid:24709220 30. Lu, HT., Chen, R., Sheu, M. et al. (2011) Rapid-acting sildenafil nasal spray using a microemulsion system: in vitro evaluation and in vivo pharmacokinetic study in rabbits. Xenobioica DOI:10.3109 / 00498254.2011.563877

[0173] Example D1 Intranasal (IN) administration and formulation of sildenafil The properties of sildenafil, including its solubility and stability, were measured in a variety of organic-aqueous mixtures.

[0174] Sildenafil is approved by the FDA as an oral tablet for the treatment of erectile dysfunction, usually in doses of 25-100 mg (1). Its active pharmaceutical ingredient, sildenafil citrate, has a molecular weight of 666.7 g / mol, and sildenafil base has a molecular weight of 474.6 g / mol (1). Sildenafil base has a pKa of 7.2 and a logP of 2.7 (12). Its solubility is approximately 7.0 mg / ml at pH 3-4, 2.1 mg / ml at pH 6, 0.11 at pH 7, and 0.03 mg / ml at pH 7 (7). Such aqueous solubility is too low to develop a suitable IN formulation. (Assuming that the bioavailability from IN administration is 2-fold better than from oral administration, a solubility of sildenafil base of approximately 42 mg / ml is required to achieve an effective concentration equivalent to at least a 25 mg oral dose. Such a concentration is calculated based on a desired spray volume of 0.150 ml per nostril, so the required concentration is 12.5 mg / 0.3 ml = 42 mg / ml for both nostrils.) Thus, despite having an excellent membrane partition coefficient (logP = 2.7), nasal administration with aqueous sildenafil is not capable of achieving an effective IN dose (equivalent to at least a 25 mg oral dose) even in the pH range where it is most soluble (pH 3-4).

[0175] However, sildenafil citrate can achieve improved solubility in certain oils (e.g., oleic acid, safflower oil) or surfactants (e.g., Tween 20, Cremophor RH60, Cremophor EL) or cosurfactants (e.g., PEG 200) (30). A microemulsion system consisting of 40% oleic acid, 10% H20, and 50% Tween 80 ethanol (weight ratio 1:4) for nasal sildenafil formulation has been found to provide rapid action (30). However, such oily solutions are not suitable for routine use by nasal drops. Therefore, new oil-free formulations consisting of simple organic-aqueous mixtures that are safe and can achieve adequate solubility and penetration are most desirable.

[0176] Example D2 Screening of solubility and stability properties of various organic-aqueous mixtures: Sildenafil As an example, the method described for the vardenafil IN spray formulation was used to first screen the aqueous solubility of saturated sildenafil at different pHs, followed by screening the permeability at different pHs. Such information is used to generate an optimal solubility and permeability combination (i.e., Jss) in aqueous systems. This information is used to provide initial clues for the desired pH, solubility, and permeability of organic aqueous solutions to be used as the desired appropriate sublingual and IN sildenafil formulations and doses. Additionally, the saturated solubility of sildenafil in various solvents was measured at approximately pH 4.0-6.0.

[0177] material Sildenafil citrate was purchased from Asian Chemical Industries Ltd (Israel) manufactured by Teva API (Israel).

[0178] The water used was obtained from a Nanopure water filtration system (Barnstead Nanopure Diamond Life Sci UV / UF system (Cat #D119310 purchased from APS Water Servives Corporation, Lake Balboa, CA, USA).

[0179] Tadalafil (TAD) 5 mg tablets were purchased from Polpharma (Poland).

[0180] Acetonitrile >= 99.5% ACS (CAS No. 75-05-8) was purchased from VWR Chemicals BDH®.

[0181] Methanol ("MeOH") was purchased from VWR Chemicals BDH®.

[0182] Ethanol 190-Proof (CAS No. 64-17-5) was purchased from EMD Millipore (Burlington, Massachusetts, USA).

[0183] Glycerin or glycerol (Lot 70K0044) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0184] Calcium lactate pentahydrate (Lot SLCB7173) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0185] Glacial acetic acid (Lot B21R026) was purchased from Alfa Aesar (Haverhill, MA, USA).

[0186] NMP (1-Methyl-2-Pyrrolidinone) (Lot 51K3683) was purchased from Sigma Aldrich (St. Louis, MO, USA).

[0187] Sodium phosphate monobasic hydrate (phosphate buffer) (Cat# BDH9298, Lot# 19E0356407) was purchased from BDH Chemicals (Radnor, PA, USA).

[0188] NaOH (sodium hydroxide) (Cat# SB0617, Lot# C26S617R0S) was purchased from Biobasic Canada Inc (Markham, Ontario, Canada).

[0189] Diethylene glycol monoethyl ether (Transcutol) (Cat# 8.03127, Lot# S7591827 831)

[0190] MultiScreen-IP Filter Plate (Cat# MAIPN4550)

[0191] Sodium phosphate dibasic heptahydrate (phosphate buffer solution) (Cat# 8210, Lot# BB-0680R)

[0192] Polyethylene glycol 400 (Cat# PX1286B-2, Lot# 60297045) was purchased from EMD Millipore (Burlington, Massachusetts, USA).

[0193] HC1 (Hydrochloric acid) (Cat# 320331; Lot# SHBG2435V)

[0194] Phosphate buffered saline (PBS, tablets) (Cat# P4417; Lot# SLCD5938) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0195] O-Phosphate, 85% (HPLC) (Cat#A260)

[0196] Triethylamine (Cat# 04884; Lot# 011464) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

[0197] Syringe filters with cellulose acetate membranes with pore size of 0.2 μm (Cat# 28145-475) were purchased from VWR (Radnor, PA, USA).

[0198] Tween 20 and Tween 80 from EMD Millipore (Burlington, Massachusetts, USA)

[0199] equipment The apparatus used in the following experiments is the same as that described in Example A2.

[0200] procedure Sildenafil aqueous solutions and several organic sildenafil aqueous solutions were screened for saturation solubility. To measure the saturation solubility of sildenafil at different pH, solutions were prepared at room temperature by the "shake flask" method using an excess amount of sildenafil citrate drug substance and pH adjusted (pH range 3.5-7.5) using a pH meter. The solutions were then filtered through a VWR 0.2μ filter. The filtrates were then measured for sildenafil concentration by HPLC, using tadalafil as the internal standard, in a manner similar to the vardenafil HPLC measurement described in Examples A1 and B1.

[0201] result The validity of the sildenafil HPLC assay was evaluated against a validated vardenafil assay (Example A2) on quality control samples and a standard curve. Similar results were obtained, with a lower limit of quantitation of 0.2ug / ml.

[0202] Table 10 shows a comparison of the saturated solubility of sildenafil in several solvents within the pH range of 3.5-7.5. The lower the pH, the higher the solubility, and above pH 4.5 the solubility drops significantly. Sildenafil J in aqueous solutions at different pH values SS A comparison of the results is shown in Figure 8. Based on these data, the sildenafil J in water ssp H maxThe estimated pH is 4.6. However, the solubility of typical aqueous organic solutions appears to be optimal at pH 4.2-4.5, which is the pH range for sildenafil J. ssp H max (pH 4.2-5.2 as shown in FIG. 8). Considering industrial applications, Tables 11 and 12 show the saturated solubility of sildenafil in different solvents at around pH 4.2 and pH 4.5. [Table 10] Saturation solubility of sildenafil in solvents with different pH* [Table 11] Saturation solubility of sildenafil in various solvents at pH 4.2* [Table 12] Saturation solubility of sildenafil in various solvents at pH 4.4*

[0203] conclusion As disclosed herein, certain organic aqueous mixed solvents, such as acetic acid / NMP / calcium lactate-aqueous mixed solvents, can significantly increase the solubility of sildenafil compared to the solubility in pure aqueous solutions (Tables 11 and 12). The solubility of sildenafil is pH-dependent. The results of this study indicate that the solubility of saturated sildenafil can be further increased by increasing the organic solvent concentration.

[0204] If the minimum solubility of sildenafil desired for an IN formulation is 42 mg / ml (calculation shown in Example D1), then the aqueous saturation solubility of sildenafil is 5 mg / ml or less at pH 4, making it an unsuitable solvent for an IN formulation of sildenafil. However, numerous other organic water-soluble solvents or mixtures have been identified (see Tables 11 and 12) that have a solubility of at least 42 mg / ml at about pH 4, and are likely to be suitable for an IN formulation of sildenafil if their permeability values ​​are comparable to or greater than those of aqueous sildenafil solutions (see Tables 13 and 14 in Example D3 below).

[0205] Example D3 Sildenafil permeability and flux screening using PAMPA This example describes the measurement of sildenafil permeability using a parallel artificial membrane permeability assay (PAMPA).

[0206] As disclosed herein, the permeability of sildenafil in different solvents at room temperature and atmospheric pressure was screened using in vitro PAMPA. PAMPA predicts the passive absorption of drugs (22-24). The unit of measurement is the apparent permeability (Papp) obtained at steady state, expressed in cm / sec, and the flux (Jss) at a specific pH, calculated from Papp and the saturation solubility, is expressed as the amount of drug crossing a unit area per second.

[0207] As disclosed herein, the effect of pH on the permeability of saturated sildenafil aqueous solutions was determined. In addition, different organic aqueous solutions were also measured to improve the permeability of saturated sildenafil at pH 4.2 and 4.6. Prior to our study, the effect of pH on the permeability of sildenafil in different organic aqueous solutions was unknown and could not be accurately predicted.

[0208] Materials and Equipment The materials and equipment were similar to those described in Example B1, and the sildenafil and solvent were the same as those described in D2.

[0209] procedure (a) Preparation of solutions Saturated solutions of sildenafil hydrochloride trihydrate (3 ml) in different solvents were prepared by adding increasing amounts of sildenafil as described in Example E2 above and adjusting to the desired pH (range 3.5-6.0 using a pH meter).

[0210] (b) Permeation test using PAMPA Sildenafil permeation studies were performed using PAMPA with receiver plates and MultiScreen-IP filter plates as described for vardenafil in Example B2. Sildenafil in different solvents at pH 4.2 and 4.5 are shown in Tables 13 and 14.

[0211] (c)HPLC analysis The concentrations of sildenafil in the donor or receiver chambers were prepared as described for vardenafil, and the HPLC assay was performed as described in Example D2.

[0212] result The mean Papp and Jss of sildenafil in different sildenafil solutions at pH 4.2 and 4.5 are shown in Tables 13 and 14. [Table 13] Effect of solvents on permeability and flux of sildenafil at pH 4.2* [Table 14] Effect of solvents on permeability and flux of sildenafil at pH 4.5*

[0213] conclusion As disclosed herein, the solubility of sildenafil is pH dependent. Aqueous sildenafil solutions exhibit increasing permeability / permeability as pH increases (corresponding to a higher proportion of theoretically predicted bound species as pH increases from 2.25 to 7.0) (FIG. 8). Drug flux for aqueous sildenafil solutions appears to be optimal near pH 4.5 or 4.6.

[0214] Based on a nasal drop volume of 150ul or 0.15ml per nostril, or 0.3ml for both nostrils, the sorum required for 1 IN dose of sildenafil is estimated to be 42mg / ml (12.5mg / 0.3ml=42mg) (see calculations in Example D1).

[0215]

number

number

[0216] Similarly, when estimated at pHmax 4.2, the water solubility of sildenafil is 3.54 mg / ml (Table 9) and the JssHmax is 1.08E-04 ug / sec / cm 2 It is.

[0217]

number

[0218] According to the formulations and methods described in Examples A1, A2, B1, and B2, the solubility is greater than 42 mg / ml with a corresponding Jss value of 1.3E-03 to 2.3E-03 ug / sec / cm 2The sildenafil organic-aqueous mixture should be eligible for an IN sildenafil formulation.

[0219] Many organic solvents with solubilities greater than 42 mg / ml at about pH 4.2 have a solubilities of 1.28E-03 ug / sec / cm 2 These formulations have been found to meet or exceed the sildenafil Jss (reference value) of 2.3E-03 ug / sec / cm and can be considered suitable formulations for IN sildenafil (see Table 11). Similarly, many organic solvents with solubilities above 42 mg / ml at pH 4.5 also have solubilities above 2.3E-03 ug / sec / cm. 2 It has been noted that these formulations meet or exceed the sildenafil Jss (reference value) of 10 mg / kg / day and, if suitable candidates are further confirmed in the Calu-3 study, they could be considered as suitable formulations for IN sildenafil (see Table 14).

[0220] Example D4 Sildenafil permeability screening using Calu-3 cell line This example describes the measurement of sildenafil permeability using the Calu-3 cell line model.

[0221] As disclosed herein, the permeability of sildenafil in different solvents at 37° C. and atmospheric pressure was screened using an in vivo cell line model, Calu-3 (a non-small cell lung cancer line).

[0222] The Papp of water-soluble drugs determined by the Calu-3 cell line model, when determined at pH 7.4, has been shown to be relevant for IN absorption in animal studies (25-26). As disclosed herein, the Calu-3 cell line model was utilized to confirm the consistency of sildenafil Papp values ​​in different organic aqueous solutions compared to water.

[0223] material The materials used were similar to the vardenafil test described in Example B2 and the sildenafil solution described in E2.

[0224] equipment The apparatus used was the same as in the vardenafil study described in Example B2.

[0225] procedure (a) Preparation of solutions Saturated solutions of sildenafil (1.5 mg / mL) were prepared in six selected solvents and adjusted to approximately pH 4.2 or pH 4.5 as previously described. After dissolving the sildenafil powder, the solutions were stirred overnight on a rotating platform. The solutions were then filtered using a 0.2 m filter. The filtrates were used for permeation studies.

[0226] (b) Calu-3 culture and monolayer preparation The culture was carried out as described above in [25-26] and in Example B2.

[0227] (c) In vitro permeability test using Calu-3 cell line model TEER measurements, growth media and procedures were similar to those described in Example B2.

[0228] (d) Preparation and measurement of HPLC assays 50 μl samples from the receptor and apical chambers were mixed with 50 μl of 50% MeOH and 10 μl of internal standard or diluted with 50 μl of medium and internal standard. The supernatant was collected after centrifugation for HPLC assay. HPLC assay was performed as in D2.

[0229] (e) Pap calculation Papp in cm / sec was calculated from samples analyzed by HPLC using the formula given in Example B2.

[0230] result A comparison of the Paap of sildenafil by PAMPA and the Papp by Calu-3 is shown in Figure 9. Due to the small number of data points, a considerable scattering of points is observed around the line of best fit. Since the purpose of this plot is to compare the Papp of sildenafil (obtained in organic aqueous solutions) with those obtained in aqueous solutions by both the PAMPA and Calu-3 methods, values ​​significantly lower than the Papp from sildenafil aqueous solutions can be easily identified in the lower left quadrant. All other points show Papp values ​​equal to or higher than those obtained from aqueous solutions. In Figure 9, there is one low point in the lower left quadrant, which corresponds to a calcium lactate (3.5%)-aqueous solution, but its magnitude is not large and there is no statistically significant difference when comparing the sildenafil Papp from a calcium lactate (3.5%)-aqueous solution determined by PAMPA or Calu-3, respectively, with the sildenafil Papp in aqueous solutions.

[0231] conclusion Since the concentration of each solution is 1.5 mg / ml, the relative Papp values ​​of the various solutions also directly reflect the relative Jss values.

[0232] Since the sildenafil PAMPA Papp at pH 4.2 is close to that at pH 4.5, the relationship between PAMPA Papp and Calu-3 Papp at pH 4.2 is expected to be similar to that shown at pH 4.5 (Figure 9). Based on the solubility and Papp requirements of sildenafil in organic aqueous solutions proposed by this method and the results of sildenafil solubility and permeability in various organic aqueous solutions, acetate / calcium lactate (1% / 3.5%) aqueous solution, acetate / calcium lactate (5% / 3.5%) aqueous solution, and acetate / NMP / calcium lactate (5% / 10% / 3.5%) aqueous solution (pH 4.2 or pH 4.5) are considered to be suitable for sildenafil IN formulations.

[0233] Example D5 Comparison of oral and intravenous administration of selective phosphodiesterase inhibitors in rats. This example describes (1) the improvement of sildenafil bioavailability when the appropriate formulation is administered IN compared to the oral route, and (2) validation of the currently proposed method in identifying the specific solubility, Papp, and concentration required for an IN sildenafil formulation.

[0234] IN administration allows the compound to bypass hepatic metabolism, resulting in faster absorption (faster peak concentration time or Tmax), higher peak concentration (Cmax), and higher bioavailability compared to oral administration, along with rapid transmucosal penetration (when using appropriate formulations). Oral administration is effective for sildenafil due to its high first-pass metabolism in the liver. Proper formulation is essential to achieve improved Cmax, Tmax, and bioavailability with IN administration. Thus, specific solubility, Papp, and concentration required for sildenafil IN formulation according to current method are required. Prior to this study, the effect of specific organic-aqueous formulations (aqueous content ≥ 50%) on sildenafil solubility and Papp suitable for IN formulation is unknown. The present D5 study confirms the application of the proposed method for sildenafil to obtain superior results from IN formulation.

[0235] procedure Sildenafil aqueous and organic aqueous solutions were prepared using the same protocol described previously. Jugular vein cannulated Sprague Dawley rats were ordered from Envigo RMS (Indianapolis, IN). Upon arrival, rats were habituated to the animal room at Western University of Health Sciences for 1 week prior to pharmacokinetic studies. Anesthesia and testing procedures were performed similar to a previously published study (29). A total of three sildenafil formulations were selected based on their solubility, permeability, and the concentrations required for an appropriate IN formulation according to the proposed method. All three formulations (see table) were administered via the IN route and one was gavaged via the oral (PO) route.

[0236] Similar to the rat study described for vardenafil (see Example C2), administration of the formulations was randomized, with two rats receiving each treatment and crossing over after one week. 200ul of blood was collected from each rat at 0, 2, 5, 10, 15, 20, 30, 45, 60, 120, and 180 minutes (the 2 minute sample was omitted for rats receiving the oral formulation). After each blood collection, the samples were centrifuged and plasma was collected and stored for analysis using liquid chromatography-tandem mass spectrometry (LC / MS / MS, Sciex API4000 and Agilent HPLC 1200 systems). For the measurements, rat plasma was used to construct a standard curve and tadalafil was used as the internal standard (IS). The assay procedure was similar to the vardenafil assay (see Example C3 for measurement of sildenafil plasma concentrations).

[0237] result The standard curve for the measurement of sildenafil concentrations in rat plasma showed excellent correlation coefficients (R 2 =0.9924-0.9976). The precision of quality control (QC) samples ranged from 96-100% for the four QC samples tested. The precision or CV ranged from 1.9-4.6%. The formulations of sildenafil and the number of rats administered IN and PO are shown in Table 15. The pharmacokinetics of the IN and oral formulations are shown in Table 16. As shown in Table 16, when normalized to mg / kg dose, the Cmax and AUC were significantly higher for the IN formulation compared to the oral formulation. When comparing the same formulation administered IN and orally, the AUC was 6-fold higher for the IN formulation. Also, the Tmax was significantly shorter for the IN formulation compared to the oral formulation. The mean difference in Cmax or Tmax between the IN and oral administrations was more than 10-fold. No significant differences in these parameters were observed among the three formulations administered IN. [Table 15] Sildenafil-containing formulations administered orally or intravenously to rats [Table 16] Pharmacokinetic parameters of sildenafil nasal spray and oral tablet in rats (data expressed as mean ± SD)

[0238] Observations and Conclusions The relative bioavailability (AUC), Cmax (peak concentration) and Tmax (time of Cmax) results between IN and oral routes are significantly different and consistent with what would be expected from the IN formulations identified by the novel method of the present invention described in Examples A1, A2, B1, B2. Thus, these rat data further confirm the validity and usefulness of the present method for identifying desirable IN formulations.

[0239] Example E Formulations that enhance the permeation and flux of one or more phosphodiesterase inhibitors and other ionizable basic / acidic drugs across mucous membranes This example describes formulations for enhancing the solubility and permeability (flux) of one or more phosphodiesterase inhibitors across mucosal membranes.

[0240] As disclosed herein, based on the methods described in Examples A1, A2, B1, B2, the formulation identified consists of the phosphodiesterase inhibitor vardenafil dissolved in an organic water-soluble solvent consisting of ethanol and PEG400 at pH 4.0, the organic water-soluble solvent enhancing the solubility of the phosphodiesterase inhibitor relative to the solubility of the phosphodiesterase inhibitor in water. The formulation includes 12% ethanol as a component. In other alternatives, the formulation may include any alcohol, such as glycerol, and may be present in any concentration between 5% and 40%, including 25% and 30%. As disclosed herein, the organic water-soluble solvent of the formulation consists of another component, PEG400, 15%. In other alternatives, the formulation may include any polyether or polyethylene glycol, such as PEG6000, in a concentration between 1% and 20%. As disclosed herein, the formulation is at pH 4.0. In other alternatives, the formulation may be at any pH between 3.5 and 7.5. As disclosed herein, the phosphodiesterase inhibitor of the formulation is vardenafil. Alternatively, the formulation may contain other drugs in a suitable Orgian aqueous solution identified according to the formulations and methods described in Examples A1, A2, B1 and B2. The formulation is then administered intranasally to a subject to treat erectile dysfunction or other suitable diseases. By intranasal administration, the formulation contacts the mucous membrane of the subject. Alternatively, by sublingual administration to a subject, the mucous membrane contacts the formulation.

[0241] With respect to the use of virtually any plural and / or singular terminology herein, one of ordinary skill in the art can translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. Various singular / plural permutations may be expressly provided herein for clarity.

[0242] Unless the context otherwise requires, the word "comprises" and variations thereof, such as "comprises" and "comprising," which are used interchangeably throughout this specification and claims with "comprises," "containing," or "characterized by," are inclusive or open-ended language and do not exclude additional, unrecited elements or method steps.

[0243] The word "consisting of" excludes any element, step, or ingredient not specified in the claim.

[0244] The phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. The present disclosure contemplates embodiments of the formulations, compositions, and methods of the invention that fall within the scope of each of these phrases. Thus, a formulation, composition, or method that consists of recited elements or steps contemplates certain embodiments in which the formulation, composition, or method consists essentially of, or consists of, those elements or steps.

[0245] When a method of using a composition or formulation (e.g., a method of treating erectile dysfunction that comprises administering the formulation or contacting the formulation with a mucosa) is disclosed herein, the corresponding composition or formulation for use is also expressly contemplated. For example, for the disclosure of a method of treating erectile dysfunction that comprises administering the formulation or contacting the mucosa with a formulation that comprises one or more phosphodiesterase inhibitors in an organic solvent, the corresponding composition or formulation for treating erectile dysfunction is also contemplated.

[0246] References throughout this specification to "one embodiment" or "one embodiment" or "one aspect" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases "in one embodiment" or "in one aspect" in various places throughout this specification are not necessarily all referring to the same embodiment.

[0247] Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0248] The various embodiments described above can be combined to provide further embodiments. These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Therefore, the claims are not limited by this disclosure.

[0249] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those of ordinary skill in the art. The various aspects and embodiments disclosed herein are illustrative and not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. A formulation, comprising: A phosphodiesterase inhibitor; Ethanol, (a) an organic water-soluble solvent comprising a polyether, diethylene glycol monoethyl ether, a medium chain glyceride, one or more saturated polyglycolized C8-C10 glycerides, or a combination thereof; and / or (b) a weak salt; The formulation, wherein the weak salt is N-methylpyrrolidone (NMP) and / or lactic acid.

2. 2. The formulation of claim 1, wherein the ethanol is present in a concentration of from 5% to 40%.

3. 2. The formulation of claim 1, wherein the organic water-soluble solvent comprises propylene glycol and / or a polyether.

4. The formulation of claim 3, wherein the polyether is polyethylene glycol.

5. The formulation of claim 4, wherein the polyethylene glycol is PEG 6000 or PEG 400.

6. The formulation of claim 3, wherein the propylene glycol and / or polyether are present in a concentration of 1% to 4%.

7. 10. The formulation of claim 1, wherein the formulation has a pH of about 3.5 to about 8.

0.

8. The formulation of claim 7, wherein the formulation has a pH greater than 4.

0.

9. The formulation of claim 1, wherein the formulation contains one or more other active ingredients for treating erectile dysfunction.

10. The formulation of claim 9, wherein the formulation comprises at least two phosphodiesterase inhibitors.

11. 2. The formulation of claim 1, wherein the phosphodiesterase inhibitor is vardenafil, sildenafil, tadalafil, or a combination thereof.

12. The formulation of claim 1, wherein the saturated solubility in the organic aqueous solvent is 20 mg / ml or more.

13. The formulation of claim 12, wherein the formulation has a steady-state flux (Jss) equal to or greater than that obtained with an aqueous formulation having a solubility of 20 mg / ml or greater at a pH of 4.0 or greater.

14. The formulation of claim 1, wherein the weak salt is present in a concentration of 1% to 40%.

15. The formulation of claim 1, wherein the lactic acid is calcium lactate.

16. Use of the formulation described in claim 1 in the manufacture of a medicine for the treatment of erectile dysfunction.

17. The use of claim 16, wherein the pharmaceutical agent is formulated for intranasal administration.

18. The use of claim 16, wherein the pharmaceutical agent is formulated for sublingual administration.

19. A method for preparing a formulation according to any one of claims 1 to 15, comprising the steps of: The method comprises: adding said phosphodiesterase inhibitor to said organic water soluble solvent; adjusting the pH of the organic aqueous solvent containing the phosphodiesterase inhibitor to about 3.5 to 8.0; A method comprising:

20. 20. The method of claim 19, wherein the solubility of the phosphodiesterase inhibitor is increased in the organic water-soluble solvent compared to the solubility of the phosphodiesterase inhibitor in water.

21. 20. The method of claim 19, wherein permeation of the phosphodiesterase inhibitor through the nasal mucosa is increased in the organic water-soluble solvent as compared to permeation of the phosphodiesterase inhibitor in water.

22. 20. The method of claim 19, wherein the bioavailability of the phosphodiesterase inhibitor is increased in the organic water-soluble solvent compared to the bioavailability of the phosphodiesterase inhibitor in water.

23. A formulation according to any one of claims 1 to 15 for use in the treatment of erectile dysfunction.

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