HK40090944A - Magnesium-containing oxytocin formulations and methods of use - Google Patents

Description

Magnesium-containing oxytocin preparations and application methods

This application is a divisional application. The original application was filed on April 12, 2017, with application number 201780036185.3, and the invention title was "Magnesium-containing oxytocin preparation and application method".

Cross-references to related applications

This application claims priority to U.S. Provisional Patent Application No. 62/321,654, filed April 12, 2016, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

This invention relates to methods for treating autism spectrum disorder, related disorders, and symptoms of such disorders, and to compositions comprising oxytocin peptides and magnesium ions.

Background Technology

Oxytocin is a naturally occurring nine-amino acid neuropeptide primarily produced in the paraventricular and supraoptic nuclei of the hypothalamus in mammals. It is released into the central nervous system via distributed neural pathways and enters the peripheral circulation via the posterior pituitary gland. Currently, the United States approves intramuscular or intravenous administration of synthetic oxytocin to produce or improve uterine contractions, thereby promoting vaginal delivery and controlling postpartum hemorrhage. From 1960 to 1997, intranasal oxytocin was approved in the United States for stimulating milk production to promote breastfeeding. Although nasal sprays were withdrawn from the U.S. market at the request of manufacturers, intranasal oxytocin is still sold in countries outside the United States, such as Switzerland, Portugal, and Brazil. The use of oxytocin peptides for the treatment of autism spectrum disorders has recently been demonstrated. See WO2004/030524A2 and WO2008/042452A1, the contents of which are incorporated herein by reference.

Autism spectrum disorder is becoming increasingly prevalent in the population and is typically identified by certain behaviors and characteristics, such as deficits in communication skills and/or social interaction, lack of eye contact, and/or inability to form and/or maintain social relationships. Children and adults diagnosed with autism spectrum disorder may exhibit one or more of these behaviors and characteristics to varying degrees. Symptoms frequently observed in individuals with autism spectrum disorder include persistent deficits in social communication and social interaction, social anxiety, and limited repetitive behaviors, interests, and activities. Other behaviors and characteristics also observed in individuals with autism spectrum disorder include aversion to physical contact, generalized anxiety, monotonous voice or inability to regulate voice volume, failure to develop peer relationships, lack of shared enjoyment and interests, and lack of social or emotional reciprocity. Other disorders presenting with social and communication deficits can include social anxiety disorder, obsessive-compulsive disorder, social (pragmatic) communication disorder, and neurodevelopmental disorders, including but not limited to attention deficit hyperactivity disorder, Prader-Willi syndrome, Timothy syndrome, Fragile-X syndrome, Rett syndrome, or Williams syndrome—which present symptoms similar to those seen in autism spectrum disorders. Individuals with autism spectrum disorder often exhibit communication deficits, such as inappropriate responses in conversation, misinterpretation of nonverbal interactions, or difficulty forming age-appropriate friendships. Furthermore, individuals with autism spectrum disorder may be overly reliant on routines, highly anxious and sensitive to changes in their environment, or intensely fixated on inappropriate objects (e.g., inanimate objects and/or narrow interests in specific topics). Moreover, the symptoms of individuals with autism spectrum disorder are wide-ranging and progressive, with some individuals exhibiting mild symptoms while others experience very severe symptoms. There are no available pharmacological treatments for the core impairments in social communication and social interaction, or restricted repetitive behaviors, interests, and activities in individuals with autism spectrum disorder and related disorders, and such treatment remains in urgent need.

Oxytocin has been shown to improve core symptoms of autism, specifically social and communication deficits and associated anxiety symptoms. Human clinical trials have demonstrated the efficacy of intranasal oxytocin in treating autism spectrum disorder, related disorders, and symptoms of these disorders. See, for example, Yatawara et al., Mol. Psychiatry 2015, 1–9; Gorka et al., Neuropsychopharmacology 2015, 40(2):278–286; Anagnostou et al., Mol. Autism 2012, 3(1):16; Guastella et al., Psychoneuroendocrinology 2009, 34(6):917–923. However, these trials have shown that the response to oxytocin treatment in patients with autism spectrum disorder and related disorders is highly variable. Therefore, there is a need for an oxytocin peptide formulation that can provide more significant efficacy in treating autism spectrum disorder and related disorders.

Summary of the Invention

Methods and compositions comprising oxytocin peptide and magnesium ions are provided for treating autism spectrum disorder, related disorders, and symptoms of such disorders. These include administration of the oxytocin peptide and magnesium ions co-administered via craniofacial mucosa (e.g., intranasal administration). The methods and magnesium-containing oxytocin peptide formulations described herein offer enhanced efficacy in treating autism spectrum disorder compared to oxytocin alone.

In one aspect, the present invention provides a method for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety, comprising administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the co-administration of oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. Oxytocin peptide and magnesium ions may be administered simultaneously or sequentially. In some embodiments, oxytocin peptide is administered simultaneously with magnesium ions at the same unit dose or at separate unit doses or formulations. In some embodiments, oxytocin peptide and magnesium ions are administered sequentially. For example, oxytocin peptide is administered some time after the administration of magnesium ions. In some embodiments, the subject is a human being.

Oxytocin peptide and magnesium ions can be administered to the subject in need via the same or different routes. In some embodiments, oxytocin peptide is administered via the craniofacial mucosa (e.g., nose, cheek, sublingual, or eye). In one embodiment, both oxytocin peptide and magnesium ions are administered intranasally in the same formulation.

In some aspects, according to the methods described herein for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety, interleukin-6 (IL-6) is used as a biomarker for the potential efficacy of administering oxytocin peptide in subjects; and for selecting subjects to apply the method. In some embodiments, the method includes measuring the level of IL-6 in a subject and administering an effective dose of oxytocin peptide and magnesium ions to a subject with elevated IL-6 levels.

In one aspect, methods for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety (including administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the co-administration of oxytocin peptide and magnesium ions produces a synergistic or enhancing effect) further include administering an effective dose of interleukin-6 (IL-6) to the subject, wherein administration of IL-6 causes an increase in oxytocin receptor expression.

In some embodiments, the oxytocin peptide is human oxytocin composed of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQ. ID NO: 1). In some embodiments, the effective dose of the oxytocin peptide is about 0.5 μg to about 2000 μg, preferably about 8 μg to about 1000 μg, more preferably about 15 μg to about 120 μg. In some embodiments, the effective dose of magnesium ions administered is about 50 μg to about 68 mg, preferably about 50 μg to about 34 mg, more preferably about 1 mg to about 3 mg. In some embodiments, the method includes administering a magnesium salt (e.g., magnesium citrate and/or magnesium chloride) to provide about 50 μg to about 68 mg of magnesium, or about 50 μg to about 34 mg of magnesium, or about 1 mg to about 3 mg of magnesium. In some embodiments, the method includes administering a quantity of magnesium citrate or magnesium chloride to provide about 50 μg to about 68 mg of magnesium, or about 50 μg to about 34 mg of magnesium, or about 1 mg to about 3 mg of magnesium. In some embodiments, the effective dose of oxytocin peptide and magnesium ions comprises about 0.5 μg to about 2000 μg, or about 15 μg to about 120 μg (e.g., about 60 μg or about 66 μg) of oxytocin peptide—administered in an aqueous solution containing about 0.11% to about 2.8% (preferably about 1.1% to about 1.6%, e.g., about 1.36%) (w/v) magnesium.

In some embodiments, the present invention provides a method for reducing one or more symptoms associated with autism spectrum disorder. Symptoms treatable by this method include any social or communication deficits treatable by oxytocin peptides, such as eye contact, social anxiety, generalized anxiety, accuracy in determining complex social cues, empathy, and deficits in communication abilities, including expressive language functions.

In some embodiments, the present invention provides a method for treating a disorder exhibiting one or more symptoms associated with autism spectrum disorder. In some embodiments, the disorder is social anxiety disorder, obsessive-compulsive disorder, social (pragmatic) communication disorder, and neurodevelopmental disorder, including but not limited to attention deficit hyperactivity disorder, Prader-Willi syndrome, Timothy syndrome, Fragile-X syndrome, Rett syndrome, or Williams syndrome, which present with symptoms similar to those exhibited in autism spectrum disorder.

In one embodiment, the present invention provides a method for treating autism spectrum disorder, comprising administering (e.g., intranasally) an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the co-administration of the oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In another embodiment, the present invention provides a method for treating autism spectrum disorder, comprising administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the effective dose of oxytocin peptide and magnesium ions is administered intranasally in the form of a liquid formulation, and the volume of the administered liquid formulation is between about 5 μL and about 1000 μL.

In one embodiment, the present invention provides a method for treating Prader-Willi syndrome, comprising administering (e.g., intranasally) an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the co-administration of the oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In another embodiment, the present invention provides a method for treating Prader-Willi syndrome, comprising administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the effective dose of oxytocin peptide and magnesium ions is administered intranasally in the form of a liquid formulation, and the volume of the administered liquid formulation is between about 5 μL and about 1000 μL.

In one embodiment, a method for treating social and communication deficits is provided, comprising administering (e.g., intranasally) an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the co-administration of the oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In one embodiment, the invention provides a method for treating social and communication deficits, comprising administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the effective dose of oxytocin peptide and magnesium ions is administered intranasally in the form of a liquid formulation, and the volume of the administered liquid formulation is between about 5 μL and about 1000 μL.

In one embodiment, a method for treating anxiety is provided, comprising administering (e.g., intranasally) an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the co-administration of the oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In one embodiment, the invention provides a method for treating anxiety, comprising administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the effective dose of oxytocin peptide and magnesium ions is administered intranasally in the form of a liquid formulation, and the volume of the administered liquid formulation is between about 5 μL and about 1000 μL.

In some of these embodiments, the effective dose of oxytocin peptide is from about 0.5 μg to about 2000 μg. In some of these embodiments, the effective dose of magnesium ions is from about 50 μg to about 68 mg. In some of these embodiments, the effective dose of oxytocin peptide and magnesium ions comprises about 15 μg to about 120 μg of oxytocin peptide administered in an aqueous solution containing about 1.1% to about 1.6% (w/v) magnesium. In some of these embodiments, the effective dose of oxytocin peptide and magnesium ions comprises about 66 μg of oxytocin peptide administered in an aqueous solution containing about 1.36% magnesium. In some of these embodiments, the weight ratio between the administered dose of oxytocin peptide and the administered dose of magnesium ions is between about 1:1 and about 1:1000. In some of these embodiments, the molar ratio between the administered dose of oxytocin peptide and the administered dose of magnesium ions is between about 1:40 and about 1:40000. In some of these embodiments, the volume of the liquid formulation administered is between about 50 μL and about 200 μL. In some of these embodiments, 1 to 4 units of the liquid formulation, about 50 μL/unit, are administered using a metering nasal device (e.g., spray or puff). In some of these embodiments, the oxytocin peptide is human oxytocin (SEQ. ID NO: 1).

In some of these embodiments, the liquid formulation is contained in an intranasal delivery device. In some of these embodiments, the intranasal delivery device is a nasal pump device. In some of these embodiments, the nasal pump device includes a container bottle attached to a pump actuator. In some of these embodiments, the pump actuator is metered to deliver a specified volume of approximately 50 μL. In some of these embodiments, the nasal pump device includes a container bottle attached to an aerosolizer. In some of these embodiments, the nasal pump device includes one or more of the following: (i) a filter to prevent backflow, (ii) a metal-free fluid path, and (iii) a gamma-radiation-stable plastic material.

Further provided is the magnesium-containing oxytocin peptide formulation described herein, in a method for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety in subjects in need. The use of the magnesium-containing oxytocin peptide formulation described herein in the preparation of a medicine for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety is also provided.

Kits are also provided that include the magnesium-containing oxytocin peptide formulation described herein, contained in an intranasal administration device such as a nasal pump and suitable packaging. The kit may further include instructions for administering the magnesium-containing oxytocin peptide formulation to subjects in need of it for the treatment of autism spectrum disorder, impairment exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety.

Attached Figure Description

Figure 1 shows the effects of saline, oxytocin, magnesium citrate, and combinations of oxytocin on social behavior in a rat model.

Figure 2 shows the effects of saline, oxytocin, magnesium citrate, and combinations of oxytocin in a rat model, as well as the effect of magnesium citrate on anxiety.

Figures 3A and 3B show the effects of magnesium citrate, oxytocin, and combinations of magnesium citrate and oxytocin on anxiety in an elevated plus maze rat model.

Detailed Implementation

This invention particularly provides a method for treating autism spectrum disorder, impairment exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety in subjects in need of treatment via craniofacial administration (e.g., intranasal administration) of the oxytocin peptide and magnesium ions described herein, or magnesium-containing oxytocin peptide formulations. The oxytocin peptide and magnesium ions are administered at an effective dose that produces a synergistic or enhancing effect compared to administration of the oxytocin peptide alone.

definition

As used herein, “oxytocin peptide” refers to a substance having biological activity associated with natural oxytocin. Oxytocin peptides can be naturally occurring endogenous peptides, fragments thereof, analogs, or derivatives. Oxytocin peptides can also be non-endogenous peptides, fragments thereof, analogs, or derivatives. In one respect, oxytocin peptide is human oxytocin. In other respects, oxytocin peptide can be an analog or derivative of human oxytocin.

As used herein, “analyte” or “derivative” refers to any peptide that is similar to naturally occurring oxytocin, wherein one or more amino acids within the peptide have been substituted, deleted, or inserted. The term also refers to any peptide in which one or more amino acids (e.g., one, two, or three amino acids) have been modified (e.g., through chemical modification). Generally, the term encompasses all peptides that exhibit oxytocin activity but may (if desired) possess different potency or pharmacological profiles.

As used herein, unless otherwise specified, the terms “treatment” or “treating” refer to a method for achieving a beneficial or desired outcome, such as clinical outcomes. For autism spectrum disorders and related disorders, beneficial or desired clinical outcomes include, but are not limited to, symptom relief and/or a reduction in the degree of symptoms, such as, a reduction in social and/or communication deficits and/or repetitive behaviors and/or anxiety. Social and communication deficits may include, but are not limited to, impairments in communication skills and/or social interaction, lack of eye contact, and/or inability to form and/or maintain social relationships.

"Synergism," "synergy," or "synergistic effect" refers to the combined action of two or more compounds in a way that complements or enhances the effect of one compound, resulting in an effect greater than that expected or anticipated by adding a given amount of the two or more compounds (if administered individually). A "synergistic effect" is said to be achieved when the combined use of two or more agents produces an overall effect (e.g., improvement of social and communication deficits and/or reduction of anxiety) greater than the individual effects expected or anticipated by adding equal amounts of any of the individual agents. A "synergistic effect" is also considered to be achieved when the combined use of two or more agents results in a faster onset of action and/or a longer duration of action when administered in equal amounts as the individual agents are used alone.

"Craniofacial mucosal delivery" refers to delivery to the mucosal surfaces of the nose, nasal passages, and nasal cavity; the mucosal surfaces of the oral cavity, including the gingiva, floor of the mouth, lips, tongue, sublingual surface (including the frenulum of the tongue and floor of the mouth); and the mucosal surfaces of the eyes or surrounding areas, including the conjunctiva, lacrimal glands, nasolacrimal ducts, and the mucosa of the upper or lower eyelids and eyes.

"Intranasal administration" or "intranasal delivery" refers to delivery to the nose, nasal passages, or nasal cavity via spray, drops, powder, gel, film, inhaler, or other means.

The "lower region of the nasal cavity" usually refers to the middle part of the nasal cavity and the protruding part of the inferior turbinate bone, and is the area of the nasal cavity that is significantly innervated by the trigeminal nerve. The "upper region of the nasal cavity" is defined by the upper third of the nasal cavity innervated by the olfactory nerve and the cribriform plate region.

As used herein, “subject” or “patient” refers to mammals, including but not limited to humans. Mammals include, but are not limited to, livestock (such as cows), locomotives, pets (such as guinea pigs, cats, dogs, rabbits, and horses), primates, mice, and rats. In one implementation, the subject is a human.

It should be noted that, as used herein, unless otherwise stated, the singular forms “a,” “one,” and “the” include plural references. Additionally, as used herein, the term “comprising” and its cognates are used in their inclusive sense; that is, equivalent to the term “including” and its corresponding cognates.

Where a numerical range is provided, it is intended that every intermediate value between the upper and lower limits of the range and any other statement or intermediate value within the stated range is included in this disclosure. For example, if a range of 1 μg to 8 μg is stated, it is intended that 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, and 7 μg, as well as ranges of values greater than or equal to 1 μg and ranges of values less than or equal to 8 μg, are also explicitly disclosed. If a range of 10-14% is stated, it is intended that 10%, 11%, 12%, 13%, and 14% are also explicitly disclosed. Furthermore, every smaller range of the stated range between any stated value or intermediate value and any other statement or intermediate value within the stated range is included in this disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and each range is also included in this disclosure if any limit, neither, or both are included in the smaller range, subject to any limit specifically excluded from the stated range. Where the scope of a statement includes one or both of the limits, the scope excluding any one or both of the included limits is also included in this disclosure.

Oxytocin peptide

Oxytocin is one of the first peptide hormones to be isolated and sequenced. Natural oxytocin is a nine-amino acid cyclic peptide hormone with two cysteine residues forming a disulfide bridge between positions 1 and 6. The amino acid sequence of human oxytocin is Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQ ID NO:1).

Methods for producing oxytocin exist, see, for example, U.S. Patent Nos. 2,938,891 and 3,076,797; furthermore, oxytocin is commercially available. A variety of peptide analogs and derivatives are available, while others may be considered for use in this invention and can be produced and tested for biological activity according to known methods. Oxytocin analogues may include, but are not limited to, 4-threonine-1-hydroxy-diaminooxytocin, 4-serine-8-isoleucine-oxytocin, 9-deamidized oxytocin, 7-D-proline-oxytocin and its deamination analogues, (2,4-diisoleucine)-oxytocin, deamination oxytocin analogues, 1-deamination-1-monocarba-E12-Tyr(OMe)]-OT(dCOMOT), 4-threonine-7-glycine-oxytocin (TG-OT), oxytocin, deamination-6-carba-oxidative toxin (dC60), L-371,257 and related series of compounds containing a triglucoethoxyphenylacetyl nucleus such as L-374,943. Other exemplary oxytocin analogues include 4-threonine-1-hydroxy-deaminated oxytocin, 9-deamidized oxytocin, oxytocin analogues containing glycine residues in place of glycamide residues, (2,4-diisoleucine)-oxytocin, oxytocin analogues with natriuretic and diuretic activities, deaminated oxytocin analogues, long-acting oxytocin analogues, and 1-deaminated-1-monocarbaz-E12-[Tyr(OMe)] -OT(dCOMOT), carbetocin, (1-butyric acid-2-(O-methyl-L-tyrosine)-1-carbazotoxin, deamin-1-monocarbazotoxin-(2-O-methyltyrosine)-oxytoxin [d(COMOT)]), [Thr4-Gly7]-oxytoxin (TG-OT), oxytocin, Ile-conopressin, deamin-6-carbazotoxin-oxidative toxin (dC60), d[Lys(8)(5/6C-fluorescein] ]VT、d[Thr(4),Lys(8)(5/6C-fluorescein)]VT、[HO(1)][Lys(8)(5/6C-fluorescein)]VT、[HO(1)][Thr(4),Lys(8)(5/6C-fluorescein)]VT、d[Om(8)(5/6C-fluorescein)]VT、d[Thr(4),Om(8)(5/6C-fluorescein)]VT、[HO(1)][Om(8)(5/6C-fluorescein)]VT、d[Thr(4),Om(8)(5/6C-fluorescein)]VT、[HO(1)][Om(8)(5/6C-fluorescein)]VT、 [5/6C-fluorescein]VT, [HO(1)][Thr(4), Om(8)(5/6C-fluorescein)]VT, and 1-deamino-oxytocin, wherein the disulfide bridge between residues 1 or 6 is replaced by a thioether, and deamino-oxytocin analogs, wherein the disulfide bond is replaced by a diselenide bond, a tellurium bond, a tellurium-selenide bond, a tellurium-sulfur bond, or a selenium-sulfur bond (e.g., peptide analogs of oxytocin described in PCT patent application WO2011/120,071 (incorporated herein by reference)). The peptides used in this invention can be peptides that can be obtained by partial substitution, addition, or deletion of amino acids in naturally occurring or natural peptide sequences. The peptides can be chemically modified, for example by carboxyl-terminal ( _-NH2 ) amidation, the application of D amino acids in the peptide, the incorporation of small non-peptide moieties, and modifications of the amino acids themselves (e.g., alkylation or esterification of R-groups in the side chains). Such analogs, derivatives, and fragments should substantially retain the desired biological activity of the natural oxytocin peptide. In some embodiments, the oxytocin analog is 4-serine-8-isoleucine-oxytocin or 9-deamidoxytocin. In some embodiments, the oxytocin analog is carbetocin. This disclosure also includes other known oxytocin analogs, such as peptide oxytocin receptor agonists described in PCT patent applications WO2012/042371 and et al. J. Med. Chem. 2014, 57:5306-5317 (the entire contents of which are incorporated herein by reference). In some embodiments, the oxytocin analog is a compound selected from compounds 1-65 described in Tables 1-3 of et al. J. Med. Chem. 2014, 57:5306-5317. In some embodiments, the oxytocin analogue is selected from compound 31 ([2-ThiMeGly7]dOT), compound 47 (carbaz-6-[Phe2,BuGly7]dOT), compound 55 (carbaz-6-[3-MeBzlGly7]dOT), and compound 57 (carbaz-1-[4-FBzlGly7]dOT, also known as merotocin).

In some embodiments, oxytocin or oxytocin analogues are isotopically labeled by having one or more atoms replaced by isotopes with different atomic masses. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen (e.g., ^2H and ^3H ), carbon (e.g., ^13C and ^14C ), nitrogen (e.g., ^15N ), oxygen (e.g., ^18O and ^17O ), phosphorus (e.g., ^31P and ^32P ), fluorine (e.g., ^18F ), chlorine (e.g., ^36Cl ), and sulfur (e.g., ^35S ). According to conventional techniques, isotopically labeled compounds can be administered to a subject or other subjects and subsequently detected, producing useful diagnostic and/or treatment management data. Furthermore, isotopically labeled compounds can be administered to a subject or other subjects in need of them, producing therapeutically advantageous absorption, distribution, metabolism, and/or elimination characteristics. All isotopic variants of oxytocin peptides (e.g., human oxytocin or its analogues or derivatives)—whether radioactive or non-radioactive—are considered.

In some embodiments, the oxytocin peptide is human oxytocin composed of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQ.IDNO:1).

"International Units" (IU, UI, or IE) are internationally recognized units of activity used to quantify vitamins, hormones, and vaccines. They define the amount of substance and provide an activity unit, as determined using defined bioassays, to standardize preparations from a variety of source materials. Similarly, USP units are defined units of dosage created by the United States Pharmacopeia and the Food and Drug Administration in collaboration to ensure the properties, strength, quality, purity, and consistency of pharmaceutical products. Generally, due to harmonizing efforts, USP units are equivalent to International Units. By convention, for oxytocin, one activity unit is typically defined as approximately equal to 2 micrograms of synthetic oxytocin peptide; or 1 mg equals 500 units (Stedman’s Medical Dictionary). Therefore, as used herein, one "IU" or "International Unit" of oxytocin peptide is an amount of oxytocin peptide having the same biological activity as approximately 2 micrograms of synthetic peptide or producing the same level of biological effect (e.g., contractile response in rat uterine strips) as approximately 2 micrograms of synthetic peptide. Analogs with weaker activity would require more material to achieve the same level of biological effect. Assays of drug efficacy are well known to those skilled in the art and may include in vitro or in vivo assays using synthetic oxytocin as a reference. (Atke and Vilhardt Acta Endocrinol. 1987:115(1):155-60; Engstrom et al. Eur. J. Pharmacol. 1998:355(2-3):203-10.)

Magnesium-containing oxytocin peptide preparations

In the method of the present invention (comprising administering an effective dose of oxytocin peptide and magnesium ions to a subject in need) for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety, the oxytocin peptide and magnesium ions may be administered in the form of a magnesium-containing oxytocin peptide formulation or composition. In one aspect, the magnesium-containing oxytocin peptide formulation or composition includes amounts of oxytocin peptide and magnesium ions that produce a synergistic or enhancing effect when used to treat autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety.

The relative ratio of oxytocin peptides and magnesium ions in magnesium-containing oxytocin peptide formulations is important for achieving optimal synergistic or enhancing effects. The optimal amounts of oxytocin peptides and magnesium ions can depend on the specific impairment or symptom, the type of synergistic or enhancing effect desired, and other factors such as the route of administration. For example, the amount of magnesium can be important for achieving a faster onset of action; the amount of oxytocin can be important for achieving a longer duration of action; and the relative ratio between oxytocin and magnesium can be important for achieving maximum improvement in social functioning, reduction in social and communication deficits, and/or reduction in anxiety.

In some embodiments, the magnesium-containing oxytocin peptide formulation or composition is a liquid formulation comprising oxytocin peptide at concentrations between about 0.01 mg/mL and about 16 mg/mL. In some embodiments, the amount of oxytocin peptide in the liquid formulation is greater than about (lower limit) 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, or 2 mg/mL. In some embodiments, the amount of oxytocin peptide in the liquid formulation is less than about (upper limit) 16, 12, 10, 8, 6, 4, 2, 1.6, 1.2, 1, 0.8, 0.6, 0.4, 0.3, 0.2, or 0.1 mg/mL. That is, the amount of oxytocin peptide in the liquid formulation is anywhere in the range of about 0.01 to 16 mg/mL, wherein the lower limit is less than the upper limit. In some embodiments, the magnesium-containing oxytocin peptide formulation or composition includes amounts between about 0.01 mg/mL and about 12 mg/mL, between about 0.05 mg/mL and about 16 mg/mL, between about 0.1 mg/mL and about 12 mg/mL, between about 0.1 mg/mL and about 8 mg/mL, between about 0.1 mg/mL and about 4 mg/mL, between about 0.1 mg/mL and about 2 mg/mL, between about 0.1 mg/mL and about 1.6 mg/mL, between about 0.1 mg/mL and about 1.2 mg/mL, between about 0.1 mg/mL and about 1 mg/mL, and about 0.1 mg/mL. Between L and approximately 0.8 mg/mL, between approximately 0.1 mg/mL and approximately 0.4 mg/mL, between approximately 0.1 mg/mL and approximately 0.3 mg/mL, between approximately 0.2 mg/mL and approximately 16 mg/mL, between approximately 0.2 mg/mL and approximately 12 mg/mL, between approximately 0.2 mg/mL and approximately 10 mg/mL, between approximately 0.2 mg/mL and approximately 8 mg/mL, between approximately 0.2 mg/mL and approximately 6 mg/mL, between approximately 0.2 mg/mL and approximately 4 mg/mL, between approximately 0.2 mg/mL and approximately 2 mg/mL, between approximately 0.2 mg/mL and approximately 1.6 mg/mL Between approximately 0.2 mg/mL and approximately 1.2 mg/mL, between approximately 0.2 mg/mL and approximately 1 mg/mL, between approximately 0.2 mg/mL and approximately 0.8 mg/mL, between approximately 0.2 mg/mL and approximately 0.6 mg/mL, between approximately 0.2 mg/mL and approximately 0.4 mg/mL, between approximately 0.2 mg/mL and approximately 0.3 mg/mL, between approximately 0.3 mg/mL and approximately 16 mg/mL, between approximately 0.3 mg/mL and approximately 12 mg/mL, between approximately 0.3 mg/mL and approximately 10 mg/mL, between approximately 0.3 mg/mL and approximately 8 mg/mL, approximately 0.3 mg/mL Oxytocin peptide at concentrations between g/mL and about 4 mg/mL, between about 0.3 mg/mL and about 3 mg/mL, between about 0.3 mg/mL and about 1 mg/mL, between about 0.3 mg/mL and about 0.5 mg/mL, between about 0.5 mg/mL and about 16 mg/mL, between about 0.5 mg/mL and about 10 mg/mL, between about 0.5 mg/mL and about 5 mg/mL, between about 0.5 mg/mL and about 1 mg/mL, between about 1 mg/mL and about 16 mg/mL, between about 1 mg/mL and about 10 mg/mL, or between about 1 mg/mL and about 5 mg/mL. In a preferred embodiment, the magnesium-containing oxytocin peptide formulation or composition comprises oxytocin peptide at concentrations between about 0.1 mg/mL and about 2 mg/mL, between about 0.15 mg/mL and about 1.5 mg/mL, or between about 0.2 mg/mL and about 1.2 mg/mL. In one embodiment, the oxytocin peptide is human oxytocin composed of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQ.ID NO:1).

In some embodiments, the magnesium-containing oxytocin peptide formulation or composition is a liquid formulation comprising an oxytocin peptide between about 5 IU/mL and about 8000 IU/mL. In some embodiments, the amount of oxytocin peptide in the liquid formulation is greater than about (lower limit) 5, 25, 50, 75, 100, 150, 200, 250, 500, 750, or 1000 IU/mL. In some embodiments, the amount of oxytocin peptide in the liquid formulation is less than about (upper limit) 8000, 6000, 5000, 4000, 3000, 2000, 1000, 800, 600, 500, 400, 300, 200, 150, 100, or 50 IU/mL. That is, the amount of oxytocin peptide in the liquid formulation is anywhere in the range of about 5 to 8000 IU/mL, wherein the lower limit is less than the upper limit. In some embodiments, the magnesium-containing oxytocin peptide formulation or composition comprises between about 500 IU/mL and about 6000 IU/mL, between about 25 IU/mL and about 8000 IU/mL, between about 50 IU/mL and about 6000 IU/mL, between about 50 IU/mL and about 4000 IU/mL, between about 50 IU/mL and about 2000 IU/mL, between about 50 IU/mL and about 1000 IU/mL, between about 50 IU/mL and about 800 IU/mL, between about 50 IU/mL and about 600 IU/mL, between about 50 IU/mL and about 500 IU/mL, and between about 50 IU/mL and about 400 IU/mL. Between L, approximately 50 IU/mL and approximately 200 IU/mL, approximately 50 IU/mL and approximately 150 IU/mL, approximately 100 IU/mL and approximately 8000 IU/mL, approximately 100 IU/mL and approximately 6000 IU/mL, approximately 100 IU/mL and approximately 5000 IU/mL, approximately 100 IU/mL and approximately 4000 IU/mL, approximately 100 IU/mL and approximately 3000 IU/mL, approximately 100 IU/mL and approximately 2000 IU/mL, approximately 100 IU/mL and approximately 1000 IU/mL, approximately 100 IU/mL and approximately 800 IU/mL, approximately 100 IU/mL Between approximately 600 IU/mL and approximately 100 IU/mL and approximately 500 IU/mL, between approximately 100 IU/mL and approximately 400 IU/mL, between approximately 100 IU/mL and approximately 300 IU/mL, between approximately 100 IU/mL and approximately 200 IU/mL, between approximately 100 IU/mL and approximately 150 IU/mL, between approximately 150 IU/mL and approximately 8000 IU/mL, between approximately 150 IU/mL and approximately 6000 IU/mL, between approximately 150 IU/mL and approximately 5000 IU/mL, between approximately 150 IU/mL and approximately 4000 IU/mL, between approximately 150 IU/mL and approximately 2000 IU/mL Oxytocin peptides between L, approximately 150 IU/mL and approximately 1500 IU/mL, approximately 150 IU/mL and approximately 500 IU/mL, approximately 150 IU/mL and approximately 250 IU/mL, approximately 250 IU/mL and approximately 8000 IU/mL, approximately 250 IU/mL and approximately 5000 IU/mL, approximately 250 IU/mL and approximately 2500 IU/mL, approximately 250 IU/mL and approximately 500 IU/mL, approximately 500 IU/mL and approximately 8000 IU/mL, approximately 500 IU/mL and approximately 5000 IU/mL, or approximately 500 IU/mL and approximately 2500 IU/mL. In a preferred embodiment, the magnesium-containing oxytocin peptide formulation or composition comprises an oxytocin peptide between about 50 IU/mL and about 1000 IU/mL, between about 75 IU/mL and about 750 IU/mL, or between about 100 IU/mL and about 600 IU/mL. In one embodiment, the oxytocin peptide is human oxytocin composed of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQ. ID NO: 1).

The amount of magnesium present in the formulation can also be expressed as a weight percentage (w/v) (grams of magnesium or ^Mg²⁺ per 100 mL of solution), as mg/mL (milligrams of magnesium or ^Mg²⁺ per milliliter of solution), or as a molar concentration (“M” - defined as the number of moles of magnesium or ^Mg²⁺ per liter of solution; or “mM” - defined as the number of millimoles of magnesium or ^Mg²⁺ per liter of solution).

In some embodiments, the magnesium-containing oxytocin peptide formulation or composition is a liquid formulation comprising magnesium or magnesium ions ( ^Mg²⁺ ) between about 1 mg/mL and about 30 mg/mL. In some embodiments, the composition comprises magnesium or magnesium ions between about 11 mg/mL and about 15 mg/mL. In some embodiments, the amount of magnesium or magnesium ions in the liquid formulation is greater than about (lower limit) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mg/mL. In some embodiments, the amount of magnesium or magnesium ions in the liquid formulation is less than about (upper limit) 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 mg/mL. That is, the amount of magnesium or magnesium ions in the liquid formulation is anywhere in the range of about 1 to 30 mg/mL, wherein the lower limit is less than the upper limit. In some embodiments, the magnesium-containing oxytocin peptide formulation or composition is a liquid formulation comprising between about 0.01 mg/mL and about 16 mg/mL (preferably between about 0.1 mg/mL and about 2 mg/mL, more preferably between about 0.15 mg/mL and about 1.5 mg/mL, or about 0.33 mg/mL) of oxytocin peptide and between about 1 mg/mL and about 30 mg/mL (or between about 3 mg/mL and about 30 mg/mL, between about 4 mg/mL and about 30 mg/mL, between about 5 mg/mL and about 30 mg/mL, between about 8 mg/mL and about 30 mg/mL, between about 10 mg/mL and about 30 mg/mL, preferably between about 11 mg/mL and about 15 mg/mL, or about 13 mg/mL, or about 12 mg/mL) of magnesium or ^Mg²⁺ . In some embodiments, the magnesium-containing oxytocin peptide formulation or composition is a liquid formulation comprising between about 50 mM and about 1500 mM of magnesium or magnesium ions ( ^Mg²⁺ ). In some embodiments, the amount of magnesium or magnesium ions in the liquid formulation is greater than about (lower limit) 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 mM. In some embodiments, the amount of magnesium or magnesium ions in the liquid formulation is less than about (upper limit) 1500, 1200, 1000, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, or 250 mM. That is, the amount of magnesium or magnesium ions in the liquid formulation is anywhere in the range of about 50 to 1500 mM, wherein the lower limit is less than the upper limit. In some embodiments, the magnesium-containing oxytocin peptide formulation or composition is a liquid formulation comprising between about 5 IU/mL and about 8000 IU/mL (preferably between about 50 IU/mL and about 1000 IU/mL, more preferably between about 75 IU/mL and about 750 IU/mL, or about 150 IU/mL) of oxytocin peptide and between about 1 mg/mL and about 30 mg/mL (preferably between about 11 mg/mL and about 15 mg/mL, or about 13 mg/mL, or about 12 mg/mL) of magnesium or Mg2 ⁺ . In some embodiments, the magnesium-containing oxytocin peptide formulation or composition is a liquid formulation comprising between about 5 IU/mL and about 8000 IU/mL (preferably between about 50 IU/mL and about 1000 IU/mL, more preferably between about 75 IU/mL and about 750 IU/mL, or about 150 IU/mL) of oxytocin peptide and between about 50 mM and about 1200 mM (or between about 100 mM and about 1200 mM, about 150 mM and about 1200 mM, about 200 mM and about 1200 mM, about 300 mM and about 1200 mM, about 400 mM and about 1200 mM, preferably between about 400 mM and about 600 mM, or about 500 mM) of magnesium or Mg2 ⁺ .

Any magnesium salt (such as a water-soluble magnesium salt) can be used to provide magnesium ions in magnesium-containing oxytocin peptide formulations. The magnesium salt used in magnesium-containing oxytocin peptide formulations can be selected based on several factors, such as the amount of free magnesium ions that can be delivered at the time of administration, the solubility of the magnesium salt in the liquid formulation medium, the acidity/basicity of the counterion, and/or the dissociation constant of the salt. For example, in liquid formulations, the magnesium salt needs to be sufficiently soluble in the liquid medium to deliver magnesium ions at the concentration required to produce a synergistic or enhancing effect with the oxytocin peptide. Other factors, such as compatibility with other substances in the formulation and the ability of the counterion to perform other functions in the formulation, can also be considered when selecting a magnesium salt. For example, magnesium citrate is sufficiently soluble in aqueous solution to provide the desired amount of magnesium or the desired magnesium ion concentration; citrate is pharmaceutically acceptable; citrate can be part of a buffer; and magnesium citrate can add a palatable flavor to the formulation. Magnesium ions in magnesium-containing oxytocin peptide formulations can be provided using one or more magnesium salts. The magnesium salt in a magnesium-containing oxytocin peptide formulation can be a magnesium salt initially used in the preparation of the formulation, or a magnesium salt formed in situ during the preparation of the magnesium-containing oxytocin peptide formulation. For example, magnesium chloride can be initially used in the preparation of the formulation; and magnesium citrate can be formed in situ after the addition of citric acid to the formulation. In such examples, magnesium ions in the magnesium-containing oxytocin peptide formulation are provided by magnesium chloride and magnesium citrate.

In some embodiments, the magnesium-containing oxytocin peptide formulation or composition comprises one or more magnesium salts selected from magnesium citrate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium lactate, magnesium stearate, magnesium oxide, magnesium carbonate, magnesium glycine, magnesium malate, magnesium taurate, magnesium gluconate, magnesium succinate, and magnesium pyrophosphate. In some embodiments, the magnesium-containing oxytocin peptide formulation or composition is a liquid formulation containing a certain amount of a magnesium salt (e.g., magnesium citrate or magnesium chloride) to provide magnesium at a concentration between about 1 mg/mL and about 30 mg/mL. In some embodiments, the composition comprises a certain amount of a magnesium salt to provide magnesium ions ( ^Mg²⁺ ) at a concentration between about 1 mg/mL and about 30 mg/mL. In some embodiments, the magnesium-containing oxytocin peptide formulation or composition is a liquid formulation containing a certain amount of one or more magnesium salts (e.g., magnesium citrate and/or magnesium chloride) to provide magnesium or magnesium ions ( ^Mg²⁺ ) at a concentration between about 1 mg/mL and about 30 mg/mL. In some embodiments, the composition includes an amount of one or more magnesium salts, thereby providing magnesium or magnesium ions at a concentration between about 11 mg/mL and about 15 mg/mL. In some embodiments, the magnesium-containing oxytocin peptide formulation or composition is a liquid formulation comprising between about 0.01 mg/mL and about 16 mg/mL (preferably between about 0.1 mg/mL and about 2 mg/mL, more preferably between about 0.15 mg/mL and about 1.5 mg/mL, or about 0.33 mg/mL) of oxytocin peptide and a certain amount of magnesium salt (e.g., magnesium citrate or magnesium chloride) to provide between about 1 mg/mL and about 30 mg/mL (or between about 3 mg/mL and about 30 mg/mL, between about 4 mg/mL and about 30 mg/mL, between about 5 mg/mL and about 30 mg/mL, between about 8 mg/mL and about 30 mg/mL, between about 10 mg/mL and about 30 mg/mL, preferably between about 11 mg/mL and about 15 mg/mL, or about 13 mg/mL, or about 12 mg/mL) of magnesium or ^Mg²⁺ . In some embodiments, the magnesium-containing oxytocin peptide formulation or composition is a liquid formulation comprising between about 5 IU/mL and about 8000 IU/mL (preferably between about 50 IU/mL and about 1000 IU/mL, more preferably between about 75 IU/mL and about 750 IU/mL, or about 150 IU/mL) of oxytocin peptide and a certain amount of one or more magnesium salts (e.g., magnesium citrate and/or magnesium chloride) to provide between about 1 mg/mL and about 30 mg/mL (preferably between about 11 mg/mL and about 15 mg/mL, or about 13 mg/mL, or about 12 mg/mL) of magnesium or Mg2 ⁺ . In some embodiments, the magnesium-containing oxytocin peptide formulation or composition is a liquid formulation comprising between about 5 IU/mL and about 8000 IU/mL (preferably between about 50 IU/mL and about 1000 IU/mL, more preferably between about 75 IU/mL and about 750 IU/mL, or about 150 IU/mL) of oxytocin peptide and a certain amount of one or more magnesium salts (e.g., magnesium citrate and/or magnesium chloride) to provide between about 50 mM and about 1200 mM (or between about 100 mM and about 1200 mM, about 150 mM and about 1200 mM, about 200 mM and about 1200 mM, about 300 mM and about 1200 mM, about 400 mM and about 1200 mM, preferably between about 400 mM and about 600 mM, or about 500 mM) of magnesium or Mg2 ⁺ .

The relative amounts of oxytocin peptide and magnesium ions in the magnesium-containing oxytocin peptide formulations or compositions described herein can be defined by a weight ratio or a molar ratio. The weight ratio between the amount of oxytocin peptide and the amount of magnesium or magnesium ions in the formulation or composition is referred to as the "OT/Mg(w) ratio". For example, in a magnesium-containing oxytocin peptide formulation or composition having an OT/Mg(w) ratio of about 1:40, for every 1 mg of oxytocin peptide present in the formulation or composition, there are about 40 mg of magnesium or magnesium ions present in the formulation or composition. The molar ratio between the amount of oxytocin peptide and the amount of magnesium or magnesium ions in the formulation or composition is referred to as the "OT/Mg(m) ratio". For example, in a magnesium-containing oxytocin peptide formulation or composition having an OT/Mg(m) ratio of about 1:1600, for every 1 μmol of oxytocin peptide present in the formulation or composition, there are about 1600 μmol of magnesium or magnesium ions present in the formulation or composition.

In some embodiments, the magnesium-containing oxytocin peptide formulation or composition has an OT/Mg(w) ratio between about 1:1 and about 1:1000. In some embodiments, the OT/Mg(w) ratio in the formulation or composition is less than about (upper limit) 1:1, 1:2, 1:5, 1:10, 1:20, 1:30, 1:40, 1:45, 1:50, 1:60, 1:80, 1:100, or 1:200. In some embodiments, the OT/Mg(w) ratio in the formulation or composition is greater than about (lower limit) 1:1000, 1:800, 1:500, 1:250, 1:200, 1:150, 1:100, 1:80, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, or 1:5. That is, the OT/Mg(w) ratio in the formulation or composition is anywhere in the range of about 1:1 to 1:1000, wherein the upper limit is greater than the lower limit. In some embodiments, the formulation or composition has an OT/Mg(w) ratio between about 1:2 and about 1:200. In some preferred embodiments, the formulation or composition has an OT/Mg(w) ratio of about 1:30, about 1:35, about 1:40, about 1:45, or about 1:50. In some embodiments, the formulation or composition has an OT/Mg(w) ratio between: about 1:2 and about 1:1000, about 1:2 and about 1:800, about 1:2 and about 1:500, about 1:2 and about 1:250, about 1:2 and about 1:150, about 1:2 and about 1:100, about 1:2 and about 1:80, about 1:2 and about 1:60, about 1:2 and about 1:50, about 1:2 and about 1:40, about 1:2 and about 1:30, and so on. Between 1:2 and approximately 1:20, between approximately 1:2 and approximately 1:10, between approximately 1:2 and approximately 1:5, between approximately 1:5 and approximately 1:1000, between approximately 1:5 and approximately 1:800, between approximately 1:5 and approximately 1:500, between approximately 1:5 and approximately 1:200, between approximately 1:5 and approximately 1:100, between approximately 1:5 and approximately 1:80, between approximately 1:5 and approximately 1:60, between approximately 1:5 and approximately 1:50, between approximately 1:5 and approximately 1:40, between approximately 1:5 and approximately 1:30, between approximately 1:5 and approximately 1: Between 20, approximately 1:5 and approximately 1:10, approximately 1:10 and approximately 1:1000, approximately 1:10 and approximately 1:800, approximately 1:10 and approximately 1:500, approximately 1:10 and approximately 1:200, approximately 1:10 and approximately 1:100, approximately 1:10 and approximately 1:80, approximately 1:10 and approximately 1:60, approximately 1:10 and approximately 1:50, approximately 1:10 and approximately 1:40, approximately 1:10 and approximately 1:30, approximately 1:10 and approximately 1:20, approximately 1: Between approximately 1:20 and approximately 1:1000, between approximately 1:20 and approximately 1:800, between approximately 1:20 and approximately 1:500, between approximately 1:20 and approximately 1:200, between approximately 1:20 and approximately 1:100, between approximately 1:20 and approximately 1:80, between approximately 1:20 and approximately 1:70, between approximately 1:20 and approximately 1:60, between approximately 1:20 and approximately 1:50, between approximately 1:20 and approximately 1:40, between approximately 1:20 and approximately 1:30, between approximately 1:30 and approximately 1:1000, between approximately 1:30 and approximately... Between 1:800, approximately 1:30 and 1:500, approximately 1:30 and 1:200, approximately 1:30 and 1:100, approximately 1:30 and 1:80, approximately 1:30 and 1:70, approximately 1:30 and 1:60, approximately 1:30 and 1:50, approximately 1:30 and 1:40, approximately 1:35 and 1:45, approximately 1:40 and 1:1000, approximately 1:40 and 1:800, approximately 1:40 and 1:500 Between approximately 1:40 and approximately 1:200, between approximately 1:40 and approximately 1:100, between approximately 1:40 and approximately 1:80, between approximately 1:40 and approximately 1:70, between approximately 1:40 and approximately 1:60, between approximately 1:40 and approximately 1:50, between approximately 1:50 and approximately 1:1000, between approximately 1:50 and approximately 1:800, between approximately 1:50 and approximately 1:50, between approximately 1:50 and approximately 1:200, between approximately 1:50 and approximately 1:100, between approximately 1:50 and approximately 1:90, approximately 1: Between approximately 1:50 and 1:80, between approximately 1:50 and 1:70, between approximately 1:50 and 1:60, between approximately 1:60 and 1:1000, between approximately 1:60 and 1:800, between approximately 1:60 and 1:500, between approximately 1:60 and 1:200, between approximately 1:60 and 1:100, between approximately 1:60 and 1:90, between approximately 1:60 and 1:80, between approximately 1:60 and 1:70, between approximately 1:80 and 1:1000, between approximately 1:80 and approximately 1:1000. The ratios are between 1:80 and 1:500, between 1:80 and 1:200, between 1:80 and 1:100, between 1:100 and 1:1000, between 1:100 and 1:800, between 1:100 and 1:500, between 1:100 and 1:200, between 1:200 and 1:1000, between 1:200 and 1:800, between 1:200 and 1:500, or between 1:500 and 1:1000. In one embodiment, the oxytocin peptide is human oxytocin composed of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQ.ID NO:1).

In some embodiments, the magnesium-containing oxytocin peptide formulation or composition has an OT/Mg(m) ratio between about 1:40 and about 1:40,000. In some embodiments, the OT/Mg molar ratio in the formulation or composition is less than about (upper limit) 1:40, 1:80, 1:100, 1:150, 1:175, 1:200, 1:250, 1:280, 1:300, 1:400, 1:500, 1:560, 1:800, 1:1000, 1:1100, 1:1200, 1:1600, 1:1700, 1:1800, 1:2000, 1:2400, 1:3200, 1:4000, or 1:8000. In some embodiments, the OT/Mg molar ratio in the formulation or composition is greater than about (lower limit) 1:40000, 1:30000, 1:20000, 1:10000, 1:7500, 1:5000, 1:4000, 1:3000, 1:2500, 1:2000, 1:1600, 1:1200, 1:1100, 1:1000, 1:800, 1:600, 1:400, or 1:200. That is, the OT/Mg(w) ratio in the formulation or composition is anywhere in the range of about 1:40 to 1:40000, where the upper limit is greater than the lower limit. In some embodiments, the formulation or composition has an OT/Mg(m) ratio between about 1:80 and about 1:8000. In some preferred embodiments, the formulation or composition has an OT/Mg(m) ratio of about 1:175, about 1:280, about 1:560, about 1:1100, about 1:1200, about 1:1400, about 1:1600, about 1:1700, about 1:1800, or about 1:2000. In some embodiments, the formulation or composition has an OT/Mg(m) ratio between: about 1:80 and about 1:40000, about 1:80 and about 1:30000, about 1:80 and about 1:20000, about 1:80 and about 1:10000, about 1:80 and about 1:7500, about 1:80 and about 1:5000, about 1:80 and about 1:3000, about 1:80 and about 1:2000, about 1:80 and about 1:16. Between 00, approximately 1:80 and approximately 1:1200, approximately 1:80 and approximately 1:800, approximately 1:80 and approximately 1:400, approximately 1:80 and approximately 1:200, approximately 1:175 and approximately 1:40000, approximately 1:175 and approximately 1:30000, approximately 1:175 and approximately 1:20000, approximately 1:175 and approximately 1:10000, approximately 1:175 and approximately 1:5000, approximately 1:175 and approximately 1:3000, approximately 1 Between approximately 1:175 and 1:2400, between approximately 1:175 and 1:2000, between approximately 1:175 and 1:1700, between approximately 1:175 and 1:1600, between approximately 1:175 and 1:1200, between approximately 1:175 and 1:1100, between approximately 1:175 and 1:800, between approximately 1:175 and 1:560, between approximately 1:175 and 1:400, between approximately 1:175 and 1:280, between approximately 1:200 and 1:175... Between 40,000, approximately 1:200 and approximately 1:30,000, approximately 1:200 and approximately 1:20,000, approximately 1:200 and approximately 1:10,000, approximately 1:200 and approximately 1:5,000, approximately 1:200 and approximately 1:3,000, approximately 1:200 and approximately 1:24,000, approximately 1:200 and approximately 1:200, approximately 1:200 and approximately 1:16,000, approximately 1:200 and approximately 1:12,000, approximately 1:200 and approximately 1:16,000, approximately 1:200 and approximately 1:12,000, approximately 1:200 and approximately 1:10,0 ... Between 800, approximately 1:200 and approximately 1:400, approximately 1:280 and approximately 1:40000, approximately 1:280 and approximately 1:30000, approximately 1:280 and approximately 1:20000, approximately 1:280 and approximately 1:10000, approximately 1:280 and approximately 1:5000, approximately 1:280 and approximately 1:3000, approximately 1:280 and approximately 1:2400, approximately 1:280 and approximately 1:2000, approximately 1:280 and approximately 1:17 Between 00:00, approximately 1:280 and approximately 1:1600, approximately 1:280 and approximately 1:1200, approximately 1:280 and approximately 1:1100, approximately 1:280 and approximately 1:800, approximately 1:280 and approximately 1:560, approximately 1:280 and approximately 1:400, approximately 1:400 and approximately 1:40000, approximately 1:400 and approximately 1:30000, approximately 1:400 and approximately 1:20000, approximately 1:400 and approximately 1:8000 Between approximately 1:400 and approximately 1:4000, between approximately 1:400 and approximately 1:3000, between approximately 1:400 and approximately 1:2400, between approximately 1:400 and approximately 1:2000, between approximately 1:400 and approximately 1:1600, between approximately 1:400 and approximately 1:1200, between approximately 1:400 and approximately 1:800, between approximately 1:560 and approximately 1:40000, between approximately 1:560 and approximately 1:30000, between approximately 1:560 and approximately 1:20000, approximately... Between 1:560 and approximately 1:8000, between approximately 1:560 and approximately 1:4000, between approximately 1:560 and approximately 1:3000, between approximately 1:560 and approximately 1:2400, between approximately 1:560 and approximately 1:2000, between approximately 1:560 and approximately 1:1700, between approximately 1:560 and approximately 1:1600, between approximately 1:560 and approximately 1:1200, between approximately 1:560 and approximately 1:1100, between approximately 1:560 and approximately 1:800, between approximately 1:800 and approximately... Between 1:40,000, approximately 1:800 and approximately 1:30,000, approximately 1:800 and approximately 1:20,000, approximately 1:800 and approximately 1:10,000, approximately 1:800 and approximately 1:5,000, approximately 1:800 and approximately 1:3,000, approximately 1:800 and approximately 1:2,400, approximately 1:800 and approximately 1:2,000, approximately 1:800 and approximately 1:1600, approximately 1:800 and approximately 1:1200, approximately 1:1100 and... Between approximately 1:40,000, between approximately 1:1100 and approximately 1:30,000, between approximately 1:1100 and approximately 1:20,000, between approximately 1:1100 and approximately 1:10,000, between approximately 1:1100 and approximately 1:5,000, between approximately 1:1100 and approximately 1:4,000, between approximately 1:1100 and approximately 1:3,000, between approximately 1:1100 and approximately 1:2,400, between approximately 1:1100 and approximately 1:2,000, between approximately 1:1100 and approximately 1:1,700. Between approximately 1:1100 and approximately 1:1600, between approximately 1:1200 and approximately 1:40000, between approximately 1:1200 and approximately 1:30000, between approximately 1:1200 and approximately 1:20000, between approximately 1:1200 and approximately 1:10000, between approximately 1:1200 and approximately 1:5000, between approximately 1:1200 and approximately 1:4000, between approximately 1:1200 and approximately 1:3000, between approximately 1:1200 and approximately 1:2400, approximately 1:120 Between 0 and approximately 1:2000, between approximately 1:1200 and approximately 1:1600, between approximately 1:1400 and approximately 1:1800, between approximately 1:1600 and approximately 1:40000, between approximately 1:1600 and approximately 1:30000, between approximately 1:1600 and approximately 1:20000, between approximately 1:1600 and approximately 1:10000, between approximately 1:1600 and approximately 1:5000, between approximately 1:1600 and approximately 1:3000, between approximately 1:1600 and approximately 1:240 Between 0, approximately 1:1600 and approximately 1:2000, approximately 1:1700 and approximately 1:40000, approximately 1:1700 and approximately 1:30000, approximately 1:1700 and approximately 1:20000, approximately 1:1700 and approximately 1:10000, approximately 1:1700 and approximately 1:5000, approximately 1:1700 and approximately 1:3000, approximately 1:1700 and approximately 1:2400, approximately 1:1700 and approximately 1:2000, approximately 1:2 Between approximately 1:000 and approximately 1:40000, between approximately 1:2000 and approximately 1:30000, between approximately 1:2000 and approximately 1:20000, between approximately 1:2000 and approximately 1:10000, between approximately 1:2000 and approximately 1:5000, between approximately 1:2000 and approximately 1:4000, between approximately 1:2000 and approximately 1:3000, between approximately 1:2000 and approximately 1:2400, between approximately 1:2400 and approximately 1:40000, between approximately 1:2400 and approximately 1:10000, between approximately 1:20 ... Between 30,000, approximately 1:2400 and approximately 1:20,000, approximately 1:2400 and approximately 1:10,000, approximately 1:2400 and approximately 1:5,000, approximately 1:2400 and approximately 1:4,000, approximately 1:2400 and approximately 1:3,000, approximately 1:3,000 and approximately 1:40,000, approximately 1:3,000 and approximately 1:30,000, approximately 1:3,000 and approximately 1:20,000, approximately 1:3,000 and approximately 1:10,000 The ratios are between approximately 1:3000 and approximately 1:4000, between approximately 1:4000 and approximately 1:40000, between approximately 1:4000 and approximately 1:30000, between approximately 1:4000 and approximately 1:20000, between approximately 1:4000 and approximately 1:10000, between approximately 1:8000 and approximately 1:40000, between approximately 1:8000 and approximately 1:30000, between approximately 1:8000 and approximately 1:20000, or between approximately 1:10000 and approximately 1:40000. In one embodiment, the oxytocin peptide is human oxytocin composed of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQ. ID NO: 1).

In some embodiments, magnesium-containing oxytocin peptide formulations or compositions comprising oxytocin peptides and magnesium ions further include one or more pharmaceutically acceptable carriers (thus constituting a pharmaceutical composition) and optionally other components such as excipients, vehicles, emulsifiers, stabilizers, preservatives, buffers, and/or other additives that may enhance stability, delivery, absorption, half-life, potency, pharmacokinetics, and/or pharmacodynamics, reduce adverse side effects, or provide other advantages for pharmaceutical application. Exemplary excipients include solubilizers, surfactants, and chelating agents. For example, formulations may include methyl-β-cyclodextrin (Me-β-CD), disodium edetate, arginine, sorbitol, NaCl, sodium methylparaben (MP), sodium propylparaben (PP), chlorobutanol (CB), benzyl alcohol, zinc chloride, ethanol, didecyl L-α-phosphatidylcholine (DDPC), polysorbate, lactose, citrate, tartrate, acetate, and/or phosphate.

Liquid carriers include, but are not limited to, water, saline, dextran aqueous solutions, and diols (especially for solutions in isotonic cases). Carriers can also be selected from various oils, including those from petroleum, animal, vegetable, or synthetic sources (e.g., peanut oil, olive oil, soybean oil, mineral oil, sesame oil, etc.). Suitable pharmaceutical excipients include, but are not limited to, starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl monostearate, sodium chloride, dry skim milk powder, glycerin, propylene glycol, water, ethanol, etc. The composition may undergo conventional pharmaceutical processes, such as sterilization, and may contain conventional pharmaceutical additives, such as preservatives, stabilizers, reducing agents, antioxidants, chelating agents, wetting agents, emulsifiers, dispersants, gelling agents, osmotic pressure regulating salts, buffers, etc. The liquid carrier may be hypotonic or isotonic with body fluids and may have a pH in the range of 3.5–8.5. The use of additives in the preparation of peptide and/or protein-based compositions, specifically pharmaceutical compositions, is well known in the art. In some embodiments, the composition has a pH of about 2 to about 7. In some embodiments, the composition has a pH of about 4 to about 7. In a preferred embodiment, the pH of the formulation/composition is about 4.5.

In some embodiments, the magnesium-containing oxytocin peptide formulation or composition may further include one or more mucosal delivery enhancers selected from (A)-(K): (A) solubilizer; (B) charge modifier; (C) pH controller; (D) degradative enzyme inhibitor; (E) mucolytic or mucolytic agent; (F) ciliostatic agent; (G) membrane permeability enhancer; (H) epithelial junction physiological regulator, such as nitric oxide (NO) stimulant, chitosan, and chitosan derivatives; (I) vasodilator; (J) selective transport enhancer; and (K) stabilizing delivery medium, carrier, support, or complex forming substance (to which the oxytocin peptide is effectively combined, associated, contained, encapsulated, or bound to stabilize the active agent for enhanced mucosal delivery). The membrane permeation enhancers in group (G) may be (i) surfactants, (ii) bile salts, (iii) phospholipids or fatty acid additives, mixed micelles, liposomes, or carriers, (iv) alcohols, (v) enamines, (iv) NO donor compounds, (vii) long-chain amphiphilic molecules, (viii) small hydrophobic permeation enhancers; (ix) sodium or salicylic acid derivatives; (x) glyceryl acetoacetate, (xi) cyclodextrin or β-cyclodextrin derivatives, (xii) medium-chain fatty acids, (xiii) chelating agents, (xiv) amino acids or their salts, (xv) N-acetyl amino acids or their salts, (xvi) enzymes that degrade into selected membrane components, (xvii) inhibitors of fatty acid synthesis, (xviii) inhibitors of cholesterol synthesis; or any combination of membrane permeation enhancers (xiv) (i)-(xviii). In various embodiments of the invention, oxytocin peptides may be combined with one, two, three, four or more of the mucosal delivery enhancers described in (A)-(K). These mucosal delivery enhancers can be mixed alone, with, or otherwise combined with pharmaceutically acceptable formulations or delivery media. The magnesium-containing oxytocin peptide formulations or compositions described herein can provide increased bioavailability of the oxytocin peptide upon delivery to the mucosal surface of a mammalian subject (e.g., in the nasal cavity).

The list of carriers and additives discussed herein is not exhaustive, and those skilled in the art can select carriers and excipients from the GRAS (Generally Recognized As Safe) list of chemicals permitted in pharmaceutical preparations, as well as those currently permitted by the U.S. Food and Drug Administration for topical and parenteral formulations, and those permitted in the future. (See also Wang et al., (1980) J. Parent. Drug Assn., 34:452-462; Wang et al., (1988) J. Parent. Sci. and Tech., 42:S4-S26).

In some embodiments, magnesium-containing oxytocin peptide formulations or compositions comprising oxytocin peptides and magnesium ions (wherein the oxytocin peptides and magnesium ions are in amounts that produce a synergistic or enhancing effect when used to treat autism spectrum disorder) further comprise one or more solvents or excipients selected from chlorobutanol, benzalkonium, methyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate, acetic acid, citric acid, glycerol, sodium chloride, sodium monohydrogen phosphate, sorbitol, and water. In some embodiments, magnesium-containing oxytocin peptide formulations or compositions further comprise chlorobutanol, acetic acid, and water.

In some embodiments, magnesium-containing oxytocin peptide formulations or compositions comprising oxytocin peptides and magnesium ions further include a chitosan-containing excipient (e.g., http://www.archimedespharma.com/productArchiDevChiSys.html). In some embodiments, magnesium-containing oxytocin peptide formulations or compositions further include about 1% of a chitosan-containing excipient. In some embodiments, chitosan glutamate is preferably used for nasal delivery—due to its excellent absorption-enhancing capabilities. In some embodiments, chitosan copolymer nanoparticles, such as nanoparticles containing chitosan glutamate and a negatively charged polymer (e.g., pentasodium tripolyphosphate), can be used. Thiolized chitosan (e.g., chitosan covalently modified with 2-iminothioane)—which has been used in microparticles containing insulin and reduced glutathione—can also be used as an excipient in the magnesium-containing oxytocin peptide formulations or compositions described herein.

In some embodiments, magnesium-containing oxytocin peptide formulations or compositions comprising oxytocin peptides and magnesium ions further include one or more gelling agents, causing the oxytocin peptide formulation to form a gel in the nasal cavity, thereby enhancing nasal absorption of the oxytocin peptide. Gelation systems that can be used in the formulations and methods described herein can include any known gelation system, such as chemically reactive pectin-based gelation systems (e.g., PecSys ^™ , Archimedes Pharma) and thermally reactive polymer gelation systems (e.g., F127, BASF). PecSys ^™ is a low-viscosity, aqueous pectin-based solution delivered as a fine mist, wherein each drop of gel contacts calcium ions in the nasal mucosa. Other low-methoxyl pectins may also be used, for example, at a concentration of about 1%. F127 contains an ethylene oxide/propylene oxide block copolymer. The gelation temperature varies depending on the component ratio and the amount of copolymer used in the final formulation. Gel formation of F127 in the human nasal cavity at approximately 18-20% wt/vol has been demonstrated, for example, in vitamin B12 gel supplements (EnerB, Nature's Bounty, NY) and gelled sumatriptan, which contain 18% wt/vol F127 and 0.3% wt/vol Carbopol (an anionic bioadhesive polymer C934P). The monomer ratios and concentrations can be adjusted for the intended oxytocin formulation to ensure gel formation at 25-37°C (typically around 34°C in the nasal cavity). If the gelation temperature is below 25°C, the formulation may gel at room temperature; if the gelation temperature is above 37°C, the formulation will not fully gel upon contact with the nasal mucosa. In some embodiments, magnesium-containing oxytocin peptide formulations or compositions may further include a mucosal adhesive such as Carbopol. The addition of a mucosal adhesive (e.g., up to 0.5% Carbopol) can further lower the gelation temperature.

In some embodiments, magnesium-containing oxytocin peptide formulations or compositions comprising oxytocin peptides and magnesium ions further include surfactants, such as nonionic surfactants (e.g., polysorbate-80), and one or more buffers, stabilizers, or tonicators. In some embodiments, magnesium-containing oxytocin peptide formulations or compositions further include propellants. The pH of the nasal spray solution is optionally between about pH 3.0 and 8.5, but the pH may be adjusted when desired to optimize the delivery of charged macromolecules (e.g., therapeutic proteins or peptides) in a substantially unionized state. The pharmaceutical solvent used may also be a slightly acidic buffered aqueous solution (pH 3-6). Suitable buffers used in these compositions are as described above or as known in the art otherwise. Other components may be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants, or gases. Suitable preservatives include, but are not limited to, phenol, methylparaben, parabens, m-cresol, sodium ethylmercuric thiosalicylate, benzalkonium chloride, etc. Suitable surfactants include, but are not limited to, oleic acid, sorbitan trioleate, polysorbate, lecithin, phosphatidylcholine, and various long-chain diglycerides and phospholipids. Suitable dispersants include, but are not limited to, ethylenediaminetetraacetic acid (EDTA). Suitable gases include, but are not limited to, nitrogen, helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon dioxide, and air. Suitable stabilizers and tonicating agents include sugars and other polyols, amino acids, and organic and inorganic salts. In some embodiments, magnesium-containing oxytocin peptide formulations or compositions further include citrate, succinate, or pyrophosphate.

In some embodiments, magnesium-containing oxytocin peptide formulations or compositions comprising oxytocin peptides and magnesium ions further include agents capable of upregulating oxytocin receptor expression, such as IL-6.

To further enhance the mucosal delivery of oxytocin peptides, enzyme inhibitors, specifically protease inhibitors, may be further included in the formulation. Protease inhibitors may include, but are not limited to, antipain, arphamenine A and B, anisole hydrochloride, AEBSF, CA-074, calpain inhibitors I and II, calpeptidase inhibitor, pepsin inhibitor A, actinomycin, aminopeptidase inhibitor, phenbutazone, boroleucine, captopril, chloroacetyl-HOLeu-Ala-Gly- _NH₂ , DAPT, diprotinin A and B, ebelactone A and B, methaqualone, leucine-inhibitory peptide, phosphonyl dipeptide, bovine trypsin inhibitor, puromycin, BBI, soybean trypsin inhibitor, benzoyl sulfonyl fluoride, E-64, chymotrypsin inhibitor, 1,10-phenanthroline, EDTA, and EGTA. Other enzyme inhibitors, such as bacitracin, may also be included in the formulation.

To enhance the delivery of oxytocin peptides and magnesium ions into or across the mucosal surface and/or absorption, absorption enhancers may be included in the formulation. These enhancers can improve the release or solubility of the composition (e.g., from the formulation delivery medium), diffusion rate, permeability and time, uptake, residual time, stability, effective half-life, peak or sustained concentration level, clearance, and other desired mucosal delivery properties (e.g., as measured at the delivery site). Therefore, enhancement of mucosal delivery can occur through any number of mechanisms, such as increasing the diffusion, transport, persistence, or stability of oxytocin peptides; increasing membrane fluidity; modulating the availability or action of calcium and other ions that modulate intracellular or bypass permeation; dissolving mucosal membrane components (e.g., lipids); altering non-protein and protein thiol levels in mucosal tissue; increasing water flow across the mucosal surface; modulating epithelial junction physiology; reducing the viscosity of mucus on the mucosal epithelium; reducing the rate of mucociliary clearance; and other mechanisms.

Mucosal absorption-enhancing compounds may include, but are not limited to, surfactants, bile salts, dihydrofusidates, bioadhesives/mucosal adhesives, phospholipid additives, mixed micelles, liposomes, or carriers, alcohols, enamines, cationic polymers, NO donor compounds, long-chain amphiphilic molecules, small hydrophobic permeability enhancers; sodium or salicylic acid derivatives, glyceryl acetoacetate, cyclodextrins or β-cyclodextrin derivatives, medium-chain fatty acids, chelating agents, amino acids or their salts, N-acetyl amino acids or their salts, mucolytic agents, enzymes specifically targeting selected membrane components, fatty acid synthesis inhibitors, and cholesterol synthesis inhibitors.

All peptides described and/or considered herein can be prepared by chemical synthesis using automated or manual solid-phase synthesis techniques commonly known in the art. Peptides can also be prepared using molecular recombination techniques known in the art.

Delivery system

Magnesium-containing oxytocin peptide formulations or compositions may be suitable for craniofacial mucosal administration (e.g., nasal, buccal, sublingual, or ocular administration). In some embodiments, the composition may further include a device for mucosal delivery. In some embodiments, the composition is suitable for buccal and/or sublingual mucosal delivery, and may further include a device for buccal and/or sublingual mucosal delivery, such as a unit-dose container, pump sprayer, dropper, plastic squeeze bottle, airless and preservative-free sprayer, nebulizer, dosing inhaler, and pressurized dosing inhaler. In some embodiments, the composition is suitable for ocular delivery, and may further include a device for conjunctival delivery, such as a dropper or plastic squeeze bottle. In some embodiments, the composition is suitable for intranasal delivery, and may further include an intranasal delivery device, such as a dropper, pump sprayer, plastic squeeze bottle, airless and preservative-free sprayer, or nasal pump device, for example, a nasal pump device including a container attached to an aerosol device.

Intranasal drug delivery has been a subject of research and development for many years, although effective delivery systems have only been devised in the last decade (Sayani and Chien, Critical Reviews in Therapeutic Drug Carrier Systems 1996, 13:85-184). Intranasal delivery offers several advantages, including relatively high bioavailability, rapid absorption kinetics, and avoidance of the first-pass effect in the liver. In some respects, intranasal administration allows for the delivery of oxytocin peptides into the nasal cavity, while in others, it allows for targeted delivery to cranial nerves in the nose and/or brain. Without being bound by any particular theory, intranasal administration of oxytocin peptides can target the olfactory nervous system or the trigeminal nervous system, or both. Oxytocin peptides can be delivered intranasally in any applicable form—including but not limited to liquid formulations, solid formulations (e.g., dry powder formulations), gel formulations, or emulsion formulations.

In embodiments where the combination of oxytocin and magnesium ions is administered intranasally, the composition can be prepared as a liquid aerosol formulation in combination with a dispersant and/or a pharmaceutically acceptable diluent. Alternatively, dry powder aerosol formulations are considered and may contain the subject compound in finely pulverized solid form and a dispersant, allowing for easy dispersion of the dry powder particles. In the case of liquid or dry powder aerosol formulations, the formulation is aerosolized into small, liquid or solid particles to ensure that the aerosolized dose reaches the mucosa of the nasal passages or lungs. The term "aerosol particles" is used herein to describe liquid or solid particles suitable for sufficiently small particle sizes for distribution to the target mucosa or alveolar membrane via the nose (in the range of about 10 micrometers) or lungs (in the range of about 2-5 micrometers). Other considerations include the structure of the delivery device, additional components in the formulation, and particle characteristics. These aspects of nasal or pulmonary administration of the drug are well known in the art, and the handling of the formulation, aerosolization methods, and the structure of the delivery device are at the level of a person skilled in the art.

In some embodiments, the magnesium-containing oxytocin peptide formulation or composition available in the methods described herein (wherein the oxytocin peptide and magnesium ions are in amounts that produce a synergistic or enhancing effect when used for the treatment of autism spectrum disorder) is administered using a device for intranasal delivery. The device can be any device suitable for intranasal delivery of the magnesium-containing oxytocin peptide formulation. In some embodiments, the device is adapted to deliver the oxytocin peptide and magnesium ions to a specific area within the nasal cavity. In some embodiments, the device is adapted to deliver the oxytocin peptide and magnesium ions to the lower two-thirds of the nasal cavity. In some embodiments, the device is adapted to deliver the oxytocin peptide and magnesium ions to the upper third of the nasal cavity. In some embodiments, the device is adapted to deliver the oxytocin peptide throughout the nasal passage.

In some embodiments, the device for intranasal delivery is a nasal pump device. In some embodiments, the nasal pump device includes a container attached to a pump actuator. In some embodiments, the pump actuator is metered to deliver a specified volume (e.g., about 5 to about 1000 μL, preferably about 50 to about 150 μL, more preferably about 50 μL or about 100 μL) at a specified droplet size. In some embodiments, the nasal pump device includes a container attached to an aerosolizer, such as the Equadel pump sold by Aptar Pharma. In some embodiments, once a threshold is reached, the device for nasal delivery functions regardless of the pressure applied to the pump. In some embodiments, the device for nasal delivery is a mucosal nebulizer that can be added to a syringe (e.g., MAD NASAL ^™ ). For delivery in large mammals, the nasal pump device may include a container attached to a pump actuator that is metered to deliver a larger volume (e.g., about 100 μL to about 600 μL, or higher).

In some embodiments, the device for intranasal delivery is designed to deliver multiple doses of a pharmaceutical formulation. For example, a nasal pump device may include a container bottle attached to a pump actuator, wherein the container bottle contains multiple doses of a liquid formulation and the pump actuator is metered to deliver a specified volume—a portion of the liquid formulation contained in the container bottle. In some embodiments, the pump actuator is metered to deliver approximately 50 μL of liquid formulation per spray. The nasal pump device may include a filter to prevent backflow in order to reduce contaminants (e.g., bacteria) entering the container bottle. In some embodiments, the nasal pump device includes a metal-free path for delivering the liquid formulation (e.g., a plastic path). In some embodiments, the pump device utilizes a plastic material stable to gamma radiation (for sterilizing the nasal device). In some embodiments, the device for intranasal delivery is equipped with a multi-dose pump that includes a microbial filter and an automatic shut-off mechanism in the pump actuator, such as the spray device described in U.S. Patent No. 5,988,449.

In some embodiments, the device for intranasal delivery is a respiratory-actuated nasal delivery device, such as those described in U.S. Patent Nos. 7,784,460 and 7,854,227. Such a device can improve delivery to a target location deep within the nasal cavity. In some embodiments, a standard-dose spray device is incorporated into a housing that allows the patient to blow into the mouthpiece to actuate the device. In some embodiments, the device consists of a conical, sealed nosepiece and mouthpiece incorporated into a conventional mechanical spray pump (e.g., the Equadel pump sold by Aptar Pharma), a rechargeable spring, and a respiratory actuation mechanism. The system can be used for single-dose or multi-dose delivery. An example of such a liquid delivery device is the OptiMist ^™ device sold by OptiNose. In use, the nosepiece of the device is inserted into the nostril and the mouthpiece is blown in. This closes the soft palate, transmits pressure to the nostril, opens a passage behind the nasal septum to provide airflow, and allows air to exit from the other nostrils (bidirectional flow). Because the device is respiratory-actuated, small particles cannot enter the lungs. Modifications to flow rate and particle size allow for targeting of specific nose regions.

In some embodiments, the device for intranasal delivery is a unit-dose metering spray device suitable for a single administration of a magnesium-containing oxytocin peptide formulation or composition. In some embodiments, the device for intranasal delivery is a multi-dose metering spray pump device suitable for repeated administration of oxytocin peptide.

Droplet size, plume volume, and flow rate can be modified to target specific nasal regions. Liquid nebulizers can provide droplet sizes between 5 and 50 micrometers to target the olfactory and/or respiratory epithelium. Larger droplets primarily travel down the nasopharynx and are swallowed, while smaller droplets are targeted to lung tissue. The Mass Median Equivalent Aerodynamic Diameter (MMAD) is used to specify the droplet size. The pH of the nasal spray is optimized to deliver charged peptides in a predominantly unionized state. The nose generally tolerates solutions with a pH of approximately 3–8. The nasal mucosa can typically absorb a volume of approximately 100 μL before saturation occurs and before the liquid begins to overflow from the nose. Therefore, plume volumes can be up to (and include) 100 μL. For use in large mammals, plume volumes can be up to (and include) 150 μL or higher (e.g., 600 μL or higher). For use in infants and young children, or for veterinary use in small animals (e.g., rodents, cats), a smaller plume volume (5-50 μL) may be used.

In some embodiments, the device for intranasal delivery is ergonomically designed to promote patient compliance, such as a pump device with a side-actuated trigger mechanism. In some embodiments, the device for intranasal delivery includes a metering spray pump operating as a closed system, which prevents air from entering the pump device, thereby preventing contamination from airborne pathogens. In some embodiments, the device for intranasal delivery includes a metering spray pump working in conjunction with a filter. Venting air is drawn in through a filter housed within the pump, keeping airborne pathogens outside the pump device. In some embodiments, the intranasal delivery device including a nasal pump device may further include microelectronic devices that can facilitate data transmission and treatment monitoring.

In some embodiments, the magnesium-containing oxytocin peptide formulation or composition comprises oxytocin peptide and magnesium ions, wherein the oxytocin peptide and magnesium ions are contained in any of the devices described herein for intranasal delivery, and wherein the concentrations of the oxytocin peptide and magnesium ions are within any of the concentration ranges described herein, as each and every combination of devices and concentrations is described separately.

method

The term “autism spectrum disorder (ASD)” or “autism” refers to a complex group of brain development disorders. These disorders manifest to varying degrees as difficulties in social interaction, verbal and nonverbal communication, and repetitive behaviors. Regarding the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), published in May 2013, all autism disorders are grouped into a single umbrella diagnosis of ASD. Previously, they were considered distinct subtypes, including autism, childhood disintegration disorder, pervasive developmental disorder-no-other-status (PDD-NOS), and Asperger syndrome. See http://www.autismspeaks.org/what-autism. Those skilled in the art will recognize that the symptoms of autism spectrum disorder overlap considerably with many other mental illnesses. Examples of disorders that exhibit symptoms similar to those shown in autism spectrum disorders include, but are not limited to, social anxiety disorder, obsessive-compulsive disorder, social (pragmatic) communication disorder, and neurodevelopmental disorders—including but not limited to attention deficit hyperactivity disorder, Prader-Willi syndrome, Timothy syndrome, Fragile-X syndrome, Rett syndrome, and Williams syndrome.

DSM-5 provides diagnostic criteria for ASD, including: (A) persistent impairment in social communication and social interaction across multiple contexts, as demonstrated by illustrative examples, either currently or historically: (1) social-emotional reciprocity. (a) Deficiencies in reciprocity, for example, ranging from failure to engage in abnormal social approaches and normal back-and-forth conversations; to reduced sharing of interests, feelings, or sentiments; to failure to initiate or respond to social interactions; (b) Deficiencies in nonverbal communication behaviors used for social interactions, for example, ranging from poor integration of verbal and nonverbal communication; to abnormal eye contact and body language or inadequate understanding and use of gestures; to a complete lack of facial expressions and nonverbal communication; and (c) Deficiencies in developing, maintaining, and understanding relationships, for example, ranging from difficulty adjusting behavior to adapt to various social situations; to difficulty sharing imaginative games or making friends; to a lack of interest in peers; and (d) Restricted, repetitive patterns of behavior, interests, or activities, as currently or historically demonstrated by at least two of the following illustrative examples: (a) Stereotyped or repetitive movements, object use, or speech (e.g., simple motor stereotypies, arranging toys or flipping objects, echolalia, idiosyncratic phrases). (1) phrases); (2) insistence on identity, inflexible adherence to routines or ritualistic patterns or verbal-nonverbal behaviors (e.g., extreme distress at small changes, difficulty in transition, rigid thought patterns, greeting rituals, need to follow the same path or eat food every day); (3) unusually restrictive or fixated interests (e.g., strong attachment or focus to unusual objects, excessively restrictive or persistent interests); and (4) hyperresponsiveness or hyporesponsiveness to sensory input or unusual interest in environmental sensory aspects (e.g., marked indifference to pain/temperature, adverse response to specific sounds or textures, excessive smelling or touching of objects, visual fascination with light or motion). See http://www.autismspeaks.org/what-autism/diagnosis/dsm-5-diagnostic-criteria.

Autism spectrum disorder (ASD) is characterized by difficulties in social interaction, communication challenges, and a tendency to engage in repetitive behaviors. However, the symptoms and their severity vary considerably across these three core areas. ASD may be associated with intellectual disability, motor coordination difficulties, and attention and physical health problems such as sleep and gastrointestinal disorders. ASD may also be associated with psychiatric symptoms, including anxiety and depression. See, for example, Kim et al., Autism 2000, 4(2):117-132.

Oxytocin is known to treat a variety of conditions, including anxiety and social and communication deficits in autism spectrum disorder (ASD). However, the efficacy of oxytocin in treating social and communication deficits in ASD has been observed to vary considerably among patients. Variations in receptor availability and receptor affinity of oxytocin may be the cause of these variations. Clinical efforts to treat ASD using commercial formulations of oxytocin (e.g., [example]) have been hampered by a lack of potency and poor tolerability. Due to the low potency and high volume of currently available oxytocin formulations, the amount of drug absorbed is insufficient to exert its efficacy when administered via nasal spray. This invention provides a method for administering oxytocin peptides in a more effective formulation and with a lower volume, enabling the delivery of an effective amount of the formulation using nasal devices intended for treating ASD, disorders exhibiting one or more symptoms associated with ASD, or social and communication deficits.

In one aspect, a method is provided for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety, comprising administering an effective amount of oxytocin peptide and magnesium ions to a subject in need, wherein the effective amount is delivered via intranasal administration in a volume readily absorbed in the nasal cavity. In some embodiments, the volume of oxytocin peptide and magnesium ions readily absorbed in the nasal cavity is between about 5 μL and about 1000 μL. In some embodiments, the molar ratio between the amount of oxytocin peptide and the amount of magnesium or magnesium ions is about 1:175, about 1:280, about 1:560, about 1:1100, about 1:1700, or about 1:2000, including any range between these ratios.

In one aspect, a method is provided comprising administering an effective amount of oxytocin peptide and magnesium ions to a subject in need, wherein the effective amount is delivered intranasally in a volume between about 5 μL and about 1000 μL. In some embodiments, the molar ratio between the amount of oxytocin peptide and the amount of magnesium or magnesium ions is about 1:175, about 1:280, about 1:560, about 1:1100, about 1:1700, or about 1:2000, including any range between these ratios. In some embodiments, the method is used to treat one or more symptoms associated with autism spectrum disorder. In some embodiments, the method is used to treat a disorder exhibiting one or more symptoms associated with autism spectrum disorder. In some embodiments, the method is used to reduce social and communication deficits. In some embodiments, the method is used to treat or reduce anxiety.

Magnesium plays a vital role in many aspects of life and health, such as energy production, oxygen uptake, central nervous system function, electrolyte balance, glucose metabolism, and muscle activity. Magnesium has also been found to be clinically effective in reducing social and communication deficits in children with autism spectrum disorder. See Mousain-Bosc et al., Magnes. Res. 2006, 19(1):53-62. The co-administration of oxytocin and magnesium ions in this invention results in synergistic or enhanced improvements in social behavior and reduction in anxiety compared to oxytocin administration alone. The underlying mechanisms of these effects are not yet clear, but may involve non-competitive blocking of N-methyl-D-aspartate (NMDA) neurotransmitter receptors, increased affinity of oxytocin receptors as allosteric modulators, or both.

In some aspects, methods are provided for treating autism spectrum disorder, including administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the co-administration of oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In some aspects, methods are provided for alleviating or reducing one or more symptoms associated with autism spectrum disorder, including administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the co-administration of oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In some aspects, methods are provided for treating a disorder exhibiting one or more symptoms associated with autism spectrum disorder, including administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the co-administration of oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In some embodiments, oxytocin peptide and magnesium ions are administered at a dose that produces an overall effect of symptom relief or reduction greater than the sum of the effects of administering equal doses of oxytocin peptide and magnesium salts alone. In some implementations, oxytocin peptide and magnesium ions are administered in a dose that produces a faster onset and/or longer duration of effect compared to administration of the individual agents in equal amounts. Examples of symptoms associated with autism spectrum disorder include, but are not limited to, persistent deficits in social communication and social interaction, social anxiety, and restricted repetitive behaviors, interests, and activities. Other behaviors and characteristics also observed in patients with autism spectrum disorder include aversion to physical contact, generalized anxiety, monotonous voice or inability to regulate the volume of one's voice, failure to develop peer relationships, lack of shared enjoyment and interests, and lack of social or emotional reciprocity. Examples of disorders presenting symptoms similar to those manifested in autism spectrum disorder include, but are not limited to, social anxiety disorder, obsessive-compulsive disorder, social (pragmatic) communication disorder, and neurodevelopmental disorders—including, but not limited to, attention deficit hyperactivity disorder, Prader-Willi syndrome, Timothy syndrome, Fragile-X syndrome, Rett syndrome, and Williams syndrome.

Prader-Willi syndrome is a complex genetic condition affecting many parts of the body and is caused by the loss of function of a gene in a specific region of chromosome 15. Individuals with Prader-Willi syndrome typically have mild to moderate intellectual disability and learning difficulties, as well as a variety of behavioral problems, including irritability, obstinacy, manipulative behavior, and compulsive behaviors such as scratching the skin. Other symptoms frequently observed in individuals with Prader-Willi syndrome include persistent deficits in social communication and interaction, anxiety and irritability, and sleep problems.

In some aspects, methods for treating Prader-Willi syndrome are provided, comprising administering an effective dose of oxytocin peptide and magnesium ions to a subject in need. In some embodiments, the co-administration of oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In some embodiments, oxytocin peptide and magnesium ions are administered at a dose that produces an overall effect greater than the sum of the effects of administering equal doses of oxytocin peptide and magnesium salts alone. In some embodiments, oxytocin peptide and magnesium ions are administered at a dose that produces a faster onset and/or longer duration of effect compared to the effect that would occur after administering equal amounts of each agent individually. In one embodiment, the present invention provides a method for treating Prader-Willi syndrome, comprising administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the effective dose of oxytocin peptide and magnesium ions is administered intranasally in a liquid formulation, and the volume of the administered liquid formulation is between about 5 μL and about 1000 μL. In some embodiments, the molar ratio between the amount of oxytocin peptide and the amount of magnesium or magnesium ions is about 1:175, about 1:280, about 1:560, about 1:1100, about 1:1700, or about 1:2000, including any range between these ratios.

In some aspects, methods are provided for alleviating or reducing one or more symptoms associated with Prader-Willi syndrome, including administering an effective dose of oxytocin peptide and magnesium ions to a subject in need. Examples of symptoms associated with Prader-Willi syndrome include, but are not limited to, persistent deficits in social communication and social interaction, anxiety and irritability, and sleep problems. In some embodiments, the co-administration of oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In some embodiments, oxytocin peptide and magnesium ions are administered at a dose that produces an overall effect of symptom relief or reduction greater than the sum of the effects of administering equal doses of oxytocin peptide and magnesium salts alone. In some embodiments, oxytocin peptide and magnesium ions are administered at a dose that produces a faster onset and/or longer duration of effect compared to that which would occur after administering equal amounts of each agent individually. Examples of symptoms associated with Prader-Willi syndrome include, but are not limited to, persistent deficits in social communication and social interaction, anxiety and irritability, and sleep problems. In one embodiment, the present invention provides a method for treating Prader-Willi syndrome, comprising administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the effective dose of oxytocin peptide and magnesium ions is administered intranasally in a liquid formulation, and the volume of the liquid formulation administered is between about 5 μL and about 1000 μL. In some embodiments, the molar ratio between the amount of oxytocin peptide and the amount of magnesium or magnesium ions is about 1:175, about 1:280, about 1:560, about 1:1100, about 1:1700, or about 1:2000, including any range between these ratios.

In some aspects, methods for treating anxiety associated with Prader-Willi syndrome are provided, including administering an effective dose of oxytocin peptide and magnesium ions to a subject in need. In some embodiments, the co-administration of oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In some embodiments, oxytocin peptide and magnesium ions are administered at a dose that produces an overall effect of relieving or reducing anxiety greater than the sum of the effects of administering equal doses of oxytocin peptide and magnesium salts alone. In some embodiments, oxytocin peptide and magnesium ions are administered at a dose that produces a faster onset and/or longer duration of effect compared to that would occur after administering equal amounts of each agent individually. In one embodiment, the invention provides a method for treating anxiety associated with Prader-Willi syndrome, including administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the effective dose of oxytocin peptide and magnesium ions is administered intranasally in a liquid formulation, and the volume of the liquid formulation administered is between about 5 μL and about 1000 μL. In some embodiments, the molar ratio between the amount of oxytocin peptide and the amount of magnesium or magnesium ions is about 1:175, about 1:280, about 1:560, about 1:1100, about 1:1700, or about 1:2000, including any range between these ratios.

In one aspect, the present invention provides a method for treating social and communication deficits, comprising administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the co-administration of oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In another aspect, the present invention provides a method for treating anxiety, comprising administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the co-administration of oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In some embodiments, oxytocin peptide and magnesium ions are administered at a dose that produces an overall effect of reducing social and communication deficits and/or anxiety greater than the sum of the effects of administering equal doses of oxytocin peptide and magnesium salt alone. In some embodiments, the molar ratio between the amount of oxytocin peptide and the amount of magnesium or magnesium ions is about 1:175, about 1:280, about 1:560, about 1:1100, about 1:1700, or about 1:2000, including any range between these ratios. In some implementations, social and communication deficits are deficiencies in communication skills and/or social interactions, lack of eye contact, and/or inability to form and/or maintain social relationships.

In some aspects, methods are provided for treating anxiety associated with autism spectrum disorder, including administering an effective dose of oxytocin peptide and magnesium ions to a subject in need. In some embodiments, the co-administration of oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In some embodiments, oxytocin peptide and magnesium ions are administered at a dose that produces an overall effect of relieving or reducing anxiety greater than the sum of the effects of administering equal doses of oxytocin peptide and magnesium salt alone. In some embodiments, oxytocin peptide and magnesium ions are administered at a dose that produces a faster onset and/or longer duration of effect compared to the effect that would occur after administering equal amounts of each agent individually. In some embodiments, the molar ratio between the amount of oxytocin peptide and the amount of magnesium or magnesium ions is about 1:175, about 1:280, about 1:560, about 1:1100, about 1:1700, or about 1:2000, including any range between these ratios.

Oxytocin peptide and magnesium ions can be administered simultaneously or sequentially. In some embodiments, oxytocin peptide and magnesium ions are administered simultaneously at the same unit dose. In some embodiments, oxytocin peptide and magnesium ions are administered simultaneously but at separate unit doses or formulations. In some embodiments, oxytocin peptide and magnesium ions are administered sequentially. In some embodiments, magnesium ions are administered to the subject in a first administration, and oxytocin peptide is subsequently administered to the subject in a second administration. In some of these embodiments, oxytocin peptide is administered between approximately 10 minutes and approximately 2 hours after magnesium ion administration. In some of these embodiments, oxytocin peptide is administered between approximately 10 minutes and approximately 2 hours, between approximately 10 minutes and approximately 1 hour, between approximately 10 minutes and approximately 30 minutes, between approximately 20 minutes and approximately 2 hours, between approximately 20 minutes and approximately 1 hour, between approximately 30 minutes and approximately 2 hours, or between approximately 30 minutes and approximately 1 hour after magnesium ion administration. In some of these embodiments, oxytocin peptide is administered approximately 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, or 120 minutes after the administration of magnesium ions. In one embodiment, the subject is first given oxytocin peptide, followed by the administration of magnesium ions. In some embodiments, the subject is a human being.

Interleukin-6 (IL-6) has been shown to induce elevated expression of oxytocin receptors in various tissues (e.g., Young et al., J. Neuroendocrinology, 1997; 9:859-65). Therefore, serum IL-6 levels can be used as a biomarker for the potential potency of oxytocin, for example, when administered intranasally with magnesium.

In some aspects, according to the methods described herein for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety, IL-6 is used as a biomarker of the efficacy of administering oxytocin peptide in a subject; and for selecting subjects for use in the method. In some embodiments, IL-6 is used to select subjects (e.g., humans) for administering oxytocin peptide (e.g., nasal administration of oxytocin peptide in combination with magnesium ions).

In some embodiments, subjects are selected for treatment based on having high levels of IL-6. The IL-6 level may be higher than a control or reference. In some embodiments, an IL-6 level is considered higher than a control or reference if an appropriate statistical analysis determines that the IL-6 level is significantly greater than the control or reference. In some embodiments, an IL-6 level is considered higher than a control or reference if the IL-6 level is greater than the control or reference by at least one standard deviation. In some embodiments, the control is an IL-6 level measured in age- and sex-matched healthy subjects. In some embodiments, the reference is a reported value of the IL-6 level, such as the reported value of IL-6 in age- and sex-matched healthy subjects. In some embodiments, the IL-6 level is determined as the level of IL-6 in a sample (e.g., a tissue or fluid sample) from the subject, including but not limited to whole blood, serum, plasma, tears, etc. The IL-6 level in the sample can be determined by any method known in the art, such as by immunoassay, e.g., an ELISA-based assay. See, for example, Yang, C-J., et al. Neuroscience 284:290-296, 2015; Emanuele, E., et al. Neuroscience letters 471(3):162-165, 2010; Ashwood, P., et al. Brain, behavior, and immunity 25(1):40-45, 2011; and Malik, M., et al. Immunobiology 216(1):80-85, 2011.

In some implementations, methods for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety include measuring the level of IL-6 in a subject (e.g., serum IL-6 level) and administering an effective dose of oxytocin peptide and magnesium ions to a subject with high IL-6 levels.

In one aspect, methods for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety (including administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the co-administration of oxytocin peptide and magnesium ions produces a synergistic or enhancing effect) further include administering an effective amount of interleukin-6 (IL-6) to the subject. In some embodiments, an effective amount of IL-6 leads to an increase in the expression of the oxytocin receptor (OTR) in the subject.

In some embodiments, according to any of the methods described herein—where IL-6 is administered to the subject—oxytocin peptide and IL-6 may be administered simultaneously or sequentially. In some embodiments, oxytocin peptide and IL-6 are administered simultaneously at the same unit dose. In some embodiments, oxytocin peptide and IL-6 are administered simultaneously but at separate unit doses or formulations. In some embodiments, oxytocin peptide and IL-6 are administered sequentially. In some embodiments, IL-6 is administered to the subject in a first administration, and oxytocin peptide is subsequently administered to the subject in a second administration. In some of these embodiments, oxytocin peptide is administered between approximately 1 minute and approximately 4 hours after administration of IL-6. In some of these embodiments, the oxytocin peptide is administered between approximately 1 minute and approximately 4 hours, approximately 10 minutes and approximately 4 hours, approximately 10 minutes and approximately 3 hours, approximately 10 minutes and approximately 2 hours, approximately 10 minutes and approximately 1 hour, approximately 10 minutes and approximately 30 minutes, approximately 20 minutes and approximately 4 hours, approximately 20 minutes and approximately 3 hours, approximately 20 minutes and approximately 2 hours, approximately 20 minutes and approximately 1 hour, approximately 30 minutes and approximately 4 hours, approximately 30 minutes and approximately 3 hours, approximately 30 minutes and approximately 2 hours, or approximately 30 minutes and approximately 1 hour after administration of IL-6. In some of these embodiments, the oxytocin peptide is administered at approximately 1 minute, approximately 10 minutes, approximately 15 minutes, approximately 20 minutes, approximately 30 minutes, approximately 45 minutes, approximately 60 minutes, approximately 90 minutes, approximately 120 minutes, approximately 150 minutes, approximately 180 minutes, approximately 210 minutes, or approximately 240 minutes after administration of IL-6. In some of these embodiments, oxytocin peptide is administered approximately 10 minutes, 15 minutes, 20 minutes, or 30 minutes after IL-6 administration. In one embodiment, oxytocin peptide is administered to the subject first, followed by IL-6. In some embodiments, the subject is a human. In some of these embodiments, magnesium ions are administered simultaneously with oxytocin peptide and/or IL-6, either before or after either or both of oxytocin peptide and IL-6.

Oxytocin peptide and magnesium ions can be administered to subjects in need via the same or different routes. In some embodiments, oxytocin peptide is administered via the craniofacial mucosa (e.g., nose, cheek, sublingual, or ocular administration). In one embodiment, both oxytocin peptide and magnesium ions are administered intranasally in the same formulation. In one embodiment, oxytocin peptide is administered via the craniofacial mucosa, and magnesium ions are administered systemically, for example, intravenously, intramuscularly, orally, subcutaneously, or intrathecally.

In some embodiments, oxytocin peptide is administered via intranasal delivery. In some embodiments, oxytocin peptide and magnesium ions are administered via intranasal delivery. Oxytocin peptide and/or magnesium ions can be delivered to the mucosal tissue within the nasal cavity using a suitable intranasal delivery device such as the nasal delivery device described herein. Suitable areas within the nasal cavity include, but are not limited to, the lower two-thirds, or the upper third, or the entire nasal passage. In some embodiments, oxytocin peptide and/or magnesium ions are delivered to the upper third of the nasal cavity. In some embodiments, oxytocin peptide and/or magnesium ions are delivered to the lower two-thirds of the nasal cavity. In some embodiments, oxytocin peptide and/or magnesium ions are specifically delivered to both the lower two-thirds and the upper third of the nasal cavity. In some embodiments, the method is provided for treating autism spectrum disorder, one or more symptoms associated with autism spectrum disorder, or a disorder exhibiting one or more symptoms associated with autism spectrum disorder, including intranasal administration of an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the co-administration of oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In some embodiments, the method is used to treat social and communication deficits or anxiety.

In some embodiments, IL-6 is administered to the subject according to any of the methods described herein, wherein IL-6 is administered intranasally. Suitable devices for intranasal delivery, such as the nasal delivery devices described herein, can be used to administer IL-6 to the mucosal tissue within the nasal cavity. In some embodiments, IL-6 is administered systemically, for example, intravenously, intramuscularly, orally, subcutaneously, or intrathecally.

In some embodiments, the oxytocin peptide is human oxytocin composed of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQ. ID NO: 1). In some embodiments, the effective dose of the oxytocin peptide is about 0.5 μg to about 2000 μg. In some embodiments, the effective dose of the oxytocin peptide is about 0.5 μg to about 1000 μg, about 1 μg to about 1000 μg, or about 1 μg to about 2000 μg. In some embodiments, the effective dose of oxytocin peptide is about 4 μg to about 1000 μg, about 8 μg to about 1000 μg, about 8 μg to about 800 μg, about 8 μg to about 500 μg, about 8 μg to about 400 μg, about 8 μg to about 300 μg, about 8 μg to about 200 μg, about 8 μg to about 100 μg, about 8 μg to about 80 μg, about 8 μg to about 50 μg, about 10 μg to about 1000 μg, about 10 μg to about 500 μg, about 10 μg to about 200 μg, about 10 μg to about 100 μg, about 16 μg to about 1000 μg, about 16 μg to about 800 μg, about 16 μg to about 500 μg, about 16 μg to about 400 μg, about 16 μg to about 200 μg, about 16 μg to about 160 μg, about 16 μg to about 120 μg, about 16 μg to about 80 μg, about 20 μg to about 1000 μg, about 20 μg to about 800 μg, about 20 μg to about 500 μg, about 20 μg to about 200 μg, about 20 μg to about 100 μg, about 30 μg to about 1000 μg, about 30 μg to about 500 μg, about 30 μg to about 300 μg, about 30 μg to about 120 μg, about 30 μg to about 90 μg, about 50 μg to about 1000 μg, about 50 μg to about 500 μg, about 50 μg to about 250 μg, about 50 μg to about 100 μg, or about 50 μg to about 80 μg. In some embodiments, the effective dose of oxytocin peptide is about 8 μg, about 16 μg, about 32 μg, about 48 μg, about 64 μg, about 80 μg, about 96 μg, about 128 μg, about 256 μg, about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 120 μg, about 150 μg, about 200 μg, about 400 μg, about 600 μg, about 800 μg, or about 100 μg. In preferred embodiments, the effective dose of oxytocin peptide is about 8 μg to about 120 μg, about 15 μg to about 120 μg, about 30 μg to about 120 μg, or about 66 μg.

In some embodiments, the effective dose of oxytocin peptide is about 0.25 IU to about 1000 IU. In some embodiments, the effective dose of oxytocin peptide is about 0.25 IU to about 500 IU, about 0.5 IU to about 500 IU, or about 0.5 IU to about 1000 IU. In some embodiments, the effective dose of oxytocin peptide is about 2 IU to about 500 IU, about 4 IU to about 500 IU, about 4 IU to about 400 IU, about 4 IU to about 250 IU, about 4 IU to about 200 IU, about 4 IU to about 150 IU, about 4 IU to about 100 IU, about 4 IU to about 50 IU, about 4 IU to about 40 IU, about 4 IU to about 25 IU, about 5 IU to about 500 IU, about 5 IU to about 250 IU, about 5 IU to about 100 IU, about 5 IU to about 50 IU, about 8 IU to about 500 IU, about 8 IU to about 400 IU, about 8 IU to about 250 IU, about 8 IU to about 200 IU, about 8 IU to about 100 IU, about 8 IU to about 80 IU, about 8 IU to about 60 IU, about 8 IU to about 40 IU, about 10 IU to about 500 IU, about 10 IU to about 400 IU, about 10 IU to about 250 IU, about 10 IU to about 100 IU, about 10 IU to about 50 IU, about 15 IU to about 500 IU, about 15 IU to about 250 IU, about 15 IU to about 150 IU, about 15 IU to about 60 IU, about 15 IU to about 45 IU, about 25 IU to about 500 IU, about 25 IU to about 250 IU, about 25 IU to about 125 IU, about 25 IU to about 50 IU, or about 25 IU to about 40 IU. In some embodiments, the effective dose of oxytocin peptide is about 4 IU, about 8 IU, about 16 IU, about 24 IU, about 32 IU, about 40 IU, about 48 IU, about 64 IU, about 128 IU, about 5 IU, about 10 IU, about 15 IU, about 20 IU, about 25 IU, about 30 IU, about 35 IU, about 40 IU, about 45 IU, about 50 IU, about 60 IU, about 75 IU, about 100 IU, about 200 IU, about 300 IU, about 400 IU, or about 50 IU. In preferred embodiments, the effective dose of oxytocin peptide is about 4 IU to about 60 IU, about 7.5 IU to about 60 IU, about 15 IU to about 60 IU, or about 30 IU.

In one implementation, the dose or amount of oxytocin in the combination is effective to provide a clinically measurable improvement in symptoms of autism spectrum disorder or related disorders. The combination of oxytocin and magnesium ions provides a synergistic or enhancing effect to improve autism spectrum disorder or related disorders. In some implementations, oxytocin is administered at a sub-therapeutic effective dose relative to the dose of oxytocin administered as a single agent. The dose of oxytocin as a single agent is partly dependent on the route of administration. Therefore, the dose of oxytocin in the combination therapies described herein will also be partly dependent on the route of administration.

The optimal dose of magnesium ions may depend on the specific impairment or symptoms, the type of synergistic or enhancing effect desired, and other factors such as the route of administration. The optimal dose can be determined in the total amount of magnesium ions administered or the concentration of magnesium ions in the administered formulation. In some embodiments, the effective dose of magnesium ions administered is from about 50 μg to about 68 mg. In some embodiments, the effective dose of magnesium ions administered is from about 50 μg to about 34 mg, or from about 1 mg to about 3 mg. In some embodiments, the effective dose of magnesium ions administered is from about 1.3 mg, or about 2.6 mg. In some embodiments, the effective dose of magnesium ions administered is from about 1.2 mg, or about 2.4 mg. In some embodiments, the effective dose of magnesium ions administered is about 50 μg to about 17 mg, about 50 μg to about 8 mg, about 50 μg to about 4 mg, about 50 μg to about 2 mg, about 50 μg to about 1 mg, about 50 μg to about 500 μg, about 100 μg to about 68 mg, about 100 μg to about 34 mg, about 100 μg to about 17 mg, about 100 μg to about 8 mg, about 100 μg to about 4 mg, about 100 μg to about 2 mg, about 100 μg to about 1 mg, about 100 μg to about 500 μg, about 200 μg to about 68 mg, about 200 μg to about 34 mg, about 200 μg to about 17 mg, about 200 μg to about 8 mg, about 200 μg to about 4 mg, about 200 μg to about 2 mg, about 200 μg to about 1 mg, about 200 μg to about 500 μg, about 500 μg Approximately 68 mg, approximately 500 μg to approximately 34 mg, approximately 500 μg to approximately 17 mg, approximately 500 μg to approximately 8 mg, approximately 500 μg to approximately 5 mg, approximately 500 μg to approximately 4 mg, approximately 500 μg to approximately 3 mg, approximately 500 μg to approximately 2 mg, approximately 500 μg to approximately 1 mg, approximately 1 mg to approximately 68 mg, approximately 1 mg to approximately 34 mg, approximately 1 mg to approximately 17 mg, approximately 1 mg to approximately 8 mg, approximately 1 mg to approximately 6 mg, approximately 1 mg to approximately 5 mg, approximately 1 mg to approximately 4 mg, approximately 1 mg to approximately 3 mg, approximately 1 mg to approximately 2 mg, approximately 1.5 mg to approximately 8 mg, approximately 1.5 mg to approximately 6 mg, approximately 1.5 mg to approximately 5 mg, approximately 1.5 mg to approximately 4 mg, approximately 1.5 mg to approximately 3 mg, approximately 1.5 mg to approximately 2 mg, approximately 1.3 mg to approximately 2.6 mg, or approximately 1.2 mg to approximately 2.4 mg. In some implementations, magnesium salts (e.g., magnesium citrate and/or magnesium chloride) are used to provide magnesium ions.

In some embodiments, the magnesium salt administered includes magnesium chloride and an effective dose of magnesium chloride hexahydrate ( _MgCl₂ · _6H₂O , MW 203.3) of about 0.48 mg to about 600 mg. In some embodiments, the effective dose of magnesium chloride hexahydrate is about 0.48 mg to about 300 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 75 mg, about 5 mg to about 150 mg, about 5 mg to about 75 mg, about 5 mg to about 50 mg, about 10 mg to about 600 mg, about 10 mg to about 300 mg, about 10 mg to about 150 mg, about 10 mg to about 75 mg, about 10 mg to about 50 mg, about 10 mg to about 30 mg, or about 12 mg to about 24 mg. In some preferred embodiments, the effective dose of magnesium chloride hexahydrate is about 6 mg, about 12 mg, about 18 mg, about 24 mg, or about 30 mg.

In some embodiments, the magnesium salt administered is magnesium citrate, and the effective dose of the magnesium salt is magnesium citrate of about 0.48 mg to about 600 mg. In some embodiments, the effective dose of magnesium citrate (e.g., dibasic anhydrous magnesium citrate, MW.214.4) is about 0.48 mg to about 300 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 75 mg, about 5 mg to about 150 mg, about 5 mg to about 75 mg, about 5 mg to about 50 mg, about 10 mg to about 600 mg, about 10 mg to about 300 mg, about 10 mg to about 150 mg, about 10 mg to about 75 mg, about 10 mg to about 50 mg, about 10 mg to about 30 mg, or about 12 mg to about 24 mg. In some preferred embodiments, the effective dose of magnesium citrate (e.g., dibasic anhydrous magnesium citrate, MW.214.4) is about 6 mg, about 12 mg, about 18 mg, about 24 mg, or about 30 mg. In some embodiments, the effective dose of magnesium citrate is about 0.48 mg to about 12 mg, about 0.5 mg to about 10 mg, about 0.5 mg to about 8 mg, about 0.5 mg to about 5 mg, about 0.5 mg to about 2.5 mg, about 0.5 mg to about 1 mg, about 1 mg to about 10 mg, about 1 mg to about 8 mg, about 1 mg to about 5 mg, about 1 mg to about 2 mg, about 2 mg to about 10 mg, about 2 mg to about 8 mg, about 2 mg to about 6 mg, about 2 mg to about 4 mg, about 3 mg to about 10 mg, about 4 mg to about 10 mg, about 4 mg to about 8 mg, about 4 mg to about 6 mg, about 5 mg to about 10 mg, about 5 mg to about 8 mg, about 5 mg to about 7 mg, about 5 mg to about 6 mg, about 6 mg to about 10 mg, about 6 mg to about 8 mg, or about 6 mg to about 7 mg. If another magnesium salt is substituted for magnesium citrate, the effective dose of that magnesium salt provides an amount of magnesium ions equal to the amount provided by magnesium citrate.

It is intended and understood that each and every dose of magnesium ions described herein may be combined with each and every dose of oxytocin peptide described herein, as if each and every combination were stated individually. For example, in some embodiments, an effective dose of oxytocin peptide is about 0.5 μg to about 2000 μg and an effective dose of magnesium ions is about 50 μg to about 68 mg of magnesium. In some embodiments, an effective dose of oxytocin peptide is about 15 μg to about 120 μg (e.g., about 60 μg or about 66 μg) and an effective dose of magnesium ions is equal to the amount of magnesium ions provided by about 10 mg to about 30 mg (e.g., about 12 mg or about 24 mg) of magnesium citrate.

In some embodiments, a method is provided for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety, comprising administering (e.g., intranasally) an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the weight ratio between the dose of the administered oxytocin peptide and the dose of the administered magnesium ions is between about 1:1 and about 1:1000, preferably between about 1:2 and about 1:200, more preferably between about 1:20, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:60, or any OT/Mg(w) ratio described herein for magnesium-containing oxytocin peptide formulations or compositions. In some embodiments, the method is provided for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety, comprising administering (e.g., intranasally) an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the molar ratio between the dose of the administered oxytocin peptide and the dose of the administered magnesium ions is between about 1:40 and about 1:40000, preferably about 1:80 to about 1: The ratio is between 8000 and more preferably about 1:175, about 1:280, about 1:500, about 1:560, about 1:800, about 1:1000, about 1:1100, about 1:1200, about 1:1400, about 1:1600, about 1:1700, about 1:1800, about 1:2000, about 1:2400, about 1:3000, or any OT/Mg(m) ratio described herein for magnesium-containing oxytocin peptide formulations or compositions. In some of these embodiments, the oxytocin peptide is human oxytocin composed of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQ.ID NO:1). In some of these embodiments, magnesium ions are provided by magnesium citrate and/or magnesium chloride. In some of these implementations, social and communication deficits are deficiencies in communication skills and/or social interactions, lack of eye contact, and/or inability to form and/or maintain social relationships.

In one embodiment, the method is provided for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety, comprising administering intranasally to a subject in need a dose of oxytocin peptide in the amount of about 0.5 μg to about 2000 μg (e.g., about 8 μg to about 300 μg, about 15 μg to about 120 μg, or about 66 μg) and a dose of magnesium or magnesium ions in the amount of about 50 μg to about 68 mg, about 50 μg to about 34 mg, about 1 mg to about 3 mg, about 1.3 mg, or about 2.6 mg. In one embodiment, the method comprises administering intranasally to a subject in need an effective amount of the magnesium-containing oxytocin peptide formulation or composition described herein. In one embodiment, the method comprises administering intranasally to a subject in need an effective amount of oxytocin peptide and magnesium ions in a liquid formulation in the amount of about 5 μL to about 1000 μL. In one embodiment, the method includes administering an effective amount of a magnesium-containing oxytocin peptide formulation or composition—comprising between about 0.01 mg/mL and about 16 mg/mL (e.g., about 0.1 mg/mL and about 16 mg/mL) of oxytocin and between about 1 mg/mL and about 30 mg/mL of magnesium or magnesium ions—to an intranasal administration to a subject in need. In another embodiment, the method includes administering an effective amount of a magnesium-containing oxytocin peptide formulation—comprising between about 0.01 mg/mL and about 16 mg/mL (e.g., about 0.1 mg/mL and about 16 mg/mL or about 0.15 mg/mL and about 1.5 mg/mL) of oxytocin and between about 1% and about 25% (by weight) of magnesium citrate (e.g., about 1% to about 15% or about 10% to about 14%) of magnesium citrate. In one embodiment, the method includes administering an effective amount of a magnesium-containing oxytocin peptide formulation—comprising between about 5 IU/mL and about 8000 IU/mL (e.g., about 50 IU/mL and about 8000 IU/mL or about 75 IU/mL and about 750 IU/mL) of oxytocin and between about 1% and about 25% (by weight) of magnesium citrate (e.g., about 1% to about 15%, about 10% to about 14%, or about 12%). In one embodiment, the method includes administering an effective amount of a magnesium-containing oxytocin peptide formulation—comprising between about 0.01 mg/mL and about 16 mg/mL (e.g., about 0.1 mg/mL and about 16 mg/mL or about 0.15 mg/mL and about 1.5 mg/mL) of oxytocin and between about 1% and about 25% (by weight) of magnesium chloride hexahydrate (e.g., about 1% to about 15%, about 8% to about 12%, or about 10%). In one embodiment, the method includes administering an effective amount of a magnesium-containing oxytocin peptide formulation—comprising between about 5 IU/mL and about 8000 IU/mL (e.g., about 50 IU/mL and about 8000 IU/mL or about 75 IU/mL and about 750 IU/mL) of oxytocin and between about 1% and about 25% (by weight) of magnesium chloride hexahydrate (e.g., about 1% to about 15%, about 8% to about 12%, or about 10%).

In some embodiments, an effective dose of oxytocin peptide and magnesium ions comprises about 0.5 μg (or 0.25 IU) to about 2000 μg (or 1000 IU) of oxytocin peptide administered in an aqueous solution containing about 0.1% to about 2.8% (w/v) magnesium. In some embodiments, an effective dose of oxytocin peptide and magnesium ions comprises about 8 μg (or 4 IU) to about 1000 μg (or 500 IU) of oxytocin peptide administered in an aqueous solution containing about 0.11% to about 1.65% (w/v) magnesium. In some embodiments, an effective dose of oxytocin peptide and magnesium ions comprises about 15 μg (or 7.5 IU) to about 120 μg (or about 60 IU) (e.g., about 60 μg or 30 IU) of oxytocin peptide administered in an aqueous solution containing about 1.1% to about 1.6% (e.g., about 1.2% or about 1.35%) magnesium. In one embodiment, an effective dose of oxytocin peptide and magnesium ions comprises about 60 μg (or 30 IU) of oxytocin peptide administered in an aqueous solution containing about 1.2% or about 1.35% magnesium.

In some embodiments, an effective dose of oxytocin peptide and magnesium ions is administered intranasally in a liquid formulation, and the volume of the administered liquid formulation is between about 5 μL and about 1000 μL. In some embodiments, the administered volume is between about 5 μL and about 500 μL, between about 5 μL and about 250 μL, between about 5 μL and about 100 μL, between about 5 μL and about 50 μL, between about 10 μL and about 1000 μL, between about 10 μL and about 500 μL, between about 10 μL and about 250 μL, between about 10 μL and about 100 μL, between about 25 μL and about 1000 μL, between about 25 μL and about 500 μL, between about 25 μL and about 250 μL, between about 25 μL and about 100 μL, between about 50 μL and about 1000 μL. Between L, approximately 50 μL and approximately 750 μL, approximately 50 μL and approximately 500 μL, approximately 50 μL and approximately 450 μL, approximately 50 μL and approximately 400 μL, approximately 50 μL and approximately 350 μL, approximately 50 μL and approximately 300 μL, approximately 50 μL and approximately 250 μL, approximately 50 μL and approximately 200 μL, approximately 50 μL and approximately 150 μL, approximately 100 μL and approximately 500 μL, approximately 100 μL and approximately 400 μL, approximately 100 μL and approximately 300 μL, or approximately 100 μL and approximately 200 μL. In some embodiments, the administered volume is about 50 μL, about 100 μL, about 150 μL, about 200 μL, about 250 μL, about 300 μL, about 350 μL, about 400 μL, about 450 μL, or about 500 μL. In some embodiments, an effective dose of oxytocin peptide and magnesium ions is administered intranasally in a liquid formulation contained in the nasal device described herein.

The combination of oxytocin peptide and magnesium ions described herein can be used to treat any social and communication deficits treatable by oxytocin, such as deficits in communication skills and/or social interaction, lack of eye contact, and/or inability to form and/or maintain social relationships. Therefore, a method for treating social and communication deficits is provided, comprising administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the social and communication deficit is a deficit in communication skills and/or social interaction, lack of eye contact, and/or inability to form and/or maintain social relationships. In one embodiment, the method comprises administering an effective dose of oxytocin peptide and magnesium ions intranasally to a subject in need. In some embodiments, the molar ratio between the amount of oxytocin peptide and the amount of magnesium or magnesium ions is about 1:175, about 1:280, about 1:560, about 1:1100, about 1:1700, or about 1:2000, including any range between these ratios.

In one embodiment, the method is provided for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety, comprising intranasally administering an effective dose of oxytocin peptide and magnesium ions to a subject in need (e.g., a human or veterinary patient), wherein the co-administration of the oxytocin peptide and magnesium ions produces a synergistic or enhancing effect. In some embodiments, the oxytocin peptide is human oxytocin composed of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQ.ID NO:1). In some embodiments, an effective dose of the oxytocin peptide is from about 0.5 μg (or 0.25 IU) to about 2000 μg (or 1000 IU), preferably from about 8 μg (or 4 IU) to about 1000 μg (or 500 IU), more preferably from about 15 μg (or 7.5 IU) to about 120 μg (or 60 IU). In some embodiments, the effective dose of magnesium ions is from about 50 μg to about 68 mg. In some embodiments, magnesium ions are provided using a magnesium salt (e.g., magnesium chloride and/or magnesium citrate) administered in an amount providing about 50 μg to about 68 mg of magnesium. In some embodiments, an effective dose of magnesium ions is provided using about 0.48 mg to about 600 mg of magnesium citrate. In some embodiments, an effective dose of magnesium ions is provided using about 0.42 mg to about 540 mg of magnesium chloride hexahydrate. In some embodiments, the effective dose of oxytocin peptide and magnesium ions comprises about 15 μg (or 7.5 IU) to about 120 μg (or 60 IU) (e.g., about 60 μg or 30 IU) of oxytocin peptide administered in an aqueous solution containing about 1.1% to about 1.54% (e.g., about 1.2% or about 1.35%) (w/v) magnesium. In some embodiments, an effective dose of oxytocin peptide and magnesium ions comprises about 10 μg to about 120 μg (e.g., about 66 μg) of oxytocin peptide administered in an aqueous solution containing about 10% to about 14% (e.g., about 12%) (w/v) magnesium citrate.

Set

This document provides kits for performing any of the methods described herein. The kits are provided for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety. In some embodiments, the kit includes, in suitable packaging, oxytocin peptides and magnesium ions, wherein the oxytocin peptides and magnesium ions are in amounts that produce a synergistic or enhancing effect when used to treat autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety, and a device for craniofacial administration (e.g., intranasal administration). The kit may further include protease inhibitors and/or at least one absorption enhancer. The kit may further include IL-6. Other kits may further include instructions for providing users and/or healthcare providers with information for performing any of the methods described herein. The kit may further include reagents/tools for measuring IL-6 levels in subjects; and optionally instructions for anticipating the efficacy of nasal oxytocin and magnesium ions.

Also provided are kits comprising a device for craniofacial administration (e.g., an intranasal administration device such as a nasal pump device) and the magnesium-containing oxytocin peptide formulation described herein, suitable for packaging. The kit may further include instructions for administering the magnesium-containing oxytocin peptide formulation to subjects in need.

The description relating to using the kit to carry out the invention generally describes how the contents of the kit are used to carry out the invention. The instructions provided in the kit of the invention are typically written instructions on a label or packaging insert (e.g., a piece of paper included in the kit), but machine-readable instructions (e.g., instructions carried on a disk or optical disc) are also acceptable.

Example

The invention can be further understood by referring to the following embodiments, which are provided by way of example and are not intended to be limiting.

Example 1: Exemplary preparation of magnesium-containing oxytocin peptide formulation

Example 1A

The hypertonic and targeted drug formulation at pH 4.5 consists of oxytocin USP (150 IU/mL); magnesium chloride USP (as a hexahydrate or anhydrous salt); citrate USP (as anhydrous or monohydrate); sodium hydroxide NF; and sterile water for injection USP. Quantitative composition is provided in Table 1. The molar ratio of oxytocin to magnesium ions in the formulation is approximately 1:1679. All components comply with the pharmacopoeia (USP/NF) requirements of the relevant monograph.

Table 1

^1. The exact amount of oxytocin used is based on the oxytocin activity as determined by the supplier’s analytical certificate.

The composition value of ^magnesium chloride represents the composition value of the hexahydrate; anhydrous salt can also be used, with the composition adjusted accordingly.

The composition values for ³ citric acid represent the composition values for the anhydrous form; the monohydrate can also be used, with the composition adjusted accordingly.

The drug product is manufactured by dissolving the ingredients in sterile water for injection, sterilely filtering it, and rapidly filling it into vials on a preservative-free pump, and is typically tested according to the FDA nasal spray guidance of July 2002.

In one embodiment, a 10-L batch of magnesium-containing oxytocin formulation is prepared according to the composition provided in Table 1 as follows: The formulation container is filled with water to approximately 60% of the desired batch volume. While stirring at ambient temperature, the required amounts of the following are added in sequence: sodium chloride, citric acid, and magnesium chloride hexahydrate. The materials dissolve readily. No heating is required; only gentle stirring is used. The pH of the solution is adjusted to 4.5 with the addition of 1N NaOH. (If over-titration occurs, 10% HCl can be used to back-titrate to pH 4.5). The required amount of oxytocin is added and stirred until dissolved. Water is added to bring the batch to the final weight/volume. Stirring continues until the solution is homogeneous.

Example 1B

The isotonic and targeted drug formulation at pH 4.5 consists of oxytocin USP (150 IU/mL); magnesium citrate; sodium chloride USP; sodium acetate trihydrate USP; glacial acetic acid USP; and sterile water for injection USP. Quantitative composition is provided in Table 2. The molar ratio of oxytocin to magnesium ions in the formulation is approximately 1:1992. The target pH of 4.5 was chosen based on optimal formulation stability at or near this pH (Hawe, et al. Pharmaceut. Res. 2009, 26:1679-1688). All components comply with the pharmacopoeia (USP/NF) requirements of the relevant monograph.

To prepare a stock oxytocin solution, lyophilized oxytocin (2 mg) was added to 1 mL of water (USP), 0.9% physiological saline, or phosphate-buffered saline in a 5 mL glass container. The solution was stirred until all the oxytocin was dissolved, and the pH was adjusted to between 3.5 and 8.5 to produce 1 mL of a 2 mg/mL (approximately 1000 IU/mL) liquid oxytocin preparation.

For use as clinical material, oxytocin and excipients are manufactured under current Good Manufacturing Practices and terminally sterilized (through aseptic filtration via a 0.2-micron membrane filter) before being filled into glass vials and sealed with a pump actuator. Various formulation concentrations can be obtained by increasing or decreasing the amount of oxytocin according to this example. Approximately 10 doses of oxytocin are obtained from this 1 mL batch volume.

Table 2

Element Concentration (mg/mL) Oxytocin USP 0.283 Magnesium citrate 120 Sodium Chloride USP 4.675 Sodium acetate trihydrate USP 6.805 Citric acid USP pH 4.5 Sterile Water for Injection (USP) qs

Example 2: Rat Social Behavior Model

Rats were treated nasally with 20 μl (10 μl/nostril) of a solution containing saline, 10 μg oxytocin, 12% magnesium citrate, and 10 μg oxytocin (molar ratio of oxytocin to magnesium ions approximately 1:1127), or 12% magnesium citrate. Eight (8) rats were used in each treatment group. Forty minutes after nasal administration, two animals from the same treatment group were paired and placed in a test chamber, and their social behaviors (sniffing, following, climbing up and down, grooming each other, and playing) were recorded for 10 minutes. The time spent on social interaction is shown in Figure 1. The results showed evidence of the enhancing effect of the combination of 12% magnesium citrate and 10 μg oxytocin on improving social behavior.

Example 3: Rat Anxiety Model

Example 3A

Rats were treated nasally with 20 μl (10 μl/nostril) of a solution containing saline, 10 μg oxytocin, 12% magnesium citrate, and 10 μg oxytocin (molar ratio of oxytocin to magnesium ions approximately 1:1127), or 12% magnesium citrate. Eight (8) rats were used in each treatment group. Fifty minutes after nasal administration, the animals were placed in an eight-arm maze, and their anxiety was assessed by the number of times the animals made open arm entries during a 5-minute time interval. The observed number of open arm entries is shown in Figure 2. The results show evidence of a synergistic effect of the combination of 12% magnesium citrate and 10 μg oxytocin in reducing anxiety.

Example 3B

Rats were treated nasally with 20 μl (10 μl/nostril) of a solution containing saline, 3% magnesium citrate, 6% magnesium citrate, 16 μg oxytocin, 10 μg oxytocin, a combination of 3% magnesium citrate and 16 μg oxytocin (molar ratio of oxytocin to magnesium ions approximately 1:176), or a combination of 6% magnesium citrate and 10 μg oxytocin (molar ratio of oxytocin to magnesium ions approximately 1:563). Eight (8) rats were used in each treatment group. Thirty minutes after nasal administration, the animals were exposed to elevated platform stress for 5 minutes, followed immediately by placement in an elevated cruciate maze for 5 minutes. Their anxiety was assessed by the number of open arm entries made during the 5-minute time period. The observed number of open arm entries is shown in Figures 3A and 3B. Animals treated with a combination of 3% magnesium citrate and 16 μg oxytocin (molar ratio of oxytocin to magnesium ions approximately 1:176) exhibited lower anxiety—as indicated by increased open arm penetrations—compared to animals treated with either 3% magnesium citrate alone or 16 μg oxytocin alone. In contrast, animals treated with a combination of 6% magnesium citrate and 10 μg oxytocin (molar ratio of oxytocin to magnesium ions approximately 1:563) exhibited greater anxiety—as indicated by decreased open arm penetrations—compared to animals treated with either 6% magnesium citrate alone or 10 μg oxytocin alone.

To further assess anxiety, the latency of entry into the open arm, the time spent in the open arm, and the number of times the closed arm was entered were measured.

These experiments were repeated with additional amounts of magnesium citrate and oxytocin—including, for example, 6% magnesium citrate alone, 20 μg oxytocin alone, and a combination of 6% magnesium citrate and 20 μg oxytocin (the molar ratio of oxytocin to magnesium ions was approximately 1:281).

Example 4: Single Subject Case Study

Subjects diagnosed with autism spectrum disorder (e.g., children) were administered a nasal liquid preparation containing 12 to 24 IU of oxytocin daily, morning and evening, for a period of 3 days. Social functioning and anxiety were assessed. Following a 4-day flushing period, subjects were administered a nasal liquid preparation containing 3% to 12% magnesium citrate daily, morning and evening, for a period of 3 days, and their social functioning and anxiety were assessed. Following a 4-day flushing period, subjects were administered a nasal liquid preparation containing a combination of 12 to 24 IU of oxytocin and 3% to 12% magnesium citrate daily, morning and evening, for a period of 3 days, and their social functioning and anxiety were assessed.

Example 5: Human Clinical Trial

This study used a double-blind, randomized, placebo-controlled, parallel design to test the efficacy of a 6-week course of intranasal administration of a combination of oxytocin and magnesium twice daily in male and female participants aged 18 to 55 years diagnosed with autism spectrum disorder. The primary power endpoint was the change in social interaction score as measured by the Autism Diagnostic Observation Schedule-II before and after the double-blind treatment period. Secondary endpoints consisted of one or more of the following:

(1) Changes in scores for communication and limitation and repetitive behaviors as measured by the Autism Diagnostic Observation Scale-II before and at the end of the double-blind treatment period;

(2) Changes in anxiety as measured by the State and Trait Anxiety Inventory, assessed before and at the end of the double-blind treatment period;

(3) Changes in depression as measured by the Center for Epidemiologic Studies Depression Scale, assessed before and at the end of the double-blind treatment period;

(4) Changes in eye gaze in response to social cues assessed before and at the end of the double-blind treatment period;

(5) Changes in facial and vocal expressions as analyzed by video recordings every two weeks during the trial period; and

(6) Changes in Clinical Global Impression and Global Assessment of Functioning scores assessed every two weeks during the trial period.

The study inclusion criteria consisted of the following:

1) Diagnosed with autism spectrum disorder based on DSM-V;

2) Qualitative abnormalities in reciprocal social interaction (domain A) exceeded the limits in the Autism Diagnostic Interview-Revised; and

3) A verbal IQ of 85 or higher and an overall IQ of 80 or higher, as measured by the Wechsler Adult Intelligent Scale-III.

The study inclusion criteria consisted of the following:

1) Primary mental illness diagnoses other than those included in criterion 1);

2) Current instability due to a comorbid psychiatric diagnosis;

3) Randomize the history of changes in the medication or dosage of psychotropic drugs within one month;

4) Currently being treated with two or more classes of psychotropic drugs;

5) Currently being treated with atomoxetine or methylphenidate;

6) History of continuous oxytocin therapy;

7) History of sensitivity to oxytocin;

8) A history of seizures or traumatic brain injury resulting in loss of consciousness lasting more than 5 minutes; and

9) History of alcoholism, substance abuse, or addiction.

Example 6: The effects of oxytocin and magnesium in social anxiety disorder

Subjects meeting the criteria for generalized social phobia in the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, were randomized to receive either a nasal placebo (saline) – Treatment A, oxytocin alone (30 IU) – Treatment B, magnesium alone (10%) – Treatment C, or oxytocin (30 IU) plus magnesium (10%) – Treatment D.

After a 1-week, single-blind, placebo-controlled run-in period, patients received a double-blind, 11-week course of treatment A, B, C, or D. Patients received treatment twice daily, approximately 12 hours apart.

Optionally, serum IL-6 levels were collected at the end of the 1-week adjustment period and at the end of the 11-week treatment course.

The number of respondents (“significant improvement” or “very good improvement”) based on the Clinical Global Impression Global Improvement Item was measured; the mean change from baseline in the total score of the Liebowitz Social Anxiety Scale was also measured. Optionally, serum IL-6 levels were correlated with the extent to which IL-6 served as a predictive biomarker for efficacy in determining efficacy.

Analyze the effects of the treatment group.

Exemplary Implementation

The present invention is further described through the following embodiments. Where appropriate and practical, each feature of the embodiments may be combined with any other embodiments.

Implementation Method 1. In one implementation, a method is provided for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety, comprising administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the co-administration of oxytocin peptide and magnesium ions produces a synergistic or enhancing effect.

Implementation Method 2. In a further embodiment of Implementation Method 1, oxytocin peptide and magnesium ions are administered simultaneously.

Implementation Method 3. In a further embodiment of Implementation Method 1, oxytocin peptide is administered before or after the administration of magnesium ions.

Implementation Method 4. In a further embodiment of any one of Implementation Methods 1 to 3, the oxytocin peptide is administered via craniofacial mucosa.

Implementation Method 5. In a further embodiment of Implementation Method 4, the oxytocin peptide is administered via intranasal administration.

Implementation Method 6. In a further embodiment of Implementation Method 5, oxytocin peptide and magnesium ions are administered via intranasal administration.

Implementation Method 7. In a further embodiment of any one of Implementation Methods 1 to 6, the effective dose of oxytocin peptide is about 0.5 μg to about 2000 μg.

Implementation Method 8. In a further embodiment of any one of Implementation Methods 1 to 7, the effective dose of magnesium ions is about 50 μg to about 68 mg.

Implementation 9. In a further embodiment of any one of Implementations 1 to 8, magnesium ions are provided using magnesium chloride and/or magnesium citrate.

Implementation 10. In a further embodiment of Implementation 1, the effective dose of oxytocin peptide and magnesium ions comprises about 15 μg to about 120 μg of oxytocin peptide administered in an aqueous solution containing about 1.1% to about 1.6% (w/v) magnesium.

Implementation Method 11. In a further embodiment of Implementation Method 1, the effective dose of oxytocin peptide and magnesium ions has a molar ratio of oxytocin to magnesium between about 1:40 and about 1:40000.

Implementation 12. In a further embodiment of any one of Implementations 1 to 11, the method is used to treat autism spectrum disorder.

Implementation 13. In a further embodiment of any one of Implementations 1 to 11, the method is used to treat a disorder that displays one or more symptoms associated with autism spectrum disorder.

Implementation 14. In a further embodiment of implementation 13, the disorder is social anxiety disorder, obsessive-compulsive disorder, social (pragmatic) communication disorder, neurodevelopmental disorder, attention deficit hyperactivity disorder, Prader-Willi syndrome, Timothy syndrome, Fragile-X syndrome, Rett syndrome, or Williams syndrome.

Implementation 15. In a further embodiment of any one of Implementations 1 to 11, the method is used to treat social and communication deficits.

Implementation 16. In a further embodiment of any one of Implementations 1 to 11, the method is used to treat anxiety.

Implementation Method 17. In a further embodiment of any one of Implementation Methods 1 to 16, the oxytocin peptide is human oxytocin (SEQ. ID NO: 1).

Implementation 18. In one embodiment, a method is provided for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety, comprising administering an effective dose of oxytocin peptide and magnesium ions to a subject in need, wherein the effective dose of oxytocin peptide and magnesium ions is administered intranasally in a liquid formulation, and the volume of the administered liquid formulation is between about 5 μL and about 1000 μL.

Implementation Method 19. In a further embodiment of Implementation Method 18, the effective dose of oxytocin peptide is about 0.5 μg to about 2000 μg.

Implementation 20. In a further embodiment of Implementation 18, the effective dose of magnesium ions is about 50 μg to about 68 mg.

Implementation 21. In a further embodiment of Implementation 18, the effective dose of oxytocin peptide and magnesium ions comprises about 15 μg to about 120 μg of oxytocin peptide administered in an aqueous solution containing about 1.1% to about 1.6% (w/v) magnesium.

Implementation 22. In a further embodiment of Implementation 18, the molar ratio of effective dose of oxytocin peptide and magnesium ions of oxytocin to magnesium is between about 1:40 and about 1:40000.

Implementation 23. In a further embodiment of Implementation 21 or 22, the volume of the liquid formulation given is between about 50 μL and about 200 μL.

Implementation 24. In a further embodiment of implementation 23, a liquid formulation is administered using a nasal dosing device at a dose of 1 to 4 units, approximately 50 μL/unit.

Implementation 25. In a further embodiment of any one of Implementations 18 to 24, the method is used to treat autism spectrum disorder.

Implementation 26. In a further embodiment of any of Implementations 18 to 24, the method is used to treat a disorder that displays one or more symptoms associated with autism spectrum disorder.

Implementation 27. In a further embodiment of implementation 26, the disorder is social anxiety disorder, obsessive-compulsive disorder, social (pragmatic) communication disorder, neurodevelopmental disorder, attention deficit hyperactivity disorder, Prader-Willi syndrome, Timothy syndrome, Fragile-X syndrome, Rett syndrome, or Williams syndrome.

Implementation 28. In a further embodiment of any of Implementations 18 to 24, the method is used to treat social and communication deficits.

Implementation 29. In a further embodiment of any of Implementations 18 to 24, the method is used to treat anxiety.

Implementation 30. In a further embodiment of any one of Implementations 18 to 29, the oxytocin peptide is human oxytocin (SEQ. ID NO:1).

Implementation 31. In a further embodiment of implementation 18, the liquid formulation is contained in an intranasal delivery device.

Implementation 32. In a further embodiment of implementation 31, the intranasal delivery device is a nasal pump device.

Implementation 33. In a further embodiment of implementation 32, the nasal pump device includes a container bottle attached to a pump actuator.

Implementation 34. In a further embodiment of implementation 33, the pump actuator is metered to deliver a specified volume of approximately 50 μL.

Implementation 35. In a further embodiment of implementation 32, the nasal pump device includes a container bottle attached to the aerosol device.

Implementation 36. In a further embodiment of any one of Implementations 32 to 35, the nasal pump device includes one or more of the following:

(i) A filter used to prevent backflow.

(ii) Metal-free fluid paths, and

(iii) Plastic materials that are stable to gamma radiation.

Implementation 37. In one embodiment, a composition comprising oxytocin peptide and magnesium ions is provided, wherein the oxytocin peptide and magnesium ions are in amounts that produce a synergistic or enhancing effect when used to treat anxiety.

Implementation 38. In a further embodiment of implementation 37, the oxytocin peptide is human oxytocin (SEQ. IDNO:1).

Implementation 39. In a further embodiment of Implementation 37, the composition is a liquid formulation containing between about 0.01 mg/mL and about 16 mg/mL of oxytocin peptide.

Implementation 40. In a further embodiment of Implementation 37, the composition is a liquid formulation containing an amount of magnesium salt, the amount of which provides magnesium between about 3 mg/mL and about 30 mg/mL.

Implementation 41. In a further embodiment of Implementation 37, the molar ratio of oxytocin peptide to magnesium ions is between about 1:40 and about 1:40000.

Implementation 42. In a further embodiment of implementation 41, the molar ratio is between about 1:40 and about 1:800.

Implementation 43. In a further embodiment of implementation 41, the molar ratio is between about 1:800 and about 1:40000.

Embodiment 44. In a further embodiment of any one of Embodiments 37 to 43, the composition further includes a device for craniofacial mucosal application.

Implementation 45. In a further embodiment of implementation 44, oxytocin peptide and magnesium ions are included in the device for craniofacial mucosal administration.

Implementation 46. In a further embodiment of implementation 45, the device is used for intranasal administration.

Although the foregoing invention has been described in detail by way of illustration and examples for purposes of clarity, it will be apparent to those skilled in the art that certain changes and modifications can be practiced without departing from the invention. Therefore, the description and examples should not be construed as limiting the scope of the invention.

All patents, patent applications, documents, and articles cited in this article are incorporated herein in their entirety through citation.

Claims (21)

1. The use of a composition comprising an effective dose of oxytocin peptide and an effective dose of magnesium ions in the manufacture of a medicament for treating autism spectrum disorder, disorders exhibiting one or more symptoms associated with autism spectrum disorder, social and communication deficits, or anxiety, wherein the medicament is formulated as an aqueous solution and administered via a craniofacial mucosa, wherein the molar ratio of the effective dose of oxytocin peptide to the effective dose of magnesium ions is between 1:800 and 1:1600, and wherein the effective dose of the oxytocin peptide and the effective dose of the magnesium ions produce a synergistic effect. 2. The application according to claim 1, wherein the molar ratio of oxytocin peptide to magnesium ions is about 1:1400. 3. The application according to claim 1, wherein the molar ratio of oxytocin peptide to magnesium ions is about 1:1200. 4. The application according to claim 1, wherein the molar ratio of oxytocin peptide to magnesium ions is about 1:1000. 5. The application according to claim 1, wherein the craniofacial delivery is intranasal. 6. The application according to claim 1, wherein the effective dose of the oxytocin peptide is between 0.5 μg and 2000 μg. 7. The application according to claim 1, wherein the effective dose of the magnesium ions is between 50 μg and 68 mg. 8. The application according to claim 1, wherein the magnesium ions are provided using magnesium chloride and/or magnesium citrate. 9. The application according to claim 1, wherein the oxytocin peptide is human oxytocin of SEQ. ID NO:1. 10. The application according to claim 1, wherein the drug is used to treat autism spectrum disorder. 11. The application according to claim 1, wherein the drug is used to treat a disorder exhibiting one or more symptoms associated with autism spectrum disorder. 12. The application according to claim 11, wherein the disorder is social anxiety disorder, obsessive-compulsive disorder, social communication disorder, attention deficit hyperactivity disorder, Prader-Willi syndrome, Timothy syndrome, Fragile-X syndrome, Rett syndrome, or Williams syndrome. 13. The application according to claim 11, wherein the disorder is a neurodevelopmental disorder. 14. The application according to claim 1, wherein the drug is used to treat social and communication deficits. 15. The application according to claim 1, wherein the drug is used to treat anxiety. 16. The application according to claim 5, wherein the drug is contained in an intranasal delivery device. 17. The application according to claim 16, wherein the intranasal delivery device is a nasal pump device. 18. The application of claim 17, wherein the nasal pump device includes a container bottle attached to the pump actuator. 19. The application of claim 18, wherein the pump actuator is metered to deliver a specified volume of about 50 μL. 20. The application of claim 19, wherein the nasal pump device includes a container bottle attached to the aerosol device. 21. The application according to any one of claims 17 to 20, wherein the nasal pump device comprises one or more of the following: (i) A filter used to prevent backflow. (ii) Metal-free fluid paths, and (iii) Plastic materials that are stable to gamma radiation.