IMMUNOMODULATORY OLIGOSACCHARIDES FOR THE TREATMENT OF

AUTISM SPECTRUM DISORDER

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Serial No. 63/069,164 filed on August 24, 2020. The entire contents of the above application are incorporated by reference herein.

FIELD OF THE INVENTION

The disclosure provides for immunomodulatory oligosaccharides, and therapeutic uses thereof for the treatment of autism spectral disorder.

BACKGROUND OF THE INVENTION

Autism Spectrum Disorders (ASD) are commonly associated with behavioral characteristics including limited social interaction, lack of verbal communication, and/or narrow and repetitive behavior patterns. Autism is one of the fastest growing developmental disorders in the U.S., with an estimated 1 million or more Americans being affected.

Autism is a complex neurodevelopmental disorder that affects 1 in 68 children in the United States, with four times as many males diagnosed than females (Christensen et al. 2016; CDC 2016). The spectrum of impairments noted in this disorder have coined the umbrella term "Autism Spectrum Disorder" (ASD). The fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V) defines ASD as having "deficits in social interaction and communication together with restricted and repetitive behaviors and interests" (American Psychiatric Association, 2013). Clinical symptoms are used to diagnose children with autism around onset at age three; a myriad of behavioral assessments are utilized for autism diagnostic purposes with the Autism Diagnostic Observation Schedule (ADOS) (Lord et al. 1989; 2000) as the standard. Despite extensive research, no definite aid for diagnosis or treatment has been detected (Loke, Hannan, and Craig 2015). Early intervention programs and special schooling are the most effective for those with this neurodevelopmental disorder and although outcomes of early intervention vary, all children benefit (Alabdali, Al-Ayadhi, and El-Ansary 2014). A combination of applied behavioral analysis (ABA) along with other educational, developmental, occupational and speech therapies are common in affected children with limited results (Frye et al. 2017). The need for other effective treatments for core symptoms of autism is highly sought after. SUMMARY OF THE INVENTION

The invention includes methods of treating a subject diagnosed with Autism Spectrum Disorder (ASD) comprising administering to said subject a composition comprising one or more oligosaccharides, or one or more human milk oligosaccharides (HMOs). In certain aspects, the HMO is selected from 3’sialyllactose, 6’sialyllactose or 2’fucosyllactose, or a pharmaceutically acceptable salt of any of thereof, or a combination of any of thereof. In further aspects, the HMO is a compound selected from Formula (I), (la), (lb), (Ic), (Id), (le), (II), (Illa), or (Illb) (described below).

Sialyllactose is a class of human milk oligosaccharides (HMOs) that appear in two different forms in human milk, 3 ’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL):

2,3 Sialyllactose (3’-SL) 2,6 Sialyllactose (6’-SL)

Sialyllactose, an oligosaccharide found in human milk, has been shown to modulate acute and chronic immune responses in both murine and human derived macrophages stimulated with LPS and various pro-inflammatory cytokines. Both forms of sialyllactose have shown reductions in interleukin (IL)-ip, IL-2, IL-4, IL-6, IL- 12, interferon (JEN) y or TNF-a in vitro, with 3’-SL exhibiting more significant reductions. In addition, 3’SL has been shown to reduce other key components that contribute to the critical nature of respiratory infections, including PDL1, COX2 and select chemokines, such as CCL2 (also known as monocyte chemoattractant protein 1 (MCP1)) and CCL5. 3 ’-Sialyllactose also modulates the gut microbiome composition to favor of beneficial Bifidobacterium spp. which correlate with the production of anti-inflammatory shortchain fatty acids. Another class of HMOs are fucosylated oligosaccharides. The primary fucosylated HMO is 2’-fuscosyllactose or 2’FL.

2’fucosyllactose has been granted generally regarded as safe (GRAS) status in the U.S. and Europe to be included in infant formula. 2’fucosyllactose has been shown to have many beneficial properties, such as affecting gut health through modulation of the gut microbiome as well as affection local gut inflammation in models of necrotizing enterocolitis and other inflammatory bowel diseases. In addition, 2’fucosyllactose has been shown to have positive effects on gut epithelial barrier function and also independent anti-inflammatory effects through the reduction in TNFa and IL-8.

An embodiment of the invention is a method of treating a subject diagnosed with an ASD with a composition comprising or consisting of 3’sialyllactose, 6’sialyllactose, 2’fucosyllactose or a combination thereof, wherein such treatment decreases a sign or symptom of said subjects ASD. In certain embodiments, such sign or symptom is anxiety or self-harm.

The methods of the invention can improve subject scores on autism assessment measures such as Vineland (such as Vineland Adaptive Behavior Score), CARS, SCQ, CBCL, SIAS, PIPPS, CAST, ADHDT, and/or ADI-R. In certain embodiments, such improvements may include, but are not limited to, autism severity, verbal communication, developmental status, and/or behavior issues such as emotional reactions. In certain additional embodiments, treatment may impact areas of autism severity, verbal communication, expressive communication, personal daily living skills, coping skills, or socialization, for example. For example, a composition of the invention can be used for treating, preventing, or ameliorating symptoms of Autism or Autism Spectrum Disorder (ASD) in a subject in need thereof. The Autism Spectrum Disorder (ASD) may comprise autism, Asperger syndrome, pervasive developmental disorder not otherwise specified (PDD- NOS), Childhood Disintegrative Disorder, syndromic autism or autism of known etiology such as fragile X syndrome, PTEN macrocephaly syndrome, RETT syndrome, tuberous sclerosis complex, Timothy syndrome, and/or Joubert syndrome. The method can improve one or more behavioral traits which can be assessed by one or more of: CARS behavior T- Score; ADI-R Reciprocal Social Interaction and/or Total score; SCQ Communication Score; VABS II Adaptive Behavior Composite, Communication Domain, Expressive Communication Subdomain, Personal Daily Living Skills Subdomain, Socialization Domain, Coping Skills Subdomain, SIAS, PIPPS, CAST, ADHDT, and/or Fine Motor Skills Subdomain score(s); and/or CBCL Emotionally Reactive T-Score.

In certain embodiments, the invention is directed to a method for treating autism spectrum disorder in a subject comprising administering to the subject, such as a child, a pharmaceutical or nutritional composition comprising an effective amount of a human milk oligosaccharide.

In a particular embodiment, the disclosure provides a method for treating autism spectrum disorder comprising administering to a subject, such as a child, an effective amount of an oligosaccharide, or a pharmaceutical composition comprising the oligosaccharide, wherein the oligosaccharide comprises a structure of Formula 1, 1(a) or II:

Formula I

Formula 1(a) Formula II or a pharmaceutically acceptable salt, solvate, or prodrug thereof, wherein: RJ-R ¹⁸ are independently selected from H, D, a halo, an unsubstituted or substituted

(Ci-Ce)alkyl, an unsubstituted or substituted (Ci-Ce)heteroalkyl, an unsubstituted or substituted (C2-Ce)alkenyl, an unsubstituted or substituted (C2-Ce)heteroalkenyl, an unsubstituted or substituted (C3-Ce)alkynyl, an unsubstituted or substituted (C3- Ce)heteroalkynyl, an unsubstituted or substituted (C4-Cs)cycloalkyl, an unsubstituted or substituted heterocycle, an unsubstituted or substituted aryl, -ROR', -RN(R')2, -RSSR', -SH, - RSOR, -RSO2R, -RSO2H, -RSO3H, -RC(=S)-R, -ROH, -RC(=O)R, -RNO2, -RSR, -RCN, - RNC, -RNNR', -RC(=O)OR', -ROC(=O)R, -RC(=O)H, -RC(=O)OH, -RC(=O)N(R') ₂ , -RN ₃ , -ROCN, -RNCO, -RONO2, -RNO, -ROP(=O)(OH) ₂ , and -RB(OH) ₂ ;

R is absent or a (Ci-Cs)alkyl; and

R' is independently selected from H, D, an unsubstituted or substituted (Ci-Ce) alkyl, an unsubstituted or substituted (Ci-Ce)heteroalkyl, an unsubstituted or substituted (C ₂ -Ce) alkenyl, an unsubstituted or substituted (C ₂ -C6)heteroalkenyl, an unsubstituted or substituted (C3-Ce)alkynyl, an unsubstituted or substituted (C3-Ce)heteroalkynyl, an unsubstituted or substituted (C4-C ₈ )cycloalkyl, an unsubstituted or substituted heterocycle, and an unsubstituted or substituted aryl. In another embodiment, the disclosure also provides a method for treating a patient diagnosed with autism spectrum disorder, comprising administering to the subject an effective amount of an oligosaccharide, or a pharmaceutical composition comprising the oligosaccharide, wherein the oligosaccharide comprises a structure of Formula 1(b) or 1(c):

Formula 1(b)

Formula 1(c) or a pharmaceutically acceptable salt, solvate, or prodrug thereof, wherein:

R ^x -R ⁶ are independently selected from H, D, a halo, an unsubstituted or substituted (Ci-Ce)alkyl, an unsubstituted or substituted (Ci-Ce)heteroalkyl, an unsubstituted or substituted (C2-Ce)alkenyl, an unsubstituted or substituted (C2-Ce)heteroalkenyl, an unsubstituted or substituted (C3-Ce)alkynyl, an unsubstituted or substituted (C3- Ce)heteroalkynyl, an unsubstituted or substituted (C ₄ -C ₈ )cycloalkyl, an unsubstituted or substituted heterocycle, an unsubstituted or substituted aryl, -ROR', -RN(R')2, -RSSR', - SH, - RSOR', -RSO2R', -RSO2H, -RSO3H, -RC(=S)-R', -ROH, -RC(=O)R', -RNO ₂ , -RSR', -RCN, -RNC, -RNNR, -RC(=O)OR', -ROC(=O)R', -RC(=O)H, -RC(=O)OH, - RC(=O)N(R') ₂ , -RN ₃ , -ROCN, -RNCO, -RONO2, -RNO, -ROP(=O)(OH) ₂ , and - RB(OH) ₂ ;

R is absent or a (Ci-Cs)alkyl; and R' is independently selected from H, D, an unsubstituted or substituted (Ci-C ₆ ) alkyl, an unsubstituted or substituted (Ci-C6)heteroalkyl, an unsubstituted or substituted (C ₂ -C6)alkenyl, an unsubstituted or substituted (C ₂ -C6)heteroalkenyl, an unsubstituted or substituted (Cs-Cejalkynyl, an un substituted or substituted (C3-C6)heteroalkynyl, an unsubstituted or substituted (C4-C ₈ )cycloalkyl, an unsubstituted or substituted heterocycle, and an unsubstituted or substituted aryl.

In yet another embodiment, the disclosure further provides a method for treating a patient diagnosed with autism spectrum disorder, comprising administering to the subject an effective amount of an oligosaccharide, or a pharmaceutical composition comprising the oligosaccharide, wherein the oligosaccharide comprises a structure of Formula 1(d), 1(e) or 11(a): Formula 1(d)

Formula 1(e)

Formula 11(a) or a pharmaceutically acceptable salt, solvate, or prodrug thereof. In a certain embodiment, a method disclosed herein comprises orally administering an oligosaccharide of the disclosure or a pharmaceutical composition comprising an oligosaccharide of the disclosure to a subj ect.

In yet a further embodiment, a method disclosed herein comprises orally administering to a subject a nutritional composition comprising at least one oligosaccharide of the disclosure.

In certain embodiments, the nutritional composition comprises or consists of 3 ’ SL, 6’SL or a combination of 3’SL and 6’SL.

In yet another embodiment, the disclosure further provides a method for treating a patient diagnosed with autism spectrum disorder comprising administering to the subject (for example, a child) an effective amount of an oligosaccharide, or a pharmaceutical composition comprising the oligosaccharide, wherein the oligosaccharide has the structure of Formula (Illa):

Formula Illa or a pharmaceutically acceptable salt, solvate, or prodrug thereof, wherein:

R -R are each independently selected from H, D, a halo, an unsubstituted or substituted (Ci-C6)alkyl, an unsubstituted or substituted (Ci-C6)heteroalkyl, an unsubstituted or substituted (C ₂ -C ₆ )alkenyl, an unsubstituted or substituted (C ₂ - C6)heteroalkenyl, an unsubstituted or substituted (C3-C6)alkynyl, an unsubstituted or substituted (C3-C6)heteroalkynyl, an unsubstituted or substituted (Ch-Csjcycloalkyl, an unsubstituted or substituted heterocycle, an unsubstituted or substituted aryl, -ROR', - RN(R') ₂ , -RSSR, -SH, - RSOR', -RSO ₂ R', -RSO ₂ H, -RSO ₃ H, -RC(=S)-R', -ROH, - RC(=O)R', -RNO ₂ , -RSR, -RCN, -RNC, -RNNR', -RC(=O)OR', -ROC(=O)R', - RC(=O)H, -RC(=O)OH, -RC(=O)N(R') ₂ , -RN ₃ , -ROCN, -RNCO, -RONO ₂ , -RNO, - ROP(=O)(OH) ₂ , and -RB(OH) ₂ ;

R is absent or a (Ci-C ₅ )alkyl;

R' is independently selected from H, D, an unsubstituted or substituted (Ci-Ce) alkyl, an unsubstituted or substituted (Ci-C6)heteroalkyl, an unsubstituted or substituted (C ₂ -Ce) alkenyl, an unsubstituted or substituted (C ₂ -C6)heteroalkenyl, an unsubstituted or substituted (C3-C6)alkynyl, an un substituted or substituted (C3-C6)heteroalkynyl, an unsubstituted or substituted (C ₄ -C ₈ )cycloalkyl, an unsubstituted or substituted heterocycle, and an unsubstituted or substituted aryl; and

R ²⁹ is an unsubstituted or substituted (Ci-C6)alkyl.

In yet another embodiment, the disclosure further provides a method for treating a patient diagnosed with autism spectrum disorder, comprising administering to the subject (for example, a child) an effective amount of an oligosaccharide, or a pharmaceutical composition comprising the oligosaccharide, wherein the oligosaccharide has the structure of Formula (Illb):

Formula Illb wherein: one, two or three of R ¹⁹ -R ²⁸ are each independently selected from the group consisting of hydrogen, an unsubstituted or substituted Ci-Ce alkyl (including, but not limited to, methyl and ethyl) and N(R’)2 (wherein R’ is as defined above), the remainder or R ¹⁹ -R ²⁸ are - OH, and R ²⁹ is substituted or unsubstituted Ci-Ce alkyl; or one, two or three of R ¹⁹ -R ²⁹ are each independently selected from NHC(O)R”, wherein R” is unsubstituted or substituted (Ci-Ce) alkyl (including, but not limited to, methyl), the remainder or R ¹⁹ -R ²⁸ are -OH, and R ²⁹ is substituted or unsubstituted C^-C ⁶ alkyl. In certain aspects, R ²⁶ is NHC(O)CH3 and R ¹⁹ -R ²⁵ and R ²⁷ -R ²⁸ are -OH, and R ²⁹ is methyl.

In a particular embodiment, the disclosure provides for a method to attenuate macrophage inflammation and/or suppress the secretion of pro-inflammatory cytokines in a subj ect suffering from a respiratory infection, comprising administering to the subj ect an effective amount of an oligosaccharide, or a pharmaceutical composition comprising the oligosaccharide, wherein the oligosaccharide comprises a structure of Formula 1, 1(a) or II. In yet another embodiment, the disclosure provides for a method to attenuate macrophage inflammation and/or suppress the secretion of pro-inflammatory cytokines in a subj ect in need thereof, comprising administering to the subj ect an effective amount of an oligosaccharide, or a pharmaceutical composition comprising the oligosaccharide, wherein the oligosaccharide comprises a structure of Formula 1(d), 1(e) or 11(a).

In certain embodiments, the methods disclosed herein comprises orally administering an oligosaccharide or HMO or a pharmaceutical composition comprising an oligosaccharide of the disclosure to a subject.

In yet a further embodiment, a method disclosed herein comprises orally administering to a subject a nutritional composition comprising at least one oligosaccharide of the disclosure.

In certain embodiments, the nutritional composition comprises or consists of 3 ’ SL, 6’ SL or a combination of 3 ’ SL and 6’ SL.In other embodiments, the nutritional composition comprise or consists of 3’SL, 6’SL or combination thereof at 145 mg/L or greater of3’SL, 6’SL or a combination of3’SL and 6’ SL.In another embodiment, the nutritional composition comprises at least 9% (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%; or any value between any of the foregoing) 3’SL, 6’SL or a combination thereof of the total oligosaccharides in the composition. In another embodiment, a pharmaceutical composition comprising the oligosaccharide of the disclosure is formulated as a tablet ora capsule.

DETAILEDDESCRIPTION OF THE INVENTION

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Thus, for example, reference to "an oligosaccharide" includes a plurality of such oligosaccharides and reference to "the therapeutic agent" includes reference to one or more therapeutic agents and equivalents thereof known to those skilled in the art, and so forth. Also, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise, ""comprises," "comprising" "include," "includes," and "including" are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of various embodiments use the term "comprising," those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of or "consisting of."

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

Although many methods and reagents are similar or equivalent to those described herein, the exemplary methods and materials are disclosed herein.

All publications mentioned herein are incorporated herein by reference in full for the purpose of describing and disclosing the methodologies, which might be used in connection with the description herein. Moreover, for terms expressly definedinthis disclosure, the definition of the term as expressly provided in this disclosure will control in all respects, even if the term has been given a different meaning in a publication, dictionary, treatise, and the like.

The term “about” as used herein, in reference to a numerical value or range, allows for a degree of variability in the value or range, for example, within 10%, within 5%, or within 4%, or within 2% of the value or range.

The term "alkyl" refers to an organic group that is comprised of carbon and hydrogen atoms that contains single covalent bonds between carbons. Typically, an "alkyl" as used in this disclosure, refers to an organic group that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 30 carbon atoms, or any range of carbon atoms between or including any two of the foregoing values. Where if there is more than 1 carbon, the carbons may be connected in a linear manner, or alternatively if there are more than 2 carbons then the carbons may also be linked in a branched fashion so that the parent chain contains one or more secondary, tertiary, or quaternary carbons. An alkyl may be substituted or un substituted, unless stated otherwise.

The term "alkenyl" refers to an organic group that is comprised of carbon and hydrogen atoms that contains at least one double covalent bond between two carbons. Typically, an "alkenyl" as used in this disclosure, refers to organic group that contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 30 carbon atoms, or any range of carbon atoms between or including any two of the foregoing values. While a C2- alkenyl can form a double bond to a carbon of a parent chain, an alkenyl group of three or more carbons can contain more than one double bond. In certain instances, the alkenyl group will be conjugated, in other cases an alkenyl group will not be conjugated, and yet other cases the alkenyl group may have stretches of conjugation and stretches of nonconjugation. Additionally, if there is more than 2 carbon, the carbons may be connected in a linear manner, or alternatively if there are more than 3 carbons then the carbons may also be linked in a branched fashion so that the parent chain contains one or more secondary, tertiary, or quaternary carbons. An alkenyl may be substituted or unsubstituted, unless stated otherwise.

The term "alkynyl" refers to an organic group that is comprised of carbon and hydrogen atoms that contains a triple covalent bond between two carbons. Typically, an "alkynyl "as used in this disclosure, refers to organic group that contains that contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or 30 carbon atoms, or any range of carbon atoms between or including any two of the foregoing values. While aC2- alkynyl can form a triple bond to a carbon of a parent chain, an alkynyl group of three or more carbons can contain more than one triple bond.

Where there is more than 3 carbons, the carbons may be connected in a linear manner, or alternatively if there are more than 4 carbons then the carbons may also be linked in a branched fashion so that the parent chain contains one or more secondary, tertiary, or quaternary carbons. An alkynyl may be substituted or unsubstituted, unless stated otherwise.

The term "aryl", as used in this disclosure, refers to a conjugated planar ring system with delocalized pi electron clouds that contain only carbon asring atoms. An "aryl" for the purposes of this disclosure encompass from 1 to 4 aryl rings wherein when the aryl is greater than 1 ring the aryl rings are j oined so that they are linked, fused, or a combination thereof. An aryl may be substituted or unsubstituted, or in the case of more than one aryl ring, one or more rings may be unsubstituted, one or more rings may be substituted, or a combination thereof.

The term "cycloalkyl", as used in this disclosure, refers to an alkyl that contains at least 3 carbon atoms but no more than 12 carbon atoms connected so that it forms a ring. A "cycloalkyl" for the purposes of this disclosure encompasses from 1 to 4 cycloalkyl rings, wherein when the cycloalkyl is greater than 1 ring, then the cycloalkyl rings are joined so that they are linked, fused, or acombination thereof. A cycloalkyl may be substituted or unsubstituted, or in the case of more than one cycloalkyl ring, one or more rings may be unsubstituted, one or more rings may be substituted, or a combination thereof.

The term "hetero-" when used as a prefix, such as, hetero-alkyl, hetero-alkenyl, hetero-alkynyl, or hetero-hydrocarbon, for the purpose of this disclosure refers to the specified hydrocarbon having one or more carbon atoms replaced by non-carbon atoms as part of the parent chain. Examples of such non-carbon atoms include, but are not limited to, N, O, S, Si, Al, B, and P. If there is more than one non-carbon atom in the heterobased parent chain then this atom may be the same element or may be a combination of different elements, such as N and O. In aparticular embodiment, a "hetero"-hydrocarbon (e. , alkyl, alkenyl, alkynyl) refers to a hydrocarbon that has from 1 to 3 C, N and/or S atoms as part of the parent chain.

The term "heterocycle," as used herein, refers to ring structures that contain at least 1 noncarbon ring atom. A "heterocycle" for the purposes of this disclosure encompass from 1 to 4 heterocycle rings, wherein when the heterocycle is greater than 1 ring the heterocycle rings are joined so that they are linked, fused, or a combination thereof. A heterocycle may be aromatic or nonaromatic, or in the case of more than one heterocycle ring, one or more rings may be nonaromatic, one or more rings may be aromatic, or a combination thereof. A heterocycle may besubstituted or unsubstituted, or in the case of more than one heterocycle ring one or more rings may be un substituted, one or more rings may be substituted, or acombination thereof. Typically, the noncarbon ring atom is N, O, S, Si, Al, B, or P. In the case where there is more than one noncarbon ring atom, these noncarbon ring atoms can either be the same element, or combination of different elements, such as N and O. Examples of heterocycles include, butare not limited to: a monocyclic heterocycle such as, aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazolidine, pyrazolidine, pyrazoline, dioxolane, sulfolane 2,3- dihydrofuran, 2, 5 -dihydrofuran tetrahydrofuran, thiophane, piperidine, 1, 2,3,6- tetrahydro-pyridine, piperazine, morpholine, thiomorpholine, pyran, thiopyran, 2,3- dihydropyran, tetrahydropyran, 1,4-dihydropyridine, 1,4-dioxane, 1,3-dioxane, dioxane, homopiperidine, 2,3,4,7-tetrahydro-lH-azepine homopiperazine, 1,3- dioxepane, 4,7- dihydro-l,3-dioxepin, and hexamethylene oxide; and polycyclic heterocycles such as, indole, indoline, isoindoline, quinoline, tetrahydroquinoline, isoquinoline, tetrahydroisoquinoline, 1,4-benzodioxan, coumarin, dihydrocoumarin, benzofuran, 2,3- dihydrobenzofuran, isobenzofuran, chromene, chroman, isochroman, xanthene, phenoxathiin, thianthrene, indolizine, isoindole, indazole, purine, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, phenanthridine, perimidine, phenanthroline, phenazine, phenothiazine, phenoxazine, 1,2-benzisoxazole, benzothiophene, benzoxazole, benzthiazole, benzimidazole, benztriazole, thioxanthine, carbazole, carboline, acridine, pyrolizidine, and quinolizidine. In addition to the polycyclic heterocycles described above, heterocycle includes polycyclic heterocycles wherein the ring fusion between two or more rings includes more than one bond common to both rings and more than two atoms common to both rings. Examples of such bridged heterocycles include quinuclidine, diazabicyclo[2.2.1]heptaneand7- oxabicyclo[2.2. l]heptane.

Theterms "heterocyclic group", "heterocyclic moiety", "heterocyclic", or "heterocyclo" used alone or as a suffix or prefix, refers to a heterocycle that has had one or more hydrogens removed there from.

The term "hydrocarbons" refers to groups of atoms that contain only carbonand hydrogen. Examples of hydrocarbonsthat can be used in this disclosure include, but are not limited to, alkanes, alkenes, alkynes, arenes, and benzyls.

The term “optionally substituted” means independent replacement of one or more hydrogen atoms with a substituent. The term "optionally substituted" alsorefersto a functional group, typically a hydrocarbon or heterocycle, where one or more hydrogen atoms may be replaced with a substituent. Accordingly, "optionally substituted" refers to a functional group that is substituted, in that one or more hydrogen atoms are replaced with a substituent, or unsubstituted, in that the hydrogen atoms are not replaced with a substituent. For example, an optionally substituted hydrocarbon group refers to an unsubstituted hydrocarbon group or a substituted hydrocarbon group. The term "substituent" refers to an atom or group of atoms substituted in place of a hydrogen atom. For purposes of this invention, a substituent would include deuterium atoms.

The term "substituted" with respect to hydrocarbons, heterocycles, and the like, refers to structures wherein the parent chain contains one or more substituents.

The term "unsubstituted" with respect to hydrocarbons, heterocycles, and the like, refers to structures wherein the parent chain contains nosubstituents. The term "nonrelease controlling excipient" as used herein, refers to an excipient whose primary function do not include modifying the duration or place of release of the active substance from a dosage form as compared with a conventional immediate release dosage form.

The term "non-release controlling excipient" as used herein, refers to an excipient whose primary function do not include modifying the duration or place of release of the active substance from a dosage form as compared with a conventional immediate release dosage form.

The term "optionally substituted" refers to a functional group, typically a hydrocarbon or heterocycle, where one or more hydrogen atoms may be replaced with a substituent. Accordingly, "optionally substituted" refers to a functional group that is substituted, in that one or more hydrogen atoms are replaced with a substituent, or unsubstituted, in that the hydrogen atoms are not replaced witha substituent. For example, an optionally substituted hydrocarbon group refers to an unsubstituted hydrocarbon group or a substituted hydrocarbon group.

The term “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” “physiologically acceptable carrier,” or “physiologically acceptable excipient” as used herein, refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each component must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenecity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. Examples of “pharmaceutically acceptable carriers” and “pharmaceutically acceptable excipients” can be found in the following, Remington: The Science and Practice of Pharmacy, 21 ^st Edition; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 5 ^th Edition; Rowe etal., Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3 ^rd Edition; Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, Fla., 2004.

The term "release controlling excipient" as used herein, refers to an excipient whose primary function is to modify the duration or place of release of the active substance from a dosage form as compared with a conventional immediate release dosage form.

The term "subject" as used herein, refers to an animal, including, but not limited to, a primate (e.g, human, monkey, chimpanzee, gorilla, and the like), rodents (e.g, rats, mice, gerbils, hamsters, ferrets, and the like), lagomorphs, swine (e.g, pig, miniature pig), equine, canine, feline, and the like. The terms "subject" and "patient" are used interchangeably herein. For example, a mammalian subject can refer to a human patient. In preferred aspects, the subject is a human patient. The terms “subject” and “patient” are used interchangeably herein. In certain aspects, the subject or patient is a child.

The term “substantially pure” as used herein in reference to a given oligosaccharide means that the oligosaccharide is substantially free from other biological macromolecules. The substantially pure oligosaccharide is at least 75% (e.g, at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

The term "therapeutically acceptable" refers to those compounds (or salts, prodrugs, tautomers, zwitterionic forms, etc. which are suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, immunogenecity, are commensurate with a reasonable benefit/risk ratio, and are effective fortheir intended use.

The terms “treat”, “treating” and “treatment”, as used herein, refers to ameliorating symptoms associated with a disease or disorder (e.g., ASD), including preventing or delaying the onset of the disease or disorder symptoms, and/or lessening the severity or frequency of symptoms of the disease or disorder.

The terms “active ingredient” and “active substance” refer to an oligosaccharide or compound, which is administered, alone or in combination with one or more pharmaceutically acceptable excipients and/or carriers, to a subject for treating, preventing, or ameliorating one or more symptoms of a disorder.

The terms “drug” or “therapeutic agent” refer to a compound, or a pharmaceutical composition thereof, which is administered to a subject for treating, preventing, or ameliorating one or more symptoms of a disorder.

The term “disorder” as used herein is intended to be generally synonymous, and is used interchangeably with, the terms “disease,” “syndrome” and “condition” (as in medical condition), in that all reflect an abnormal condition of the body or of one of its parts that impairs normal functioning and is typically manifested by distinguishing signs and symptoms.

An "oligosaccharide" is a saccharide polymer containing a small number (typically three to ten) of simple sugars (monosaccharides). A “human milk oligosaccharide” is an oligosaccharide found in human milk. As used herein, the term “human milk oligosaccharide” includes natural or native oligosaccharides found in human milk, as well as pharmaceutically acceptable salts, derivatives, prodrugs, and solvates thereof. The term “natural human milk oligosaccharide” or “natural HMO” refers to human milk oligosaccharides naturally found in human milk. Natural human milk oligosaccharides (HMOs) are separated into different classes including, for example, sialylated human milk oligosaccharides (which include sialyllactoses; sialyllactoses are sialylated oligosaccharides that comprise a lactose) and fucosylated oligosaccharides (which include “fucosy llactoses”; fucosyllactoses are fucosylated oligosaccharides that comprise a lactose). HMOs include natural sialylated human milk oligosaccharides and fucosylated oligosaccharides, as well as non-naturally occurring derivatives thereof. Non-limiting examples of sialyllactoses are 3’-SL and 6’-SL. A non-limiting example of a Fucosyllactose is 2’-FL. The terms “3’-SL” and “3’SL” are used interchangeably herein. Similarly, the terms “6’-SL” and “6’SL” are used interchangeably herein. Sialyllactoses have been shown to modulate acute and chronic immune responses in both murine and human derived macrophages stimulated with LPS and various pro-inflammatory cytokines. Both 3’SL and 6’SL have shown reductions in interleukin (IL)-ip, IL-2, IL-4, IL-6, IL- 12, interferon (IFN) y or TNF-a in vitro, with 3’-SL exhibiting more significant reductions. In addition, 3’SL has been shown to reduce other key target proteins, including PDL1, COX2 and select chemokines, such as CCL2 (also known as monocyte chemoattractant protein 1 (MCP1)) and CCL5. In vivo data in mouse models of rheumatoid arthritis, which include an LPS challenge, sialyllactose has shown benefit in clinical assessments of disease when administered orally.

Fucosylated oligosaccharides are a class of human milk oligosaccharides (HMOs) that have been associated with the production of anti-inflammatory short-chain fatty acids. Fucosylated oligosaccharides include, for example, 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose, lacto-N-fucopentaoses (that is to say lacto-N-fucopentaose I, lacto-N- fucopentaose II, lacto-N-fucopentaose III and lacto-N-fucopentaose V), lacto-N- difucohexaose I, fucosyllacto-N-hexaose, Difucosyllacto-N-hexaose I and Difucosyllacto-N- neohexaose II. In certain aspects, the fucosylated non-digestible oligosaccharide is 2’- fucosyllactose (2’-FL). In certain aspects, the fucosylated oligosaccharide is 2'- fucosyllactose (2'-FL), 3-fucosyllactose (3 ’-FL), difucosyllactose (DFL). In yet further aspects, the fucosylated oligosaccharide is 2’ -FL. As used herein, a “fucosylated oligosaccharide” is an oligosaccharide having the three sugar unit backbone, wherein each of the sugar units (fucose (Fuc), galactose (Gal), and glucose (Glc)) can be independently either in its native form or in a modified form. For example, the modified form of a sugar unit can be a sugar unit, in which at least one or more (e.g., 1, 2, 3, or more) of the hydroxyl groups is replaced with hydrogen, alkyl or a functional group; such as, for example, hydrogen, substituted or unsubstituted Ci-Ce alkyl (e.g., methyl, ethyl), or substituted or unsubstituted amine group.

The terms 2’-fucosyllactose or “2’ -FL” and “2’FL” are used interchangeably herein. Derivatives of natural HMOs can be chemically modified as compared to the natural HMO. HMOs include, but are not limited to, compounds having a structure of Formula I, 1(a), 1(b), 1(c), 1(d), 1(e), II, 11(a), 111(a) or 111(b).

“Autism Spectrum Disorder” is defined herein as having deficits in social interaction and communication together with restricted and repetitive behaviors and interests consistent with fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V). Autism Spectrum Disorder (ASD) may comprise autism, Asperger syndrome, pervasive developmental disorder not otherwise specified (PDD-NOS), Childhood Disintegrative Disorder, syndromic autism or autism of known etiology such as fragile X syndrome, PTEN macrocephaly syndrome, RETT syndrome, tuberous sclerosis complex, Timothy syndrome, and/or Joubert syndrome. Behavioral traits include one or more of: CARS behavior T-Score; ADI-R Reciprocal Social Interaction and/or Total score; SCQ Communication Score; VABS II Adaptive Behavior Composite, Communication Domain, Expressive Communication Subdomain, Personal Daily Living Skills Subdomain, Socialization Domain, Coping Skills Subdomain, and/or Fine Motor Skills Subdomain score(s); Achenbach System of Empirically Based Assessment (ASEBA), Attention-Deficit/Hyperactivity Disorder Test (ADHDT), Childhood Asperger Syndrome Test (CAST), Penn Interactive Peer Play Scale, (PIPPS), Social Interaction Assessment Scale (SIAS) and/or CBCL Emotionally Reactive T-Score. In certain embodiments, the above methods and uses may include an initial step of identifying a subject in need of treatment based on the subject having impairment in one or more behavioral traits descried above. It is a particular benefit of the treatment to reduce anxiety in a subject, such as improving the anxiety subscale score in the Children's Behavior Checklist (CBCL)/ Achenbach System of Empirically Based Assessment (ASEBA), such as at week 8 and/or week 16 of the beginning of treatment. It is a particular benefit of the treatment to reduce hyperactivity subscale score in a subject, such as in the Attend on - Deficit/Hyperactivity Disorder Test (ADHDT), such as at week 8 and/or week 16 of the beginning of treatment. It is a particular benefit of the treatment to improve communicative and interactive behaviors, such as in the Penn Interactive Peer Play Scale, (PIPPS), such as at week 8 and/or week 16 of the beginning of treatment. It is a particular benefit of the treatment to improve behavior assessment, such as in Childhood Asperger Syndrome Test (CAST), such as at week 8 and/or week 16 of the beginning of treatment. Additionally, improvement in the subject’s microbiota (gut and/or oral) composition is desirable. Improvement in, or avoidance of, gastrointestinal symptoms, such as constipation, diarrhea, stool consistency, stool smell, flatulence and abdominal pain is desirable, such as, for example, at weeks 8 and 16 of the beginning of treatment.

In a preferred embodiment the composition is administered to a patient diagnosed with autism spectrum disorder but has not been diagnosed with other neurodevelopmental disorders or psychiatric diseases.

Oral administration of the oligosaccharides of the disclosure provide for systemic circulation of the oligosaccharides both in infantsand adults. Unlike other drug products approved by the FDA, the oligosaccharides described herein can not only be administered to treat a disease or disorder in an adult subj ect, but can also be administered to pregnant females, infants, and subjects who have impaired organ function (e.g., liver disfunction, kidney failure). Due to the oligosaccharides of the disclosure having little to no adverse effects in humans, this form of therapy could be used as a preventive, as a first line therapy option, or as an adjunct to existing therapies that would be well tolerated by patients of either sex. Preferred subjects are children, such as children about 4 years of age. In additional aspects, the subject is about 18 years of age or less, about 15 years of age or less, about 13 years of age or less, about 10 years of age or less, about 8 years of age or less, about 6 years of age or less, or about 5 years of age or less. Subjects, however, can also include adults.

In certain aspects, the composition is not a mammalian milk. In further aspects, the composition is not derived from mammalian milk or not derived from human milk.

In certain embodiments, the one or more human milk oligosaccharides is selected from lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-hexaose (LNH), lacto-N- neohexaose (LNnH), 2’fucosyllactose (2’FL), 3’fucosyllacose (3 ’FL), lacto-difucotetraose (LDFT), lacto-N-fucopenaose II/III (LNFP II/III), lactose-N-fucopentaose I (LNFP I), lacto- N-difuco-hexaose I (LNDFH I), lacto-N-difuco-hexaose II (LNDFH II), difucosyl-para- lacto-N-neohexaose (DFpLNnH), difucosyllacto-N-hexaose c (DFLNH c), 3’sialyllactose (3’SL), 6’sialyllactose (6’SL), LS-tetrasaccharide a (LSTa), LS-tetrasaccharide b (LST b), LS-tetrasaccharide c (LST c), 3'-sialyl-N-acetyllactosamine (3’SLN), 6'-sialyl-N- acetyllactosamine (6’SLN), or disialyllacto-N-tetraose (DSLNT). For example, the human milk oligosaccharide can be selected from 2’FL, 3 ’FL, 3’SL, 6’SL, LNT, or LNnT. In certain aspects, the one or more human milk oligosaccharides are selected from 2’fucosyllactose, 3’sialyllactose or 6’sialyllactose. The composition can, for example, comprise 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more by mass one or more human milk oligosaccharides. The composition can additionally comprise a mixture of two, three, four or five human milk oligosaccharides; for example, the composition can comprise a mixture selected from: i. 2’FL and LNT; ii. 2’FL and LNnT; iii. 2’FL, 3 ’FL, 3’SL, 6’SL and LNT; iv. 3’SL and 6’SL; or v. 6’SL and LNT.

In additional aspects, the composition comprises a mixture of one neutral core and one neutral fucosylated human milk oligosaccharide or a mixture of one neutral and one acidic human milk oligosaccharide or a mixture of one neutral fucosylated and one acidic human milk oligosaccharide. For example, the composition can comprise a mixture of 4: 1 2’FL:LNnT.

In yet further aspects, the oligosaccharide or HMO administered to the patient is a compound having a structure of Formula 1, 1(a), 1(b), 1(c), 1(d), 1(e), II, 11(a), 111(a) or 111(b). In additional aspects, the sialyllactose compound is a compound selected from a compound of Formula 1, 1(a) and/or II.

As described herein, the subject is administered a composition comprising one or more oligosaccharides or human milk oligosaccharides. The composition can comprise 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more by mass one or more human milk oligosaccharide. In certain aspects, the composition is not human milk. In additional aspects, the composition is not derived from human milk.

In certain aspects, the one or more human milk oligosaccharides are selected from lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-hexaose (LNH), lacto-N- neohexaose (LNnH), 2’fucosyllactose (2’FL), 3’fucosyllacose (3 ’FL), lacto-difucotetraose (LDFT), lacto-N-fucopenaose II/III (LNFP II/III), lactose-N-fucopentaose I (LNFP I), lacto- N-difuco-hexaose I (LNDFH I), lacto-N-difuco-hexaose II (LNDFH II), difucosyl-para- lacto-N-neohexaose (DFpLNnH), difucosyllacto-N-hexaose c (DFLNH c), 3’sialyllactose (3’SL), 6’sialyllactose (6’SL), LS-tetrasaccharide a (LSTa), LS-tetrasaccharide b (LST b), LS-tetrasaccharide c (LST c), 3'-sialyl-N-acetyllactosamine (3’SLN), 6'-sialyl-N- acetyllactosamine (6’SLN), or disialyllacto-N-tetraose (DSLNT), or a combination of any of thereof. In yet other aspects, the one or more human milk oligosaccharides are selected from 2’FL, 3 ’FL, 3’SL, 6’SL, LNT, or LNnT, or a combination of any of thereof.

The composition administered to the subject can comprise one HMO or can comprise a mixture of two, three, four, five or more HMOs. In certain aspects, the composition comprises one HMO and the HMO is selected from the group consisting of 2’FL, 3 ’FL, 3’SL, 6’SL, LNT, or LNnT.

In further aspects, the composition comprises a mixture of 2’FL and at least one other HMO. In certain aspects, the composition comprises 2’FL and LNT; 2’FL and LNnT; 2’FL, 3 ’FL, 3’SL, 6’SL and LNT. The composition comprising 2’FL and LNT includes a 4: 1 mixture of 2’FL and LNT; such a composition is GRAS (generally regarded as safe) and is available from Glycom, Lyngby, Denmark. A composition comprising 2’FL, 3 ’FL, 3’SL, 6’SL and LNT is sold by Jennewein Biotechnologie and is GRAS.

In a particular embodiment, the disclosure provides for an oligosaccharide having the structure of Formula 1, 1(a) or II:

Formula I

Formula 1(a) Formula II or a pharmaceutically acceptable salt, solvate, or prodrug thereof, wherein, R -R are independently selected from H, D, a halo, an unsubstituted or substituted (Ci-C6)alkyl, an unsubstituted or substituted (Ci-C6)heteroalkyl, an unsubstituted or substituted (C ₂ - C ₆ )alkenyl, an unsubstituted or substituted (C ₂ - C6)heteroalkenyl, an unsubstituted or substituted (C3-C6)alkynyl, an unsubstituted or substituted (C ₃ -C ₆ )heteroalkynyl, an unsubstituted or substituted (C ₄ -C ₈ )cycloalkyl, an unsubstituted or substituted heterocycle, an unsubstituted or substituted aryl, -ROR', - RN(R') ₂ , -RS SR', -SH, - RSOR', -RSO ₂ R', -RSO ₂ H, -RSO ₃ H, -RC(=S)-R', -ROH, - RC(=O)R', -RNO ₂ , -RSR, -RCN, -RNC, -RNNR', -RC(=O)OR', -ROC(=O)R', - RC(=O)H, RC(=O)OH, -RC(=O)N(R') ₂ , -RN ₃ , -ROCN, -RNCO, -RONO ₂ , -RNO, - ROP(=O)(OH) ₂ , and-RB(OH) ₂ ; R is absent or a(Ci-C5)alkyl; R' is independently selected from H, D, an unsubstituted or substituted (Ci -Ce) alkyl, an unsubstituted or substituted (Ci- C6)heteroalkyl, an unsubstituted or substituted (C ₂ -C ₆ )alkenyl, an unsubstituted or substituted (C ₂ -C ₆ )heteroalkenyl, an unsubstituted or substituted (C3-C6)alkynyl, an unsubstituted or substituted (C3- C6)heteroalkynyl, an unsubstituted or substituted (C4-C8)cycloalkyl an unsubstituted or substituted heterocycle, and an unsubstituted or substituted aryl.

In a certain embodiment, the disclosure provides for an oligosaccharide having the structure of Formula 1(b) or 1(c):

Formula 1(b)

Formula 1(c) or a pharmaceutically acceptable salt, solvate, or prodrug thereof, wherein, RZ-R ⁶ are independently selected from H, D, a halo, an unsubstituted or substituted (Ci-C ₆ )alkyl, an unsubstituted or substituted (Ci-C ₆ )heteroalkyl, an unsubstituted or substituted (C2-C6)alkenyl, an unsubstituted or substituted (C2- C6)heteroalkenyl, an unsubstituted or substituted (C3-C6)alkynyl, an unsubstituted or substituted (C3-C6)heteroalkynyl, an unsubstituted or substituted (C4-Cs)cycloalkyl, an unsubstituted or substituted heterocycle, an unsubstituted or substituted aryl, -ROR', - RN(R') ₂ , -RS SR', -SH, - RSOR', -RSO ₂ R', -RSO ₂ H, -RSO ₃ H, -RC(=S)-R', -ROH, - RC(=O)R', -RNO ₂ , -RSR', -RCN, -RNC, -RNNR', -RC(=O)OR', -ROC(=O)R', - RC(=O)H, -RC(=O)OH, -RC(=O)N(R') ₂ , -RN ₃ , -ROCN, -RNCO, -RONO ₂ , -RNO, - ROP(=O)(OH) ₂ , and-RB(OH) ₂ ; R is absent or a(Ci-Cs)alkyl; R’ is independently selected from H, D, an unsubstituted or substituted (Ci -Ce) alkyl, an unsubstituted or substituted (Ci- C6)heteroalkyl, an unsubstituted or substituted (C ₂ -C6)alkenyl, an unsubstituted or substituted (C ₂ -C6)heteroalkenyl, an unsubstituted or substituted (C ₃ -C6)alkynyl, an unsubstituted or substituted (C ₃ - C ₆ )heteroalkynyl, an unsubstituted or substituted (C ₄ - Cs)cycloalkyl, an unsubstituted or substituted heterocycle, and an unsubstituted or substituted aryl.

In a particular embodiment, the disclosure provides for a method disclosed herein which comprises administering a 3’- sialyllactose (3’SL)-based oligosaccharide disclosed herein or a pharmaceutical composition comprising a 3 ’-sialyllactose (3’SL)- based oligosaccharide disclosed herein.

In another embodiment, the disclosure also provides for a method disclosed herein which comprises administering one or more oligosaccharides having the structure of Formula 1, 1(a) and/or II:

Formula I

Formula 1(a) Formula II or a pharmaceutically acceptable salt, solvate, or prodrug thereof, wherein,

1 18

R -R are independently selected from H, D, a halo, an un substituted or substituted (Ci-C ₆ )alkyl, an unsubstituted or substituted (Ci-C ₆ )heteroalkyl, an unsubstituted or substituted (C2-C6)alkenyl, an unsubstituted or substituted (C2- C ₆ )heteroalkenyl, an unsubstituted or substituted (C ₃ -C ₆ )alkynyl, an unsubstituted or substituted (C3-C6)heteroalkynyl, an unsubstituted or substituted (C4-C8)cycloalkyl, an unsubstituted or substituted heterocycle, an unsubstituted or substituted aryl, -ROR', - RN(R') ₂ , -RSSR, -SH, - RSOR', -RSO ₂ R', -RSO ₂ H, -RSO ₃ H, -RC(=S)-R', -ROH, -

RC(=O)R', -RNO2, -RSR', -RCN, -RNC, -RNNR', -RC(=O)OR', -ROC(=O)R', - RC(=O)H, -RC(=O)OH, -RC(=O)N(R') ₂ , -RN ₃ , -ROCN, -RNCO, -RONO ₂ , -RNO, - ROP(=O)(OH) ₂ , and-RB(OH) ₂ ;

R is absent or a(Ci-C5)alkyl; R' is independently selected from H, D, an unsubstituted or substituted (Ci-C ₆ ) alkyl, an unsubstituted or substituted (Ci- C6)heteroalkyl, an unsubstituted or substituted (C2-C6)alkenyl, an unsubstituted or substituted (C2-C6)heteroalkenyl, an unsubstituted or substituted (C3-C6)alkynyl, an unsubstituted or substituted (C3-C6)heteroalkynyl, an unsubstituted or substituted (C4-C8)cycloalkyl, an unsubstituted or substituted heterocycle, and an unsubstituted or substituted aryl.

In an alternate embodiment, the disclosure further provides for a method disclosed herein which comprises administering a pharmaceutical composition which comprises one or more oligosaccharides having the structures of Formula 1, 1(a) and/or II or a pharmaceutically acceptable salt, solvate, or prodrug thereof. In another embodiment, the disclosure also provides for a method disclosed herein which comprises administering one or more oligosaccharides having the structure of Formula 1(b) and/or 1(c):

Formula 1(b)

Formula 1(c) or a pharmaceutically acceptable salt, solvate, or prodrug thereof, wherein,

R -R are independently selected from H, D, a halo, an unsubstituted or substituted (Ci-C6)alkyl, an unsubstituted or substituted (Ci-C6)heteroalkyl, an unsubstituted or substituted (C ₂ -C ₆ )alkenyl, an unsubstituted or substituted (C ₂ - C6)heteroalkenyl, an unsubstituted or substituted (C3-C6)alkynyl, an unsubstituted or substituted (C3-C6)heteroalkynyl, an unsubstituted or substituted (C4-Cs)cycloalkyl, an unsubstituted or substituted heterocycle, an unsubstituted or substituted aryl, -ROR', - RN(R') ₂ , -RSSR, -SH, - RSOR', -RSO ₂ R', -RSO ₂ H, -RSO ₃ H, -RC(=S)-R', -ROH, - RC(=O)R', -RNO ₂ , -RSR, -RCN, -RNC, -RNNR', -RC(=O)OR', -ROC(=O)R', - RC(=O)H, -RC(=O)OH, -RC(=O)N(R') ₂ , -RN ₃ , -ROCN, -RNCO, -RONO ₂ , -RNO, - ROP(=O)(OH) ₂ , and-RB(OH) ₂ ;

R is absent or a(Ci-C ₅ )alkyl;

R' is independently selected from H, D, an unsubstituted or substituted (Ci-Ce) alkyl, an unsubstituted or substituted (Ci- C ₆ )heteroalkyl, an unsubstituted or substituted (C ₂ -C6)alkenyl, an unsubstituted or substituted (C ₂ -C6)heteroalkenyl, an unsubstituted or substituted (C3-C6)alkynyl, an unsubstituted or substituted (C3- C6)heteroalkynyl, an unsubstituted or substituted (C4-Cs)cycloalkyl, an unsubstituted or substituted heterocycle, and an unsubstituted or substituted aryl.

In an alternate embodiment, the disclosure further provides for a method disclosed herein which comprises administering a pharmaceutical composition which comprises one or more oligosaccharides having the structure of Formula 1(b) and/or 1(c) or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In yet another embodiment, the disclosure provides for a method disclosed herein which comprises administering one or more oligosaccharide having the structures of Formula 1(d), 1(e) and/or 11(a):

Formula 1(d) Formula 1(e) Formula 11(a) or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In an alternate embodiment, the disclosure also provides for a method disclosed herein which comprises administering a pharmaceutical composition which comprises one or more oligosaccharides of Formula 1(d), 1(e) and/or 11(a) or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In a further embodiment, said oligosaccharide is substantially a single enantiomer, a mixture of about 90% or more by weight of the (-)-enantiomer and about 10% or less by weight of the (+)-enantiomer, a mixture of about 90% or more by weight of the (+)- enantiomer and about 10% or less by weight of the (-)-enantiomer, substantially an individual diastereomer, or a mixture of about 90% or more by weight of an individual diastereomer and about 10% or less by weight of any other diastereomer.

The oligosaccharides disclosed hereinmay be enantiomerically pure, such as a single enantiomer or a single diastereomer, or be stereoisomeric mixtures, such as a mixture of enantiomers, a racemic mixture, or a diastereomeric mixture. As such, one of skill in the art will recognize that administration of an oligosaccharide in its (R) form is equivalent, for oligosaccharides that undergo epimerization in vivo, to administration of the oligosaccharide in its (S) form. Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate using, for example, chiral chromatography, recrystallization, resolution, diastereomeric salt formation, orderivatizationinto diastereomeric adducts followed by separation.

When the oligosaccharide disclosed herein contains an acidic or basic moiety, it may also be disclosed as a pharmaceutically acceptable salt (See, Berge etal., J Pharm. Sci. 1977, 66, 1-19; and “Handbook of Pharmaceutical Salts, Properties, and Use,” Stah and Wermuth, Ed.; Wiley-VCH and VHCA, Zurich, 2002).

Suitable acids for use in the preparation of pharmaceutically acceptable salts include, but are not limited to, acetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, boric acid, (+)- camphoric acid, camphorsulfonic acid, (+)- (1 S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamic acid, dodecyl sulfuric acid, ethane- 1,2- disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid, a-oxo-glutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, (+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, lauric acid, maleic acid, (-)-L-malic acid, malonic acid, (±)-DL- mandelic acid, methanesulfonic acid, naphthal ene-2-sulfonic acid, naphthal ene-l,5-disulfonic acid, 1- hydroxy-2 -naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, perchloric acid, phosphoric acid, L- pyroglutamic acid, saccharic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid.

Suitable bases for use in the preparation of pharmaceutically acceptable salts, including, but not limited to, inorganic bases, such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, or sodium hydroxide; and organic bases, such as primary, secondary, tertiary, and quaternary, aliphatic and aromatic amines, including L-arginine, benethamine, benzathine, choline, deanol, diethanolamine, diethylamine, dimethylamine, dipropylamine, diisopropylamine, 2-(diethylamino)- ethanol, ethanolamine, ethylamine, ethylenediamine, isopropylamine, N-methyl- glucamine, hydrabamine, IH-imidazole, L-ly sine, morpholine, 4-(2-hydroxy ethylmorpholine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, l-(2- hydroxyethyl)-pyrrolidine, pyridine, quinuclidine, quinoline, isoquinoline, secondary amines, triethanolamine, trimethylamine, triethylamine, N-methyl-D- glucamine, 2- amino-2-(hydroxy methyl)- 1,3 -propanediol, and tromethamine.

The oligosaccharide as disclosed herein may also be designed as a prodrug, which is a functional derivative of the oligosaccharide as disclosed herein and is readily convertible into the parent oligosaccharide in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent oligosaccharide. They may, for instance, be bioavailable by oral administration whereas the parent oligosaccharide is not.

The prodrug may also have enhanced solubility in pharmaceutical compositions over theparent oligosaccharide. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. See Harper, Progress in Drug Research 1962, 4, 221-294; Morozowich et al. in “Design of Biopharmaceutical Properties through Prodrugs and Analogs,” Roche Ed., APHA Acad. Pharm. Sci. 1977; “Bioreversible Carriers in Drug in Drug Design, Theory and Application,” Roche Ed., APHA Acad. Pharm. Sci. 1987; “Design of Prodrugs,” Bundgaard, Elsevier, 1985; Wang et al., Curr. Pharm. Design 1999, 5, 265-287; Pauletti et al., Adv. Drug. Delivery Rev. 1997, 27, 235-256; Mizen et al., Pharm. Biotech. 1998, 11, 345-365; Gaignault et al., Pract. Med. Chem. 1996, 671- 696; Asghamej ad in “Transport Processes in Pharmaceutical Systems,” Amidon et al., Ed., Marcell Dekker, 185-218, 2000; Balant et al., Eur. J. Drug Metab. Pharmacokinet. 1990, 15, 143-53; Balimane and Sinko, Adv. Drug Delivery Rev. 1999, 39, 183-209; Browne, Clin. Neuropharmacol. 1997, 20, 1-12; Bundgaard, Arch. Pharm. Chem. 1979, 86, 1-39; Bundgaard, Controlled Drug Delivery 1987, 17, 179-96; Bundgaard, Adv. Drug Delivery Rev. 1992, 8, 1-38; Fleisher et al., Adv. Drug Delivery Rev. 1996, 19, 115-130; Fleisher et aL, Methods Enzymol. 1985, 112, 360-381; Farquhar et al., J. Pharm. Sci. 1983, 72,324-325; Freeman et al., J. Chem. Soc., Chem. Commun. 1991, 875- 877; Friis and Bundgaard, Eur. J. Pharm. Sci. 1996, 4, 49-59; Gangwar et al., Des. Biopharm. Prop. Prodrugs Analogs, 1977, 409- 421;

Nathwani and Wood, Drugs 1993, 45, 866-94; Sinhababu and Thakker, Adv. Drug Delivery Rev. 1996, 19, 241-273; Stella et al., Drugs 1985, 29, 455-73; Tan etal., Adv. Drug Delivery Rev. 1999, 39, 117-151; Taylor, A dv. Drug Delivery Rev. 1996, 19, 131- 148; Valentino and Borchardt, Drug Discovery Today 1997, 2, 148-155; Wiebe and Knaus, Adv. Drug Delivery Rev. 1999, 39, 63-80; Waller et al., Br. J. Clin. Pharmac. 1989, 28, 497-507.

The oligosaccharide may be produced by biotechnological means using enzymebased fermentation technology (recombinant or natural enzymes) or microbial fermentation technology. In the latter case, microbes may either express their natural enzymes and substrates or may be engineered to produce respective substrates and enzymes. Single microbial cultures and/or mixed cultures may be used. Alternatively, the oligosaccharides may be produced by chemical synthesis from lactose and other substrates.

Biotechnological approaches have made it possible for the large scale, cost-efficient production of target oligosaccharides.

Precisely, the oligosaccharides disclosed herein can be produced in high yields in aqueous media by fermentation of genetically modified bacteria, yeasts or other microorganisms. See, for example, W0200104341; W02007101862,W02010070104; W02010142305;W02012112777; Priem e/a/., Glycobiology 12:235 (2002); Drouillard etal.,Angew. Chem. Int. Ed. 45: 1778 (2006); Han et al.,Biotechnol. Adv. 30: 1268 (2012); Lee e/ al., Microb. Cell Fact. 11 :48 (2012); Baumgartner et al., Microb. Cell Fact. 12:40 (2013); andWO 2014135167A1. Alternatively, the oligosaccharides of the disclosure can be synthesized based upon methods described in WO2011100980A1; W02012007588A1; W02012127410A1; WO20 12155916 A 1 ; WO2013044928 A 1 ; and US9102966B2. LNT can be synthesized as described in WO 2012/155916 and WO 2013/044928, a mixture ofLNT and LNnT can be made as described in WO 2013/091660, 2'-FL can be made as described in WO 2010/115934 and WO 2010/115935, 3-FL can be made as described in WO 2013/139344, 6'-SL and salts thereof can be made as described in WO 2010/100979, sialylated oligosaccharides can be made as described in WO 2012/113404 and mixtures of human milk oligosaccharides can be made as described inWO 2012/113405. As examples of enzymatic production, sialylated oligosaccharides can be made as described in WO 2012/007588, fucosylated oligosaccharides can be made as described inWO 2012/127410. With regard to biotechnological methods, WO 2001/04341 and WO 2007/101862 describe how to make oligosaccharides optionally substituted by fucose or sialic acid using genetically modified E.coli.

In a certain embodiment, the disclosure provides for a nutritional composition that comprises one or more oligosaccharides (e. , 3’SL, 6’SL, or 2’FL or derivatives thereof) disclosed herein along with one or more foodgrade agents. In certain embodiments, the nutritional composition comprises or consists of 3’SL, 6’SL, or 2’FL or a combination thereof. In other embodiments, the nutritional composition comprise or consists of 3’SL, 6’SL or 2’FL or a combination thereof at 145 mg/L or greater of 3’SL, 6’SL, or 2’FL or a combination thereof.

In another embodiment, the nutritional composition comprises at least 9% (e.g. , 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%; or any value between any of the foregoing) 3’SL, 6’SL or 2’FL or a combination thereof of the total oligosaccharides inthe composition. Examples of foodgrade agents that can be used with the oligosaccharides disclosed herein, include, but are not limited to, milk (e.g., cow's milk, almond milk, soy milk), yogurt, maltodextrin, milk protein concentrate, Sucromalt, glycerine, cocoa powder, soy protein isolate, fructose, vegetable or animal oils (e.g., high oleic safflower oil, soy oil, canola oil), plant sterol esters, HMSs/HMOs, soy lecithin, carrageenan, taurine, L-camitine, vitamins and/or minerals (e.g., sodium ascorbate, potassium citrate, sodium phosphate, calcium citrate, choline chloride, potassium chloride, sodium citrate, magnesium oxide, alpha-tocopheryl acetate, zinc sulfate, ferrous sulfate, niacinamide, calcium pantothenate, vitamin A palmitate, citric acid, manganese sulfate, pyridoxine hydrochloride, vitamin D3, copper sulfate, thiamine mononitrate, riboflavin, beta carotene, folic acid, biotin, potassium iodide, chromium chloride, sodium selenate, sodium molybdate, phytonadione, vitamin B12, magnesium chloride, calcium phosphate).

Also disclosed herein are pharmaceutical compositions comprising one or more oligosaccharides of the disclosure (e.g, 3’SL, 6’SL, and/or 2’FL or derivatives thereof), or a pharmaceutically acceptable salt, solvate, or prodrug thereof, as an active ingredient, combined with a pharmaceutically acceptable vehicle, carrier, diluent, or excipient, or a mixture thereof; in combination with one or more pharmaceutically acceptable excipients or carriers.

Disclosed herein are pharmaceutical compositions in modified release dosage forms, which comprise one or more oligosaccharides (e.g, 3’SL, 6’SL, and/or 2’FL or derivatives thereof) of the disclosure, or a pharmaceutically acceptable salt, solvate, or prodrug thereof; and one or more release controlling excipients or carriers as described herein. Suitable modifiedrelease dosage vehicles include, but are not limited to, hydrophilic or hydrophobic matrix devices, water-soluble separating layer coatings, enteric coatings, osmotic devices, multiparticulate devices, and combinations thereof. The pharmaceutical compositions may also comprise non-release controlling excipients or carriers.

Further disclosed herein are pharmaceutical compositions in enteric coated dosage forms, which comprise one or more oligosaccharides (e.g, 3’SL, 6’SL, and/or 2’FL or derivatives thereof) as disclosed herein, or a pharmaceutically acceptable salt, solvate, or prodrug thereof; and one or more release controlling excipients or carriers for use in an enteric coated dosage form. The pharmaceutical compositions may also comprise nonrelease controlling excipients or carriers.

Further disclosed herein are pharmaceutical compositions in effervescent dosage forms, which comprise one or more oligosaccharides (e.g, 3’SL, 6’SL, and/or 2’FL or derivatives thereof) as disclosed herein in substantially pure form (e.g, lacking other oligosaccharides found in milk), or a pharmaceutically acceptable salt, solvate, or prodrug thereof; and one or more release controlling excipients or carriers for use in an effervescent dosage form. The pharmaceutical compositions may also comprise nonrelease controlling excipients or carriers.

Additionally, disclosed are pharmaceutical compositions in a dosage form that has an instant releasing component and at least one delayed releasing component, and is capable of giving a discontinuous release of one or more oligosaccharides (e. ., 3 ’ SL6’ SL, and/or, 2’FL or derivatives thereof) disclosed herein in the form of at least two consecutive pulses separated in time (e. , separated in time from 0.1 up to 24 hours or a few days). The pharmaceutical compositions comprise an oligosaccharide as disclosed herein, or a pharmaceutically acceptable salt, solvate, or prodrug thereof; and one or more release controlling and non-release controlling excipients or carriers, such as those excipients or carriers suitable for a disruptable semi-permeable membrane and as swellable substances.

Disclosed herein also are pharmaceutical compositions in a dosage form for oral administration to a subject, which comprise one or more oligosaccharides (e.g, 3 ’ SL,6’ SL, and/or 2’FL or derivative thereof) as disclosed herein, or a pharmaceutically acceptable salt, solvate, or prodrug thereof; and one or more pharmaceutically acceptable excipients or carriers, enclosed in an intermediate reactive layer comprising a gastric juice-resistant polymeric layered material partially neutralized with alkali and having cation exchange capacity and a gastric juice-resistant outer layer.

Provided herein are pharmaceutical compositions that comprise about 0.1 to about 1000 mg or up to 2000 mg or up to 3000 mg (or any value between 0.1 - 3000 mg), about 1 to about 500 mg, about 2 to about 100 mg, about 1 mg, about 2 mg, about 3 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 100 mg, about 500 mg of one or more oligosaccharides as disclosed herein, in the form of immediate release tablets for oral administration. The pharmaceutical compositions further comprise inactive ingredients such as flavoring agents, copovidone, ethylcellulose, magnesium stearate, mannitol, and silicon dioxide.

Provided herein are pharmaceutical compositions that comprise about 0. 1 to about 1000 mg or up to 2000 mg or up to 3000 mg (or any value there between), about 1 to about 500 mg, about 2 to about 100 mg, about 1 mg, about 2 mg, about 3 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 100 mg, about 500 mg of one or more oligosaccharides as disclosed herein, in the form of extended release tablets for oral administration. The pharmaceutical compositions further comprise inactive ingredients such as ethylcellulose, dibutyl sebacate, polyvinyl pyrroliodone, sodium stearyl fumarate, colloidal silicon dioxide, and polyvinyl alcohol.

The pharmaceutical compositions disclosed herein may be disclosed in unit-dosage forms or multiple-dosage forms. Unit-dosage forms, as used herein, refer to physically discrete units suitable for administration to human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the oligosaccharide sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carriers or excipients. Examples of unit-dosage forms include ampoules, syringes, and individually packaged to capsules. Unit-dosage forms may be administered in fractions or multiples thereof. A multiple-dosage form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dosage form. Examples of multiple- dosage forms include vials, bottles of tablets or capsules, or bottles of pints or gallons.

The oligosaccharides as disclosed herein may be administered alone, or in combination with one or more other oligosaccharides disclosed herein, and/or one or more other active ingredients. The pharmaceutical compositions that comprise an oligosaccharide disclosed herein may be formulated in various dosage forms for oral, parenteral, and topical administration. The pharmaceutical compositions may also be formulated as a modified release dosage form, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release, and gastric retention dosage forms.

These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art (see, Remington: The Science and Practice of Pharmacy, supra; Modified-Release Drug Delivery Technology, Rathbone et al., Eds., Drugs and the Pharmaceutical Science, Marcel Dekker, Inc.: New York, N.Y., 2002; Vol. 126).

The pharmaceutical compositions disclosed herein may be administered at once, or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations.

In the case wherein the patient's condition does not improve, upon the doctor's discretion the administration of the oligosaccharides may be administered chronically, that is, for an extended period of time, including throughout the duration of the patient's life in order to ameliorate or otherwise control or limit the symptoms of the patient's disease or condition.

Once improvement of the patient's condition has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained. Patients can, however, require intermittent treatment on a long-term basis upon any recurrence of symptoms.

The pharmaceutical compositions disclosed herein may be formulated in solid, semisolid, or liquid dosage forms for oral administration. As used herein, oral administration also includes buccal, lingual, and sublingual administration. Suitable oral dosage forms include, but are not limited to, tablets, capsules, pills, troches, lozenges, pastimes, cachets, pellets, medicated chewing gum, granules, bulk powders, effervescent or non-effervescent powders or granules, solutions, emulsions, suspensions, solutions, wafers, sprinkles, elixirs, and syrups. In addition to the oligosaccharides, the pharmaceutical compositions may contain one or more pharmaceutically acceptable carriers or excipients, including, but not limited to, binders, fillers, diluents, disintegrants, wetting agents, lubricants, glidants, coloring agents, dye-migration inhibitors, sweetening agents, and flavoring agents.

Binders or granulators impart cohesiveness to a tablet to ensure the tablet remaining intact after compression. Suitable binders or granulators include, but are not limited to, starches, such as com starch, potato starch, and pre-gelatinized starch (e. , STARCH 1500); gelatin; sugars, such as sucrose, glucose, dextrose, molasses, and lactose; natural and synthetic gums, such as acacia, alginic acid, alginates, extract of Irish moss, Panwar gum, ghatti gum, mucilage of isabgol husks, carboxymethyl cellulose, methylcellulose, polyvinylpyrrolidone (PVP), Veegum, larch arabogalactan, powdered tragacanth, and guar gum; celluloses, such as ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC); microcrystalline celluloses, such as AVICEL-PH-101, AVICEL-PH- 103, AVICELRC-581, AVICEL-PH-105(FMC Corp., Marcus Hook, Pa.); and mixtures thereof. Suitable fillers include, but are not limited to, talc, calcium carbonate, microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. The binder or filler may be present from about 50 to about 99% by weight in the pharmaceutical compositions disclosedherein.

Suitable diluents include, but are not limited to, dicalcium phosphate, calcium sulfate, lactose, sorbitol, sucrose, inositol, cellulose, kaolin, mannitol, sodium chloride, dry starch, and powdered sugar. Certain diluents, such asmannitol, lactose, sorbitol, sucrose, and inositol, when present in sufficient quantity, can impart properties to some compressed tablets that permit disintegration in the mouth by chewing. Such compressed tablets can be used as chewable tablets.

Suitable disintegrants include, but are not limited to, agar; bentonite; celluloses, such as methylcellulose and carboxymethylcellulose; wood products; natural sponge; cation- exchange resins; alginic acid; gums, such as guar gum and Veegum HV; citrus pulp; cross-linked celluloses, such as croscarmellose; cross- linked polymers, such as crospovidone; cross-linked starches; calcium carbonate; microcrystalline cellulose, such as sodium starch glycolate; polacrilin potassium; starches, such as corn starch, potato starch, tapioca starch, and pre-gelatinized starch; clays; aligns; and mixtures thereof. The amount of disintegrant in the pharmaceutical compositions disclosedherein varies upon the type of formulation, and is readily discernible to those of ordinary skill in the art. The pharmaceutical compositions disclosed herein may contain from about 0.5 to about 15% or from about 1 to about 5% by weight of a disintegrant. It should be understood that many carriers and excipients may serve several functions, even within the same formulation. The pharmaceutical compositions disclosed herein may be formulated as compressed tablets, tablet triturates, chewable lozenges, rapidly dissolving tablets, multiple compressed tablets, or enteric-coating tablets, sugar- coated, or film-coated tablets.

Enteric-coated tablets are compressed tablets coated with substances that resist the action of stomach acid but dissolve or disintegrate in the intestine, thus protecting the active ingredients from the acidic environment ofthe stomach. Enteric-coatings include, but are not limited to, fatty acids, fats, phenylsalicylate, waxes, shellac, ammoniated shellac, and cellulose acetate phthalates.

Sugar-coated tablets are compressed tablets surrounded by a sugar coating, which may be beneficial in covering up obj ecti enable tastes or odors and in protecting the tablets from oxidation. Film-coated tablets are compressed tablets that are covered with a thin layer or film of awater-soluble material. Film coatings include, but are not limited to, hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate. Film coating imparts the same general characteristics as sugar coating. Multiple compressed tablets are compressed tablets made by more than one compression cycle, including layered tablets, and press-coated or dry-coated tablets.

The tablet dosage forms may be prepared from the active ingredient in powdered, crystalline, or granular forms, alone or in combination with one or more carriers or excipients described herein, including binders, disintegrants, controlled-release polymers, lubricants, diluents, and/or colorants. Flavoring and sweetening agents are especially useful in the formation of chewable tablets and lozenges.

The pharmaceutical compositions disclosed herein may be formulated as soft or hard capsules, which can be made from gelatin, methylcellulose, starch, orcalcium alginate. The hard gelatin capsule, also known as the dry-filled capsule (DFC), consists of two sections, one slipping over the other, thus completely enclosing the active ingredient. The soft elastic capsule (SEC) isa soft, globular shell, such as a gelatin shell, which is plasticized by the addition of glycerin, sorbitol, or a similar polyol. The soft gelatin shells may contain a preservative to prevent the growth of microorganisms. Suitable preservatives are thoseas described herein, including methyl- and propyl-parabens, and sorbic acid. The liquid, semisolid, and solid dosage forms disclosed herein may be encapsulated in a capsule. Suitable liquid andsemisolid dosage forms include solutions and suspensions in propylene carbonate, vegetable oils, or triglycerides. Capsules containing such solutions can be prepared as described in U.S. Pat. Nos. 4,328,245; 4,409,239; and 4,410,545. The capsules may also be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient.

The pharmaceutical compositions disclosed herein may be formulated in liquid and semisolid dosage forms, including emulsions, solutions, suspensions, elixirs, and syrups. An emulsion is a two-phase system, in which one liquid is dispersed in the form of small globules throughout another liquid, which can be oil-in- water or water-in-oil. Emulsions may includea pharmaceutically acceptable non-aqueous liquids or solvent, emulsifying agent, and preservative.

Suspensions may include a pharmaceutically acceptable suspending agent and preservative. Aqueous alcoholicsolutions may include a pharmaceutically acceptable acetal, such as a di(lower alkyl) acetal of a lower alkyl aldehyde (the term “lower” means an alkyl having between 1 and 6 carbon atoms), e. , acetaldehyde diethyl acetal; and a water-miscible solvent having one or more hydroxyl groups, such as propylene glycol and ethanol. Elixirs are clear, sweetened, and hydroalcoholic solutions. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may also contain a preservative. For a liquid dosage form, for example, a solution in a polyethylene glycol may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g. , water, to be measured conveniently for administration.

Other useful liquid and semisolid dosage forms include, but are not limited to, those containing the active ingredient(s) disclosed herein, and a dialkylated mono- or poly - alkylene glycol.

The pharmaceutical compositions disclosed herein for oral administration may be also formulated in the forms of liposomes, micelles, microspheres, or nano systems. Micellar dosage forms can be prepared as described in U.S. Pat. No. 6,350,458.

The pharmaceutical compositions disclosed herein may be formulated as non- effervescent or effervescent, granules and powders, to be reconstituted into a liquid dosage form. Pharmaceutically acceptable carriers and excipients used in the non- effervescent granules or powders may include diluents, sweeteners, and wetting agents.

Pharmaceutically acceptable carriers and excipients used in the effervescent granules or powders may include organic acids and a source of carbon dioxide.

Coloring and flavoring agents can be used in all of the above dosage forms.

The pharmaceutical compositions disclosed herein can be formulated as an oral nutritional composition. An oral nutritional composition can contain sources of protein, lipids and/or digestible carbohydrates can be in solid, powdered, or liquid forms. The composition can be designed to be the sole source of nutrition or a nutritional supplement. Suitable protein sourcesinclude intact, hydrolyzed, and partially hydrolyzed protein, which can be derived from any suitable source such as milk (e.g, easin, whey), animal (e.g, meat, fish), cereal (e.g, rice, corn), and vegetable (e.g, soy, potato, pea), insect (e.g, locust) and combinations of these sources. Examples of the source of protein include whey protein concentrates, whey protein isolates, whey protein hydrolysates, acid.

The pharmaceutical compositions disclosed herein may be formulated as immediate or modified release dosage forms, including delayed-, sustained, pulsed-, controlled, targeted-, and programmed- release forms.

The pharmaceutical compositions disclosed herein may be co-formulated with other active ingredients which do not impair the desired therapeutic action, or with substances that supplement the desired action.

The pharmaceutical compositions disclosed herein may be administered parenterally by injection, infusion, or implantation, for local or systemic administration. Parenteral administration, as used herein, include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrastemal, intracranial, intramuscular, intrasynovial, and subcutaneous administration.

The pharmaceutical compositions disclosed herein may be formulated in any dosage forms that are suitable for parenteral administration, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and solid forms suitable for solutions or suspensions in liquid prior to inj ection. Such dosage forms can be prepared according to conventional methods known to those skilled in the art of pharmaceutical science (see, Remington: The Science and Practice of Pharmacy, supra).

The pharmaceutical compositions intended for parenteral administration may include one or more pharmaceutically acceptable carriers and excipients, including, but not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, cryoprotectants, lyoprotectants, thickening agents, pH adjusting agents, and inert gases.

The pharmaceutical compositions disclosed herein may be formulated for single or multiple dosage administration. The single dosage formulations are packaged in an ampule, a vial, or a syringe. The multiple dosage parenteral formulations must contain an antimicrobial agent at bacteriostatic or fungistatic concentrations. All parenteral formulations must be sterile, as known and practiced in the art.

The pharmaceutical compositions may be formulated as a suspension, solid, semisolid, or thixotropic liquid, for administration as an implanted depot. In one embodiment, the pharmaceutical compositions disclosed herein are dispersed in a solid inner matrix, which is surrounded by an outer polymeric membrane that is insoluble in body fluids but allows the active ingredient in the pharmaceutical compositions diffuse through.

The pharmaceutical compositions disclosed herein may be administered topically to the skin, orifices, or mucosa. The topical administration, as used herein, include (intra)dermal, conjunctival, intracorneal, intraocular, ophthalmic, auricular, transdermal, nasal, vaginal, ureteral, respiratory, and rectal administration.

The pharmaceutical compositions disclosed herein may be formulated in any dosage forms that are suitable for topical administration for local or systemic effect, including emulsions, solutions, suspensions, creams, gels, hydrogels, ointments, dusting powders, dressings, elixirs, lotions, suspensions, tinctures, pastes, foams, films, aerosols, irrigations, sprays, suppositories, bandages, dermal patches. The topical formulation of the pharmaceutical compositions disclosed herein may also comprise liposomes, micelles, microspheres, nanosystems, and mixtures thereof.

Pharmaceutically acceptable carriersand excipients suitable for use in the topical formulations disclosed herein include, but are not limited to, aqueous vehicles, water- miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, penetration enhancers, cryoprotectants, lyoprotectants, thickening agents, and inert gases.

The pharmaceutical compositions disclosed herein may be administered intranasally or by inhalation to the respiratory tract. The pharmaceutical compositions may be formulated in the form of an aerosol or solution for delivery using a pressurized container, pump, spray, atomizer, such as an atomizer using electrohydrodynamics to produce a fine mist, or nebulizer, alone or in combination with a suitable propellant, such as 1,1, 1,2- tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. The pharmaceutical compositions may also be formulated as a dry powder for insufflation, alone or in combination with an inert carrier such as lactose or phospholipids; and nasal drops. For intranasal use, the powder may comprise a bioadhesive agent, including chitosan or cyclodextrin.

The pharmaceutical compositions disclosed herein may be formulated as a modified release dosage form. As used herein, the term “modified release” refers to a dosage form in which the rate or place of release of the active ingredient(s) is different from that of an immediate dosage form when administered by the same route.

Modified release dosage forms include delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-programmed-release, and gastric retention dosage forms. The pharmaceutical compositions in modified release dosage forms can be prepared using a variety of modified release devices and methods known to those skilled in the art, including, but not limited to, matrix controlled release devices, osmotic controlled release devices, multiparticulate controlled release devices, ion- exchange resins, enteric coatings, multilayered coatings, microspheres, liposomes, and combinations thereof. The release rate of the active ingredient(s) can also be modified by varying the particle sizes and polymorphism of the active ingredient(s).

The pharmaceutical compositions disclosed herein in a modified release dosage form may be prepared by methods known to those skilled in the art, including direct compression, dry or wet granulation followed by compression, melt-granulation followed by compression.

Generally, the amount of an oligosaccharide disclosed herein required to be administered to the person can vary depending upon factors such as the risk and condition severity, the age of the person, the form of the composition, and other medications being administered to the person. It would be expected that an oligosaccharide described herein should be well tolerated irrespective of the age and condition of the subj ect. The dosage of oligosaccharide to be administered can readily be set by a medical practitioner and would generally be in the range from about 10 mg to about 20 g per day, in certain embodiments from about 10 mg to about 15 g per day, from about 100 mg to about 10 g per day, in certain embodiments from about 500 mg to about 10 g per day, in certain embodiments from about 1 g to about 7.5 g per day. An appropriate dose can be determined based on several factors, including, for example, the body weight and/or condition of the patient being treated, the severity of the condition, being treated, other ailments and/or diseases of the person, the incidence and/or severity of side effects and the manner of administration. Appropriate dose ranges can be determined by methods known to those skilled in the art. During an initial treatment phase, the dosing can be higher (for example 200 mg to 20 g per day, preferably 500 mg to 15 g per day, more preferably 1 g to 10 g per day, in certain embodiments 2.5 g to 7.5 g per day). During a maintenance phase, the dosing can be reduced (for example, 10 mg to 10 g per day, preferably 100 mg to 7.5 g per day, more preferably 500 mg to 5 g per day, in certain embodiments 1 g to 2.5 g per day).

Depending on the disorder to be treated and the inj ection in suitable dosage unit with pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.

The dose may be in the form of one, two, three, four, five, six, or more sub-doses that are administered at appropriate intervals per day. The dose or sub-doses can be administered in the form of dosage units containing from about 0.01 to about 2 grams, from about 0.05 to about 1 gram, or from about 10 to about 500 milligrams active ingredient(s) per dosage unit.

In certain embodiments, an appropriate dosage level is about 0.01 to about 5 g/kg patient body weight per day (mg/kg per day), about 0.01 to about 1 g/kg per day, about 0.01 to about .5 g/kg per day, or about 0.1 to about 500 mg/kg per day, which may be administered in single ormultiple doses. A suitable dosage level may be about 0.1 to about 500 mg/kg per day, about 0.1 to about 250 mg/kg per day, or about 0.1 to about 100 mg/kg per day. Within this range the dosage may be about 0.01 to about 0.1, about 0.1 to about 1.0, about 1.0 to about 10, or about 10 to about 100 mg/kg per day.

The oligosaccharides disclosed herein may also be combined or used in combination with other agents useful in the treatment, prevention, or amelioration of one or more symptoms of an autoimmune disorder and/or inflammatory disorder. Or, by way of example only, the therapeutic effectiveness of one of the oligosaccharides described herein may be enhanced by administration of an adjuvant (z.e., by itself the adjuvant may only have minimal therapeutic benefit, but in combination with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced). Additional therapeutic agents can include probiotics, prebiotics, and products administered to improve gastrointestinal health.

Such other agents, adjuvants, or drugs, may be administered, by a route and in an amount commonly used therefore, simultaneously or sequentially with an oligosaccharide as disclosed herein. When an oligosaccharide as disclosed herein is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to an oligosaccharide disclosed herein may be utilized, but is not required. Accordingly, the pharmaceutical compositions disclosed herein include those that also contain one or more other active ingredients or therapeutic agents, in addition to an oligosaccharide disclosed herein.

For use in the therapeutic applications described herein, kits and articles of manufacture are also described herein. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic.

For example, the container(s) can comprise one or more oligosaccharides described herein, optionally in a composition or in combination with another agent as disclosed herein. The container(s) optionally have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits optionally comprise an oligosaccharide with an identifying description or label or instructions relating to its use in the methods described herein.

In certain embodiments, a container consists of 3’SL, 6’SL, 2’FL or a combination thereof.

In other embodiments, the container comprises or consists of 3’SL, 6’SL, 2’FL or a combination thereof at 145 mg/L or greater. In another embodiment, the container comprises a composition that is at least 9% (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%; or any value between any ofthe foregoing) 3 ’ SL, 6’ SL, 2’FL or a combination thereof of the total oligosaccharides in the composition.

A kit will typically comprise one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and/or devices) desirable from a commercial and user standpoint for use of an oligosaccharide described herein. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package insert with instructions for use. A set of instructions will also typically be included.

A label can be on or associated with the container. A label can be on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e. , as a package insert. A label can be used to indicate that the contents are to be used for a specific therapeutic application. The label can also indicate directions for use of the contents, such as in the methods described herein. These other therapeutic agents may be used, for example, in the amounts indicated in the Physicians' Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.

The following examples are intended to illustrate but not limit the disclosure. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used.

EXAMPLES

EXAMPLE 1

A 12 year-old boy, diagnosed with ASD with frequent anxiety and episodes of self harm, was given 40-50 mg/kg per day 3’-sialyllactose (Glycom, food grade, GRAS) dosed orally once a day. His father measured his heart rate before and after treatment and noticed a reduction in heart rate when his son was taking 3’-sialyllactose daily. In addition, his father noted a significant decrease in anxiety and self-harm episodes while taking daily amounts of 3’-sialyllactose.

EXAMPLE 2

Clinical Study Attributes

The ASD diagnosis can be confirmed upon inclusion in the study according to the Diagnostic Statistical Manual (DSM-V) by clinical psychologists using the ADOS test at baseline. Inclusion criteria can include that participants have ASD and be within 3-5 years of age at the start and during the trial period.

Exclusion criteria can include: (i) allergies to milk, rice or nuts; (ii) major medical problems, including cardiac, liver, endocrine or renal disease; (iii) history of seizure disorder or gross neurological deficit; (iv) concomitant treatment with psychiatric medication; (v) current diet supplementation with N-acetylcysteine, alpha lipoic acid, whey protein or higher than regular multivitamin doses of vitamin B 12 or folic acid; or (vi) acute illness. Subjects in both groups can continue taking multi-vitamins, probiotics and other medications/supplements.

All behavioral assessments can be conducted by clinical psychologists. Behavioral assessments can be scheduled to avoid overwhelming the child with excessive testing and to minimize evaluation errors. At the end of this visit, children were randomized to either placebo or intervention. Parents were given a diary to measure adverse effects and/or unusual events.

A visit is scheduled at week 8 to collect the remaining unused products, parents' diaries, and to provide new product for next period. Compliance and adverse events can be recorded. The behavioral tests can be repeated by the same individuals in the same sequence as baseline. A follow-up assessment can be conducted at week 12.

All primary and secondary outcomes can be obtained at baseline and study end. Diaries given to parents and designed to collect any side effect and/or unusual events, can be collected. All study staff, participants, and parents/legal guardians can be blinded to treatment allocation.

Behavioral analysis in areas of autism behavior and severity, communication, developmental status and behavioral problems can be conducted throughout the study by trained assessors. Behavioral issues can be measured by the Child Behavior Checklist 11/2-5 LDS (CBCL). The CBCL is an instrument used to rate a child's problem behaviors and competencies (Achenbach 1991; Achenbach and Rescorla 2000) it was completed by the child's caregiver.

It will be understood that various modifications may be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are within the scope of the following claims. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference. The relevant teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.