(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/054475 Al 7 April 2016 (07.04.2016) P O P C T

(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every A01N 47/46 (2006.01) A61P 25/08 (2006.01) kind of national protection available): AE, AG, AL, AM, A61K 31/26 (2006.01) A61P 25/28 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (21) International Application Number: DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/US2015/053657 HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (22) International Filing Date: KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, 2 October 2015 (02. 10.2015) MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (25) Filing Language: English SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (26) Publication Language: English TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 62/059,594 3 October 2014 (03. 10.2014) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (71) Applicant: THE JOHNS HOPKINS UNIVERSITY TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, [US/US]; 3400 N . Charles St., Baltimore, Maryland 21218 TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, (US). DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (72) Inventors: TALALAY, Paul; 55 12 BoxhiU Ln, Baltimore, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Maryland 212 10 (US). ZIMMERMAN, Andrew; 38 GW, KM, ML, MR, NE, SN, TD, TG). Daniels St., Hopedale, Massachusetts 01747 (US). Published: (74) Agent: MCGOWAN, Malcolm; Cermak Nakajima & McGowan LLP, 127 S. Peyton St., Suite 200, Alexandria, — with international search report (Art. 21(3)) Virginia 223 14 (US).

(54) Title: COMPOSITIONS AND METHODS FOR TREATING AUTISM SPECTRUM DISORDERS (57) Abstract: The instant disclosure features, among other Fig. 4 things, compositions and methods for treating an autism spectrum disorder in a human. The compositions comprise an effective amount of: (1) an isothiocyanate (e.g., sulforaphane or a derivative thereof) or (2) a glucosinolate, and optionally, an enzyme, to thereby treat an autism spec trum disorder and/or reduce the severity of at least one symptom of the disorder. Methods for preparing such com positions are also featured.

© weeks weeks v o o COMPOSITIONS AND M ETHODS FOR TREATING AUTISM SPECTRUM DISORDERS

Background

[001] Autism Spectru m Disorder (ASD) encompasses a group of com plex disorders of brain development. These disorders are cha racterized, in varying degrees, by difficulties in socia l interaction, verba l and nonverba l com munication and repetitive behaviors. These disorders include, but are not li mited to, autistic disorder, childhood disintegrative disorder, pervasive developmental disorder-not otherwise specified (PDD-NOS), and Asperger syndrome. ASD affects 1-2% of predomina ntly male individua ls. ASD is an enormous medical and economic problem in the United States and, at present, there is no approved, mechanism-based treatment.

Summary

[002] The disclosure is based, at least in part, on the resu lts from a clinical study demonstrating that patients with moderate t o severe autism exhibited marked improvements in aberra nt behavior and verba l com munication following treatment with sulfora phane. In fact, on subsca le analysis of Clinica l Globa l Impression-I mprovement (CG I-I) scale scores after treatment with sulforapha ne, 46%, 54%, and 42% of su lfora pha ne reci pients were much or very-m uch improved on social interaction, aberra nt behavior, and verba l com munication, respectively, as com pared t o 0%, 9%, and 0%, respectively, for placebo recipients. These studies indicate that com positions com prising an isothiocyanate (e.g., sulforapha ne or derivatives of sulfora pha ne) possessing sulforapha ne activity, are useful for treating autism spectrum disorders in humans.

[003] Accordingly, in one aspect, the disclosure featu res a method for treati ng an autism spectru m disorder in a huma n. The method com prises ad ministering to the huma n an effective amount of an isothiocya nate, such as su lforapha ne or a derivative thereof, to thereby treat the disorder.

[004] In another aspect, the disclosu re featu res a method for treating an autism spectru m disorder in a huma n. The method com prises administering t o the huma n an effective amount of an isothiocya nate (e. g., sulfora phane or a derivative thereof) to thereby treat the disorder, wherei n the huma n has not experienced a seizure within one yea r prior to administering the sulforapha ne or derivative thereof. [005] In yet another aspect, the disclosure featu res a method for treating an autism spectru m disorder in a huma n, which method com prises admi nistering t o the human : (a) an effective amount of an isothiocya nate (e. g., su lfora pha ne or a derivative thereof) t o thereby treat the disorder; and (b) an effective amount of an anti-seizure drug.

[006] In another aspect, the disclosu re featu res a method for treating an autism spectru m disorder in a huma n, the method com prisi ng ad ministeri ng t o the huma n an effective amou nt of an isothiocya nate (e.g., sulfora pha ne or a derivative thereof) t o thereby treat the disorder, wherein the huma n is not concu rrently bei ng treated with a drug associated with increased risk of seizure.

[007] In another aspect, the disclosu re featu res a method for treating an autism spectrum disorder, which method com prises: (i) ad ministeri ng t o the huma n an effective amou nt of an isothiocya nate (e. g., sulfora pha ne or a derivative thereof) t o thereby treat the disorder; and (ii) monitoring the huma n for the occurrence of seizures.

[008] In another aspect, the disclosu re featu res a method for treating an autism spectru m disorder in a huma n, the method com prising administering t o the huma n an effective amou nt of an isothiocya nate (e. g., sulfora pha ne or a derivative thereof) t o thereby treat the disorder, wherei n the isothiocya nate (e. g., the sulfora pha ne or derivative thereof) is t o be ad ministered in conjunction with a reduced ca lorie diet.

[009] In some embodiments of any of the methods descri bed herein, the autism spectru m disorder is autistic disorder, chi ld hood disintegrative disorder, pervasive developmenta l disorder-not otherwise specified (PDD-N OS), or Asperger syndrome.

[0010] In some embodiments of any of the methods described herein, the effective amount of an isothiocya nate (e.g., sulforapha ne or a derivative thereof) can be, e.g., between 25 and 75 µιηοΙ if the huma n weighs 100 pounds or less; between 75 and 125 µιηοΙ if the human weighs between

101 t o 199 pounds; or between 125 and 175 µιηοΙ if the human weighs more tha n 200 pounds.

The effective amount can be administered daily. In some embodiments, the effective amount of an isothiocyanate (e.g., sulfora phane or a derivative thereof) ca n be 50 µιηοΙ, if the human weighs 100 pounds or less; between 100 µιηοΙ if the human weighs between 101-199 pounds; or

150 µιηοΙ if the huma n weighs more than 200 pounds. In some embodiments, the effective amount is 5 t o 15 mg, 15 t o 25 mg, 20 t o 30 mg, or 5 t o 50 mg, e.g., daily. In some embodiments, the effective amou nt (e.g., the effective daily amou nt) is 5 t o 15 mg if the human weighs less tha n 100 pounds; 15 t o 25 mg if the human weighs between 101 and 199 pou nds, or 20 t o 35 mg if the huma n weighs more tha n 200 pounds. In some embodi ments, the effective amount is 4 t o 12.5 mg, 9 t o 25 mg, 12.5 t o 38 mg, or 3 to 45 mg, e.g., daily. In some embodiments, the effective amou nt (e.g., the effective daily amount) is 4 t o 12.5 mg if the huma n weighs less tha n 100 pounds; 9 t o 25 mg if the huma n weighs between 101 and 199 pounds, or 12.5 t o 38 mg if the human weighs more tha n 200 pounds.

[0011] In some embodiments of any of the methods herei n, the effective amou nt is sufficient t o produce at least a 30 (e. g., at least a 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, o r 90) % decrease in tota l Aberrant Behavior Checklist (ABC) and/or Socia l Responsiveness Sca le (SRS) scores (see worki ng exam ples) for a huma n with ASD relative t o the score(s) prior t o treatment with the effective amount of sulfora pha ne or derivative thereof.

[0012] In some embodi ments of any of the methods described herein, the effective amount is administered as one dose. In some embodiments, the amount is administered as more than one

(e. g., at least two, t hree, four, or five or more) dose(s). For exa mple, in some embodi ments, the effective amount of an isothiocyanate (e. g., sulfora phane or a derivative thereof) is administered t o a patient once dai ly as a single dose (e. g., a single ca psule or t ablet com prising between 10 t o

200 µιηοΙ of an isothiocya nate (e.g., su lfora pha ne or a derivative thereof)). In another exa mple, the isothiocyanate (e. g., sulfora phane or derivative thereof) ca n be ad ministered t o a patient as more tha n one dose per day, such that the total amount administered in one day is the effective amount (e. g., two or three capsules or tablets t aken daily).

[0013] In some embodiments of any of the methods descri bed herein, the isothiocya nate (e.g., sulforapha ne o r derivative thereof) is admi nistered t o the huma n for at least fou r (e. g., at least five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, or 18) weeks. In some embodiments of any of the methods described herei n, the isothiocya nate (e.g., sulfora phane o r derivative thereof) is administered t o the human for at least three (e. g., at least four, five, six, seven, eight, nine, 10, 12, 18, 24, 36, 48, o r 60) months. In some embodi ments of any of the methods described herein, the isothiocya nate (e.g., sulfora pha ne or derivative thereof) is t o be ad ministered for the lifetime of the huma n with the autism spectrum disorder.

[0014] In some embodiments of any of the methods described herei n, the human has moderate t o severe autism . In some embodi ments, the huma n has severe autism .

[0015] In some embodiments, any of the methods described herein ca n further com prise determi ning whether the huma n has an autism spectru m disorder. Methods for making such a determi nation are known in the art (e.g., Diagnostic and Statistica l Manual of Menta l Disorders

(DSM ), e .g., DSM-V), descri bed herei n, and exem plified in the working exa mples.

[0016] In some embodi ments of any of the methods described herein, the effective amount is sufficient t o reduce the severity of one or more behavioral sym ptoms of the disorder. In some embodiments of any of the methods described herein, the effective amount is sufficient t o reduce the severity of one or more symptoms of the disorder by at least 10 (e.g., at least 15, 20,

25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80) %. Methods for measuring the efficacy of a treatment for autism are known in the art, described herei n, and exemplified in the working exam ples.

[0017] In some embodiments of any of the methods descri bed herein, the huma n is a male.

[0018] In some embodiments of any of the methods descri bed herein, the huma n is an adolescent or an infant. In some embodi ments of any of the methods descri bed herein, the huma n is between 13 t o 30 years of age. In some embodi ments of any of the methods described herein, at least six months (e.g., at least seven months, at least eight months, at least nine months, at least one yea r, at least 18 months, or at least two yea rs) prior t o ad ministering the sulfora pha ne or derivative thereof, the huma n has not had a seizure (e.g., has not reported experiencing a seizu re). In some embodiments, the human is not overweight. In some embodiments, the huma n is not obese. In some embodi ments, the huma n is not hypertensive.

In some embodi ments, the human is not at an increased risk for ca rdiovascu la r disease.

[0019] In some embodiments, any of the methods described herein can further include monitoring a human for an improvement in one or more symptoms of the disorder.

[0020] In yet another aspect, the disclosure featu res a pha rmaceutica l com position com prisi ng: a pha rmaceutica lly-acceptable ca rrier and an effective amount of an isothiocya nate (e. g., sulforapha ne or a derivative thereof). In some embodi ments, the effective amount is between

25 µιηοΙ and 200 µιηοΙ (e.g., between 50 and 150 µιηοΙ ) of an isothiocya nate (e.g., sulfora pha ne or a derivative thereof).

[0021] In another aspect, the disclosu re featu res a pha rmaceutica l composition com prisi ng an effective dose of an isothiocya nate (e.g., sulforapha ne or a derivative thereof), wherein the effective dose comprises 50 µιηοΙ or more. In some embodi ments, the effective dose is no more tha n 300 (e. g., 275, 250, 225, 200, 175, 150, 125, 100, or 75) µηηοΙ of the active ingredient

(e. g., an isothiocya nate, such as sulfora pha ne or a derivative thereof) .

[0022] In another aspect, the disclosu re featu res a pha rmaceutica l unit dosage form of an isothiocya nate (e. g., sulfora pha ne or a derivative thereof), wherein the dosage form com prises between 10 µιηοΙ and 200 µιηοΙ of an isothiocya nate (e.g., sulfora pha ne or a derivative thereof) . In some embodiments, the dosage form com prises between 25 µιηοΙ and 100 µιηοΙ of an isothiocya nate (e. g., sulfora phane or a derivative thereof). In some embodi ments, the dosage form com prises between 25 µιηοΙ and 75 µιηοΙ of an isothiocya nate (e. g., su lforapha ne or a derivative thereof). In some embodiments, the dosage form comprises between 25 µιηοΙ and 50 µιηοΙ of an isothiocya nate (e.g., sulfora pha ne or a derivative thereof) . In some embodiments, the dosage form com prises between 10 µιηοΙ and 50 µιηοΙ of an isothiocyanate

(e. g., sulfora pha ne or a derivative thereof). In some embodiments, the dosage form comprises between 10 µιηοΙ and 100 µιηοΙ of an isothiocya nate (e.g., sulfora pha ne or a derivative thereof) . In some embodiments, the dosage form com prises between 10 µιηοΙ and 25 µιηοΙ of an isothiocya nate (e. g., sulfora phane or a derivative thereof).

[0023] In another aspect, the disclosu re featu res a food, beverage, medica l food, or a dieta ry supplement comprisi ng between 25 µιηοΙ and 200 µιηοΙ of an isothiocya nate. In another aspect, the disclosure featu res a medical food or a dieta ry supplement com prisi ng an effective dose of an isothiocya nate, wherein the effective dose comprises 50 µιηοΙ . The isothiocya nate ca n be, e .g., sulfora pha ne or a derivative thereof. The medica l foods and/or dieta ry supplements ca n optiona lly incl ude a la bel identifyi ng the products as such and/or providing exempla ry dosages and dosing schedu les for the product. [0024] Additional suita ble isothiocya nate content for the pharmaceutica l com positions, unit dosage forms, food, beverage, medica l foods, dieta ry supplements, and other compositions embraced by the disclosu re are described herein (infra).

[0025] The clinica l results set forth herei n also indicate that su lfora pha ne precursor com pou nds, e.g., glucora pha nin, are also useful for the treatment of an autism spectrum disorder. Accordingly, in another aspect, the disclosure featu res a food or beverage (or medica l food or dieta ry supplement) com position com prisi ng a glucosinolate and, optiona lly, an enzyme

(e. g., an isolated glucosinolate compou nd and an isolated enzyme). The enzyme is ca pable of converting the glucosinolate t o a thiocya nate or an isothiocya nate (e.g., conversion in the gut of the human). The glucosinolate ca n be a sulforapha ne precursor, such as glucora pha nin

(infra), or a glucosi nolate precursor t o a sulforapha ne derivative, such as any of the sulforapha ne derivatives descri bed herein .

[0026] In some embodiments of any of the com positions or methods descri bed herei n, the enzyme ca n be a myrosinase. In some embodiments, the enzyme ca n be a thioglucosidase, a glutathione transferase, an NAD( P)H :qui none reductase, or a glucu ronosyltra nsferase. In some embodiments of any of the compositions or methods descri bed herein, a com position (e. g., a food, beverage, medica l food, or dieta ry su pplement) described herei n ca n contai n more tha n one (e. g., at least two, three, fou r, or five) different enzyme(s) (e.g., different isolated enzymes).

[0027] In some embodiments of any of the compositions or methods described herein, the composition com prises an amount of the glucosinolate and, optiona lly, an enzyme sufficient t o produce in a human each day: between 25 and 75 µιηοΙ of an isothiocyanate if the huma n weighs

100 pounds or less; between 75 and 125 µιηοΙ of an isothiocyanate if the human weighs betweenlOl to 199 pou nds; or between 125 and 175 µιηοΙ of the isothiocyanate if the human weighs more than 200 pou nds. The isothiocyanate can be, e.g., sulforaphane or a derivative thereof.

[0028] In some embodi ments of any of the methods or compositions described herein, the glucosinolate is glucoraphanin . In some embodi ments, the com position com prises a myrosinase. [0029] In some embodiments, a com position (e. g., a food, beverage, medica l food, or dieta ry supplement composition described herein) comprises, or is, a food bar, yogu rt, ice crea m, fruit, fruit puree, or cerea l. The food or beverage com position ca n be or comprise, e.g., a dessert or a confection.

[0030] In some embodiments, a com position (e. g., a food, beverage, medica l food, or dieta ry supplement composition described herein) comprises, or is, a dai ry product, vegeta ble juice, or fruit juice.

[0031] Any of the com positions, e.g., food, beverage, medica l food, or dieta ry supplements described herein ca n be used for treating, preventing, and/or ameliorating one or more sym ptoms of an autism spectrum disorder.

[0032] In yet another aspect, the disclosu re features a method for treating an autism spectrum disorder in a huma n, which method com prises adm inistering t o the human a com position in an amount effective to treat the autism spectru m disorder, wherein the com position com prises a glucosinolate (e. g., an isolated glucosinolate) and, optiona lly, at least one enzyme (e. g., an isolated enzyme) ca pable of converting the glucosinolate t o a thiocyanate or an isothiocya nate.

In some embodiments, the composition can be, e.g., a food, beverage, medica l food, dietary supplement, or other com position described herein. In some embodiments, the enzyme is myrosinase. In some embodi ments, the enzyme (e.g., isolated enzyme) is a thioglucosidase, a glutathione transferase, an NAD( P)H :quinone reductase, or a glucu ronosyltra nsferase. In some embodiments, the com position com prises more tha n one different enzyme.

[0033] In some embodiments of any of the methods or com positions described herei n, the sulforapha ne or derivative thereof is su bsta ntia lly ena ntiopu re, e.g., for the ( ?) enantiomer. In some embodiments, the sulfora phane or derivative thereof is a racemic mixture of both the ( ?) and (5) enantiomers. In some embodiments, the com position is an extract from a cruciferous pla nt (e. g., broccoli, broccoli sprouts, ca uliflower, or ka le) that is, optiona lly, enriched for sulforapha ne.

[0034] In some embodiments, the pha rmaceutica l com position, unit dosage forms, medica l foods, or dieta ry supplements described herein can be a ca psule or tablet com prises between

100 to 500 mg of cruciferous pla nt (or pla nt seed or sprout) extract, e.g., an extract from broccoli sprouts seeds, such as BroccoSprouts™ seeds. In some embodi ments, the pha rmaceutica l com position or unit dosage form com prises about 250 ± 50 mg (e.g., approxi mately 232 mg) of cruciferous plant (or pla nt seed or sprout) extract, e.g., an extract from broccoli sprouts seeds, such as BroccoSprouts™ seeds.

[0035] The isothiocya nate com pou nd ca n be, e.g., any of those described herei n.

[0036] In some embodi ments of any of the methods, com positions, or dosage forms descri bed herein, the sulforaphane derivative has any one of the structura l form ulas ( II) to (VI I) shown below.

[0037] In some embodiments of any of the methods, com positions, or dosage forms described herein, the sulfora pha ne derivative has at least 10 (e. g., at least 15, 20, 25, 30, 35, 40, 45, 50,

55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99, 100, or more) % of the in vitro or in vivo biologica l activity of an eq uiva lent mola r amount of sulforapha ne t o induce a protective response in cel ls

(e. g., norma l huma n fibroblasts or murine liver cel ls), e.g., promote expression of stress responsive genes.

[0038] In some embodiments, any of the compositions, pha rmaceutica l com positions and dosage forms descri bed herein are for use in treating an autism spectru m disorder, e.g., autism, such as moderate t o severe autism .

[0039] Unless otherwise defined, all technica l and scientific terms used herein have the sa me mea ning as com mon ly understood by one of ordina ry ski ll in the art t o which this disclosure pertains. Preferred methods and materia ls are descri bed below, although methods and materia ls si mila r or eq uiva lent t o those descri bed herei n ca n also be used in the practice or testing of the presently disclosed methods and com positions. All publications, patent applications, patents, and other references mentioned herei n are incorporated by reference in thei r enti rety.

[0040] Other featu res and adva ntages of the present disclosure, e.g., methods for treating an autism spectru m disorder in a huma n, wil l be appa rent from the following descri ption, the exa mples, and from the claims. Brief Description of the Drawings

[0041] Fig. 1 is a table providing the baseline physiological characteristics of patients in the study.

[0042] Fig. 2 depicts the schedule used for a study of the effects of sulforaphane in ASD.

[0043] Figs. 3A-3F are a series of graphs depicting the changes in total ABC (Aberrant Behavior

Checklist) and SRS (Social Responsiveness Scale) scores of patients during the clinical study. Forty male ASD participants who were treated daily with either placebo (initially n = 14) or sulforaphane (initially n = 26) for 4, 10, and 18 weeks, followed by a terminal 4-week untreated period (22 weeks). Fig. 3A (ABC) and 3D (SRS) show all observations. Means of changes in raw, unadjusted total scores (±S.E.M.) at 4, 10, 18, and 22 weeks are shown in Fig. 3B for ABC and 3 E for SRS. Reductions in ABC score upon sulforaphane treatment were -20.2% (P =0.035), -31.5%

(P = 0.002) and -33.6% (P < 0.001), at 4, 10, and 18 weeks, respectively. The corresponding changes in SRS were -12.2% (P = 0.29), -12.2% (P = 0.080) and -16.8% (P = 0.017). Fig. 3C (ABC) and 3 F (SRS) show the changes in total scores at all time-points for placebo- and sulforaphane- treated participants. All changes were calculated from the initial values for each individual participant at time 0 (the means of the two values obtained at screening and at enrollment).

[0044] Fig. 4 is a series of line graphs depicting the changes in Aberrant Behavior Checklist

(ABC) subscores for irritability, lethargy, stereotypy, and hyperactivity. After 4, 10, and 18 weeks of treatment with sulforaphane o r placebo, and a 4-week untreated recovery period (22 weeks). Raw, unadjusted mean values of changes (± S.E.M.) for sulforaphane- and placebo- treated participants are shown. Changes were significant at the 95% confidence level (*) for both irritability and lethargy at 10 and 18 weeks of treatment.

[0045] Figs. 5A and 5 B are line graphs depicting total scores for (Fig. 5A) Aberrant Behavior

Checklist (ABC) and (Fig. 5B) Social Responsiveness Scale (SRS) of individual placebo- and sulforaphane-treated participants at baseline and after 18 weeks. At 18 weeks, total ABC scores were available for 35 (10 placebo and 25 sulforaphane), and total SRS scores for 37 (11 placebo and 26 sulforaphane). Only the differences for sulforaphane treatment were significant at 18 weeks, thus a change in score of from 62.4 t o 45.0 on the ABC scale (Fig. 5A) was significant (P <

0.001), and a change in score of from 121.5 t o 105.2 on the SRS scale (Fig. 5B) was significant (P < 0.001). Means for the subjects shown, at 1 and 18 weeks respectively, for placebo treatment, were 62.4 and 62.6 on the ABC scale, and 121.5 and 117.5 on the SRS scale.

[0046] Figs. 6A and 6 B are each tables depicting the effect of sulforaphane treatment on total scores and changes in total scores. Fig. 6A provides the ABC (Aberrant Behavior Checklist) and

SRS (Social Responsiveness Scale) total scores of participants who completed at least one post- intervention measurement (n=40). The ABC and SRS scores and changes thereof from baseline are the raw, unadjusted values while the P-values are from the linear mixed model adjusting for repeated measures. Fig. 6B provides the CGI-I (Clinical Global Impression-Improvement) scores at 18 weeks for the 37 subjects for whom scores were available.

[0047] Fig. 7 is a flow diagram of progress through phases of the study.

[0048] Fig. 8 is a table providing the summary of adverse events reported and laboratory results obtained from the patient visit at 18 weeks.

[0049] Fig. 9 is a table providing the complete laboratory studies (including hematology, chemistry, and urinalysis) for the patients in the study.

[0050] Fig. 10 is a series of chemical structures of exemplary sulforaphane derivative compounds.

Detailed Description

[0051] The disclosure features, among other things, compositions and methods for treating an autism spectrum disorder in a human. While in no way intended t o be limiting, exemplary compositions, pharmaceutical compositions, dosage forms, dosage regimens, and methods for treatment are set forth below.

Glucosinolates

[0052] Glucosinolates refer t o a class of compounds that contain both sulfur and nitrogen and are derived from glucose and an amino acid. These compounds, naturally found in a variety of cruciferous plants, are converted t o isothiocyanates by enzymes, such as thioglucosides, for example, myrosinase. Generally, in plant cells, myrosinase and glucosinolates are separated in the cell and if the cell is damaged, such as by insect predation, with loss of com partmenta lization, myrosinase or other simila rly acting enzymes comes into contact with glucosi nolates, which are then converted t o isothiocya nates. Myrosinase (EC 3.2. 1.147, CAS®

Registry Number: 9025-38-1) is known t o those of ski ll in the art, as are simila rly acting enzymes that convert precursor molecu les, such as glucosinolates, t o more active com pounds, such as isothiocya nates, e.g., sulfora pha ne. It is understood that glucosinolates can be either natura lly- occurring com pounds (e. g., those prod uced by pla nts) or synthetic (e. g., analogs or derivatives of glucosinolates found in pla nts), or a mixture of natu ral and synthetic compou nds.

[0053] The disclosu re also features com positions com prisi ng a glucosi nolate and one or more enzymes, and/or one or more types of enzymes, and optional ly co-factors or other enzymes in the meta bolic pathway, for the treatment of autism and autism spectrum disorders. Such enzymes ca n be, but are not limited to, myrosinase, thioglucosidases, glutathione tra nsferases,

NAD( P)H :qui none reductase (Q.R) and glucu ronosyltra nsferases, which have si mila r activities or are in related pathways. For exa mple, as known in the art, in the presence of water, myrosi nase cleaves the glucose group from a glucosinolate. The remaining molecule then converts t o a thiocya nate, an isothiocya nate or a nitri le; these are the active substa nces that serve as defense for the pla nt. Thus, com positions featuring a glucosinolate (e. g., an isolated glucosi nolate, such as glucorapha nin or a derivative thereof) and an enzyme (e. g., an isolated enzyme, such as myrosinase) allow for the conversion of the glucosinolate t o an isothiocya nate

(e. g., sulfora pha ne or a derivative thereof) in vivo. The com positions ca n be form ulated t o contain an amou nt of the glucosi nolate and, optiona lly, an enzyme such that an effective amount of an isothiocyanate (e. g., su lfora pha ne or a derivative thereof) is prod uced in the huma n who has consu med the com position or t o whom the com position was ad ministered .

Exem pla ry effective amou nts of sulforapha ne and/or derivatives thereof are described herei n.

Isothiocyanates

[0054] An isothiocya nate (ITC) refers t o a phytochemica l com pou nd com prisi ng an -N=C=S functiona l group. These com pou nds have been associated with a num ber of positive properties, incl uding, e.g., inhibiting ca ncer development (e.g., anti-oxida nt properties) as well as having anti-i nflam matory and anti-microbia l properties. Brown and Ha mpton (2011) Biochim Biophys Acta 1810(9) :888-894. As discussed above, isothiocyanates exist as precursor molecules, glucosi nolates, in cruciferous vegeta bles and chemica lly generated, e.g., by myrosinase following damage t o pla nt tissue (such as chewi ng or cutting) or by the action of intestina l bacteria . Singh and Singh (2012) Carcinogenesis 33(10) : 1833-1842. For exa mple, the glucosi nolate precursor of phenethyl isothiocya nate (PEITC) is gluconasturtiin [e.g., in watercress). Su lfora pha ne, by contrast, is derived from glucora pha nin (4-methylsulfinylbutyl glucosi nolate; 1-thio-, l-(5-(methylsulfinyl)-N-(sulfoxy)penta nimidate)), which is found in cruciferous vegeta bles.

[0055] Exem pla ry isothiocyanates include, e.g., PEITC, benzyl-ITC (BITC), and sulforaphane.

Additiona l exem pla ry isothiocyanates include, without limitation, 1-adama ntyl isothiocyanate; 1- naphthyl isothiocya nate; 2,4,6-trimethylphenylisothiocya nate; 2,4,6-trichlorophenyl isothiocya nate; 2,4-dichlorophenyl isothiocya nate; 2,4-dimethoxyphenyl isothiocya nate; 2,4- xylyl isothiocya nate; 2,5-dichlorophenyl isothiocya nate; 2,5-difluorophenyl isothiocya nate; 2,5- dimethoxyphenyl isothiocya nate; 2,6-difluorophenyl isothiocyanate; 2,6-dimethylphenyl isothiocya nate; 2-(methylthio)phenyl isothiocya nate; 2-(trifluoromethyl) phenyl isothiocya nate;

2-bromophenyl isothiocya nate; 2-chloro-4-nitrophenyl isothiocya nate; 2-chloro-5-

(trifl uoromethyl)phenyl isothiocya nate; 2-chloroethyl isothiocya nate; 2-ch lorophenyl isothiocya nate; 2-ethyl phenyl isothiocya nate; 2-fl uorophenyl isothiocya nate; 2-iodophenyl isothiocya nate; 2-methoxy-4-nitrophenyl isothiocya nate; 2-methoxy-5-methylphenyl isothiocya nate; 2-methoxyphenyl isothiocya nate; 2-na pthyl isothiocya nate; 2-phenethyl isothiocya nate; 2-phenylethyl isothiocya nate; phenethyl isothiocya nate; 3,3,5- tri methylcycohexyl isothiocya nate; 3,4,5-trimethoxyphenyl isothiocya nate; 3,4-dichlorophenyl isothiocya nate; 3,5-bis(trifl uoromethyl)phenyl isothiocya nate; 3,5-di-tert-buryl-4- hydroxyphenyl isothiocya nate; 3,5-dichlorophenyl isothiocya nate; 3-(methylthio)propyl isothiocya nate; 3-(trifluoromethyl)phenyl isothiocya nate; 3-bromophenyl isothiocya nate; 3- chlorophenyl isothiocya nate; 3-cya nophenyl isothiocya nate; 3-fl uorophenyl isothiocya nate; 3- methoxyphenyl isothiocya nate; 3-methoxypropyl isothiocya nate; 3-nitrophenyl isothiocya nate;

3-pyridyl isothiocya nate; 4-(methylthio)phenyl isothiocya nate; 4-(trifluoromethyl) phenyl isothiocya nate; 4-bromo-2-ch lorophenyl isothiocya nate; 4-bromophenyl isothiocya nate; 4- chlorophenyl isothiocyanate; 4-cyanophenyl isothiocyanate; 4-dimethylamino-l-naphthyl isothiocyanate; 4-ethylphenyl isothiocyanate; 4-fluorophenyl isothiocyanate; 4-iodophenyl isothiocyanate; 4-isopropylphenyl isothiocyanate; 4-methoxyphenyl isothiocyanate; 4-methyl-

2-nitrophenyl isothiocyanate; 4-methylphenyl isothiocyanate; 4-nitrophenyl isothiocyanate; 5- chloro-2-methylphenyl isothiocyanate; m-tolyl isothiocyanate; o-tolyl isothiocyanate; p-tolyl isothiocyanate; tert-butyl isothiocyanate; acetyl isothiocyanate; benzoyl isothiocyanate; ethyl isothiocyanate; cyclohexyl isothiocyanate; hexyl isothiocyanate; methallyl isothiocyanate; methyl isothiocyanate; pentyl isothiocyanate; and 2,3-dichlorophenyl isothiocyanate (see, e.g.,

U.S. Patent No. 7,105,190, the disclosure of which, as it relates to exemplary isothiocyanate compounds and methods for producing them, is incorporated herein by reference in its entirety). Yet additional exemplary ITC compounds are set forth in U.S. Patent Application

Publication No. 2009/0291989, the disclosure of which, as it relates to the structures of ITC compounds and methods for making the compounds, is incorporated herein by reference in its entirety.

[0056] Sulforaphane (l-isothiocyanato-4-methylsulfinylbutane) is an exemplary widely- consumed isothiocyanate found in cruciferous vegetables, such as cabbage, broccoli, broccoli sprouts, Brussels sprouts, cauliflower, cauliflower sprouts, and water cress. In the plant, sulforaphane is present in bound form as glucoraphanin, a glucosinolate. The compound has been shown to promote expression of genes responsive to stress from oxidation, inflammation,

DNA damage, and radiation (Zhang et al. (1994) Proc Natl Acad Sci USA 91(8):3147-3150; Zhang et al. (1992) Proc Natl Acad Sci USA 89(6):2399-2403). Sulforaphane has the following chemical structure:

(structural formula I)

[0057] Methods for producing or obtaining sulforaphane are known in the art, described herein, and exemplified in the working examples. For example, the compound can be isolated from pla nts in which it is produced . The worki ng exa mples describes isolation of su lfora pha ne from broccoli sprout extract (SF-BSE). See also Egner et al. (2014) Cancer Prev Res 7:813) and

Azizi et al. (2011) J Chinese Chem Soc 58:906-910. Methods for extracting isothiocya nates from glucosi nolate-containi ng pla nts are also described in, e.g., U.S. Patent Application Publication

No. 20060127996.

[0058] In addition, various synthetic methods for producing su lfora pha ne are known in the art.

Sch mid and Ka rrer (1948) Helvetica Chimica Acta 31(6) :1497-1505 . Methods for chemica lly synthesizi ng su lfora pha ne are also descri bed in, e.g., Internationa l Patent Application

Pu blication No. W O 2013/179057 and Chen et al. (2011) Synthesis 24:3991-3996, the methods from which are incorporated herei n by reference in thei r entirety. De Nicola et al. f urther describes a gra m-sca le synthetic method for enantiopu re ?-su lfora phane from Tuscan black ka le seeds. (2014) Molecules 19(6) :6975-6986.

[0059] Isolated isothiocya nates, such as su lfora pha ne, can be sensitive t o oxidation and degradation. Thus, in some embodiments, an isothiocyanate (e. g., sulforaphane or a derivative

(a nalog) thereof) ca n be provided or produced in a sta bilized form . Sta bilized forms of sulforapha ne are descri bed in, e.g., U.S. Patent Application Publication No. 20080176942, the disclosu re of which as it pertains t o making and usi ng stabilized su lfora phane com positions, is incorporated herein by reference in its enti rety. Methods for stabilizing sulfora pha ne ca n involve, e.g., the formation of sulfora pha ne-cyclodextrin com plexes. For exa mple, U.S. Patent

No. 7,879,822 describes a synthetic process for prepa ring sulforapha ne followed by its subsequent sta bilization by the formation of a sulfora pha ne-cyclodextri n com plex. The disclosu re of this patent, as it relates t o the methods for making and prepa ring stabilized sulforapha ne com positions, is incorporated herein by reference in its entirety.

[0060] Derivatives of sulforapha ne are known in the art and described in, e.g., U.S. Patent

Application Publication No. 20130123203, e.g., at pages 4 and 5, the disclosu re of which is incorporated herein by reference in its entirety. Derivatives of su lfora pha ne are also described in, e.g., Hu et al. (2013 ) Eur J Med Chem 64:529-539 and Kiel basinski et al. (2014) Eur J Med

Chem 76:332-342, the chemica l structures from which, as well as methods for synthesizing such derivatives of sulforapha ne, are incorporated herei n by reference in their entirety. Derivatives (a nalogs) of su lfora pha ne are further descri bed in, e.g., U.S. Patent Appl ication Publication No.

20130142739 and Zha ng and Tang (2007) Acta Pharmacol Sin 28(9) :1343-1354 (in particular,

Table 1 titled "SF analogs: relation of structure t o ind ucer activity" ), the disclosu res of each of which are incorporated by reference in their entirety.

[0061] Exem pla ry derivatives of sulforapha ne incl ude any one of the followi ng com pounds:

(structu ral form ulas II, III, IV, V, VI, and V II, respectively).

[0062] In some embodiments, the derivative of su lfora pha ne is one of the com pou nds depicted in paragra ph [0127] of U.S. Patent Application Publication No. 2013/0123203.

[0063] Suita ble derivatives for use in the methods and com positions described herein ca n also be fou nd in, e.g., U.S. Patent No. 5,411,986, the disclosu re of which (pa rticu la rly Table 3 incl usive of the structures) is incorporated herein by reference in its enti rety. In some embodiments, the derivative com prises or is: 6-isothiocya nato-2-hexa none; exo-2-acetyl-6- isothiocya natonorbornane; exo-2-isothiocya nato-6-methylsu lfonylnorborna ne; 6- isothiocya nato-2-hexa nol; l-isothiocya nato-4-dimethylphosphonylbuta ne; exo-2-(l'- hydroxyethyl)-5-isothiocya natonorbornane; exo-2-acetyl-5-isothiocya natonorborna ne; 1- isothiocya nato-5-methylsulfonylpenta ne; and cis- or tra ns-3-

(methylsulfonyl)cyclohexyl methylisothiocya nate. In some embodi ments, the derivative has one of the structures depicted in Fig. 10.

[0064] In some embodiments, an isothiocyanate (e. g., a sulforapha ne derivative or analog) has at least 15 {e. g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99, 100, or more) % of the activity of an eq uiva lent molar amount of sulfora pha ne t o induce a protective response in cells. For exam ple, Zha ng and Tang (supra) describes a com parison of the biologica l activity of sulforapha ne (both isolated from pla nts and chemica lly synthesized) and severa l analogs to induce the expression by cel ls in vitro of NAD(P)H :q uinone oxidoreductase 1 (NQ.01), a phase 2 enzyme associated with inhi bition of carcinogenesis. U.S. Patent Application

Pu blication No. 20130123203 (supra) describes severa l in vitro studies cha racterizing the ability of sulforapha ne t o ind uce expression of HSP70, HSP40, and HSP90 in norma l human fibroblasts, e.g., at 5 micromola r (see paragraphs 279 and 284). Thus, in some embodiments, an isothiocyanate (e. g., an ana log of sulfora pha ne) for use or inclusion in the compositions and methods described herein has at least 15 (e. g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,

75, 80, 85, 90, 95, 98, 99, 100, or more) % of the activity of an equivalent mola r amount of sulforapha ne t o induce expression (e.g., mRNA or protein expression) by cel ls of one or more of these heat shock proteins or NQ.01 in vitro. Methods for measuring mRNA or protein expression

(e. g., qua ntitative reverse transcription-polymerase chain reaction, Northern Blot, Western Blot, or enzyme-li nked immunosorbent assay (ELISA)) are known in the art and described in, e.g.,

Sa mbrook et. al., Molecular Cloning: A La boratory Manua l (2nd Ed .), Vol. 1-3, Cold Spri ng Ha rbor

La boratory, Cold Spring Ha rbor, N.Y., 1989. Su lfora phane was also shown t o stim ulate mitochondria l biogenesis in XALD fibroblast cel ls, unfolded protei n response protei ns (e.g.,

ATF4, CHO P, and elongation initiation factor 2 (el F2a) in vitro (see paragra phs 231 t o 243 of

U.S. Patent Application Publication No. 20130123203. Therefore, in some embodiments, an isothiocya nate com pou nd (e. g., a sulfora phane derivative) has at least 15 (e.g., at least 20, 25,

30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99, or 100) % of the activity of sulforapha ne t o ind uce expression by a cell of an unfolded protei n response protei n in vitro.

[0065] Hu et al. (2006) Cancer Lett 243(2) :170-192 describes enhanced expression of heat shock protei ns and 26S proteasome subunits in the liver of mice treated with sulfora pha ne at

90 mg/kg. Accordingly, in some embodi ments, an isothiocya nate com pound (e.g., a sulforapha ne derivative) described herein possesses at least 15 (e. g., at least 20, 25, 30, 35, 40,

45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99, 100, or more) % of the activity of su lfora pha ne t o induce expression of these proteins or other stress responsive genes (protein prod ucts) in the liver of mice, e.g., when administered by the sa me route and at simi la r mola r dosages.

Additiona l methods for com paring the biologica l activity of an isothiocya nate com pound t o sulforapha ne (or a su lfora phane derivative t o sulforapha ne) in vitro and in vivo are known in the art and described in, e.g., U.S. Patent Application Publication No. 20130123203, the disclosu re of such methods is incorporated herein by reference in its enti rety.

[0066] In some embodiments, the su lforapha ne or derivative (a nalog) is a racemate of ( ?) and

(5) enantiomers. In some embodiments, the su lfora pha ne or derivative thereof is substa ntial ly enantiopu re in the ( ?) form. As used herein, substa ntia lly ena ntiopure refers t o a prepa ration of an isothiocya nate (e. g., sulfora phane or a derivative thereof) that is at least 65 (e. g., 66, 67, 68,

69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,

95, 96, 97, 98, 99, or 100) % of one enantiomer, e.g., the (R) form . See, e.g., Khia r et al. (2009) J

Org Chem 74(16) :6002-6009. In some embodi ments, the prepa ration is substa ntia lly enantiopu re for ( ?)-su lfora pha ne (De Nicola et al., supra).

Plant Sources

[0067] As described above, glucosinolates can be isolated from plant sources. Methods for the extraction of natural products as sources for com pounds such as sulforapha ne, include methods for extraction from pla nt sou rces in contrast t o those prod uced by chemica l synthetic methods, such as from plant sou rces such as cruciferous vegeta bles include, but are not limited t o homogenization of the vegetables in cold water, lyophilization, extraction of the resulta nt powder with acetonitrile, filtration and evaporative concentration. See, e.g., U.S.

Patent Application Pu blication No. 20130123203 . Other methods for extraction of com pou nds from pla nts are known in the art and include, for exa mple, extractions of seeds and sprouts t o produce com pou nds of the present invention, such as t aught by U.S. Pat. No. 5,725,895, which is herein incorporated in its enti rety. Known methods for extracti ng natura l prod ucts, particula rly from crucife rous pla nts, com prise extraction methods com prising boi ling wate r extraction of desired com pounds.

[0068] Pla nt sources suita ble for use in the methods and com positions disclosed herein may be any portion of a cruciferous plant, including, but not li mited t o cells, seeds, sprouts, leaves, sta lks, roots, flowers and other plant structu res. Pla nt sou rces include, but are not limited to, plants from the family Cruciferae, such as Brassiceae, and including Brassicinae. For example, the plant source may be Brassica oleracea selected from the group of varieties of acephala

(kale, collards, wild cabbage, curly kale), medullosa (marrowstem kale), ramosa (thousand head kale), alboglabra (Chinese kale), botrytis (cauliflower, sprouting broccoli), costata (Portuguese kale), gemmifera (Brussels sprouts), gongylodes (kohlrabi), italica (broccoli), palmifolia (Jersey kale), sabauda (savoy cabbage), sabellica (collards), and selensia (borecole), among others.

[0069] Useful broccoli cultivars t o be used in the method and compositions disclosed herein are

Saga, DeCicco, Everest, Emerald City, Packman, Corvet, Dandy Early, Emperor, Mariner, Green

Comet, Green Valiant, Arcadia, Calabrese Caravel, Chancellor, Citation, Cruiser, Early Purple

Sprouting Red Arrow, Eureka, Excelsior, Galleon, Ging a, Goliath, Green Duke, Greenbelt, Italian

Sprouting, Late Purple Sprouting, Late Winter Sprouting White Star, Legend, Leprechaun,

Marathon, Mariner, Minaret (Romanesco), Paragon, Patriot, Premium Crop, Rapine (Spring

Raab), Rosalind, Salade (Fall Raab), Samurai, Shogun, Sprinter, Sultan, Taiko, and Trixie.

However, many other broccoli cultivars are suitable.

[0070] Useful cauliflower cultivars t o be used in the method and compositions disclosed herein are Alverda, Amazing, Andes, Burgundy Queen, Candid Charm, Cashmere, Christmas White,

Dominant, Elby, Extra Early Snowball, Fremont, Incline, Milkyway Minuteman, Rushmore, S-207,

Serrano, Sierra Nevada, Siria, Snow Crown, Snow Flake, Snow Grace, Snowbred, Solide, Taipan,

Violet Queen, White Baron, White Bishop, White Contessa, White Corona, White Dove, White

Flash, White Fox, White Knight, White Light, White Queen, White Rock, White Sails, White

Summer, White Top, Yukon. However, many other cauliflower cultivars are suitable.

[0071] Methods for isolating glucosinolates from plant sources are known in the art. See, e.g.,

Devi and Thangam (2010) Adv Biol Res 4(6):309-313 and Bjerg and S0rensen (1987) "Isolation of

Intact Glucosinolates by Column Chromatography and Determination of Purity", Glucosinolates in Rapeseeds: Analytical Aspects, World Crops: Production, Utilization, Description, Volume 13, pages 59-75. In some embodiments, the glucosinolates can be obtained from hot water extracts as described, e.g., U.S. Patent No. 6,242,018. See also Cohen et al. (2000) J Natl Cancer

Inst 92:61-68; Fahey et al. (1997) Proc Natl Acad Sci USA (1997) 94:10367-10372; Talalay et al.

(2003) Adv Enzyme Regul 43:121-134; Shirai et al. (1997) Cancer Res 57:195-198; and Habig et al. (1974) J Biol Chem 249:7130-7139, the disclosu re of each of which, in particu la r as it relates t o isolation or extraction of glucosinolates from pla nt sou rces, is incorporated herei n by reference in its enti rety.

Thera peutic Com positions

Pharmaceutical Compositions

[0072] Also featured herein are pha rmaceutical com positions contai ning an effective amou nt of an isothiocya nate (e. g., sulfora phane or a derivative thereof), such as any of those descri bed herein. Fu rther provided are com positions com prisi ng an effective amount of a glucosinolate

(e. g., a sulforapha ne or sulforapha ne derivative precursor, such as glucora pha nin) and, optiona lly, an enzyme ca pable of converting a glucosinolate t o a thiocya nate or an isothiocyanate (e. g., sulforaphane or a derivative thereof). These com positions can be prepared in a manner well known in the pha rmaceutica l art, and ca n be administered by a variety of routes, dependi ng upon whether loca l or systemic treatment is desired and upon the area to be treated .

[0073] Administration may be topica l (including tra nsderma l, epiderma l, ophtha lmic and to mucous mem bra nes including intra nasa l, vaginal and recta l delivery), pulmona ry (e. g., by inha lation or insufflation of powders or aerosols, including by nebulizer; intratrachea l or intranasa l), ora l, or parentera l. Parenteral ad ministration includes intravenous, intraarteria l, subcutaneous, intra peritonea l, intra muscu la r or injection or infusion; or intracra nia l, e.g., intratheca l or intraventricula r, administration . Pa rentera l admi nistration ca n be in the form of a single bol us dose, or may be, for exa mple, by a contin uous perfusion pum p. Pha rmaceutica l com positions and form ulations for topica l administration may incl ude transderma l patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.

Conventiona l pha rmaceutica l ca rriers, aqueous, powder or oily bases, thickeners and the li ke may be necessa ry or desira ble.

[0074] This disclosu re also provides pha rmaceutica l com positions which contain, as the active ingredient, a com pou nd provided herein or a pharmaceutica lly accepta ble sa lt thereof, in com bination with one or more pha rmaceutica lly accepta ble ca rriers (excipients). In making the com positions provided herei n, the active ingredient is typica lly mixed with an exci pient, dil uted by an exci pient or enclosed withi n such a ca rrier in the form of, for exa mple, a ca psule, sachet, paper, or other contai ner. When the excipient serves as a dil uent, it ca n be a solid, semi -solid, or liquid materia l, which acts as a vehicle, ca rrier or medi um for the active ingredient. Thus, the com positions ca n be in the form of t ablets, pills, powders, lozenges, sachets, cachets, elixi rs, suspensions, emulsions, sol utions, syrups, aerosols (as a solid or in a liquid medium), ointments containi ng, for exa mple, up t o 10% by weight of the active com pound, soft and hard gelati n ca psu les, su ppositories, sterile injecta ble solutions, and sterile packaged powders.

[0075] In prepa ring a form ulation, an active com pou nd ca n be milled t o provide the appropriate particle size prior t o com bining with the other ingredients. If an active com pou nd is substantia lly insoluble, it ca n be milled t o a particle size of less than 200 mesh. If an active com pou nd is substa ntia lly water sol uble, the particle size can be adjusted by milling t o provide a su bsta ntia lly uniform distri bution in the formu lation, e.g. about 40 mesh .

[0076] The com pou nds provided herei n may be mil led using known mil li ng procedu res such as wet mil li ng t o obtain a particle size appropriate for t ablet formation and for other form ulatio n types. Finely divided (na nopa rticu late) prepa rations of the com pounds provided herei n ca n be prepa red by processes known in the art, e.g., see Internationa l Patent Application Pu blication

No. WO 2002/000196. Some exa mples of suita ble exci pients include lactose, dextrose, sucrose, sorbitol, man nitol, sta rches, gum acacia, ca lciu m phosphate, alginates, tragaca nth, gelati n, ca lcium si licate, microcrysta lline cell ulose, polyvi nylpyrrolidone, cellu lose, water, syrup, and methyl cellu lose. The form ulations can additiona lly incl ude : lubricating agents such as t alc, magnesi um stearate, and minera l oil; wetting agents; emulsifying and suspending agents; preservi ng agents such as methyl- and propyl hydroxy-benzoates; sweetening agents; and flavori ng agents. The com positions provided herein ca n be form ulated so as to provide quick, sustai ned or delayed release of the active ingredient after ad ministration t o the patient by employing proced ures known in the art.

[0077] For prepa ring solid compositions such as t ablets, the principal active ingredient is mixed with a pharmaceutica l exci pient to form a solid preform ulation com position conta ining a homogeneous mixture of a com pound provided herein . When referring t o these preform ulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules.

[0078] The tablets or pills provided herein can be coated or otherwise compounded t o provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves t o resist disintegration in the stomach and permit the inner component t o pass intact into the duodenum or t o be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0079] The liquid forms in which the compounds and compositions provided herein can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0080] In some embodiments, the compounds provided herein are formulated for intravenous administration. Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor

ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.

The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorga nisms ca n be achieved by various antibacteria l and antifunga l agents, for exa mple, parabens, chlorobuta nol, phenol, ascorbic acid, thimerosa l, and the like. In many cases, it will be preferable t o include isotonic agents, for exa mple, suga rs, polya lcohols such as mannitol, sorbitol, and/or sodium chloride in the com position. Prolonged absorption of the injectable com positions ca n be brought about by incl uding in the com position an agent that delays absorption, for exa mple, alumi num monostea rate and gelatin .

[0081] Sterile injecta ble solutions ca n be prepa red by incorporating the active com pound in the required amount in an appropriate solvent with one or a com bination of ingredients enumerated above, as required, followed by filter sterilization. Genera lly, dispersions are prepa red by incorporating the active com pound into a sterile vehicle, which contains a basic dispersion medium and the requi red other ingredients from those enumerated above. In the case of sterile powders for the prepa ration of steri le injecta ble solutions, the preferred methods of prepa ration are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-fi ltered solution thereof.

[0082] In one embodi ment, the com pounds provided herein are prepared with ca rriers that will protect the com pounds agai nst rapid elimination from the body, such as a control led release formu lation, incl udi ng impla nts and microenca psu lated delivery systems. Biodegrada ble, biocom patible polymers ca n be used, such as ethylene vinyl acetate, polya nhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid . Such fo rmulations ca n be prepa red using standa rd tech niq ues, or obtained com mercia lly, e.g., from Alza Corporation and

Nova Pha rmaceutica ls, Inc. Liposoma l suspensions (incl udi ng li posomes t argeted t o selected cells with monoclona l anti bodies to cel lula r antigens) ca n also be used as pha rmaceutica lly accepta ble ca rriers. These ca n be prepa red accordi ng t o methods known to those skil led in the art, for exa mple, as descri bed in U.S. Patent No. 4,522,811.

[0083] The com positions ad ministered t o a patient ca n be in the form of pha rmaceutica l com positions descri bed above. These com positions ca n be sterilized by conventional sterilization techniques, or may be steri le filtered. Aqueous sol utions ca n be packaged for use as is, or lyophilized, the lyophilized prepa ration bei ng co mbined with a steri le aqueous ca rrier prior t o administration . The pH of the compound prepa rations typica lly will be between 3 and

11, more prefera bly from 5 t o 9 and most preferably from 7 t o 8. It wil l be understood that use of certai n of the foregoi ng excipients, carriers, or sta bilizers will result in the formation of pha rmaceutica l sa lts.

[0084] The compositions can be formu lated in a unit dosage form, each dosage containing, e .g., from about 5 t o about 300 µιηοΙ, about 25 t o about 200 µιηοΙ, of the active ingredient. The term

"unit dosage forms" refers t o physical ly discrete units suitable as unita ry dosages for human subjects and other mammals, each unit contai ning a predetermined qua ntity of active materia l ca lculated t o produce the desired therapeutic effect, in association with a suita ble pharmaceutica l excipient. In some embodiments, the unit dosage form comprises at least 5 (e. g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 11, 115, 120, 125,

130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240,

250, 260, 270, 280, 290, 300, 310, 320, 3330, 340, 350, 360, 370, 380, 390, or 400 or more µιηοΙ of isothiocyanate (e. g., sulforaphane or a derivative thereof). In some embodiments, the unit dosage form com prises between 5-100 µιηοΙ of an isothiocyanate (e. g., sulforaphane or a derivative thereof). In some embodiments, the unit dosage form com prises between 10-50 µιηοΙ of an isothiocya nate (e. g., sulforaphane or a derivative thereof). In some embodiments, the unit dosage form com prises between 15-50 µιηοΙ of an isothiocyanate (e. g., sulforaphane or a derivative thereof). In some embodiments, the unit dosage form com prises between 25-50 µιηοΙ of an isothiocya nate (e. g., sulforaphane or a derivative thereof) . In some embodiments, the unit dosage form com prises 50 ± 10 µιηοΙ of an isothiocya nate (e. g., sulforaphane or a derivative thereof). In some embodi ments, the unit dosage form com prises 50 ± 25 µιηοΙ of an isothiocyanate (e. g., sulforaphane or a derivative thereof). In some embodi ments, the unit dosage form com prises between 50-150 µιηοΙ of an isothiocyanate (e. g., sulfora pha ne or a derivative thereof). In some embodiments, the unit dosage form com prises between 25-150

µιηοΙ of an isothiocya nate (e. g., sulforaphane or a derivative thereof). In some embodiments, the unit dosage form com prises between 50-200 µιηοΙ of an isothiocya nate (e.g., sulfora pha ne or a derivative thereof). In some embodiments, the unit dosage form is no greater tha n 400 (e. g., no greater tha n 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, or 75) µηηοΙ of an isothiocya nate (e. g., sulfora pha ne or a derivative thereof).

[0085] In some embodiments, the effective daily dose is at least 10 (e. g., at least 15, 20, 25, 30,

35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,

145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270,

280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 or more) µηηοΙ of an isothiocya nate (e. g., sulfora pha ne or a derivative thereof). In some embodi ments, the effective daily dose com prises 50 ± 25 µιηοΙ of an isothiocya nate (e.g., sulfora pha ne or a derivative thereof) . In some embodiments, the effective dai ly dose is between 25 and 75 µιηοΙ if the patient weighs 100 pou nds or less; between 75 and 125 µιηοΙ if the patient weighs between

101 to 199 pounds; or between 125 and 175 µιηοΙ if the patient weighs more tha n 200 pounds.

[0086] In some embodiments, the com positions provided herein contai n from about 10 t o about 50 mg of the active ingredient. One having ordinary skill in the art w ill appreciate that this embodies com positions containi ng about 5 to about 55, about 15 t o about 45, about 25 t o about 50, about 10 t o about 30, about 30 t o about 60, about 20 t o about 70, or about 10 t o about 20 mg of the active ingredient.

[0087] In some embodiments, the su lforapha ne or derivative thereof is substantia lly enantiopu re, e.g., for the ( ?) ena ntiomer. In some embodiments, the su lfora phane or derivative thereof is a racemic mixture of both the ( ?) and (5) ena ntiomers. In some embodi ments, the com position is an extract from a cruciferous pla nt (e. g., broccoli, broccoli sprouts, cau liflower, or ka le) that is, optiona lly, enriched for sulfora pha ne.

[0088] In some embodiments, the pha rmaceutica l com position or unit dosage forms descri bed herein ca n be a ca psule or tablet comprises between 100 t o 500 mg of cruciferous pla nt (or pla nt seed or sprout) extract, e.g., an extract from broccoli sprouts, such as BroccoSprouts™. In some embodiments, the pha rmaceutica l com position or unit dosage form com prises about 250

± 50 mg (e. g., approximately 232 mg) of cruciferous pla nt (or pla nt seed or sprout) extract, e.g., an extract from broccoli sprouts, such as BroccoSprouts™.

[0089] Simila r dosages may be used of the com pounds described herein in the methods and uses provided herein . [0090] The active com pound can be effective over a wide dosage range and is genera lly ad ministered in a pharmaceutica lly effective amount. It wil l be understood, however, that the amount of the compound actua lly admi nistered will usua lly be determined by a physicia n, accordi ng to the relevant circumstances, includi ng the condition to be treated, the chosen route of admi nistration, the actua l com pound admi nistered, the age, weight, and response of the individual patient, the severity of the patient's sym ptoms, and the like.

[0091] The amount of com pou nd or com position administered to a patient wil l vary dependi ng upon what is being administered, the purpose of the ad ministration, such as prophylaxis or therapy, the state of the patient, the manner of admi nistration, and the like. In thera peutic applications, com positions ca n be ad ministered to a patient already suffering from a disease in an amount sufficient to cure or at least partia lly arrest the sym ptoms of the disease and its com plications. Effective doses will depend on the disease condition being treated as wel l as by the judgment of the attendi ng cli nicia n depending upon factors such as the severity of the disease, the age, weight and genera l condition of the patient, and the like.

[0092] The thera peutic dosage of a com pound provided herein ca n vary according to, for exam ple, the particula r use for which the treatment is made, the manner of administration of the compound, the hea lth and condition of the patient, and the judgment of the prescribi ng physician. The proportion or concentration of a compound provided herein in a pharmaceutica l com position ca n vary depending upon a num ber of factors including dosage, chemica l cha racteristics (e. g. , hydrophobicity), and the route of administration. For exa mple, the com pounds provided herein can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the com pound for parentera l administration . The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overal l health status of the particu la r patient, the relative biologica l efficacy of the compound selected, form ulation of the excipient, and its route of administration. Effective doses ca n be extra polated from dose-response curves derived from in vitro or ani mal model test systems.

Food Compositions [0093] In some embodiments, a glucosi nolate (e.g., a precursor of sulforapha ne or sulforapha ne derivative) and/or an isothiocya nate (e. g., su lfora phane or a derivative thereof) may be used in the form of a food (e.g., solid food or drin k (e. g., beverage)), medica l food, or supplement (e. g., nutritiona l supplement or dieta ry supplement). A solid food product refers t o an edible, ingesti ble composition, exa mples of which include, but are not limited to, fruits and vegeta bles. For exa mple, the food com position can com prise an isolated glucosinolate and, optiona lly, an isolated enzyme, wherein the enzyme is ca pable of converting the glucosinolate t o a thiocya nate or an isothiocyanate. The enzyme ca n be any of those descri bed herein (e. g., myrosinase) or otherwise known in the art t o have such functiona l activity.

[0094] As used herein, the term "isolated", e.g., as applied t o a substance (e. g., a glucosinolate, an isothiocyanate, or an enzyme), refers t o a substa nce that has been separated or purified from com ponents (e. g., proteins or other natura lly-occu rring biologica l or organic molecu les) which natura lly accom pany it, e.g., proteins, lipids, ca rbohyd rates, and nucleic acid in plant material .

Typically, a substa nce is isolated when it constitutes at least 60 (e.g., at least 65, 70, 75, 80, 85,

90, 92, 95, 97, or 99) %, by weight, of the tota l amount of materia l in a sam ple. For exa mple, a glucosinolate (e. g., a sulfora pha ne precursor, such as glucoraphanin) can be isolated from a plant in which the glucosinolate is natu rally produced . Li kewise, an isothiocyanate compound ca n be isolated from pla nt materia l. The isolated glucosi nolate (e.g., a sulfora phane precursor, such as glucoraphanin), isothiocya nate, and/or enzyme ca n then be added t o a food, beverage, medica l food, dieta ry supplement, or other product in a specified amount (e.g., an effective amount) for consu mption by, or administration to, a huma n, e.g., for treating an autism spectrum disorder.

[0095] In some embodiments, the food, beverage, medica l food, or dieta ry supplement com position com prises an amount of a glucosinolate (e. g., a su lfora pha ne precu rsor, such as glucora pha nin) and enzyme sufficient t o prod uce in the huma n each day: between 25 and 75

µιηοΙ of an isothiocya nate if the human weighs 100 pounds or less; between 75 and 125 µιηοΙ of an isothiocya nate if the huma n weighs between 101 t o 199 pou nds; or between 125 and 175

µιηοΙ of the isothiocya nate if the huma n weighs more than 200 pou nds. [0096] In some embodi ments, the com position (e. g., food, beverage, medica l food, or dieta ry supplement composition) comprises, or is, a food bar, yogurt, ice crea m, fruit, fruit puree, or cerea l. The com position ca n be a dessert or a confection . Beverage or other liquid com positions ca n contai n a dairy product, fruit juice, or vegeta ble juice.

[0097] As used herei n, the term "medica l food" refers t o a food that is form ulated to be consumed or ad ministered entera lly under the supervision of a physicia n and which is intended for the specific dieta ry management of a disease or condition for which distinctive nutritiona l requi rements, based on recognized scientific princi ples, are established by medica l evaluation

(from section 5(b) of the Orpha n Drug Act, 21 U.S.C. § 360ee(b)(3)). Thus, medica l foods are disti nguished from the broader category of foods for specia l dieta ry use and from foods that make hea lth clai ms by the requi rement that medica l foods be intended t o meet distinctive nutritiona l requirements of a disease or condition, used under medica l supervision, and intended for the specific dieta ry management of a disease or condition . Medica l foods are not just those foods recom mended by a medica l professiona l as part of an overa ll diet t o manage sym ptoms or reduce the risk for a disease or condition . Accordingly, medica l foods do not incl ude all foods fed t o a human (e.g., a huma n with an autism spectru m disorder) .

[0098] Likewise, it is also understood that medica l foods requi re some processing or form ulation for use in a human, rather tha n merely a food used in its natura l state. Medica l foods must be:

(a) a food for ora l or tube feeding; (b) labeled for the dieta ry management of a specific condition

(e. g., an autism spectrum disorder); and (c) intended t o be used under medical supervision (see, e.g., U.S. Food and Drug Administration, Guidance for Industry: Frequently Asked Questions

About Medical Foods, Center for Food Safety and Applied Nutrition, May 2007, Revised August

2013).

[0099] As used herei n, the term "dieta ry supplement" mea ns "a product intended to supplement the diet that bea rs or contains one or more of the followi ng dieta ry ingredients: (A) a vita min; (B) a minera l; (C) an herb or other botanica l; (D) an amino acid; (E) a dieta ry substance for use by man t o supplement the diet by increasing the tota l dieta ry inta ke; or (F) a concentrate, meta bolite, constituent, extract, or com bination of any ingredie nt described in cla use (A), (B), (C), (D), or (E)." 21 U.S.C. § 321(ff). It is understood that a dieta ry supplement is not food in its natu ral state. Dietary supplements ca n also be extracts or concentrates, and may be fou nd in many forms such as t ablets, capsu les, softgels, gelca ps, liquids, or powders.

[00100] Dieta ry supplements and medica l foods ca n, optiona lly, be labeled as such, e.g., as requi red by applica ble laws.

[00101] A food, beverage, dieta ry supplement, or medical food described herei n may contain, e.g., from about 5 to about 300 µιηοΙ , about 25 t o about 200 µιηοΙ , of an active ingredient, such as an isothiocya nate (e. g., sulfora pha ne or a derivative thereof). In some embodiments, the food, beverage, dieta ry su pplement, or medica l food com prises at least 5 (e. g., at least 10, 15,

20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130,

135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250,

260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 or more) µηηοΙ of an isothiocya nate (e. g., sulfora pha ne or a derivative thereof). In some embodi ments, the food, beverage, dieta ry su pplement, or medica l food com prises between 5-100 µιηοΙ of an isothiocya nate (e. g., sulfora pha ne or a derivative thereof). In some embodi ments, the food, beverage, dieta ry su pplement, or medica l food com prises between 10-50 µιηοΙ of an isothiocya nate (e. g., sulfora pha ne or a derivative thereof). In some embodi ments, the food, beverage, dieta ry supplement, or medica l food com prises between 15-50 µιηοΙ of an isothiocya nate (e. g., sulfora pha ne or a derivative thereof). In some embodi ments, the food, beverage, dieta ry su pplement, or medica l food com prises between 25-50 µιηοΙ of an isothiocya nate (e. g., sulfora pha ne or a derivative thereof). In some embodi ments, the food, beverage, dieta ry su pplement, or medica l food com prises 50 ± 10 µιηοΙ of an isothiocya nate

(e. g., sulfora pha ne or a derivative thereof). In some embodiments, the food, beverage, dieta ry supplement, or medica l food comprises 50 ± 25 µιηοΙ of an isothiocya nate (e. g., su lforapha ne or a derivative thereof). In some embodiments, the food, beverage, dietary supplement, or medica l food com prises between 50-150 µιηοΙ of an isothiocyanate (e. g., su lforapha ne or a derivative thereof). In some embodi ments, the food, beverage, dieta ry supplement, or medica l food com prises between 25-150 µιηοΙ of an isothiocya nate (e.g., sulforapha ne or a derivative thereof) . In some embodiments, the food, beverage, dieta ry supplement, or medica l food com prises between 50-200 µιηοΙ of an isothiocyanate (e. g., su lforapha ne or a derivative thereof) . In some embodiments, the food, beverage, dieta ry su pplement, or medica l food com prises no greater tha n 400 (e.g., no greater tha n 350, 325, 300, 275, 250, 225, 200, 175,

150, 125, 100, or 75) µιηοΙ of an isothiocya nate (e. g., su lfora phane or a derivative thereof).

[00102] In some embodiments, the food, beverage, dietary supplement, or medical food is

la beled such that the effective daily dose is at least 10 (e.g., at least 15, 20, 25, 30, 35, 40, 45, 50,

55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155,

160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,

310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 or more) µηηοΙ of an isothiocya nate (e.g.,

sulforapha ne or a derivative thereof). In some embodiments, the effective daily dose

com prises 50 ± 25 µιηοΙ of an isothiocya nate (e.g., sulforapha ne or a derivative thereof). In

some embodiments, the effective daily dose is between 25 and 75 µιηοΙ if the patient weighs

100 pounds or less; between 75 and 125 µιηοΙ if the patient weighs between 101 t o 199

pounds; or between 125 and 175 µιηοΙ if the patient weighs more tha n 200 pounds.

[00103] In some embodi ments, the food, beverage, dieta ry supplement, or medical food

provided herein contains from about 10 t o about 50 mg of the active ingredient. One having

ordina ry ski ll in the art wil l appreciate that this embodies com positions containing about 5 t o

about 55, about 15 t o about 45, about 25 to about 50, about 10 t o about 30, about 30 to about

60, about 20 t o about 70, or about 10 t o about 20 mg of the active ingredient.

[00104] In some embodiments, the sulfora phane or derivative thereof is substa ntia lly

ena ntiopure, e.g., for the ( ?) ena ntiomer. In some embodiments, the sulfora pha ne or derivative

thereof is a racemic mixtu re of both the ( ?) and (5) enantiomers. In some embodiments, the

com position is an extract from a cruciferous plant (e.g., broccoli, broccoli sprouts, ca uliflower,

or ka le) that is, optiona lly, enriched for a glucosinolate (e.g., a glucora phanin, such as

sulforapha ne).

[00105] In some embodi ments, the medica l food or supplement described herein ca n be a

ca psu le or t ablet com prises between 100 t o 500 mg of cruciferous plant (or plant seed or

sprout) extract, e.g., an extract from broccoli sprouts, such as BroccoSprouts™. In some

embodiments, the medica l food or supplement com prises about 250 ± 50 mg (e.g.,

approxi mately 232 mg) of cruciferous plant (or pla nt seed or sprout) extract, e.g., an extract from broccoli sprouts, such as BroccoSprouts™. Methods for making such products are described herein and exemplified in the working exa mples.

Applications

Autism Spectrum Disorders

[00106] Also featured herei n are methods for treating an autism spectru m disorder.

McPartland and Volkma r (2012) Handb Clin Neurol 106:407-418. Autism spectru m disorder

(ASD) and autism are both genera l terms for a group of com plex disorders of brain development. These disorders are cha racterized, in varying degrees, by difficulties in socia l interaction, verba l and nonverba l com munication and repetitive behaviors. These disorders incl ude, but are not li mited to, autistic disorder, chi ld hood disintegrative disorder, pervasive developmenta l disorder-not otherwise specified (PDD-NOS) and Asperger synd rome.

[00107] Classical autism is a highly varia ble neu rodevelopmenta l disorder. It is typica lly diagnosed during infancy or early child hood. Accordi ng to the criteria set out in the DSM - IV

(see working exa mples), diagnosis of autism requires a triad of sym ptoms t o be present: incl uding (i) impai rments in socia l interaction, (ii) impai rments in com munication and (iii) restricted and repetitive interests and behaviors. Other dysfu nctions, such as atypica l eating, are also com mon but are not essentia l for diagnosis. Social impairments include : (i) impairments in the use of multiple nonverba l behaviors (e.g. eye contact) t o regu late socia l interaction; (ii) fai lure t o develop peer relationships appropriate t o developmenta l level; (iii) lack of sponta neous seeki ng t o share enjoyment, interests, or achievements; (iv) lack of socia l or emotiona l reci procity. Com munication impai rments in autism may present in one or more of the fol lowing ways: delay in (or tota l lack of) the development of spoken la nguage; marked impairment in the ability t o initiate or sustai n a conversation; stereotyped and repetitive use of la nguage; and/or a lack of sponta neous make-believe play.

[00108] Asperger synd rome or Asperger Disorder is simila r t o autism, and sha res certai n featu res. Li ke autism, Asperger syndrome is also characte rized by impairment in socia l interaction and this is accom panied by restricted and repetitive interests and behavior. Thus, diagnosis of Asperger syndrome is characterized by the sa me triad of impai rments as autism . However, Aspe rger synd rome is typical ly less severe in sym ptomology tha n cla ssica l autism and Aspe rger's patients may f unction with self-sufficiency and lead re latively norma l lives.

[00109] Childhood disintegrative disorder (CDD), also known as Heller syndrome, is a condition in which chi ldren develop norma lly for at least two yea rs, but then demonstrate a severe loss of socia l, com munication and other ski lls. Chi ldhood disintegrative disorder is very much like autism and both involve norma l development followed by significa nt loss of la nguage, socia l play and motor skil ls. Diagnosis of CDD is dependent on dra matic loss of previously acqui red ski lls in two or more of the fol lowi ng areas: la nguage, socia l skil ls, play, motor skills (such as a d ramatic decline in the ability t o walk, cli mb, grasp, etc.), bowel or bladder control (despite previously being toi let- trai ned). The loss of developmenta l ski lls may be abrupt and t ake place over the cou rse of days t o weeks or may be more grad ua l.

[00110] Pervasive Developmental Disorder - Not Otherwise Specified (PDD-NOS) is an ASD that describes patients exhibiting some, but not all, of the sym ptoms associated with other well defined ASDs. The key criteria for diagnosis of an ASD incl ude difficu lty socia lizing with others, repetitive behaviors, and heightened sensitivities t o certain sti muli. These are all found in the

ASDs descri bed above. However, autism, Asperger syndrome, and chi ldhood disi ntegrative disorder all have other features that enable their specific diagnosis. When specific diagnosis of one of these fou r disorders ca nnot be made, but ASD is appa rent, a diagnosis of PDD-N OS is made. Such a diagnosis may result from symptoms sta rti ng at a later age tha n is applica ble for other conditions in the spectru m.

[00111] ASD ca n be associated with intellectua l disa bility, difficulties in motor coordination and attention and physica l hea lth issues such as sleep and gastrointesti nal disturbances. Some persons with ASD excel in visua l skil ls, music, math and art.

Methods for Treatment

[00112] The compositions described herei n ca n be ad ministered to a subject, e.g., a huma n subject, using a variety of methods that depend, in part, on the route of admi nistration. The route ca n be, e.g., ora l, inhalation, intravenous injection or infusion (IV), subcuta neous injection (SC), intra peritonea l (IP) injection, or intra muscu la r injection (IM ). [00113] The terms "thera peutica lly effective amount" or "thera peutica lly effective dose," or simi la r terms used herein are intended to mea n an amou nt of an agent (e.g., a glucosinolate

(e. g., glucora phanin), sulfora pha ne, or a derivative thereof) that w ill elicit the desired biologica l or medica l response (e. g., an improvement in one or more sym ptoms of an autism spectru m disorder). Toxicity and thera peutic efficacy of such com positions ca n be determi ned by known pha rmaceutica l proced ures in cel l cultures or experimenta l animals. These proced ures ca n be used, e.g., for determining the LD50 (the dose letha l to 50% of the popu lation) and the ED50

(the dose thera peutica lly effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the thera peutic index and it ca n be expressed as the ratio LD50/ED50. A com pou nd that exhi bits a high therapeutic index is preferred . Whi le com positions that exhibit toxic side effects may be used, ca re should be t aken t o design a delivery system that targets such com pounds t o the site of affected tissue and t o minimize potentia l damage t o norma l cells and, thereby, red uce side effects.

[00114] The data obtained from the cell cultu re assays and anima l studies ca n be used in formu lating a range of dosage for use in humans. The dosage of such compounds lies genera lly withi n a range of circu lati ng concentrations of the com pou nds that include the ED50 with little or no toxicity. The dosage may vary withi n this range depending upon the dosage form employed and the route of admi nistration utilized.

[00115] A dose ca n be form ulated in anima l models t o achieve a circu lating plasma concentration range that includes the IC50 (i. e., the concentration of the antibody which achieves a half-maxima l inhibition of sym ptoms) as determined in cell cultu re. Such information ca n be used t o more accurately determine useful doses in huma ns. Levels in plasma may be measu red, for exam ple, by high performa nce liquid chromatography. In some embodiments, e.g., where loca l ad ministration (e. g., t o the eye or a joint) is desired, cel l culture or animal modeling ca n be used to determine a dose req uired t o achieve a therapeutica lly effective concentration within the loca l site.

[00116] In some embodiments, a food, beverage, dieta ry supplement, medical food, or pha rmaceutical composition described herein can be administered or consumed with a mea l. A mea l represents particularly a standa rd meal, namely breakfast, lunch or dinner. Food may increase the absorption of sulfora phane or derivative by the gut. The increase in ora l absorption and systemic bioavaila bility a compound is determined by measuring the plasma concentration of calcitonin achieved after administration of the drug at various interva ls prior t o a mea l and at mea ltime. Typically, the plasma concentration is measured at predetermined periods after the administration of the drug so as to determine the maximum plasma concentration (Cmax) and the tota l amount absorbed as determined by the area under the curve (AUC).

[00117] In some embodi ments, a food, beverage, dieta ry su pplement, medica l food, or pha rmaceutica l com position described herei n is to be ad ministered to a human in conjunction with a low ca lorie or low fat diet.

[00118] In some embodi ments, a food, beverage, dieta ry supplement, medica l food, or pha rmaceutica l com position is t o be adm inistered t o the human in conju nction with one or more other therapies of an autism spectru m disorder. For exa mple, sulfora pha ne or a derivative thereof ca n be ad ministered t o a huma n along with one or more of risperidone; aripiprazole; a selective serotoni n reupta ke inhibitor (SSRI), such as fluoxetine and sertra li ne; naltrexone; ola nza pine; fluvoxa mine; clomipra mine; haloperidol; thiodazi ne; f luphenazi ne; chlorpromazi ne; ola nza pine (Zyprexa ®); ziprasidone (Geodon ®); and methylphenidate . Such medici nes fall under categories of anti-depressa nts, anti-a nxiety drugs, antipsychotics, or inattention/hyperactivity drugs. In some embodi ments, an isothiocya nate (e. g., sulfora phane or a derivative thereof) is t o be adm inistered with an anti-seizu re drug. The anti-seizure drug ca n be, e.g., ca rba mazepine (Tegretol ®), la motrigi ne (La micta l®), topi ramate (Topa max®), or valproic acid (Depa kote ®).

[00119] In some embodi ments, a food, beverage, dieta ry supplement, medica l food, or pha rmaceutica l com position is not t o be administered if a human is bei ng ad ministered concu rrently with (or, e.g., less tha n 30 (e. g., less tha n 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,

18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) day(s) prio r to) ad ministration of a drug associated with increased risk for seizures.

[00120] As used herein, a subject "in need of prevention," "in need of treatment," or "in need thereof," refers t o one, who by the judgment of an appropriate medica l practitioner (e. g., a doctor, a nurse, or a nurse practitioner in the case of huma ns; a veterinaria n in the case of non - huma n mammals), would reasona bly benefit from a given treatment.

[00121] The term "preventi ng" is art-recognized, and when used in relation t o a condition, is wel l understood in the art, and incl udes admi nistration of a com position which reduces the frequency of, or delays the onset of, sym ptoms of a medica l condition in a subject relative t o a subject which does not receive the composition.

[00122] A therapeutic composition described herein (e. g., a food, beverage, dieta ry supplement, medical food, pharmaceutica l com position, or any other composition com prising an isothiocya nate, a glucosinolate, an enzyme, sulforapha ne, or a derivative thereof) ca n replace o r augment a previously or currently administered thera py. For exa mple, upon treati ng with su lfora phane or a derivative thereof, administration of the one or more additional active agents can cease or diminish, e.g., be administered at lower levels. In some embodiments, ad ministration of the previous therapy ca n be maintained . In some embodiments, a previous therapy w ill be maintained until the level of the isothiocya nate (e. g., su lfora pha ne or a derivative thereof) reaches a level sufficient t o provide a therapeutic effect. The two thera pies ca n be ad ministered in com bination .

[00123] Monitoring a subject (e. g., a human patient) for an improvement in an autism spectru m disorder, as defi ned herein, mea ns eva luating the subject for a cha nge in a disease parameter, e.g., an improvement in one or more sym ptoms of a given disorder. Methods for eva luating patients with an autism spectru m disorder, including eva luati ng improvements in thei r sym ptoms fol lowi ng a treatment, are known in the art and exemplified in the working exa mples. For exa mple, a medica l practitioner ca n look for cha nges in Aberra nt Behavior

Checklist (ABC) scores, Socia l Responsiveness Scale (SRS) scores, Clinica l Globa l Impression-

Improvement (CGI-I) scores, o r Clinica l Globa l Impression-Severity (CGI-S) scores from baseli ne t o a predetermined poi nt in time following initiation of treatment. The sym ptoms of autism spectru m disorders are wel l known in the art of medicine. In some embodiments, the eva luation is performed at least one (1) hou r, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks o r more, after an ad ministration. The subject ca n be eva luated in one or more of the following periods: prior t o begi nni ng of treatment; during the treatment; or after one or more elements of the treatment have been admi nistered.

Eva luation ca n include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered . It ca n also incl ude eva luating the need t o add or drop a selected thera peutic moda lity, e.g., adding or dropping any of the treatments for an autism spectrum disorder described herein .

[00124] The following exa mples are intended t o illustrate, not t o li mit, this disclosure.

Exa mples

Exa mple 1. Materia ls and Methods

[00125] Preparation of Sulforaphane-rich Broccoli Sprout Extracts. Su lfora pha ne-rich broccoli sprout extract (SF-BSE) was prepa red by the Cu llman Chemoprotection Center at Joh ns

Hop kins Unive rsity essentia lly as descri bed in Egner et al. (2014) Cancer Prev Res 7:813). In brief, specia lly selected BroccoSprouts™ seeds were surface-disi nfected and grown (sprouted) for three days in a com mercia l sprouting faci lity under controlled light and moistu re conditions. A boiling water extract was prepa red, filtered, cooled and treated with the enzyme myrosinase (fro m dai kon sprouts) in order t o convert precu rsor glucosinolates t o isothiocyanates, and then lyophi lized at a food processing faci lity (Oregon Freeze Dry, Albany,

Oregon) . The lyophilized powder (216 µιηοΙ SF/g powder) was enca psulated into #1 gelca ps by

ALFA Specia lty Pha rmacy (Col umbia, M aryla nd) . Each ca psu le conta ined 50 µιηοΙ SF (232 mg of BSE); placebo ca psules were filled with microcrystal line cel lulose. The powders (bul k and ca psules) were maintai ned at -20^C and repeated ly checked for microbia l conta m inants and

SF titer prior t o conveya nce t o the study site pha rmacy t o be dispensed t o patients.

[00126] Study Protocol . Written informed consent was obtained from all participants involved in the study descri bed herein either directly, or from parents or ca regivers. All participants met criteria for autistic disorder. See American Psychiatric Association (2000) Diagnostic and

Statistica l Man ual of Menta l Disorders (DSM -IV; 4th ed .) (America n Psychiatric Association,

Arlington). Forty-fou r male ASD patients were enrolled. The Autism Diagnostic Observation

Sched ule (ADOS), performed by a trained psychologist/tester (in 43 participa nts) and/or DSM - IV (supra) checklist of sym ptoms performed by a trained physicia n (2 partici pants), were used t o confirm the diagnosis of autism at the screening visit. All partici pants were moderately t o severely autistic o n the CGI-S (Cli nica l Globa l Impression Severity Scale; Choq ue et al. (2014) J

Autism Dev Disord 44(7) :1773-1778), with varied cognitive ca pacity (Fig. 1).

[00127] Eligibility criteria included : (1) male gender, (2) age 13 t o 30, (3) no concu rrent chronic illness, (4) no history of active seizu res within one yea r prior t o sta rting treatment, and (5) norma l liver, rena l, and thyroid functions. Pa rticipa nts continued their regu la r medications, if any, during the study.

[00128] Pa rticipants were assigned t o receive either placebo o r sulfora pha ne according t o com puter-generated random ly-perm uted blocks of three assignments with sulfora phane and placebo treatments allocated in a 2:1 ratio in two strata defi ned by parent-reported history of im provement in behavior duri ng febrile illness. Physicia ns and study staff were blind t o grou p assign ment. Forty-fou r subjects were selected t o provide at least 80% power t o test the prima ry hypothesis for the Socia l Responsiveness Sca le (SRS) usi ng a two-tai led two- sa m ple t - test with a = 0.05 and assumi ng that the true difference in average cha nge in SRS was 15 units with sta ndard deviation of 16 units. This is rough ly twice the average magnitude of natura l cha nge observed over 1 yea r among male chi ldren and adolescents with ASD (Constantino et al. (2009) Dev Psychopathol 21(1) :127-138) .

[00129] The study involved seven visits: (i) screeni ng, (ii) randomization and initiation of treatment, (iii) a visit at 24 hou rs post initiation of treatment, and then follow-u p visits at (iv) 4,

(v) 10, and (vi) 18 weeks after the f irst dose. Treatment was disconti nued after the 18-week visit, and participa nts returned at 22 weeks. Medica l history, physica l exa m including vita l signs, adverse event reporting, and SRS, ABC (Aberrant Behavior Checklist; M arsh burn et al.

(1992) JAutism Dev Disord 22(3) :357-373), and CGI-I (Cli nica l Globa l Impression Im provement;

The OSU Resea rch Unit on Pediatric Psychopha rmacology (2005) OSU Autism Rating Sca le. The

Ohio State University, Colu m bus, Ohio) were performed (Fig. 2). At the 4-, 18-, and 22-week visits, hematology, chemistry, and urinalysis were performed . [00130] All f amilies were contacted after the final partici pant com pleted follow-up and asked for thei r impressions of the study and their chi ld's progress whi le under treatment. They were then informed whether he received su lfora pha ne or placebo.

[00131] Administration of Medication and Protocol Schedule . Ca psules of sulforapha ne-rich broccoli sprout extracts were maintai ned at -20°C, and checked periodica lly microbiologica lly, and for sulfora pha ne titer (Egner et al. (2011) Cancer Prev Res 4(3) :384-395). Indistinguisha ble placebo ca psules contained microcrysta lli ne cel lulose. Sulfora phane or placebo was ad ministered daily for 18 weeks. The participa nts were dosed accordi ng t o body weight: 50

µιηοΙ (1 ca psu le) of sulforaphane for <100 lb, 100 µιηοΙ (2 ca psules) for 101-199 lb, and 150

µιηοΙ (3 ca psu les) for >200 lb. Placebo reci pients received equiva lent numbers of capsu les accordi ng t o thei r weight. Ca psules were dispensed t o partici pants in sea led bottles with instructions t o keep them in a household freezer.

[00132] Behavioral Outcome Measures. The Aberra nt Behavior Checklist (ABC) is a parent- or ca regiver-reported 58-item questionnaire, designed t o assess medication effects; each item is scored on a sca le of increasi ng severity from 0 t o 3 (Marshbu rn et al., supra). ABC also assesses severa l subdomains (irrita bility, letha rgy, stereotypy, and hyperactivity). The Socia l

Responsiveness Sca le (SRS) is a parent- o r ca regiver-reported 65-point socia l com munication q uestionnai re that covers 5 subscales (awa reness, cognition, com munication, motivation, and autistic mannerisms) (Consta nti no et al., supra). Each SRS item is rated o n a sca le of 1 t o 4; the total score was our pri mary efficacy endpoint. The Ohio Autism Cli nica l Globa l Impression

Severity Sca le (CG I-S, also designated OACIS-S, and only measured at screeni ng) (Choq ue et al., supra; OSU Autism Rating Sca le, supra) is a cli nicia n-rated assessment of the severity of autistic behavior (in increasing order of severity from 1 t o 7) and incl udes the fol lowi ng su bdomains: globa l autism severity, socia l interaction, aberrant behavior, repetitive or ritua listic behaviors, verbal and non-verba l com munication, hyperactivity/i nattention, anxiety, sensory sensitivities and restricted/na rrow interests. The Ohio Autism Clinica l Globa l Impressions Improvement

Sca le (CGI-I o r OACIS-I) is a clinician-rated assessment of how much the patient's behavior has cha nged during an intervention . [00133] Statistical Evaluation . Forty-fou r subjects were origi nally enrolled and randomized t o sulforapha ne treatment (n = 29) or placebo (n = 15); 4 subjects discontinued participation in the study before the first (4-week) return visit. Behavior scores for the remaining 40 partici pants, who com pleted at least part of the outcome measure eva luations (14 placebo and

26 sulforapha ne), are described in our prima ry results and shown in Figs. 3(A-F), 4, 5(A and B), and 6 (A and B). To compensate for incidenta l changes in ABC/SRS scores due t o norma l fluctuation, these scores were obtained at both screening and randomization visits, and their averages were used to com pare with subsequent ABC/SRS scores. The prima ry analysis used the differences between scores of individ uals at 4, 10, 18, and 22 weeks from thei r respective average pre-treatment values. The difference between the sulfora pha ne and placebo treatment grou ps in the cha nge in ABC and SRS scores from baseline t o 18 weeks, and their reversion t o baseline at 22 weeks, was the measu re of efficacy.

[00134] Each outcome was modeled in a sha red-baseline mixed effects genera l linea r model with fixed effects for visit and the interaction of post-randomization visit and treatment group and random partici pant-specific intercepts and slopes with unstructu red cova ria nce. The absence of a main effect for treatment (i.e., a "sha red baseline") properly reflects the true state of the popu lation sa mpled prior t o randomization and has the adva ntage of adjusti ng for any cha nce differences at baseli ne in a man ner simila r to ANCOVA (Lia ng and Zeger (2000)

Indian JStat 62(1) :134-148). Linea r contrasts of least-squa re mea ns were used t o esti mate cha nges from baseline betwee n treatment and control groups at each fol low-up visit. Given its assu mptions, the mixed model yields esti mates that are unbiased as long as loss t o fol low-up and missing test scores are predicta ble from observed scores under assum ptions of the model .

An intention-to-treat analysis that included all 44 participa nts led t o simi la r conclusions.

[00135] Statistica l analyses were performed with SAS ® 9.3 softwa re (SAS Institute, Ca ry, North

Ca roli na), and Stata ® v.11.2 (Statacorp, College Station, Texas).

[00136] Safety and Tolerance. Adverse event monitoring and docu mentation by severity, d uration, and relatedness were performed by a physicia n at each follow-up visit. Study drug pause and stop rules were form ulated as follows: In case of any significa nt adverse events, o r if la boratory values were above the study eligi bility rules (i.e., AST o r ALT > 1.5x upper li mit of normal, serum creatinine > 1.2 mg/dL or TSH outside normal limits), study medication was t o be stopped for 2 weeks and subjects re-evaluated. If laboratory studies returned t o normal and no more adverse events were noted, the medication was t o be restarted. Otherwise, the study medication was t o be stopped indefinitely. Study drug safety and adherence t o the protocol were monitored at quarterly meetings of a Data Safety Monitoring Board constituted by 3 members at the Lurie Center for Autism.

[00137] Quality of Data Certification. An on-site FDA Good Clinical Practice Raw Data Audit was performed on May 19-21, 2014. The inspection was performed by Philippe Ourisson, Ph.D.

(Manager, Quality Assurance and Scientific Support), and confirmed by Paul Swidersky

(President), Quality Associates, Inc., 8161 Maple Lawn Boulevard, Fulton, MD. The folder of each of the 44 subjects was reviewed. Every informed consent form was examined. All inclusion/exclusion criteria were verified. Most baseline physicals and all subject evaluations through week 22 were examined, then transcribed to a spreadsheet. No evidence was obtained that there were significant errors in collecting the data and transferring the results to the spreadsheets.

[00138] Statistical Analysis of Outcome Measures and Intention-to-Treat Analysis. The study hypotheses concerned differences between the sulforaphane and placebo treatment groups in the average change in ABC and SRS scores from baseline to 18 weeks, and their reversion t o baseline at 22 weeks. CGI-I scores were examined as a secondary outcome at the same time points. Of the 44 subjects originally enrolled and randomized t o sulforaphane treatment (n =

29) or placebo (n = 15), 4 subjects discontinued participation in the study before the first return visit, 4 weeks after treatment initiation. In the body of the paper, change scores calculated as the change from baseline t o each follow-up time point for each participant were analyzed. The

4 participants who discontinued early were not included in the main analyses.

[00139] An alternative, intention-to-treat (ITT) analysis included all randomized participants.

Each outcome was modeled in a mixed effects general linear model with fixed effects for visit and the interaction of post-randomization visit and treatment group and random participant- specific intercepts and slopes with unstructured covariance. The absence of a main effect for treatment (i.e., a "shared baseline") properly reflects the true state of the population sampled prior t o randomization and has the advantage of adjusting for any chance differences at baseline in a manner similar t o ANCOVA. The model was used to estimate the difference between treatment and control in mean change from baseline at each time point. Given its assumptions, the mixed model yields estimates that are unbiased as long as missing test scores are predictable from observed scores. In this model, the baseline scores (the mean of scores at the screening and randomization visits) of the 4 participants lost t o follow-up were included in the analysis, with all participants analyzed according to their assigned treatment group without regard t o whether they continued treatment. The test statistic used was the difference between the two treatment groups in the average change in SRS from baseline t o 18 weeks.

We used a two-tailed test at a = 0.05. To quantify the change in efficacy after 4 weeks without treatment we estimated the difference in the treatment-placebo comparison at 22 versus 18 weeks, by a simple linear contrast of the estimated model parameters.

[00140] By mixed model analysis of the ITT sample, the sulforaphane group had significantly greater improvement in their overall SRS and ABC scores compared to the placebo group. After

18 weeks of treatment, the sulforaphane group had a reduction in SRS total score of 15.3 units from baseline compared t o a decrease of 3.2 units in the placebo group (difference = 12.1 units, P = 0.01). After subjects stopped the medication, the SRS scores tended t o revert t o the mean (although incompletely), with the average decrease of SRS total score from baseline t o

22 weeks being 6 units in the sulforaphane group and 2.9 units in the placebo group (difference

= 3.1 units, P = 0.70).

[00141] The effect of sulforaphane on ABC total scores was comparable, with a decrease of

17.6 units among participants randomized t o active treatment compared t o a decrease of 0.08 unit in the placebo group (difference = 17.5 units, P = 0.0001). As with SRS scores, ABC total scores also tended to revert t o baseline after stopping treatment, such that the average decrease of total ABC score from baseline t o 22 weeks was 8.8 units in the sulforaphane group, compared to 0.08 unit in the placebo group (difference = 8.9 units, P = 0.28).

[00142] Guided by the power analysis and sample size calculation discussed in the paper

(indicating power t o detect a 15-point change in total SRS score), a positive response t o treatment was defined post hoc as a 30% decrease from baseline on SRS and ABC scores. After conservatively considering the 4 dropouts (1 on placebo and 3 on sulforaphane) as not meeting the 30% reduction threshold, ABC scores at 18 weeks were available for 39 participants (11 placebo and 28 sulforaphane), and SRS scores at 18 weeks were available for 41 participants (12 placebo and 29 sulforaphane). Additionally, 3 participants on sulforaphane inexplicably had >30% reduction in their ABC scores from baseline, whereas they were not improved either on CGI-I

(OACIS-I) or post-study parent interviews. These 3 participants were conservatively considered as not meeting the 30% reduction threshold. With that adjustment, 31% (9/29) participants on sulforaphane had a positive response on SRS compared t o 0% (0/15) participants on placebo

(Fisher exact test, P = 0.018). Similarly, 44.8% (13/29) of participants taking sulforaphane had a positive response on ABC compared t o 13% (2/15) patients on placebo (P = 0.048). Also consistent with the "per-protocol" findings reported in the paper, an ITT analysis suggested that more participants in the sulforaphane compared to the placebo group experienced a positive response on several subdomains of the CGI-lmprovement scale. After 18 weeks of treatment,

41% (12/29) participants taking sulforaphane were "much improved" or "very much improved" on the social interaction subdomain of the CGI-I as compared to 0% (0/15) of participants on placebo (P = 0.003). Forty-eight percent (14/29) of participants on sulforaphane were "much improved" or "very much improved" on the aberrant or abnormal behavior subdomain of the

CGI-I as compared t o 6.7% (1/15) of placebo participants (P = 0.007). Thirty-eight percent

(11/29) participants on sulforaphane were "much improved" or "very much improved" on the verbal communication subdomain of the CGI-I as compared t o 6.7% (1/15) of placebo participants (P = 0.035).

[00143] In conclusion, the intention-to-treat data analysis is presented as the most conservative approach t o evaluation of the data, and results in similar findings that confirm the per-protocol analysis. Moreover, whether those participants who dropped out of the study are included or excluded does not detract from the statistical validity of sulforaphane's effects in this clinical trial of young men with ASD. Example 2. Results of the Study

[00144] Participant Characteristics. More than 90% of all scheduled tests were completed on the 40 participants who received placebo or sulforaphane treatment and returned for the first return visit (week 4). Twenty-two participants (6 placebo, 16 sulforaphane) were also tested at

22 weeks, 4 weeks after treatment ended (Fig. 7). Four participants (1 placebo, 3 sulforaphane) were lost t o follow-up prior t o their first on-treatment visit.

[00145] Participants, all male, were 13-27 years old at enrollment (median: 17 years). A history of behavioral improvements with fever was given by a large majority (32 of 40; 80%) of participants. Participants in sulforaphane and placebo groups were well matched, and did not differ at baseline with respect t o various demographic, behavioral and clinical features, behavioral outcome score measures, abnormalities in physical examination, blood chemistries, hematology, and urinalysis (Fig. 1).

[00146] Analysis of Outcome Measures. The total and the changes in total ABC and SRS behavioral scores of the 26 sulforaphane-treated and 14 placebo recipients from enrollment t o the 18-week end of treatment and after a 4-week recovery period are shown in Figs. 2-6.

Treatment group mean ABC scores differed significantly at 4, 10, and 18 weeks (Figs. 3B and 3 E for ABC and SRS, respectively). At 18 weeks there was a 34% reduction in ABC and a 17% reduction in SRS scores, and these trended toward non-significant differences upon cessation of treatment (Figs. 3B, 3E, 6A, and 6B).

[00147] Significantly greater improvement was observed among participants randomized t o sulforaphane at 4, 10, and 18 weeks for irritability, lethargy, stereotypy and hyperactivity subscales of the ABC, and in awareness, communication, motivation and mannerisms subscales of SRS (Figs. 4, 6A, and 6B). After stopping sulforaphane treatment, both ABC and SRS subscores tended t o revert toward baseline.

[00148] On subscale analysis of CGI-I scale scores at 18 weeks (Figs. 6A and 6B), 46% (12/26),

54% (14/26), and 42% (11/26) of sulforaphane recipients were much or very-much improved on social interaction, aberrant behavior, and verbal communication, respectively, compared t o

0% (0/11; P = 0.007), 9% (1/11; P = 0.014), and 0% (0/11; P = 0.015), respectively, for placebo recipients. [00149] Individual changes in total ABC and SRS scores from basal levels t o 18 weeks are shown in

Figs. 5A and 5B. A positive response was defined post-hoc as a 30% decrease from baseline in total

ABC and SRS scores. Thirty-five percent (9/26) of participants on sulforaphane had a positive response on SRS compared to 0% (0/11) on placebo (Fisher exact test P= 0.036), and 60% (15/25) of participants receiving sulforaphane had a positive response on ABC compared to 20% (2/10) on placebo (P = 0.059).

[00150] Clinical impressions made during the study, while blind t o group assignment, were that

13 of the 40 participants improved noticeably with respect t o sociability and behavior, usually observable by 4 weeks; all were receiving sulforaphane. In queries to families and caregivers, before unblinding, 17 of 26 whose sons had taken sulforaphane reported gradual changes within the first month of treatment and correctly surmised their group assignment, whereas the remaining 9 on sulforaphane, and all but 1 of 14 who received placebo, were not improved, and believed that their sons had not received sulforaphane. Positive responses t o sulforaphane were spontaneously reported by parents and caretakers, who commented (before disclosure of treatment category) on improved social responsiveness, behavioral compliance and calmness in the subjects with ASD who were taking the active compound.

[00151] Safety and Adverse Events. Sulforaphane treatment effectively improved core aberrant behaviors of ASD, and was safe and well-tolerated (Fig. 8). Notably, none of the laboratory results were outside normal ranges at any time point (Fig. 9). Unexpectedly, the sulforaphane group gained significantly more weight over the 18-week period, compared to placebo (4.31 vs.

0.31 lb, P = 0.056). Pulse rate was lower in the sulforaphane group both at baseline and during the study. Thirty-six adverse events were noted during the trial. Vomiting, increased aggressions, abdominal pain, increased flatulence, irritability, constipation, diarrhea, fever, headache and exacerbation of seasonal allergies were reported in 12-19 percent of participants on sulforaphane; their incidence was the same in the placebo groups (P > 0.10).

[00152] Two participants had single unprovoked seizures: one after 3 weeks on sulforaphane, with an undisclosed history of recent seizures; the other 3 weeks after discontinuing treatment and a past (more than 1 year) history of well-controlled seizures with anti-epileptic drugs. Although patients with autism are predisposed t o seizures, we cannot rule out the possibility of seizures as a possible adverse effect of sulforaphane in ASD.

[00153] While the present disclosure has been described with reference t o the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made t o adapt a particular situation, material, composition of matter, process, process step or steps, t o the objective, spirit and scope of the present disclosure. All such modifications are intended t o be within the scope of the disclosure. What is claimed is:

1. A method for treating an autism spectrum disorder in a human, the method comprising administering to the human daily a composition comprising an effective amount of an isothiocyanate, to thereby treat the disorder, wherein the effective amount is: between 25 and

75 µ τ ο Ι if the human weighs 100 pounds or less; between 75 and 125 µ τ ο Ι if the human weighs between 101 to 199 pounds; or between 125 and 175 µ τ ο Ι if the human weighs more than 200 pounds.

2. The method of claim 1, wherein the amount is 50 µ τ ο Ι, if the human weighs 100 pounds or less; 100 µ τ ο Ι if the human weighs between 101-199 pounds; or 150 µ τ ο Ι if the human weighs more than 200 pounds.

3. The method of claim 1 or 2, wherein the amount is administered daily as one dose.

4 . The method of claim 1 or 2, wherein the amount is administered daily as more than one dose.

5 . The method of any one of claims 1 t o 5, wherein the isothiocyanate is administered to the human for at least 18 weeks.

6. The method of any one of claims 1 to 5, wherein the disorder is autism.

7 . The method of claim 6, wherein the human has moderate t o severe autism.

8. The method of any one of claims 1to 7, wherein the amount is effective to reduce the severityof one or more behavioral symptoms of the disorder.

9 . The method of any one of claims 1 to 8, wherein the human has a medical history of behavioral improvements with fever.

10. The method of any one of claims 1 to 9, wherein the isothiocyanate is sulforaphane or a derivative thereof.

11. The method of claim 10, wherein the sulforaphane is enantiopure for the ( ?) enantiomer.

12. The method of any one of claims 1 to 11, wherein the isothiocyanate has been extracted from a cruciferous vegetable or seeds thereof.

13. The method of any one of claims 1 to 9 or 12, wherein the isothiocyanate has any one of the structural formulas (II) t o (Lll). 14. The method of any one of claims 1 to 13, wherein the isothiocyanate has at least 50% of the activity of an equivalent molar amount of sulforaphane to induce expression of a heat shock protein in cells in vitro.

15. The method of any one of claims 1 to 14, wherein the composition is a pharmaceutical composition.

16. The method of any one of claims 1 to 14, wherein the composition is a medical food or a dietary supplement.

17. The method of any one of claims 1 to 14, wherein the composition is a food.

18. A pharmaceutical composition comprising: a pharmaceutically-acceptable carrier and between 50 µιηοΙ and 150 µιηοΙ of an isothiocyanate.

19. A pharmaceutical composition comprising an effective dose of an isothiocyanate, wherein the effective dose comprises 50 µιηοΙ .

20. The pharmaceutical composition according to claim 18 or 19, wherein the isothiocyanate is sulforaphane or a derivative thereof.

21. A pharmaceutical unit dosage form comprising between 25 µιηοΙ and 200 µιηοΙ of an isothiocyanate.

22. The pharmaceutical unit dosage form according to claim 21, wherein the isothiocyanate is sulforaphane or a derivative thereof.

23. A medical food or a dietary supplement comprising between 25 µιηοΙ and 200 µιηοΙ of an isothiocyanate.

24. A medical food or a dietary supplement comprising an effective dose of an

isothiocyanate, wherein the effective dose comprises about 50 µιηοΙ .

25. The medical food or dietary supplement according to claim 23 or 24, wherein the

isothiocyanate is sulforaphane or a derivative thereof.

26. A method for treating an autism spectrum disorder in a human, the method comprising

administering to the human an effective amount of an isothiocyanate to thereby treat the

disorder, wherein the human has not experienced a seizure within one year prior to

administering the isothiocyanate. 27. A method for treating an autism spectrum disorder in a human, the method comprising administering to the human: (a) an effective amount of an isothiocyanate t o thereby treat the disorder; and (b) an effective amount of an anti-seizure drug.

28. A method for treating an autism spectrum disorder in a human, the method comprising administering to the human an effective amount of an isothiocyanate t o thereby treat the disorder, wherein the human is not concurrently being treated with a drug associated with increased risk of seizure.

29. A method for treating an autism spectrum disorder in a human, the method comprising:

(i) administering t o the human an effective amount of an isothiocyanate t o thereby treat the disorder; and

(ii) monitoring the human for the occurrence of seizures.

30. A method for treating an autism spectrum disorder in a human, the method comprising administering to the human an effective amount of an isothiocyanate t o thereby treat the disorder, wherein the isothiocyanate is t o be administered in conjunction with a reduced calorie diet.

31. The method of any one of claims 26 t o 30, wherein the effective amount is: between 25 and 75 µιηοΙ if the human weighs 100 pounds or less; between 75 and 125 µιηοΙ if the human weighs between 101 t o 199 pounds; or between 125 and 175 µιηοΙ if the human weighs more than 200 pounds.

32. The method of claim 31, wherein the effective amount is 50 µιηοΙ, if the human weighs

100 pounds or less; between 100 µιηοΙ if the human weighs between 101-199 pounds; or 150

µιηοΙ if the human weighs more than 200 pounds.

33. The method of any one of claims 26 t o 32, wherein the amount is administered daily as one dose.

34. The method of any one of claims 26 t o 32, wherein the amount is administered daily as more than one dose.

35. The method of any one of claims 26 t o 34, wherein the isothiocyanate is administered t o the human for at least 18 weeks.

36. The method of any one of claims 26 t o 35, wherein the disorder is autistic disorder. 37. The method of claim 36, wherein the human has moderate to severe autism.

38. The method of any one of claims 26 to 37, wherein the amount is effective to reduce the severity of one or more behavioral symptoms of the disorder.

39. The method of any one of claims 26 to 38, wherein the isothiocyanate has been extracted from a cruciferous vegetable or seeds thereof.

40. The method of any one of claims 26 to 39, wherein the isothiocyanate is sulforaphane or a derivative thereof.

41. The method of claim 40, wherein the sulforaphane is enantiopure for the ( ?) enantiomer.

42. The method of any one of claims 26 to 39, wherein the isothiocyanate has any one of the structural formulas (II) to (Lll).

43. The method of any one of claims 26 to 42, wherein the isothiocyanate has at least 50% of the activity of an equivalent amount of sulforaphane to induce expression of a heat shock protein in cells in vitro.

44. The method of claim 27, wherein the anti-seizure drug is selected from the group consisting of carbamazepine, lamotrigine, topiramate, and valproic acid.

45. A method for treating an autism spectrum disorder in a human, the method comprising administering to the human a composition in an amount effective to treat the autism spectrum disorder, wherein the composition comprises an isolated glucosinolate and at least one isolated enzyme, and wherein the enzyme is capable of converting the glucosinolate to a thiocyanate or an isothiocyanate.

46. The method of claim 45, wherein the composition is a food or a beverage.

47. The method of claim 45, wherein the composition is a medical food.

48. The method of claim 45, wherein the composition is a dietary supplement.

49. The method of any one of claims 45 to 48, wherein the enzyme is myrosinase.

50. The method of any one of claims 45 to 49, wherein the enzyme is a thioglucosidase, a glutathione transferase, an NAD(P)H:quinone reductase, or a glucuronosyltransferase.

51. The method of any one of claims 45 to 50, wherein the composition comprises more than one different enzyme. 52. The method of any one of claims 45 t o 51, wherein the composition comprises an amount of the glucosinolate and enzyme sufficient to produce in the human each day: between 25 and 75 µιηοΙ of an isothiocyanate if the human weighs 100 pounds or less; between 75 and 125 µιηοΙ of an isothiocyanate if the human weighs between 101 t o 199 pounds; or between 125 and 175 µιηοΙ of the isothiocyanate if the human weighs more than

200 pounds.

53. The method of claim 52, wherein the isothiocyanate is sulforaphane or a derivative thereof.

54. A food or beverage composition comprising an isolated glucosinolate and an isolated enzyme, wherein the enzyme is capable of converting the glucosinolate t o a thiocyanate or an isothiocyanate.

55. The food or beverage composition of any one of claims 45 t o 48, wherein the enzyme is myrosinase.

56. The food or beverage composition of any one of claims 45 t o 49, wherein the enzyme is a thioglucosidase, a glutathione transferase, an NAD(P)H:quinone reductase, or a glucuronosyltransferase.

57. The food or beverage composition of any one of claims 54 t o 56, wherein the composition comprises more than one different enzyme.

58. The food or beverage composition of any one of claims 54 t o 56, wherein the composition comprises an amount of the glucosinolate and enzyme sufficient t o produce in the human each day: between 25 and 75 µιηοΙ of an isothiocyanate if the human weighs 100 pounds or less; between 75 and 125 µιηοΙ of an isothiocyanate if the human weighs between

101 to 199 pounds; or between 125 and 175 µιηοΙ of the isothiocyanate if the human weighs more than 200 pounds.

59. The food or beverage composition of claim 52, wherein the isothiocyanate is sulforaphane or a derivative thereof.

60. The food or beverage composition of any one of claims 54 t o 59, wherein the food comprises a food bar, yogurt, ice cream, fruit, fruit puree, or cereal. 61. The food or beverage composition of any one of claims 54 to 59, wherein the food is a dessert or confection.

62. The food or beverage composition of any one of claims 54 to 59, wherein the beverage comprises a dairy product, fruit juice, or vegetable juice.

INTERNATIONAL SEARCH REPORT International application No.

PCT/US 15/53657

A . CLASSIFICATION O F SUBJECT MATTER IPC(8) - A01N 7/46; A61K 31/26; A61P 25/08, 25/28 (2015.01) CPC - A01 47/46; A6 1 31/26; G01N 33/6896 According to International Patent Classification (IPC) or to both national classification and IPC

B . FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols) IPC(8) Classifications: A0 N 47/46; A61K 31/26; A61 P 25/08, 25/28 (2015.01) CPC Classifications: A01N 47/46; A61K 31/26; G01N 33/6896

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) PatSeer (US, EP, WO, JP, DE, GB, CN, FR, KR, ES, AU, IN, CA, INPADOC Data); Google Scholar; Google; ProQuest; EBSCO; autism spectrum disorder, isothiocyanate, sulforaphane, glucosinolate, myrosinase, seizure

C . DOCUMENTS CONSIDERED T O B E RELEVANT

Category* Citation o f document, with indication, where appropriate, of the relevant passages Relevant to claim No.

W O 2014/008361 A2 (NUTRAMAX LABORATORIES, INC.) 09 January 2014; paragraphs 18-19, 20/18-19, 21-24, [00029], [00053], [00058] 25/23-24

1-2, 3/1-2, 4/1-2, 31/26-30, 32/31/26-30

W O 2013/067040 A 1 (THE JOHNS HOPKINS UNIVERSITY) 10 May 2013; paragraphs [0033], 26, 28, 45-48, 49/45-48, [0036], [0051], [0062], [0097], [00154]-[00155] 54, 55/45-48

1-2, 3/1-2, 4/1-2, 27, 29-30, 31/26-30, 32/31/26-30, 44

(DANIELSSON, S et al.) Epilepsy in young adults with autism: a prospective population-based 27, 29, 31/27, 31/29, follow-up study of 120 individuals diagnosed in childhood. Epilepsia. 2005. Vol 46(6); 32/31/27, 32/31/29, 44 page 921, left column, 2nd-5th paragraphs; table 2

(AMARENDRA) Why do autistic children need to lose weight? 2 1 July 2012 [Retrieved on 14 30, 31/30, 32/31/30 November 2015]. Retrieved from the Internet: ; page 1, 'physical', 'obsession'; page 2, 'ways to prevent overeating and weight gain'

I Further documents are listed in the continuation of Box C . | | See patent family annex.

* Special categories of cited documents: "T" later document published after the international filing date or priority "A" document defining the genera] state of the art which is not considered date and not in conflict with the application but cited to understand to be of particular relevance the principle or theory underlying the invention "E" earlier application or patent but published on or after the international "X" document of particular relevance; the claimed invention cannot be filing date considered novel or cannot be considered to involve an inventive "L" document which may throw doubts on priority claim(s) or which is step when the document is taken alone cited to establish the publication date of another citation or other "Y" document of particular relevance; the claimed invention cannot be special reason (as specified) considered to involve an inventive step when the document is "O" document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such combination means being obvious to a person skilled in the art "P" document published prior to the international filing date but later than "&" document member of the same patent family the priority date claimed Date of the actual completion o f the international search Date of mailing of the international search report

13 November 2015 (13.1 1.2015) 0 4 JAN 2016

Name and mailing address of the ISA/ Authorized officer Mail Stop PCT, Attn: ISA/US, Commissioner for Patents Shane Thomas P.O. Box 1450, Alexandria, Virginia 22313-1450 Facsimile No. 571-273-8300

Form PCT/ISA/210 (second sheet) (January 201 5) INTERNATIONAL SEARCH REPORT International application No.

PCT/US15/53657

Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)

This international search report has not been established in respect of certain claims under Article 7(2)(a) for the following reasons:

1. □ Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely:

□ Claims Nos.: because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically:

3. Claims Nos.: 5-17, 33-43, 50-53, 56-62 because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box No. Ill Observations where unity of invention is lacking (Continuation of item 3 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:

1. I I As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims.

2. As all searchable claims could be searched without effort justifying additional fees, this Authority did not invite payment of additional fees.

3. □ As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos.:

No required additional search fees were timely paid by the applicant. Consequently, this international search report restricted to the invention first mentioned in the claims; it is covered by claims Nos.:

The additional search fees were accompanied by the applicant's protest and, where applicable, the payment of a protest fee. The additional search fees were accompanied by the applicant's protest but the applicable protest fee was not paid within the time limit specified in the invitation. No protest accompanied the payment of additional search fees.

Form PCT/ISA/2 10 (continuation of first sheet (2)) (January 2015)