CN112822997A - Compositions and methods for treating autism - Google Patents

Abstract

Translated fromChinese

本发明提供了治疗孤独症的组合物和方法。具体地，本发明涉及通过施用α‑甲基‑DL‑酪氨酸来治疗孤独症的核心症状。The present invention provides compositions and methods for treating autism. In particular, the present invention relates to the treatment of core symptoms of autism by administering alpha-methyl-DL-tyrosine.

Description

Compositions and methods for treating autism

Cross Reference to Related Applications

This application claims priority and benefit from U.S.patent application 16/040,405 filed on 2018, 7/19, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to compositions and methods for treating autism (autism). In particular, the present invention relates to the treatment of the core symptoms of autism by administering alpha-methyl-DL-tyrosine.

Background

Autism Spectrum Disorder (ASD) or autism is defined in the Diagnostic and statistical Manual of Mental illness V (Diagnostic and statistical Manual of Mental Disorders V, DSM-5) as "social communication and social interaction difficulties, as well as restricted and repetitive patterns of behavior, interests or activities". Childhood autism is more prevalent than childhood cancer, juvenile diabetes and Acquired Immune Deficiency Syndrome (AIDS) in children, taken together, with an estimated prevalence of 150 million in the United States (US), 300 million children in europe, and tens of millions elsewhere throughout the world. Autism also represents a significant economic burden in both children and adults. More disturbingly, childhood autism appears to be growing at a rate of 10-17% per year without clear reasons. Finally, there are currently no approved drugs for the treatment of the core symptoms of autism. Autism is a serious disease that represents a huge economic burden and an area of unmet medical need.

Autism was originally described by doctor Leo Kanner in 1943, who described a 5 year old patient as: "... the words have a special literal, rigidifying meaning to him that he completely dismisses others and immediately looks for objects when in the room, such as Lai, m.c., et al austism, lanct, 2014.383 (9920): p.896-910.

Originally, autism was considered a form of schizophrenia in children. However, in the mid 1980 s autism was found to be a genetic disease and was considered to have a genetic cause. Autism is currently diagnostically defined as "social communication and social interaction difficulties, as well as restricted and repetitive patterns of behavior, interests or activities" in the diagnostic and statistical manual for mental disorders, version 5 (DSM-5) (2013). The diagnostic criteria in DSM-5 for the "social communication and social interaction difficulties" feature of ASD include: (1) social-emotional reciprocity deficiencies, e.g., from an unusual social approach and an inability to talk back and forth normally; reduced sharing of interest, mood, or emotion; to the point where it is impossible to initiate or respond to social interactions; (2) defects in nonverbal communication behavior for social interactions, e.g., poor communication from integrated verbal and nonverbal communication; eye-to-eye communication and body language abnormalities or defects in the understanding and use of gestures; to complete lack of facial expressions and nonverbal communication; and (3) the drawback of developing, maintaining, and understanding interpersonal relationships, for example, from difficulties in adjusting behavior to accommodate various social environments; difficulty in sharing imaginative games or making friends; to the peer (peer) without interest.

Autism is often manifested early in children and is associated with a variety of comorbidities, including epilepsy, Fragile-X syndrome (Fragile-X syndrome), Retts syndrome (Retts syndrome), attention deficit/hyperactivity disorder (ADHD), abnormal sensory or motor responses, sleep disorders (disturbed sleep), cognitive decline, anxiety, and aggression.

In 2010, the CDC reported a rate of U.S. autism of 1 out of 68 children, with boys being 5 times more susceptible than girls. This means that 1 of the 42 boys was diagnosed with autism. Since the assessment of autism prevalence in 2014, this figure has increased by about 30% and more than twice the rate reported in only 12 years, and this problem is growing rapidly. In New Jersey, the observed autism rate was 1 out of 46 children, meaning that 1 of the 29 boys born in New Jersey may have autism.

The financial cost of nurturing autistic children was estimated to be 320 ten thousand dollars more than the cost of nurturing non-autistic children in 2014, and this has not considered the cost of maintaining the population as an adult once their family is no longer able to do so. The social costs of autism are extensive and profound and many have never been explored. For example, information on the utility of healthcare for autistic patients was developed only in 2017 (mid-2017), and their need for psychiatric care and care for high incidence of autism-related complications was found to be well beyond the general population or other subpopulations of psychiatric patient populations. Also, school stops and the rate of exclusions were significantly higher in the autistic population up to 2017, 9 months, and increased as the number of autistic patients increased.

Recently, attention has been focused on autism-related mortality. Although autism is not generally considered a fatal disease, many researchers have reported a significantly increased mortality rate for the autistic population, the leading cause of death being suicide. A matched-pair case cohort study based on Swedish National Patient and Cause of Death registries (Swedish National Patient Registry and the house of Death Registry) noted deaths in 1987 to 2009 and found that autistic patients had a 256% higher mortality rate than the general population. The average age at death for the general population was 70.2 years and for autistic patients 58.39, suicide was associated with better performing patients. A review of 1,706 children and adolescents reported an 18% increase in the risk of suicidal ideation or attempts in autism. In a study of canada, suicide was attempted in 35% of patients with Asperger's syndrome. The same is true in japan, australia, the united kingdom and belgium. In a review of the PubMed literature in france, a total of 21.3% of autistic patients were found to report suicidal ideation or attempted suicide, noting that the method used is often violent ".

A large number of resources are used to study the causes of autism. Genetic, dietary, developmental, pharmacological, environmental and behavioral factors are all considered to be potential causes of the syndrome.

Purified single-enantiomer representation of the known molecule (alpha-methyl-L-tyrosine) (currently represented by

Sold) inhibits tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis due to enzyme-mediated conversion of L-tyrosine to DOPA (dihydroxyphenylalanine). The first step in catecholamine synthesis is highly stereospecific for L-tyrosine. L-AMPT (methylated L-tyrosine) acts as a competitive inhibitor of tyrosine hydroxylase: (

Prescribing Information，2015；FDA Summary Basis of Approval[SBA]for

1979)。

Was initially approved by the FDA in 1979 and was prescribed for patients with pheochromocytoma for: (1) preoperative preparation of a surgical patient; (2) the operation is contraindicated for the management of patients; and (3) chronic treatment of patients with malignant pheochromocytoma. For adults andchildren 12 years and older, advised

The initial dose was 250mg orally four times a day. The dose may be increased by 250mg to 500mg per day up to a maximum of 4.0 g/day in 4 divided doses. Although it is not limited to

Are commercially available, but a method for treating autism or its core symptoms by administering a racemic mixture of alpha-methyl-DL-tyrosine (i.e., DL-AMPT) has not been developed by those of ordinary skill in the art.

In view of general concern (a) the prevalence of autism has risen dramatically; (b) the devastating effects of the disease on children and their families; and (c) autism represents a sudden public health event, placing a tremendous social and economic burden on our social community, educational, medical, and mental health systems. Therefore, there is an urgent need for therapeutically effective methods for treating autism, particularly the core symptoms of the disease.

Summary of The Invention

In one aspect, the present invention provides a method for treating autism in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of α -methyl-DL-tyrosine and a pharmaceutically acceptable carrier. In an exemplary embodiment, the composition comprises α -methyl-DL-tyrosine in an amount of about 50mg (w/w) to about 500mg (w/w).

In another aspect, the present invention provides a method for providing a plasma concentration of a therapeutic agent for a long period of time to treat autism in a subject in need thereof, the method comprising administering to the subject a therapeutic agent at a concentration of about 50mg (w/w) to about 500mg (w/w) three times daily, wherein the therapeutic agent is α -methyl-DL-tyrosine, wherein the plasma concentration is about 500ng/ml to 5000ng/ml, and wherein the period of time is at least 1 week.

In another aspect, the present invention provides a method for treating an autism-related clinical profile in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of α -methyl-DL-tyrosine, thereby treating the autism-related clinical profile in the subject. In an exemplary embodiment, the clinical characteristic is a social communication deficit, a social interaction deficit, a social motivation deficit, lethargy and social withdrawal, inappropriate speech, hyperactivity, stereotyped behavior, irritability and excitement, restrictive behavior, repetitive behavior, cerebellar behavior, sexual behavior (semester behavior), compulsive behavior, self-disabling behavior, or a combination thereof.

Other features and advantages of the present invention will become apparent from the following detailed description examples and the accompanying drawings. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Drawings

FIG. 1 depicts the results of the Conners Parent Rating Scale Data (Conners Parent Rating Scale Data).

Fig. 2 illustrates patient registration and treatment.

Figure 3 shows an example of the maximal change from baseline in vital signs recorded in patients in study HT 02-121 (example 6) and indicates that these changes are transient and far from achieving the orthostatic criterion.

Figure 4 shows the weekly, total mean and random patient-wise L1-79 plasma concentrations in study HT 02-121 from open tags 100mg and 200mg TID (except day 1). 1 onday 1, L-79 plasma concentrations were assessed 1 hour after dosing.

Figure 5 shows weekly (excluding day 1), total mean and random patient-wise L1-79 plasma concentrations in study HT 02-121 from the open label 100mg TID set.

Figure 6 shows weekly (excluding day 1), total mean and random patient-wise L1-79 plasma concentrations in study HT 02-121 from the open label 200mg TID set.

Fig. 7 graphically shows CGI-overall severity (CGI-S) of L1-79 compared to placebo over time.

Fig. 8 graphically shows CGI-overall improvement over time (CGI-I) for L1-79 compared to placebo.

FIG. 9 shows the change in CGI-S from baseline at week 4 (ITT population, sketch).

Figure 10 shows the change in CGI-S from baseline toweek 4 by patient (ITT population, sketch).

FIG. 11 shows responder analysis (ITT population, sketch) of CGI-S atweek 4/LOCF.

Figure 12 shows the change in normalized socialization score for VABS II fromweek 0 to 8 from baseline (ITT population, sketch).

Figure 13 shows the change from baseline in the VABS II normalized communication score fromweek 0 to week 8 (ITT population, sketch).

Figure 14 shows the change in total ADOS-2 score fromweek 0 toweek 4 relative to screening (ITT population, sketch).

Figure 15 shows the change in total ADOS-2 restriction and repetitive behavior score fromweek 0 toweek 4 relative to screening (ITT population, sketch).

Figure 16 shows the change in total ADOS-2 social impact score fromweek 0 toweek 4 relative to screening (ITT population, sketch).

Figure 17 shows the change in ADOS-2 total score by patient from screening to week 4 (ITT population, sketch).

Figure 18 shows the change in SRS-2 total T score from baseline fromweek 0 to week 8 (ITT population, sketch).

FIG. 19 shows the change in social communication and traffic T scores from baseline for SRS-2DSM-5 fromweek 0 to week 8 (ITT population, sketch).

Fig. 20 shows the change in SRS-2 social communication T score from baseline fromweek 0 to week 8 (ITT population, sketch).

Figure 21 shows the change in SRS-2 social motivation T score from baseline fromweek 0 to week 8 (ITT population, sketch).

FIG. 22 shows the change in SRS-2DSM-5 restrictive and repetitive behavior fromweek 0 toweek 8 from baseline (ITT population, sketch).

Figure 23 shows the change in SRS-2 total T score from baseline toweek 4 by patient (ITT population, sketch).

Figure 24 shows the change in social communication and traffic T score from baseline toweek 4 for SRS-2DSM-5 by patient (ITT population, sketch).

Figure 25 shows the change in SRS-2 social communication T score from baseline toweek 4 by patient (ITT population, sketch).

Figure 26 shows the change in SRS-2 social motivation T score from baseline toweek 4 by patient (ITT population, sketch).

FIG. 27 shows responder analysis of SRS-2 total T scores atweek 4/LOCF (ITT population, sketch).

FIG. 28 shows responder analysis (ITT population, sketch) of SRS-2DSM-5 social communication and communication T scores atweek 4/LOCF.

FIG. 29 shows responder analysis (ITT population, sketch) of SRS-2 social communication T score atweek 4/LOCF.

FIG. 30 shows responder analysis (ITT population, sketch) of SRS-2 social motivation T scores atweek 4/LOCF.

Figure 31 shows the change from baseline in ABC-C lethargy and social withdrawal fromweek 0 to 8 (ITT population, sketch).

Figure 32 shows the change in ABC-C inappropriate speech from baseline fromweek 0 to week 8 (ITT population, sketch).

Figure 33 shows the change in ABC-C hyperactivity and non-compliance from baseline betweenweeks 0 and 8 (ITT population, sketch).

Figure 34 shows the change in ABC-C stereotyped behavior from baseline fromweek 0 to week 8 (ITT population, sketch).

Figure 35 shows the change in ABC-C irritability and agonism from baseline fromweek 0 to week 8 (ITT population, sketch).

Figure 36 shows the change in ABC-C lethargy and social withdrawal from baseline to the fourth week by patient (ITT population, sketch).

Figure 37 shows the change in ABC-C inappropriate speech by patient from baseline to fourth week (ITT population, sketch).

FIG. 38 shows the responder analysis (ITT population, sketch) of the ABC-C lethargy and social withdrawal aspects ofweek 4/LOCF.

FIG. 39 shows a responder analysis (ITT population, sketch) of ABC-C inappropriate speech atweek 4/LOCF.

FIG. 40 shows the change in RBS-R total score from baseline fromweek 0 to week 8 (ITT population, sketch).

Figure 41 shows the change in RBS-R restrictive behaviour from baseline atweeks 0 to 8 (ITT population, sketch). The mean improvement in the total score of RBS-R was recorded by a reduction in the L1-79200 mg score compared to placebo.

Figure 42 shows the change in RBS-R ceremonic behavior from baseline fromweek 0 to week 8 (ITT population, sketch).

FIG. 43 shows the change in RBS-R identity behavior fromweek 0 toweek 8 from baseline (ITT population, sketch).

FIG. 44 shows the change from baseline in RBS-R compulsion fromweek 0 to week 8 (ITT population, sketch).

Figure 45 shows the change from baseline in RBS-R stereotypical behavior fromweek 0 to week 8 (ITT population, sketch).

FIG. 46 shows the change in RBS-R self-disabling behavior fromweek 0 toweek 8 from baseline (ITT population, sketch).

Figure 47 shows the change in RBS-R total score from baseline toweek 4 by patient (ITT population, sketch).

Figure 48 shows responder analysis (defined as improvement or no deterioration) of RBS-R total score atweek 4 or LOCF.

Detailed Description

The inventive subject matter may be understood more readily by reference to the following detailed description, which forms a part of this disclosure. It is to be understood that this invention is not limited to the particular products, methods, conditions or parameters described and/or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention.

Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, a noun without a quantitative modification indicates one or more.

As used above and throughout the disclosure, the following terms and abbreviations are understood to have the following meanings unless otherwise indicated.

In the present disclosure, nouns that are not defined by a quantitative term include plural references, and reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" is a reference to one or more such compounds and equivalents thereof known to those skilled in the art, and so forth. The term "plurality," as used herein, refers to more than one. When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

As used herein, the terms "component," "composition of compounds," "compound," "drug," "pharmacologically active agent," "therapeutic agent," "therapy," "treatment," or "drug" are used interchangeably herein to refer to a compound or composition of matter that, when administered to a subject (human or animal), induces a desired pharmacological and/or physiological effect through a local and/or systemic effect.

As used herein, the following terms are defined as follows:

ABA application behavior analysis

ABC-C abnormal behavior checklist-community

ADHD attention deficit hyperactivity disorder

ADIR autism diagnosis interview examination

ADME absorption, distribution, metabolism and elimination

ADOS-2 autism diagnostic observation protocol, second edition

AE adverse events

AIDS acquired immunodeficiency syndrome

ASD autism spectrum disorder

AST aspartate aminotransferase

ALT alanine aminotransferase

AUC concentration area under time Curve

AUC_0-8Area under concentration time curve of 0 to 8 hours

AUC_0-12Area under concentration time curve of 0 to 12 hours

AUC_0-∞Area under the concentration time curve from 0 to infinity

AUC_0-tzArea under concentration time curve from 0 to the last quantifiable concentration

CFR U.S. Federal regulations

CGI-I clinical Global impression-Global improvement

CGI-S clinical Global impression-disease severity

Cmax maximum concentration

CNS central nervous system

DL-AMPT racemic (D and L isomers) alpha-methyl-p-tyrosine

DOPA Dihydroxyphenylalanine

DSM-5 diagnostic and statistical manual for mental disorders V

EI early intervention

EOP2 end of second phase

FDA United states food and drug administration

FDASIA U.S. food and drug administration safety and innovation

FOBs function observation and evaluation scale

Good GMP production practice

HBV hepatitis B virus

Hemorrhagic cerebrovascular accident of HCVA

HED human equivalent dose

HIV human immunodeficiency virus

IEP individualization education program

L isomer of L-AMPT alpha-methyl-p-tyrosine

LC-MS/MS liquid chromatography mass spectrometry

LOCF last observed value conversion method

lQTS Long QT syndrome

Maximum tolerated dose of MTD

NDA new drug application

Noael has no visible adverse effect level

Pharmacodynamics of PD

PK pharmacokinetics

PND postpartum day

PSP pediatric study program

RBS-R repetitive behavior Scoring-revision

SBA approved digest basis

SRS-2 social response Scale, second edition

t_1/2Half life

TdP Torsade’s de Pointes

TID three times daily

TK pharmacokinetics

TQT complete QT

Upper normal limit of ULN

USAN adopted name

VABS II Welan Adaptation behavior Scale, second edition

For autism, for example, a negative impact or symptom may include any object of the diagnostic criteria specified for autism spectrum disorders in american psychiatric society's diagnostic and statistics manual fifth edition (DSM-5), the contents of which are incorporated herein by reference, such as a deficiency in socio-emotional reciprocity (e.g., from abnormal social patterns and inability to talk back and forth normally; to decreased sharing of interests, emotions, or emotions; to inability to initiate or respond to social interactions); defects in nonverbal communication behavior for social interactions (e.g., from poor comprehensive verbal and nonverbal communication; to eye contact and body language abnormalities or defects in understanding and use of gestures; to complete lack of facial expressions and nonverbal communication); deficiencies in developing, maintaining, and understanding interpersonal relationships (e.g., from difficulty in adjusting behavior to accommodate various social environments; difficulty in sharing imaginative games or contacting friends; inability to be interested in peers); styling or repetitive motion, using objects or speech (e.g. simple sports styling, arranging toys or turning objects, imitating speech, idiosyncratic wording); insights into the same, hard-to-stick practices, or stylized patterns of verbal or non-verbal behavior (e.g., extreme pain with small changes, difficult transitions, rigid thinking patterns, stylized greetings, the need to take the same route or eat the same food each day); a high limit of abnormality in intensity or attention, a fixed interest (e.g., a strong attachment or addiction to unusual items, an over-limit or a persistent interest); and excessive or insufficient response to sensory input or an abnormal interest in environmental perception (e.g., apparent apathy to pain/temperature, adverse reaction to specific sounds or textures, excessive smelling or touching of objects, visual fascination of lights or movements).

Some patient subpopulations of the autism spectrum include those diagnosed with asperger's disorder (i.e., asperger's syndrome) or social communication disorder, exhibiting symptoms of Attention Deficit Hyperactivity Disorder (ADHD) (e.g., inattention, hyperactivity, and impulsivity) and/or twitching (motor or vocational twitching). See, for example, DSM-5.

The assessment of the symptoms of autism or any symptom of the present disclosure can be performed using methods known in the art. For example, one method of assessing autism symptoms is based on a Clinical Global Impression (CGI) scale of changes from baseline in various psychological tests. These tests may include the abnormal Behavior Checklist-Community (ABC-C), the conners parental rating scale, and the Autism Diagnostic Observation protocol (ADOS). Autism symptoms can also be assessed from individual clinical observations of clinicians, from image information obtained from visits at regularly scheduled clinics, and from information provided over time by the subject's caregiver. The compositions and methods of the present disclosure result in at least a 1 point reduction in at least one dimension of the conners parental rating scale assessment score.

As used above and throughout the disclosure, the term "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired result in the treatment of the associated disorder, condition or side effect. It will be appreciated that the effective amount of a component of the invention will vary from patient to patient, not only with respect to the particular compound, component or composition selected, the route of administration, and the ability of the component to bring about the desired result in the individual, but also with respect to factors such as the disease state or severity of the condition being alleviated, the individual's hormone level, age, sex, weight, condition of the patient and the severity of the pathological condition being treated, concurrent medications and special diets being followed by the particular patient, and other factors which will be recognized by those skilled in the art, the appropriate dosage being determined by the attending physician. Dosage regimens may be adjusted to provide improved therapeutic response. An effective amount is also one in which the therapeutically beneficial effect outweighs any toxic or detrimental effects of the components.

The invention also provides pharmaceutical compositions comprising a compound of the invention and one or more pharmaceutically acceptable carriers. "pharmaceutically acceptable carrier" includes any excipient that is non-toxic to the cells or mammal to which it is exposed at the dosages and concentrations employed. The pharmaceutical composition may comprise one or more additional therapeutic agents.

"pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complication, commensurate with a reasonable benefit/risk ratio.

Examples of pharmaceutically acceptable carriers include, for example, but are not limited to, solvents, dispersion media, buffers, coatings, antibacterial and antifungal agents, wetting agents, preservatives, buggers, chelating agents, antioxidants, isotonic agents, and absorption delaying agents.

Examples of pharmaceutically acceptable carriers also include, for example, but are not limited to, water; brine; phosphate buffered saline; glucose; glycerol; alcohols such as ethanol and isopropanol; phosphates, citrates and other organic acids; ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; EDTA; salt-forming counterions, such as sodium; and/or nonionic surfactants such as TWEEN, polyethylene glycol (PEG), and PLURONICS; isotonic agents, for example, sugars, polyalcohols (e.g., mannitol and sorbitol), and sodium chloride; and combinations thereof.

In the present invention, the disclosed compounds can be prepared in the form of pharmaceutically acceptable salts. "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues (e.g., amines); alkali metal or organic salts of acidic residues (e.g., carboxylic acids); and so on. Pharmaceutically acceptable salts include, for example, the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic and the like. These physiologically acceptable salts are prepared by methods known in the art, for example by dissolving the free amine base with an excess of acid in aqueous alcohol (aqueous alcohol), or by neutralizing the free carboxylic acid with an alkali metal base (e.g. hydroxide) or an amine.

The compounds described herein may be prepared in alternative forms. For example, many amino-containing compounds can be used or prepared as acid addition salts. Generally, such salts improve the isolation and handling properties of the compounds. For example, depending on the reagents, reaction conditions, and the like, the compounds described herein may be used or prepared, for example, as their hydrochloride or tosylate salts. Isomorphic crystalline forms, all chiral and racemic forms, N-oxides, hydrates, solvates and acid salt hydrates are also considered to be within the scope of the present invention.

Certain acidic or basic compounds of the present invention may be present as zwitterions. All forms of the compounds, including free acids, free bases, and zwitterions are contemplated within the scope of the present invention. It is well known in the art that compounds containing both amino and carboxyl groups are generally present in equilibrium in their zwitterionic forms. Thus, any compound described herein that contains, for example, both an amino group and a carboxyl group also includes reference to its corresponding zwitterion.

The term "stereoisomers" refers to compounds having the same chemical composition but differing arrangements of atoms or groups in space. The term "enantiomer" refers to stereoisomers that are mirror images of each other that are not superimposable.

The term "administering" refers to either directly administering a compound or composition of the invention, or administering a prodrug, derivative, or analog that will form an equivalent amount of the active compound or substance in vivo.

The term "inhibitor" as used herein includes compounds that inhibit the expression or activity of a protein, polypeptide or enzyme, and does not necessarily mean complete inhibition of expression and/or activity. Conversely, inhibiting includes inhibiting the expression and/or activity of a protein, polypeptide, or enzyme to an extent and for a period of time sufficient to produce the desired effect.

Without intending to be bound by any particular mechanism of operation, it is believed that the tyrosine hydroxylase inhibitors according to the invention act by reducing the amount of epinephrine secreted into the blood stream.

Tyrosine hydroxylase inhibitors are well known in the art and are described fully, for example, in the following: U.S. patent application publications US 2015/0290279, US 2015/0216827, US 2015/0111937, US 2015/0111878, US 2013/0184214 and US 20130183263; US patents US 8,481,498, US 9,308,188 and US 9,326,962; and PCT patent application publication WO2015061328, which is incorporated by reference herein in its entirety. Any suitable tyrosine hydroxylase inhibitor known to those skilled in the art may be used.

In certain embodiments, the tyrosine hydroxylase inhibitor is a tyrosine derivative. Tyrosine derivatives may be capable of existing in different isomeric forms, including stereoisomers and enantiomers. Tyrosine derivatives may for example be present in both the L form or the D form. The tyrosine derivative may also be present, for example, in racemic form.

Representative tyrosine derivatives include, for example, one or more of the following: methyl (2R) -2-amino-3- (2-chloro-4-hydroxyphenyl) propionate, D-tyrosine ethyl ester hydrochloride, methyl (2R) -2-amino-3- (2, 6-dichloro-3, 4-dimethoxyphenyl) propionate, H-D-tyrosine (tBu) -allyl ester hydrochloride, methyl (2R) -2-amino-3- (3-chloro-4, 5-dimethoxyphenyl) propionate, methyl (2R) -2-amino-3- (2-chloro-3-hydroxy-4-methoxyphenyl) propionate, methyl (2R) -2-amino-3- (4- [ (2-chloro-6-fluorophenyl) methoxy ] phenyl) propionate, and mixtures thereof, Methyl (2R) -2-amino-3- (2-chloro-3, 4-dimethoxyphenyl) propionate, methyl (2R) -2-amino-3- (3-chloro-5-fluoro-4-hydroxyphenyl) propionate, diethyl 2- (acetylamino) -2- (4- [ (2-chloro-6-fluorobenzyl) oxy ] benzylmalonate, methyl (2R) -2-amino-3- (3-chloro-4-methoxyphenyl) propionate, methyl (2R) -2-amino-3- (3-chloro-4-hydroxy-5-methoxyphenyl) propionate, methyl (2R) -2-amino-3- (2, methyl 6-dichloro-3-hydroxy-4-methoxyphenyl) propionate, methyl (2R) -2-amino-3- (3-chloro-4-hydroxyphenyl) propionate, H-DL-tyrosine methyl ester hydrochloride, H-3, 5-diiodo-tyrosine methyl ester hydrochloride, H-D-tyrosine methyl ester hydrochloride, methyl D-tyrosine ester hydrochloride, H-D-tyrosine methyl ester hydrochloride, H-D-tyrosine methyl ester hydrochloride, (2R) -2-amino-3- (4-hydroxyphenyl) propionate, methyl (meth) acrylate, and the like, (2R) -2-amino-3- (4-hydroxyphenyl) methyl ester hydrochloride, methyl (2R) -2-amino-3- (4-hydroxyphenyl) propionate hydrochloride, methyl (2R) -2-azanyl-3- (4-hydroxyphenyl) propionate hydrochloride, 3-chloro-L-tyrosine, 3-nitro-L-tyrosine, ethyl 3-nitro-L-tyrosine hydrochloride, DL-m-tyrosine, DL-o-tyrosine, Boc-tyrosine (3, 5-I2) -OSu, Fmoc-tyrosine (3-N02) -OH, alpha-methyl-L-tyrosine, alpha-methyl-D-tyrosine and alpha-methyl-DL-tyrosine. In certain embodiments of the invention, the tyrosine derivative is α -methyl-L-tyrosine as shown below:

in other embodiments, the tyrosine derivative is α -methyl-D-tyrosine. In other embodiments, the tyrosine derivative is a racemic form of α -methyl-DL-tyrosine as shown below:

in a particular embodiment, the tyrosine derivative is a structural variant of α -methyl-L-tyrosine or α -methyl-DL-tyrosine. Structural variants of α -methyl-L-tyrosine or α -methyl-DL-tyrosine are well known in the art and are fully described in, for example, U.S. patent 4,160,835, which is incorporated herein by reference in its entirety.

In one embodiment, the tyrosine derivative of the present invention is an aryl alanine compound having the formula:

wherein R is₁Is hydrogen, methyl or ethyl ester group, or alkyl of 1 to 4 carbon atoms; r₂Is hydrogen, lower alkyl, lower alkene, succinimide, or alkyl of 1-4 carbon atoms; r₃Is a substituted benzene ring of the general formula

Wherein Y is₁Para and is hydrogen, hydroxy, methyl ether, dimethyl ether, trimethyl ether, or unsubstituted or halogen-substituted benzyl; y is₂And Y₃Are the same or different, and wherein Y is₂And Y₃One or both of which are in the meta or ortho position, and wherein Y is₂And Y₃Is hydrogen, hydroxy, halogen, methyl ether or nitro; and R is₄Is hydrogen, acetyl, tert-butoxycarbonyl or fluorenylmethoxycarbonyl.

In some embodiments, Y1 and Y2 are the same or different and are selected from the group consisting of hydrogen, cyanoamino, carboxyl, cyano, thiocarbamoyl, aminomethyl, guanidino, hydroxyl, methanesulfonamido, nitro, amino, methanesulfonyloxy, carboxymethoxy, formyl, methoxy, and substituted or unsubstituted 5-or 6-membered heterocyclic rings containing carbon and one or more nitrogen, sulfur or oxygen atoms, specific examples of such heterocyclic rings being pyrrol-1-yl, 2-carboxypyrrol-1-yl, imidazol-2-ylamino, indol-1-yl, carbazol-9-yl, 4, 5-dihydro-4-hydroxy-4-trifluoromethylthiazol-3-yl, 4-trifluoromethylthiazol-2-yl, imidazol-2-yl and 4, 5-dihydroimidazol-2-yl such that (a) Y1 and Y2 cannot both be hydroxyl, (b) Y1 and Y2 cannot both be hydrogen, and (c) when one of Y1 and Y2 is hydrogen, the other cannot be hydroxyl.

In one example, R₃Is a substituted or unsubstituted benzoheterocycle having the formula:

wherein the benzo-heterocycle is selected from indol-5-yl, 1- (N-benzoylcarbamoylimino) -indol-5-yl, 1-carbamoyliminoindol-5-yl, 1H-2-oxindol-5-yl, indol-5-yl, 2-mercaptobenzimidazol-5 (6) -yl, 2-aminobenzimidazole-5 (6) -yl, 2-methanesulfonamido-benzimidazol-5 (6) -yl, 1H-benzo

Oxazol-2-on-6-yl, 2-aminobenzothiazol-6-yl, 2-amino-4-mercaptobenzothiazol-6-yl, 2, 1, 3-benzothiadiazol-5-yl, 1, 3-dihydro-2, 2-dioxo-2, 1, 3-benzothiadiazol-5-yl, 1, 3-dihydro-1, 3-dimethyl-2, 2-dioxo-2, 1, 3-benzothiadiazol-5-yl, 4-methyl-2 (1H) -oxoquinolin-6-yl, quinoxalin-6-yl, 2-hydroxyquinoxalin-7-yl, 2-mercaptobenzothiazol-6-yl, 2-amino-4-mercaptobenzothiazol-6-yl, 2, 1, 3-benzothiadiazol-5-yl, 1, 3-dihydro, 2, 3-dihydroxyquinoxalin-6-yl and 2, 3-dihydro-3 (4H) -oxo-1, 4-benzo

Oxazin-7-yl.

In another example, R₃Is a substituted or unsubstituted heterocycle having the formula:

wherein the heterocycle is selected from 5-hydroxy-4H-pyran-4-on-2-yl, 2-hydroxypyridin-4-yl, 2-aminopyridin-4-yl, 2-carboxypyridin-4-yl, or tetrazolo [1, 5-a ] pyridin-7-yl.

In a particular embodiment, the tyrosine hydroxylase inhibitor is daminomycin (aquayamycin). In one example, the daminomycin is a compound of the formula.

In another specific embodiment, the tyrosine hydroxylase inhibitor is oudemanone. In one example, the agroeybendum element is a compound of the formula.

Other suitable tyrosine hydroxylase inhibitors known to those skilled in the art may also be used. Examples of other tyrosine hydroxylase inhibitors include, but are not limited to, for example, cycloheximide, anisomycin, 3-iodo-L-tyrosine, pyraratine, phenylcarbonyl derivatives with catechol or triphenol ring systems, such as phenethylamine and gallic acid derivatives, 4-isopropyltropolone (4-isopyropolone), 2- (4-thiazolyl) benzimidazole, 8-hydroxyquinoline, phenanthroline (o-phenantroline), 5-iodo-8-hydroxyquinoline, bilirubin, 2, 9-dimethyl-1, 10-phenanthroline, α - α' -bipyridine, dibenzo [ f, h ] s]Quinoxaline, 2, 4, 6-tripyridyl-s-triazine, 3-amino-4H-pyrroloiso-triazine

Oxazole-5 (6H) -carboxylic acid ethyl ester, alpha-nitroso-beta-naphthol, sodium diethyldithiocarbamate, ethylenediaminetetraacetic acid (see R Hochster, Metabolic innovations V4: A Comprehensive treat 52Elsevier (2012)).

In a particular embodiment, the tyrosine hydroxylase inhibitor is L1: 79, which refers to the racemic form of alpha-methyl-DL-tyrosine or D, L alpha-methyl-p-tyrosine (abbreviated DL-AMPT).

alpha-methyl-DL-tyrosine inhibits the activity of Tyrosine Hydroxylase (TH), which catalyzes the first conversion of catecholamine biosynthesis, i.e. the conversion of tyrosine to Dihydroxyphenylalanine (DOPA), which is the rate-limiting step in catecholamine synthesis. alpha-methyl-DL-tyrosine is a tyrosine analogue that competes competitively for TH and is excreted almost invariably in urine.

In preliminary tests, it was observed that Demser (i.e., α -methyl-L-tyrosine) had a beneficial effect on the symptoms of intestinal leakage, but this effect was transient. A greater physiological effect and half-life of the racemic mixture (i.e. a-methyl-DL-tyrosine) was observed. This may be due to the inability of the D amino acid isomer to be transported by the different L amino acid transport systems present in the body, or by renal excretion which allows the L isomer to persist for a longer period of time.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount or dose of a tyrosine hydroxylase inhibitor (e.g., alpha-methyl-DL-tyrosine) and a pharmaceutically acceptable carrier.

As described herein, the effective dosage of the compositions of the present invention for treating autism will vary depending on a number of different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is an adult or child, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Therapeutic doses can be titrated using routine methods known to those skilled in the art to optimize safety and efficacy.

The pharmaceutical compositions of the present invention may comprise a "therapeutically effective amount". A "therapeutically effective amount" is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. The therapeutically effective amount of the molecule may vary according to a variety of factors, such as the disease state, age, sex, and weight of the individual and the ability of the molecule to elicit a desired response in the individual. A therapeutically effective amount is also one in which the therapeutically beneficial effect outweighs any toxic or detrimental effect of the molecule.

As used herein, the term "treatment" refers to a therapeutic treatment, including prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change associated with a disease or disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of a disease or disorder, stabilization of a disease or disorder (i.e., wherein the disease or disorder is not exacerbated), delay or slowing of progression of the disease or disorder, amelioration or palliation of the disease or disorder, and remission (whether partial or complete), whether detectable or undetectable, of the disease or disorder. Those in need of treatment include those already with the disease or condition as well as those predisposed to the disease or condition or those for which the disease or condition is to be prevented.

The dosage regimen may be adjusted to provide the best desired response (e.g., therapeutic or prophylactic response).

In one example, a single bolus may be administered. In another example, several divided doses may be administered over time. In yet another example, the dosage may be proportionally reduced or increased depending on the urgency of the treatment situation. Dosage unit form, as used herein, refers to physically discrete units suitable as unitary dosages for the treatment of mammalian subjects. Each unit may contain a predetermined amount of active compound calculated to produce the desired therapeutic effect. In some embodiments, the dosage unit form of the invention is determined and directly dependent on the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved.

The composition of the present invention may be administered only once, or it may be administered multiple times. For multiple doses, the composition may be administered, for example, three times per day, twice per day, once every two days, twice per week, weekly, biweekly, or monthly.

It is noted that dosage values may vary with the type and severity of the condition to be alleviated. It will also be understood that the specific dosage regimen for any particular subject will be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges given herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

"administration" to a subject is not limited to any particular delivery system, and can include, but is not limited to, oral (e.g., in capsules, suspensions, or tablets), parenteral (including subcutaneous, intravenous, intramedullary, intra-articular, intramuscular, or intraperitoneal injections), topical, or transdermal. Administration to the host may be in a single dose or repeated administration and be in any of a variety of physiologically acceptable salt forms and/or with acceptable pharmaceutical carriers and/or additives (as previously described) as part of a pharmaceutical composition. Again, physiologically acceptable salt forms and standard Pharmaceutical formulation techniques are well known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co.).

In one aspect, the dosage of tyrosine hydroxylase inhibitor may be from about 1mg to about 4 g. In a particular embodiment, the dosage of tyrosine hydroxylase inhibitor may be from about 10mg to about 1500 mg. In some suitable embodiments of the invention, the composition comprises the following amount of tyrosine hydroxylase inhibitor (i.e., alpha-methyl-DL-tyrosine): about 50mg (w/w) to about 1500mg (w/w); about 50mg (w/w) to about 500mg (w/w); about 75mg (w/w) to about 350mg (w/w); about 90mg (w/w) to about 350mg (w/w); about 100mg (w/w) to about 300mg (w/w); or from about 100mg (w/w) to about 200mg (w/w). In one embodiment, the composition comprises a tyrosine hydroxylase inhibitor (i.e., alpha-methyl-DL-tyrosine) in an amount of about 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 500, 750, 1000, or 1500mg (w/w /). As used herein, "composition" refers to any composition comprising a pharmaceutically effective amount of one or more active ingredients (e.g., a tyrosine hydroxylase inhibitor, another acne treatment agent, or a combination thereof).

In some embodiments, multiple compositions with different dosages are administered simultaneously or sequentially. For example, in one embodiment, a first composition comprising α -methyl-DL-tyrosine in an amount of about 200mg (w/w) and a second composition comprising α -methyl-DL-tyrosine in an amount of about 100mg (w/w) are administered simultaneously. In another embodiment, a first composition comprising α -methyl-DL-tyrosine in an amount of about 200mg (w/w) and a second composition comprising α -methyl-DL-tyrosine in an amount of about 100mg (w/w) are administered sequentially.

In one aspect, the composition is administered for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In another aspect, the composition is administered for the duration of a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 week dosing period. In another aspect, the composition is administered for a duration of the dosing period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

In another aspect, the present invention provides a method for obtaining a plasma concentration of a therapeutic tyrosine hydroxylase inhibitor drug (e.g., α -methyl-DL-tyrosine) over a long period of time for the treatment of autism in a subject in need thereof, the method comprising administering to the subject a therapeutic drug at a concentration of about 50mg (w/w) to about 500mg (w/w) three times daily, wherein the plasma concentration is about 500ng/ml to 5000ng/ml, and wherein the period of time is at least 1 week. In one embodiment, the medicament is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks in order to achieve the desired plasma concentration. In a particular embodiment, the medicament is administered for a duration of about 1 day to about 4 weeks, about 2 days to about 4 weeks, or about 1 week to about 4 weeks in order to achieve the desired plasma concentration. In some embodiments, the drug is administered for a treatment regimen duration of more than 6 months in order to achieve the desired plasma concentration.

Administration of the compositions of the present invention can result in plasma concentrations of about 500ng/ml to 5000 ng/ml; about 800ng/ml to 2500ng/ml, or about 1400ng/ml to 1800 ng/ml.

The pharmaceutical compositions of the present invention can be formulated in a variety of ways, including, for example, solid, semi-solid, and liquid dosage forms, such as capsules, tablets, pills, powders, liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. In some embodiments, the composition is in the form of an injectable or infusible solution. The composition is in a form suitable for oral, topical, intravenous, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, or transdermal administration. In a particular embodiment, the composition is in the form of a capsule. In another particular embodiment, the composition is in the form of a tablet.

Administration of the pharmaceutical composition may be by a variety of routes, including oral, nasal, subcutaneous, intravenous, intramuscular, transdermal, vaginal, rectal, or any combination thereof. Transdermal administration can be achieved using, for example, oleic acid, 1-methyl-2-pyrrolidone, dodecylnonyloxyethylene.

In one aspect, the invention provides for administering to a subject a therapeutically effective amount of a first tyrosine hydroxylase inhibitor (e.g., alpha-methyl-DL-tyrosine) in combination with a therapeutically effective amount of a second tyrosine hydroxylase inhibitor (e.g., alpha-methyl-L-tyrosine). In another aspect, the present invention provides for administering to a subject a therapeutically effective amount of one or more tyrosine hydroxylase inhibitors (e.g., alpha-methyl-DL-tyrosine and/or alpha-methyl-L-tyrosine) in combination with a therapeutically effective amount of other agents useful for treating autism.

In some of these aspects, gamma-aminobutyric acid (GABA) and a tyrosine hydroxylase inhibitor (e.g., alpha-methyl-DL-tyrosine) may be administered. GABA may be administered concurrently with the tyrosine hydroxylase inhibitor. In other aspects, GABA may be administered separately from the tyrosine hydroxylase inhibitor, e.g., at another time of day. In some aspects, GABA is administered at bedtime. Typically, the dosage of GABA is from about 5mg to about 30mg, e.g., 5, 10, 15, 20, 25 or about 30mg of GABA, with 15mg of GABA being particularly preferred.

In still other of these aspects, a p 4503 a4 promoter is administered in addition to a tyrosine hydroxylase inhibitor (e.g., α -methyl-DL-tyrosine) and optionally GABA. Preferred p 4503 a4 promoters include 5, 5-diphenylhydantoin, valproic acid, and carbamazepine.

Those subjects of the autism spectrum, including those diagnosed with asperger's disorder (asperger's syndrome) or social communication disorder, and also having symptoms of ADHD and/or tics, may be treated using the methods of the present disclosure. In these aspects, an effective amount of a tyrosine hydroxylase inhibitor and an effective amount of a beta adrenergic agonist (also referred to as a beta agonist) can be administered to a subject. The tyrosine hydroxylase inhibitor may be any tyrosine hydroxylase inhibitor described herein, with alpha-methyl-DL-tyrosine being particularly preferred. Beta-adrenergic agonists are known in the art and include, for example, salbutamol, levalbuterol, fenoterol, formoterol, isoproterenol, metaproterenol, salmeterol, terbutaline, clenbuterol, isoetharine, pirbuterol, procaterol, ritodrine, epinephrine, and combinations thereof. Salbutamol is a particularly preferred beta adrenergic agonist.

In certain of these aspects, the tyrosine hydroxylase inhibitor is administered concurrently with the beta adrenergic inhibitor. In other aspects, the beta adrenergic inhibitor is administered separately from the alpha-methyl-DL-tyrosine, e.g., at another time of day.

In accordance with the present disclosure, the described methods for treating a disease or condition can be used in combination with therapeutic methods that are also known to be effective for treating the same disease or condition. For example, autistic behavior and symptoms can be treated with compounds that affect autonomic neurotransmission (e.g., amphetamine, methylphenidate, etc.), psychotropic drugs (e.g., risperidone), neurotransmitter reuptake inhibitors (e.g., fluoxetine), compounds that stimulate glutaminergic transmission (e.g., LY2140023), and/or compounds that affect cholinergic neurotransmission (e.g., galantamine). Accordingly, the present disclosure also relates to methods of treating autism in a patient by administering an effective amount of a tyrosine hydroxylase inhibitor (e.g., alpha-methyl-DL-tyrosine) and an effective amount of a compound that affects autonomic neurotransmission, a psychotropic drug, a neurotransmitter reuptake inhibitor, a compound that stimulates glutaminergic transmission, and/or a compound that affects cholinergic neurotransmission. In one embodiment, the present invention provides a method of treating autism in a patient by administering a therapeutically effective amount of a tyrosine hydroxylase inhibitor (e.g., alpha-methyl-DL-tyrosine) and a therapeutically effective amount of a Central Nervous System (CNS) agent.

Diseases or conditions treated by the compositions of the present invention include, for example, autism or a disease or disorder associated therewith.

The autonomic nervous system involves symptoms similar to those seen in autism. Autism Spectrum Disorder (ASD) is associated with abnormal findings in autonomic related structures, including the brain island and amygdala. In autism, autonomously related changes are seen, such as an increase in basal heart rate and a decreased heart rate due to psychosocial challenges. Autism-autonomic connection is exemplified by the consequences of Respiratory Sinus Arrhythmia (RSA), which include social difficulties, language difficulties, and delays in cognitive development.

It is hypothesized that the chronically overactivated autonomic system is a relevant factor for autism based on excessive anxiety levels, which leads to autism, physiological hyperarousal, and other relevant factors. Anxiety is probably the largest complication associated with autism and it can lead to other features of the disease that are associated with central nervous system structures that are related to autonomic function. Phenotypically, autism and anxiety have stereotyped repetitive behaviors and limited interest, evasive behavior, and verbal problems. The relationship between anxiety and autonomic nervous symptoms reported as other sequelae of elevated heart rate, sweating, and "fight or escape" responses reveals the role of peripheral nervous system function in autism. However, this may be secondary to central autonomic activation. Central functions may manifest as elevated emotional responsiveness and exaggerated threat perception or reduced suppression of fear responses, which are associated with the above-described central structure in which autonomic and emotional responses overlap.

There is a great deal of evidence that autism is associated with cholinergic function of the central nervous system, particularly with the various alpha-subtype nicotinic receptors, particularly in the cerebellum. However, since autonomic function is generally regarded as an equilibrium of cholinergic and catecholaminergic systems, the perceived increase or decrease in cholinergic function may be indicative of a change in the dynamic equilibrium of these systems with a catecholaminergic base. It is believed that therapeutic alterations in ASD can be achieved through acetylcholine-based manipulation through administration of alpha-methyl-DL-tyrosine or manipulation of the balance of dopamine, norepinephrine, or epinephrine-mediated mechanisms.

The present invention also relates to the treatment of the core symptoms of ASD by administering alpha-methyl-DL-tyrosine to a subject in need thereof.

Without intending to be bound by any particular mechanism of operation, it is believed that tyrosine hydroxylase inhibitors (e.g., α -methyl-DL-tyrosine) administered according to the present invention modulate catecholaminergic pathways associated with autism, and more particularly, such pathways that are involved in the core symptoms of ASD, including catecholaminergic function in the CNS and gastrointestinal tract. Thus, the known effects of catecholamines on the endocrine and neuroendocrine systems, including but not limited to reducing the amount of epinephrine secreted into the bloodstream, which may alleviate the intensity of irritability and agitation, as well as other core symptoms of ASD, are modulated by the administration of tyrosine hydroxylase inhibitors (e.g., α -methyl-DL-tyrosine).

In one aspect, the present invention provides a method for treating the core symptoms of Autism Spectrum Disorder (ASD) in a subject in need thereof, the method comprising administering a therapeutically effective amount of a racemic mixture of α -methyl-DL-tyrosine.

In another aspect, the present invention provides a method for treating an autism-related clinical feature in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a tyrosine hydroxylase inhibitor (e.g., alpha-methyl-DL-tyrosine), thereby treating the autism-related clinical feature in the subject.

Examples of clinical features associated with autism include, for example, but are not limited to, lack of social communication, lack of social interaction, lack of social motivation, lethargy and social withdrawal, inappropriate speech, hyperactivity, stereotyped behavior, irritability and agitation, restrictive behavior, repetitive behavior, ceremonial behavior, identity behavior, compulsive behavior, self-disabling behavior, or combinations thereof.

In one embodiment, the clinical profile is assessed based on changes from baseline in one or more psychological tests. Examples of psychological tests include, but are not limited to, a Clinical Global Impression (CGI) rating scale, a naval adaptive behavior scale (VABS), an autism diagnosis observation protocol (ADOS), a Social Response Scale (SRS), an abnormal behavior checklist-community (ABC-C), a Repetitive Behavior Scale (RBS), a Conners Parent Rating Scale (CPRS), or a combination thereof. In a particular embodiment, the clinical profile meets the requirements in the Diagnostic and Statistical Manual of Mental Disorders V (DSM-V) standards.

The terms "subject", "individual" and "patient" are used interchangeably herein and refer to an animal, such as a human, for whom treatment (including prophylactic treatment) with a pharmaceutical composition according to the invention is provided. As used herein, the term "subject" refers to both humans and non-human animals. The terms "non-human animal" and "non-human mammal" are used interchangeably herein and include all vertebrates, such as mammals, e.g., non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, horses, and non-mammals, e.g., reptiles, amphibians, chickens and turkeys.

In one embodiment, the subject is a human patient aged 3 to 21 years, aged 5 to 21 years, or aged 6 to 17 years. In another embodiment, the subject is an adult patient.

All patents and references cited in this specification are incorporated herein by reference in their entirety.

Also provided herein are kits comprising one or more of the molecules or compositions described herein. The following examples are provided to supplement the present disclosure and to provide a better understanding of the subject matter described herein. These examples should not be considered as limiting the described subject matter. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included therein without departing from the true scope of the present invention.

Examples

Example 1

200 patients were initially screened. The 30 subjects who met the study criteria agreed. Nine (9) subjects had high blood glucose levels (hyperglycemia) before consent to the study.

High blood glucose levels (hyperglycemia) are defined as fasting plasma blood glucose levels of 126mg/dl or higher in two different cases. The mean patient age was 62 years and the median patient age was 60 years. 6 patients were female and 3 patients were male. 5 patients were between 50 and 60 years of age and 4 patients were over 60 years of age.

The patients in the study were administered a treatment regimen comprising: tyrosine hydroxylase inhibitor (i.e., alpha-methyl-DL tyrosine), melanin promoter (i.e., melanotan II), p 4503 a4 promoter (i.e., 5-diphenylhydantoin), and leucine aminopeptidase inhibitor (i.e., N- [ (2S, 3R) -3-amino-2-hydroxy-4-phenylbutyryl ] -L-leucine). These compounds were administered on each of five days per week for a period of six weeks with one or two days off between weekly cycles. Blood glucose levels were monitored in all subjects every two weeks. Blood glucose levels were determined by daily blood glucose testing followed by laboratory blood glucose testing every two weeks.

After about two to four weeks, all nine subjects had normal blood glucose levels: defined as a fasting plasma blood glucose level of 125mg/dl or less in two different cases.

Overall, subjects were well-tolerated for the above-mentioned treatments, had no treatment-related adverse events, and had recorded 100% of the treatment responses.

Example 2

Patients were screened and evaluated for the extent to which they met the DSM-V criteria for autism spectrum disorders. These subgroups meeting the criteria are administered treatment regimens comprising: tyrosine hydroxylase inhibitor (i.e., alpha-methyl-DL tyrosine), at a dose of 50 to 100mg, 3 times per day. Another subgroup also included a treatment regimen with a daily dose of 30mg of p 4503 a4 promoter (i.e., 5-diphenylhydantoin). Gamma-aminobutyric acid at a dose of 15mg is optionally administered to both subgroups at bedtime to aid sleep and to quiet activities (ticks) and repetitive behaviors (e.g. bruxism). Vasopressin is administered as needed to assist brain management. After each administration of the treatment regimen, the subject is again assessed for changes in the extent to which the DSM-5 criteria are met.

Example 3

Blind, randomized, placebo-controlledphase 2 study for use of AMPT in the treatment of autism

Research and design: blinded, randomized, 2-group, 8-week treatment period followed by additional follow-up visits with treatment discontinuation for the next 18 weeks for a total of 26 weeks of study participation. The treatment group consisted of LI-79 alone or placebo.

Sample amount: L1-79N-30 and placebo N-10.

Study population: the investigator considered autistic patients over 12 years of age who met the inclusion criteria and performed well.

The major inclusion criteria were: endorsement of informed consent, normal clinical laboratory values, DSM-5 compliance with autism diagnosis, qualified ADOS score, sufficiently high functionality to complete the regimen, no psychotropic drugs for at least 2 weeks, ABC-C score > 12.

The main exclusion criteria were: fragile X syndrome, epilepsy, use of free replacement drugs, any comorbidity (co-morbidity), other psychiatric disorders, outside of laboratory values.

Experiment treatment: LI-79 is a medicament

In racemic form. It will be administered daily for 8 weeks and then continued onweeks 10, 13 and 26.

Non-experimental treatment: a placebo.

Dosage and administration: randomized LI-79 or placebo will be administered orally at a dose of 90mg TID.

Evaluation protocol: patients will receive the above protocol for 8 weeks with weekly treatment assessments at 1 to 8 weeks, followed by post-treatment follow-up visits atweeks 10, 13 and 26.

And (3) safety measurement: regularly arranging complete historical records and physical examination, vital signs,CBC, differential, platelet count, urinalysis, serum enzymes include: total protein, albumin, glucose, BUN, creatinine, direct and total bilirubin, alkaline phosphatase, phosphorus, calcium, aspartate aminotransferase ("AST"), alanine aminotransferase ("ALT"), sodium, potassium, chloride, bicarbonate, T₄TSH and adverse event assessment. The stand alone DMC will oversee the performance of the test to ensure patient safety.

Duration of study: up to 50 weeks (12 weeks enrollment, 38 weeks treatment and follow-up).

Study endpoint: the primary endpoint will be the attending physician's assessment as reflected in the Clinical Global Impression (CGI) scale, which is based on the following changes: self-baseline in various psychological tests, including abnormal behavior checklist-community (ABC-C), conners parental rating scale, and autism diagnosis observation protocol (ADOS), as well as their personal observations in the clinic, and videotaping information taken at regularly scheduled clinical visits (according to the protocol) and provided by caregivers during the course of the study.

Example 4

α -methyl-DL tyrosine (AMPT) was administered to 3 patients. The patients in this group were eligible for and treated with the DSM-5 definition of autism. See table 1.

TABLE 1

Patient numbering	Age (age)	Sex	Dosage form	Adverse events
					01-001	3	Male sex	90TID	Is free of
01-002	15	Male sex	90TID	Is free of
					01-003	11	Male sex	90TID	Is free of

Abnormal behavior checklists-communities (ABC-C), autism diagnostic observation regimens (ADOS), Conans Parental Rating Scale (CPRS) and global clinical impression scale were used to assess the disease. Video is taken at each visit.

Data for two of these patients three weeks prior to the observation are shown in the table below. The conars parental rating scale data is shown in fig. 1. These clinical improvements appear to start rapidly and are durable. Sustained improvement over weeks and months has been observed. No adverse events were reported except mild fatigue in both patients on the first day of dosing.

TABLE 2

TABLE 3

TABLE 4

Example 5

Evaluation of L1-79 administration in patients with autism

L1-79 was used anecdotally in two patients, followed by a more structured assessment in another 8 patients with autism. All patients were administered a starting dose of L1-79 of 90mg TID for at least 3 months. During the evaluation, doses up to 400mg TID were used. A summary of the available data on these 10 patients is provided in the clinical efficacy summary, the use of L1-79 for the treatment of autism, filed in IND128673, serial No. 0005. Despite the small sample size and the open label related to L1-79, the results were encouraging with a continuing improvement in ABC-C range and an average reduction of 30% in ADOS-2 and one child exhibiting a 60% reduction in ADOS-2, which resulted in a loss of his diagnosis of autism. In addition, longer treatments result in a greater magnitude of therapeutic benefit to the core symptoms of autism. Furthermore, when study drug L1-79 was discontinued, not all of the benefits subsided. These data indicate that L1-79 has the potential to ameliorate the core symptoms of autism.

Example 6

Randomized, double-blind placebo-controlled 4-week study in male patients diagnosed with autism

The purpose of this clinical study (referred to herein as "study HT 02-121"; i.e., example 6) was to determine whether L1-79 is a well-tolerated and clinically useful agent for the treatment of ASD, as well as to evaluate PK and Pharmacodynamics (PD) for four weeks with TID dosing of L1-79. Although the preliminary study of example 5 included open label treatment with L1-79 for at least three months, the study was only conducted for 4 weeks, based on the limitations of available toxicological data. Based on the shorter duration, the response to L1-79 in study HT 02-121 was expected to be smaller compared to the longer duration pilot study.

The clinical study was a randomized, double-blind, placebo-controlled two-cohort, 4-week dose escalation study incorporating two open label treatment groups to assess the safety and efficacy of L1-79100 mg and 200mg TID in male patients aged 12 to 21 years diagnosed with autism. The first cohort of 20 patients consisted of three groups of patients: five patients in the first group received open label L1-79L 00mg TID and performed PK and EKG assessments. The remaining two groups in the cohort consisted of 15 patients randomized on a 2: 1 basis to receive L1-79100 mg TID or placebo. PK and security data from open label L1-79100 mg TID set are submitted to FDA for review and acceptance prior to second queue inclusion. The second queue is programmatically identical to the first queue. The 20 patients of the second cohort consisted of the same three groups of patients, but the dose used was 200mg TID, instead of 100mg TID. The major inclusion criteria are as follows: (1) males between 13 and 21 years of age; (2) DSM-5 compliant diagnosis of autism spectrum disorder confirmed by an autism diagnosis interview examination (ADIR) and an ADOS-2 score consistent with autism diagnosis; (3) the study period must be stable with no more than one concomitant medication and there are no planned changes in psychosocial interventions.

The major exclusion criteria were as follows: (1) sexually active men, (2) have a history of fragile-X syndrome, Rett syndrome, or any other comorbidity including, but not limited to, cancer, genetic disease, or any disease or syndrome requiring drug treatment, (3) DSM-5 diagnosis of schizophrenia, schizoaffective disorder, alcohol use disorder, or Attention Deficit Hyperactivity Disorder (ADHD), (4) have any active medical problem, including epilepsy and asthma, (5) uncontrolled complications including, but not limited to, persistent or active infection, symptomatic cardiovascular disease, liver disease, kidney disease, skeletal muscle disease, Human Immunodeficiency Virus (HIV), hemorrhagic cerebrovascular accident (HCVA), Hepatitis B Virus (HBV) or psychiatric/social conditions that would limit compliance with research requirements, (6) any disease requiring long-term treatment, (7) any disease requiring treatment with immunosuppressive agents; and/or (8) current or lifetime diagnosis of severe psychiatric disorders.

Open label patients

The first five patients in each cohort were assigned to receive the active drug in an open label manner. Patients were treated as randomized patients, except for: (1) blood and urine samples were drawn at baseline and 1 hour after dosing, and at 1 hour after dosing attreatment visits 1, 2, 3, and 4 weeks to determine PK of L1-79, (2) EKG was assessed at baseline andtreatment visits 1, 2, 3, and 4 weeks and within 3 days before follow-upvisits 1 and 4 weeks after dosing, (3) vital signs, physical examination, and ASD assessments were performed atvisit 1 week after dosing, (4) blood and urine samples were drawn for safety analysis at baseline and atvisit 1 hour after dosing, and at follow-upvisits 1 week and 4 weeks after dosing.

Randomized patients

Randomized patients were treated as open label patients, except for three: (1) no blood sample was drawn for PK; (2) no ECG was performed; and (3) these patients had no 1 week follow-up visit, only 4 weeks follow-up visit. The time and event protocol for this study is provided in table 5 below.

TABLE 5 time and event scheme

Safety endpoint

Safety was the primary endpoint of the study. The following safety endpoints were evaluated: ECG, physical examination, laboratory evaluations (hematology, chemistry and urinalysis), vital signs including orthostatic blood pressure, Adverse Events (AE) and concomitant medications.

Efficacy endpoint

The primary efficacy endpoint was the determination of clinical improvement recorded by investigators according to the Clinical Global Impression (CGI) rating scale. Additional efficacy endpoints included: (1) change from baseline in socialization and communication range of the wenlan adaptive behavior scale, second edition (VABS II) parental/caregiver rating scale; (2) changes from baseline in ADOS-2 total and sub-scores, (3) changes in the social response scale, second edition (SRS-2) total and subscales, (4) changes in the ABC-C range, (5) changes in the repeat behavior scale-revision (RBS-R) total and subscales. Only descriptive statistics were planned in view of the exploratory design of the study.

Results

Study population

Patient recruitment and scheduling is summarized in figure 2.A total of 42 patients were randomized and received at least one dose of study drug. One patient was randomized to open label L1-79200 mg TID, but parents were asked to voluntarily withdraw the study atweek 0/baseline after receiving a single dose of study drug in the clinic. Thirty-nine patients completed the study. One patient treated with double-blind L1-79200 mg TID withdrawed from the study due to AE (see table 8) and one patient treated with placebo voluntarily withdrawn from the study. Demographic characteristics are summarized in table 6.

TABLE 6 demographic characteristics

¹One patient was randomized to open label L1-79200 mg TID and received only a single dose of study drug. The table does not contain data from this patient.

²There was significant confusion between the two randomized patients (subjects 01-008-01-009) who were serially enrolled at site 01 and received the number of drug cassettes on the same day. Since there is sufficient uncertainty about the treatment actually received by both patients, a conservative analysis of the data was performed by omitting both patients from the efficacy analysis. Data from both patients were included in the study population and safety analysis (however, no AE was reported by both subjects).

Safety feature

Concomitant medication and physical examination

Most patients did not take CNS drugs during the study. The following CNS drugs were used during the study: clonidine (n-2),

(n＝1)、

(n-3), lorazepam (n-1),

(n-1) and

(n-1). No clinically significant physical examination findings were reported during the study.

Adverse events

The incidence of AEs by major system organ category and preferred entries is shown in table 7, and a list of AEs reported during the study is shown in table 8. All AEs were mild to moderate in intensity and self-limiting.

TABLE 7 incidence of adverse events by major System organ categories and preferred entries

TABLE 8 list of adverse events

DB is double blind; OL ═ open label

¹AE reported at 4 weeks follow-up visit

²AE reported before treatment

Of the patients who experienced AE (34.1%, 14 of 41 patients), most experienced AE of mild (24.4%, 10 of 41 patients) to moderate (14.6%, 6 of 41 patients) intensity. Only three AEs were reported by more than one patient. Two patients in the open label 100mg group and one patient in the double-blind placebo group reported intentional self-injury as treating an emergency AE. Open label two patients in the 100mg group reported stressful as a treatment emergency AE. For one of the patients (02-002) reporting intentional self-injury and irritability, these symptoms appeared within days of withdrawal from active treatment (i.e., after completion of week 4). For another patient (02-001), symptoms began at the time of active drug treatment. The presence of urinary cystine crystals atvisit week 4 in two patients in the double-blind 200mg group was reported as an untreated emergency AE.

No Serious Adverse Events (SAE) or deaths were reported during the study. Due to adverse events, a total of 2 patients were withdrawn from the study: 1 patient in the double-blind 200mg group experienced a treatment emergency AE of grand mal seizure and 1 patient in the double-blind placebo group experienced a treatment emergency AE of intentional self-injuring and agonizing.

Vital signs

Orthostatic vital signs were collected at each visit as described in study HT 02-121. No changes were observed that met the orthostatic hypotension criteria (i.e., a decrease in systolic pressure of 20mm Hg, a decrease in diastolic pressure of 10mm Hg, or an increase in heart rate of 30 times/min). A small number of subjects showed no symptomatic drop in systolic and/or diastolic blood pressure and an increase in heart rate atweek 1 orweek 2 of treatment, but these were remitting atweek 4 and mild in nature (e.g., systolic blood pressure drop of 10mm Hg, diastolic blood pressure drop of 5mm Hg and heart rate increase of 15 to 24 times/min). Fig. 3 provides an example of the maximum change from the baseline and demonstrates that these changes are transient, far below the criterion of erectility.

ECGS and laboratory evaluation

No clinically significant changes from baseline EKG were noted in any of the patients. Urinalysis did not reveal findings associated with clinical symptoms. Notably, crystalluria was observed in half (50%) of the patients taking L1-79 compared to 30% of placebo-treated patients. Of the population that appeared to have crystallized urine during the study, approximately half (50%) had concentrated urine (specific gravity > 1.025). Most of the crystallized urine consists of calcium oxalate crystals. There were no symptoms associated with crystalluria.

One patient presented with a brief mild elevation of asymptomatic amylase at week 4 (146U/L, upper limit of normality [ ULN ] 125U/L), which was resolved by the 4-week follow-up visit.

No significant elevation of AST or ALT or any other chemical parameter was noted in the safe population.

Hematological parameter evaluation revealed no significant deviation in the safe population.

Pharmacokinetics

Pharmacokinetics were assessed in open label 100mg and 200mg TID patients. Onday 1, L-79 concentrations were assessed one hour after dosing. Subsequently, randomized L1-79 plasma concentrations were assessed atweeks 1, 2, 3, and 4. Plasma concentrations of 100mg and 200mg TID were 0 (< 2.5) to 736ng/mL and 23 to 1680ng/mL, respectively, 1 hour after administration onday 1. A summary of the individual patients and the combined ensemble-averaged total plasma concentrations of L1-79 is shown in figure 4. As shown in fig. 5 and 6, the overall random L1-79 plasma concentrations were relatively stable fromweek 1 toweek 4.

Efficacy

At the beginning of the study, long-term adolescent and reproductive toxicology data were lacking. Applicants conducted a short study lasting 28 days, with limits on the number, sex, and age of patients admitted to the study. As a result, the study was not designed or driven to demonstrate the statistical significance of any efficacy endpoints. Thus, as expected, none of the results measured statistical significance. Anticipated and observed are positive trends in a variety of devices consistent with improvement in the core symptoms of autism. In fact, there are a variety of efficacy measures that demonstrate similar indicators of improvement in treating the target cardiac social range affected by ASD. In addition, only the efficacy data for patients receiving blind treatment are presented.

Measurement of results

Duringcohort 1 andcohort 2 of the present study, there were some differences in how the questionnaires were managed. In the preliminary study, all tables were completed at the study site in the presence of investigators. Incohort 1 of the study, VABS-II, SRS-2, ABC-C and RBS-R were filled out by patient/patient's family at home prior to the treatment visit and returned at the treatment visit to expedite study performance. VABS II, SRS-2, ABC-C and RBS-R duringcohort 1 were not evaluated at the treatment visit and as a result, it was difficult for the family members to fill out the form correctly. To improve the quality and integrity of the questionnaires duringcohort 2, all questionnaires (except ADOS-2[ both cohorts managed by an externally certified ADOS-2 test administrator ] and CGI [ completed by investigators in both cohorts ] were completed by the primary investigator with the assistance of the patient/patient's family at the pre-specified treatment visit. Sincecohort 2 data was more reliable, the discussion of results focused on comparing L1-79200 mg to placebo, with the exception of ADOS-2 and CGI, where L1-79100 mg and 200mg were compared to placebo.

After the study was completed, it was found that the base anchor for VABS-II was not always properly established. Furthermore, only two sub-ranges (communication and socialization) were completed for the patient, so that no range or total score could be obtained. However, the results regarding the extent of VABS II communication and socialization are shown below.

A list of efficacy measurements is summarized below (table 9). For those measurements that indicated a positive trend in favor of L1-79, three sets of numbers are shown for each efficacy measurement. These numbers consist of: a) line graphs showing comparative changes between 3 treatment groups from screening/baseline to 4 weeks (in some cases followed by 4 weeks post treatment time points), b) individual patient response plots, and c) responder analysis plots. For those efficacy measurements that did not show a positive trend in favor of L1-79, only line graphs showing comparative changes between the 3 treatment groups from screening/baseline to week 4 (in some cases followed by 4 weeks post treatment time points) are shown.

Responder definition:

in addition to analyzing the data by observing the magnitude of change in each efficacy endpoint of patients exposed to the active drug as compared to placebo, it is also important to define a "responder" population. Generally, responder analysis is aimed at focusing on the number of patients showing any benefit, rather than the overall variation in the study population. Generally, responders are defined as patients whose target symptoms show a pre-specified improvement at the efficacy endpoint. This definition is in accordance with accepted standards for judging intermediate and long term outcomes of experimental treatments studied in almost every disorder, including autism. However, it does not take into account the need for a precise definition in the study of short duration of diseases with amplitude of symptomatic severity (such as autism). In ASD, the behavioral, social and emotional regulation of children is usually subject to a mild to moderate period of improvement or worsening, which is associated with a variety of determinable and yet undetermined factors (Boso, 2010; Jyonouchi, 2011). Thus, in a short duration study, the "baseline" measurement may actually be at the peak, midpoint, or lowest point of one of these amplitudes. If the target symptoms under study are measured at baseline, they are at the peak or midpoint, then the short duration study may only have an opportunity to demonstrate initial efficacy by preventing the target symptoms from returning to their lowest point of amplitude. To illustrate this possibility in some study patients, the responder definition included patients who exhibited short-term stability or improvement in target symptoms. Accordingly, for each efficacy endpoint that exhibited a positive trend, a responder analysis was performed as defined herein.

A tabular summary of efficacy measurements from screening/baseline changes is provided in table 9.

Table 9 summary comparison of mean change from screening/baseline in force measurements atweek 4 or LOCF for placebo.

LOCF (local locked Loop) last observation value conversion method

Clinical Global Impression (CGI) rating scale

The CGI-overall severity (CGI-S) and CGI-overall improvement (CGI-I) over time of L1-79 compared to placebo are shown in graphical form in fig. 7 and 8. The mean CGI-S change from baseline atweek 4 or the last observation for push to effect (LOCF) is shown in fig. 9 and indicates an average 0.5 point increase in overall CGIs for patients in the 200mg group compared to CGIs in the placebo and 100mg groups.

A summary of patient changes from baseline toweek 4 in CGI-S is shown in fig. 10. In addition, responder analysis (defined as improvement) for CGIS atweek 4 or LOCF is shown in fig. 11.

Responders to CGI-S are defined as improving only (rather than improving or not changing) because, unlike other measurements taken weekly, CGI-S requires the clinician to give an overall assessment of the severity of clinical symptoms of the patient based on all outcome measurements and the patient' S movement throughout the study.

Responder analysis of CGI-S atweek 4 showed a clear dose response improvement trend for the L1-79100 mg and 200mg groups compared to placebo.

Wen lan adaptive behavioral Scale, second edition (VABS II)

The changes from baseline over time in the VABS II normalized socialization score and the VABS II normalized communication score for L1-79 compared to placebo are shown graphically in fig. 12 and 13, respectively.

Autism diagnostic observation protocol, second edition (ADOS-2)

Changes over time in the ADOS-2 total score screening, restricted and repeat behavior total score and social impact total score compared to placebo for L1-79 are shown graphically in fig. 14, 15 and 16. Although not generally used as a measure of outcome, the change in ADOS-2 over a short period of time is consistent with the earlier shown open label pilot study of longer duration.

The consistency of this effect among multiple patients is shown by the summary of the change from screening toweek 4 in the ADOS-2 total score shown in figure 17.

Social response Scale, second edition (SRS-2)

The changes from baseline over time in SRS-2 total T score, SRS-2DSM-5 social, communication and interaction T score, SRS-2 social communication T score, SRS-2 social motivation T score, and SRS-2DSM-5 restriction and repetitive behavior T score compared to placebo are shown graphically in fig. 18, fig. 19, fig. 20, fig. 21, and fig. 22, respectively.

The overall SRS-2, SRS-2DSM-5 social communication and interaction, SRS-2 social communication and SRS-2 social motivation T scores in the L1-79200 mg treatment group improved by nearly 8 points or more on average (fig. 18, fig. 19, fig. 20, and fig. 21). The social communication and interaction scale consists of the DSM-5 standard, which compensates for the social communication and social interaction deficiencies required for ASD diagnostics. It is of considerable interest to find that patients with a baseline score of 60 to 83 (8 points or more drop) are likely to exhibit categorical changes in clinical severity because the "normal range" (lower than 60T score), the "mild range" (60 to 65T score), the "medium range" (66 to 75T score) and the "severe range" (76T score or higher) of symptom severity are defined within these T score ranges.

A summary of the change from baseline toweek 4 in SRS-2 total T score, SRS-2DSM-5 social, communication and interactive T score, SRS-2 social communication T score and SRS-2 social motivation T-score for each patient is shown in fig. 23, fig. 24, fig. 25 and fig. 26, respectively.

Both the magnitude and number of responders observed at L1-79200 mg were greater for SRS-2 total, SRS-2DSM-5 social communication and interaction, SRS-2 social communication and SRS-2 social motivation T score compared to placebo (fig. 27, 28, 29 and 30). For the SRS-2 total T score, 3 patients in the L1-79200 mg group had sufficient improvement to change the severity classification compared to 2 in both the L1-79100 mg group and the placebo group. For the SRS-2 social motivation T score, 6 patients in the L1-79200 mg group exhibited a categorical change in clinical severity compared to 4 and 3 patients in the 100mg and placebo groups, respectively. For the SRS-2DSM-5 social, communication, and interactive T scores, 4 patients in the 200mg group had an alteration in severity classification compared to 2 and 3 patients in the 100mg and placebo groups, respectively. Similarly, for the SRS-2 social communication T score, 4 patients in the 200mg group showed improved classification compared to 3 in both the placebo and 100mg groups.

Responder analyses (defined as improvement or no deterioration) atweek 4 or LOCF for SRS-2 total T score, SRS-2DSM-5 social, communication and interaction T score, SRS-2 social communication T score and SRS-2 social motivation T score are shown in figures 27, 28, 29 and 30, respectively.

Figures 27 to 30 show that the percentage of responders at L1-79200 mg (as defined by improvement or lack of deterioration) was greater compared to placebo; see "responder definitions" (section above).

Abnormal behavior checklist-Community (ABC-C)

The changes from baseline over time in the range of ABC-C lethargy and social withdrawal, ABC-C inappropriate speech, ABC-C hyperactivity and noncompliance, ABC-C stereotyped behavior, and ABC-C excitability and agonism for L1-79 versus placebo are shown graphically in fig. 31, fig. 32, fig. 33, fig. 34, and fig. 35, respectively.

A summary of the patient's changes from baseline toweek 4 in the ABC-C range of lethargy and social withdrawal and ABC-C range of inappropriate speech are shown in fig. 36 and 37, respectively. Although the mean change between the 200mg and placebo groups did not differ from the baseline score, responder analysis did indicate a greater trend for patients in the 200mg group to respond to improved or no worsening in behavior compared to placebo, as shown in fig. 38 and 39, respectively.

As with SRS-2, both the number of responders and the magnitude of the response indicate a trend toward social and verbal range differentiation to ABC-C for those patients given L1-79200 mg. Also noteworthy, the stability or improvement of these ranges was observed in 90% to 100% of patients with L1-79200 mg, which are known to vary significantly in intensity over a shorter period of time.

Repeat behavior Scale-revision (RBS-R)

The changes from baseline over time in RBS-R total score, RBS-R limiting behavior, RBS-R instrumental behavior, RBS-R equal behavior, RBS-R compulsive behavior, RBS-R stereotyped behavior, and RBS-R self-injurious behavior for L1-79 compared to placebo are shown graphically in fig. 40, fig. 41, fig. 42, fig. 43, fig. 44, fig. 45, and fig. 46.

There was an average improvement in the total score of RBS-R as indicated by a reduction in the L1-79200 mg score compared to placebo. (FIG. 40).

A summary of the patient's change from baseline toweek 4 in the RBS-R total score is shown in figure 47. In addition, responder analysis (defined as improvement or no deterioration) of RBS-R total scores atweek 4 or LOCF is shown in figure 48.

In agreement with ABC-C lethargy and social withdrawal and ABC-C inappropriate speech range, the RBS-R total score showed a clear trend of greater magnitude of reduction in restricted and repetitive behavior of L1-79200 mg compared to placebo. This was further demonstrated by a responder analysis, which showed that 91% of patients treated with L1-79200 mg had improved or stable symptoms compared to 78% treated with placebo.

General conclusion

Currently available treatments for children and adults with ASD are directed only to the incidental symptoms associated with the disorder (irritability, agitation, impulsivity, hyperactivity), and have the side effects of requiring monitoring of metabolic parameters by blood tests. Although phlebotomy (phlebotomy) is unpleasant for most neurologic children and adults, it is quite traumatic for those who fail to adequately convey the extreme sensory sensitivity of their fear. In fact, this is a trap for these individuals, since the many abnormal behaviors they exhibit are most likely due to the inability to interact, communicate and contact with others. Providing them with treatments that not only improve the core symptoms responsible for these behaviors, but also the potential to treat without invasive monitoring would greatly contribute to improving the quality of life of these children.

The results from study HT 02-121 provide proof-of-concept that L1-79 was shown to provide benefits in treating the core symptoms of ASD. ASD is defined primarily as persistent deficits in social communication and social interaction, as well as restricted and repetitive patterns of behavior, interests, or activities. Preliminary evidence from multiple independent assessments dedicated to measuring both social and behavioral intensity of these core symptoms suggests a consistent trend that is repeatable across multiple devices. While the studies are of limited scale and short duration, excluding any expectation or ability to show statistically significant improvement in the resulting measurements used, the agreement between the various measurements utilized in the present study is highly encouraging. Blind assessments by the study participating clinicians showed a change of nearly 1 point in CGI-S compared to baseline over less than one month of treatment. Similarly, the ADOS-2 score for patients receiving L1-79 increased by approximately 1 point from baseline over the same period.

Previous experience with L1-79 open label administration in 10 patients with autism (see example 5) clearly demonstrated that L1-79 has the potential to ameliorate the core symptoms of autism. In study HT 02-121, multiple independent potency measurements (including CGI-S, ADOS-2, SRS-2, ABC-C, and RBS-R) demonstrated sustained improvement in the social range of target scores affected by ASD despite short treatment periods and small patient numbers. L1-79 was safe and well tolerated over a 28 day treatment period. At the end of the study, many parents hoped their children to continue to receive treatment with L1-79. As a result, round table videos were taken at two clinical sites, intended to allow these parents to discuss the effects of L1-79 on their children with autism directly with the FDA.

Example 7

L1-79 randomized, double-blind, placebo-controlled adaptation test for the treatment of core deficits in social communication function and adolescents and adults with autism spectrum disorders

Completed study HT 02-121 (example 6) is a phase II safety study of L1-79 for treatment of autism. Study HT 02-121 was a randomized, double-blind, placebo-controlled, two-cohort, 4-week dose escalation study incorporating 2 open-label treatment groups to evaluate the safety and efficacy of L1-79100 mg and 200mg TID in male patients aged 13 to 21 years with autism. The results from study HT 02-121 provide proof-of-concept that L1-79 was shown to provide benefits in treating the core symptoms of ASD. Preliminary evidence from multiple independent assessments dedicated to measuring both social and behavioral intensity of these core symptoms suggests a consistent trend that is repeatable across multiple devices. While the limited size and short duration of the study precludes any desire or ability to show a statistically significant improvement in the resulting measurements used, the agreement between the various measurements utilized in the present study is highly encouraging. After reviewing the results from study HT 02-121, the FDA allowed L1-79 to begin the registration trial to gain market approval and granted the L1-79 IND128673 Rapid channel name (Fast Track designation).

As a result, the inventors are proposing an adaptation test approach in a phase III environment that will allow a convenient and thorough approach to evaluate L1-79 as a treatment for treating core deficits defined in social interactions and interactions. Based on the results from study HT 02-121 (example 6), two phase III randomized, double-blind, parallel group, placebo-controlled clinical studies (study 301 and study 302) using an adaptation design were presented to evaluate the best inclusion criteria, sample amounts and outcome measures during the first part of study 301, quantifying the safety and efficacy of administering LI-79 at 200 or 300mg TID doses to subjects diagnosed with autism in a prospective randomized and double-blind fashion based on their autism diagnostic observation protocol 2(ADOS-2) results.

Most of the clinical experience with L1-79 for the treatment of autism was with doses of 100 to 200mg TID. The study of example 8 (also referred to herein as "study 301") attempted to quantify the safety and efficacy of L1-79 administration at 200 or 300mg TID doses to subjects diagnosed with autism in a prospectively randomized, double-blind fashion based on their autism diagnostic observation protocol-2 (ADOS-2) results.

The dosage form is a capsule containing 100mg DL- α -methyl-p-tyrosine capsules (hereinafter referred to as "L1-79"). The expected administration regimen of L1-79 for the phase III clinical study (study 301 and study 302) was oral administration of 2 or 3capsules 3 times per day (TID).

The principle of research and design:

ASD are obstacles characterized by defective social interactions and the presence of behavioral, interesting or activity limitations, repetitive patterns during child development (Swedo, s.e., Baird, g., Cook, e.h., Happe', f.g., Harris, j.c., Kaufmann, w.e., Wright, Harry, H. (Eds.) (2013) neuro-elastic disorders, in American Psychiatric Association, diagnostic and statistical management (5 th.) American Psychiatric Association). Recent literature suggests that the peripheral and autonomic nervous system is involved in children and adults with ASD (Baker, 2017; Fenning, 2017). As a result, electrodermal (electrodermal) skin tests may serve as important biomarkers for patient populations more likely to respond to treatments targeting the sympathetic nervous system.

The main aims are as follows:

the main objective of this study was to evaluate the efficacy, safety and tolerability of L1-79 compared to placebo for the treatment of core deficits in social communication and interaction in adolescents and adults with Autism Spectrum Disorder (ASD).

Secondary objective:

secondary goals of the study included the following:

1. evaluating optimal inclusion criteria, sample amounts and result measurements during a first portion of the adaptation test;

2. evaluating the effect of L1-79 compared to placebo in reducing repeat and limiting behavior in ASD;

3. evaluating the effect of L1-79 compared to placebo in reducing abnormal behavior in ASD, including hyperactivity, agitation and irritability; and

4. additional modeling of Pharmacokinetics (PK) and Pharmacodynamics (PD) of L1-79 in a subset of subjects was performed by time-intermittent sparse sampling.

Research and design:

eligible participants will be adolescents and adults aged 12 to 21 years who, based on clinician interviews and assessment of the ASD symptoms of ADOS-2, and a clinical global severity impression (CGI-S) rating of 4 or higher (moderate or higher), meet diagnostic and statistical manual for psychiatric disorders of ASD-release 5 (DSM-5) criteria.

Subjects will be randomized to placebo, L1-79200 mg or L1-79300 mg three times per day (TID) groups at a 1: 2 ratio.

Subjects in the 200 and 300mg groups will begin to take 200mg TID. Subjects in the 300mg group will titrate up to 300mg TID after 7 days.

Screening evaluations would include ADOS-2, DSM-5 and CGI-S review of ASD standards.

The adaptive design will be used with the proposed interim analysis for making decisions on the following criteria; potential endpoints for the primary outcome, modifications to the study population and estimated sample size have been defined. The data monitoring committee and independent statistical team will perform interim analyses and suggest to the sponsor based on predetermined criteria for the changes proposed in the study. The sample size recalculation will depend on the primary outcome and potential study population to be determined in the interim analysis. Sample size recalculation will be based on blind data.

The event schema is provided in table 30.

TABLE 30 event schema

TABLE 30 event schema

Checking an table-community by using ABC-C as an abnormal behavior; ADOS-2 ═ autism diagnostic observation scale-2; ASD ═ autism spectrum disorder; clinical results of CGI-C

Global change impression; CGI-S is an impression of overall clinical severity; DSM-5 ═ diagnostic and statistical manual for psychiatric disorders-5 th edition;

PSI is the index of parental stress; RBS-R ═ repetitive behavior scale-revision; SAS — Spence anxiety scale; SSP ═ Spence anxiety scale; SRS-2 ═ social

Response scale-2; VABS-3 ═ wenlan adaptation behavior scale-version 3; WASI-2 ═ Weschler intelligence reduction scale

¹Including review of previous/ongoing medications

²Complete examination of skin, head, eye, ear, nose, throat, neck, thyroid,Assessment of lung, heart, abdomen, lymph nodes, extremities and body weight

³Height was measured only at screening.

⁴Partial inspection to update the results of the inspection at screening

⁵Including respiratory rate, oral temperature, sitting and standing upright blood pressure and pulse

⁶Including amphetamines, barbiturates, cocaine metabolites, opioids, benzodiazepines

Steroids, cannabinoids and cotinine

⁷For women only after menstrual onset

⁸Subjects will only have a random PK for one ofvisits 2, 3 or 4.

⁹Including serum chemistry, hematology (including coagulation), and urinalysis

¹⁰AdOS-2 will be videotaped, potentially serving as an additional review by blind reviewers for mutual social interaction

¹¹Adapting to behavioral composite range only

Inclusion criteria were:

1. the subject must be a male or female adolescent or adult up to 21 years of age

2. The subject must be between 13 and 21 years of age

3. Women after a menstrual initial tide (if appropriate) must undergo birth control.

4. ASD were diagnosed based on assessment tools using DSM-5 criteria [ e.g., autism symptom scale (ASRS), childhood autism scale-2 (CARS2), or autism interview-revision (ADI-R) ] and confirmed by ADOS-2(CGI-S score 4 or higher).

5. The subjects had to be stable with no more than one concomitant medication and had no planned changes in psychosocial intervention during the trial

6. Subjects and caregivers must be willing and able to participate in a testing procedure sufficient to obtain a test effectiveness score as used herein.

7. The subject must have a caregiver who has recognized that they are more than a year, spend at least 10 hours per week with them, and would like to accompany them with each appointment.

8. The subject must appear to the investigator to be sufficiently tolerant and able to meet the requirements of the trial. For example, patients who are intolerant of blood draw or ECG will not be qualified candidates for the present study.

9. The subject must be able to swallow the capsule

10. The subject and its caregiver must be willing to sign an informed consent form, or have an informed consent form provided by their legal guardian or agent. All subjects < 18 years of age or those who are unable to self-care must have the caregiver's consent.

Exclusion criteria:

1. sexually active males and females

2. Uncontrolled complications include, but are not limited to, persistent or active infection, symptomatic cardiovascular disease, liver disease, kidney disease, skeletal muscle disease, HIV, HCVA, HBV, or psychiatric/social conditions that would limit compliance with research requirements.

3. Any disease requiring treatment with an immunosuppressant

4. A diagnosis of fragile-X syndrome, Rett syndrome, or other neurological disorder that may underlie the symptoms of autism in a subject (e.g., congenital or acquired brain injury, brain malformation, stroke, neurogenetic disorder, or metabolic disorder).

DSM-5 diagnosis of schizophrenia, schizoaffective disorder, alcohol use disorder or ADHD, current or lifetime diagnosis of severe psychotic disorder (e.g., bipolar disorder, etc.);

6. there are any active chronic medical problems including, but not limited to, uncontrolled seizures, heart disease, cancer, asthma, genetic disease or any disease or syndrome requiring sustained drug therapy.

7.A subject in need of more than one drug for treatment of autism, or a subject that has not been weaned to its lowest tolerable drug dose.

8. A subject suffering from any disease requiring treatment with an immunosuppressive drug.

9. There are out of range liver or kidney function tests or other unexplained abnormal laboratory values that investigators consider clinically meaningful.

10. Are unwilling or unable to give informed consent to any subject or caregiver.

Study population:

350 patients are planned.

Test product, dosage and mode of administration:

the encapsulation was 100 and 200mg of D-L α -methyltyrosine (L1-79). Two dose groups will be tested, 200mg TID (one 200mg dose combined with placebo) and 300mg TID (one 200mg capsule and one 100mg capsule).

Duration of treatment:

12 weeks (84 days)

And (3) evaluating the efficacy:

baseline assessments not included in the efficacy measurements included the Weschler mental reduction Scale (WASI-2) and Spence Anxiety Scale (SAS). The electrodermal test will be performed at baseline to determine if greater autonomic nervous system variability is relevant to treatment response. (see research principles). The primary outcome measure would be the change from baseline atweek 12 on a 5-element composite measure containing the Socialization (SOC), Communication (COM), and Daily Living Skills (DLS) ranges of the Adaptive Behavior Composite (ABC) from the wenlan adaptive behavior scale-version 3 (VABS-3), and the socialization, communication and interaction (SCI), and restricted interest and repetitive behavior (RRB) subscales of the social response scale-2 (SRS-2). The primary secondary outcome measure will be based on the change from baseline CGI-S atweek 12.

Additional secondary outcome measures will assess the change from baseline score atweek 12 in a gated manner (to be determined during interim analysis):

1. abnormal behavior checklist-community (ABC-C) social withdrawal/lethargy and inappropriate speech scope

ABC-C, irritability, hyperactivity and stereotypy range

3. Repeat behavior Scale-revision (RBS-R)

4. Parental Stress Index (PSI) profile

5. Sensory characteristics-simple form (SSP)

Spence Anxiety Scale (SAS)

ADOS-2 Total score (ADOS-2 will be videotaped at the end of screening and study, potentially for blind review of bi-directional interactivity.)

And (3) safety evaluation:

safety assessments include physical examination, orthostatic measurement of blood pressure and pulse, standard hematology and clinical chemistry assessments, concomitant drug use, urinalysis, ECG, and spontaneously reported adverse events.

Evaluation of PK:

to obtain a better understanding of PK and PD in patients with ASD by L1-79, sparse sampling will be used to assist in modeling PK parameters and comparing them to data obtained in more intensive studies conducted in young healthy adults. Sparse sampling will limit the number of phlebotomy procedures, which is an important consideration in children with ASD, which are often much more traumatic to a blood drawing procedure than peers who do not have ASD.

The statistical method comprises the following steps:

the adaptation design will evaluate three components of the study; the result variables, potential modifications to the inclusion criteria and recalculation of sample size based on decisions made at interim analysis times. Modification of inclusion criteria will be based on a potential ASD internal-table evaluation of baseline severity of the following features known to directly affect treatment and intervention strategies for patients with ASD: 1) severity of anxiety is assessed by a Weschler intelligence reduction scale and severity of destructive behavioral symptoms based on assessment of the Spence anxiety scale, based on an ABC-C irritability and hyperactivity scale. In addition, a skin reactivity test was performed to determine that autonomic sensitivity would be used as an exploratory biomarker for responder phenotypes (see reasonable reasons for the principles of research for these subgroups). The selection of primary results will be based on a conditional capability analysis of a five-factor composite measure containing Socialization (SOC), Communication (COM) and Daily Living Skills (DLS) ranges of the Adaptive Behavior Composite (ABC) from the wenlan adaptive behavior scale-version 3 (VABS-3) and socialization, communication and interaction (SCI) and restricted interest and repetitive behavior (RRB) subscales of the social response scale-2 (SRS-2). After selecting the results and inclusion criteria, the sample size will be recalculated based on the blind data.

It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.

Claims (28)

1. A method for providing a plasma concentration of a therapeutic agent for a long period of time to treat autism in a subject in need thereof, the method comprising administering to the subject a daily concentration of about 50mg (w/w) to about 1500mg (w/w) of the therapeutic agent, wherein the therapeutic agent is α -methyl-DL-tyrosine, wherein the plasma concentration is about 500ng/ml to 5000ng/ml, and wherein the period of time is at least 1 week.

2. The method of claim 1, wherein the concentration of the therapeutic agent is about 90mg (w/w /).

3. The method of claim 1, wherein the concentration of the therapeutic agent is about 100mg (w/w /).

4. The method of claim 1, wherein the concentration of the therapeutic agent is about 200mg (w/w /).

5. The method of claim 1, wherein the concentration of the therapeutic agent is about 250mg (w/w /).

6. The method of claim 1, wherein the concentration of the therapeutic agent is about 300mg (w/w /).

7. The method of claim 1, wherein the concentration of the therapeutic agent is about 350mg (w/w /).

8. The method of claim 1, wherein the therapeutic agent is administered in multiple divided doses.

9. The method of claim 1, wherein the therapeutic agent is administered for a duration of about 1 week to about 4 weeks or more.

10. The method of claim 1, wherein the plasma concentration is about 800ng/ml to 2500 ng/ml.

11. The method of claim 1, wherein the plasma concentration is about 1400ng/ml to 1800 ng/ml.

12. A method for treating an autism-related clinical profile in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of α -methyl-DL-tyrosine, thereby treating the autism-related clinical profile in the subject.

13. The method of claim 12, wherein the clinical characteristic is a deficit in social communication, a deficit in social interaction, a deficit in social motivation, lethargy and social withdrawal, inappropriate speech, hyperactivity, stereotyped behavior, irritability and agitation, restrictive behavior, repetitive behavior, ceremonial behavior, identity behavior, compulsive behavior, self-disabling behavior, or a combination thereof.

14. The method of claim 12, wherein the clinical profile meets the requirements of diagnostic and statistical manual V (DSM-V) standards for psychiatric disorders.

15. The method of claim 12, wherein the clinical profile is assessed based on changes from baseline in one or more psychological tests.

16. The method of claim 15, wherein the psychological test is based on a Clinical Global Impression (CGI) rating scale, a wenlan adaptation behavior scale (VABS), an autism diagnostic observation protocol (ADOS), a Social Response Scale (SRS), an abnormal behavior checklist-community (ABC-C), a Repetitive Behavior Scale (RBS), a Conners Parents Rating Scale (CPRS), or a combination thereof.

17. The method of claim 12, wherein the composition comprises α -methyl-DL-tyrosine in an amount of about 50mg (w/w) to about 1000mg (w/w).

18. The method of claim 12, wherein the composition comprises α -methyl-DL-tyrosine in an amount of about 90mg (w/w /).

19. The method of claim 12, wherein the composition comprises α -methyl-DL-tyrosine in an amount of about 100mg (w/w /).

20. The method of claim 12, wherein the composition comprises α -methyl-DL-tyrosine in an amount of about 200mg (w/w /).

21. The method of claim 12, wherein the composition comprises α -methyl-DL-tyrosine in an amount of about 250mg (w/w /).

22. The method of claim 12, wherein the composition comprises α -methyl-DL-tyrosine in an amount of about 300mg (w/w /).

23. The method of claim 12, wherein the composition comprises α -methyl-DL-tyrosine in an amount of about 350mg (w/w /).

24. The method of claim 12, wherein the composition is administered in multiple divided doses.

25. The method of claim 12, wherein the composition is administered three times daily.

26. The method of claim 12, wherein the composition is administered for at least 1 week.

27. The method of claim 12, wherein the composition is administered for a duration of about 1 week to about 4 weeks.

28. A method for providing a plasma concentration of a therapeutic agent for a long period of time to treat autism in a subject in need thereof, the method comprising administering to the subject a daily concentration of about 50mg (w/w) to about 500mg (w/w) of the therapeutic agent, wherein the therapeutic agent is α -methyl-DL-tyrosine, wherein the plasma concentration is about 500ng/ml to 5000ng/ml, and wherein the period of time is at least 1 week.

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