(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/210540 Al 07 December 2017 (07.12.2017) W ! P O PCT

(51) International Patent Classification: LEIBEL, Rudolph, L.; 464 Riverside Dr. # 1, New York, A61K 31/221 (2006.01) A61K 31/198 (2006.01) NY 10027 (US). COTTER, Sara; 1634 Walnut Avenue, A23L 1/302 (2006.01) Wilmette, IL 60091 (US). (21) International Application Number: (74) Agent: DAVITZ, Michael, A. et al; Leason Ellis LLP, One PCT/US2017/035655 Barker Avenue, Fifth Floor, White Plains, NY 10601 (US). (22) International Filing Date: (81) Designated States (unless otherwise indicated, for every 02 June 2017 (02.06.2017) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (25) Filing Language: English CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, (26) Publication Language: English DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, KP, KR, (30) Priority Data: KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, 62/345,133 03 June 2016 (03.06.2016) MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, 62/375,662 16 August 2016 (16.08.2016) PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (71) Applicants: THE TRUSTEES OF COLUMBIA SD, SE, SG, SK, SL, SM, ST, SV, SY,TH, TJ, TM, TN, TR, UNIVERSITY IN THE CITY OF NEW YORK TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. [US/US]; 412 Low Memorial Library, 535 West 116th (84) Designated States (unless otherwise indicated, for every Street, New York, NY 10027 (US). LEVO THERA¬ kind of regional protection available): ARIPO (BW, GH, PEUTICS, INC. [US/US]; 1701 E. Lake Avenue, Suite GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, 260, Glenview, IL 60025 (US). UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (72) Inventors: BURNETT, Lisa, Cole; 1380 Riverside Drive TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, Apt. 8E, New York, NY 10033 (US). EGLI, Dieter; 116 EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, Pinehurst Avenue, Apt. R43, New York, NY 10033 (US). MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,

(54) Title: METHODS OF TREATING PRADER-WILLI SYNDROME

Rationale for treatment in PWS individuals with agents that increase cA P levels and block its degradation

o o FIG. 2

(57) Abstract: The present invention relates to methods for regulating prohormone convertase (PCI ) and compounds and treatments which increase PCI levels, for treating Prader-Willi Syndrome (PWS). O

[Continued on nextpage] WO 2017/210540 Al llll II II 11III I II I II I III I II II III II I II

TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG).

Published: METHODS OF TREATING A - SYNDROME

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority t U.S. Provisional Patent Application No. 62/345,133 filed me 20 ; U.S. Provisional Application No. 62/375,662 filed A g s 16, 2 6, each of which is incorporated by reference ts entirety.

GOVERNMENT SUPPORT

This work was supported in pa t b grant from th National Institute of Health R ID . 524 con equently, the U.S may have certain rights in the present invention.

FIELD OF THE INVENTION

The present invention relates to methods for regulating prohormone convertase (PCI) a compounds and treatments which increase PC levels

BACKGROUND O F THE INVENTION

Prader-Willi syndrome (PWS) is caused by a oss of paternally expressed ge es in an imprinted region of chromosome 15q. Among the canonical pnenotypes are ! p rphagic obesity, central hypogonadism and low growth hormone. Rare niicrodeletion PWS patients define a 9 1 fo minimum critical deletion region encompassing 3 genes, including the non- coding D 6. We have found that N .L 2 a d PC are d wnregu a ed in PWS iPSC- derived neurons as compared t unaffected controls. Nhlh2 and csk transcript levels are reduced so hypothalami of fasted Snordl s" mice.

Deficiency of lh in m ce causes obesity, hypogonadism, and growth failure. Nhlh2 promotes expression of the prohormone convertase, (PCI). Hu an and mice deficient in PC display hyperphagic obesity, hypogonadism, decreased growth hormone, and diabetes du to impaired prohormone processing. For example, S r ( " * mice display in vivo functional defect in prohormone processing of proins m . proGf H, and p ghre in associated with reductions in PCI.

Currently there are no treatments for PWS patients and effective treatments and mode systems are urgently needed. SUMMARY OF THE INVENTION

The methods of the present invention provide for regulating prohormone converiase by administering art effective amount of a phosphodiesterase inhibitor (P.DE4 inhibitor or PDB4i). Expression of the prohormone convettase may be pregu a ed by administration of a therapeutically e ve amount o a PDE4 inhibitor. The P E inhibitor a be administered to cell or to patient w h Prader-Willi syndrome. The PDE4 inhib or may he administered to an obese subject t is also expected that these methods will be se& for treating patients with Schaaf-Yang Syndrome and Autism Spectrum Disorder.

The PDE4 inhibitor ay be administered orally, intravenously, subc aneo s y intrathecaliy, topically, intrauasaily, or to the lungs.

The P E4 inhibitor can include, theophylline, roflumilast, apreinilast, ibdulast, GSK35627S, M 52, BMX as well as combinations of these drags.

n certain embodiments, th P >E4 inhibitor can include any of the inhibitors from Tables 1A or Table IB.

In additional embodiments, combinations of P E4 inhibitors ma be used n the present methods.

The methods of the present invention also include administering a therapeutically effective amount of a adenylate cyclase activator. The aden ate -cyclase activator can be administered to a cell or to a patient with Prader-Willi syndrome. The adenylate cyclase activator may be administered to an obese subject.

The adenylate cyclase activator can foe administered orally, intravenously, intrathecaliy, intranasaliy, topically, or to the lungs. The adenylate cyclase activator can include, Forsko n F , FD2, FD3, FD4, FD5 ( 4 7) FD6 as well as combinations of these drugs.

The FDE4 inhibitor may a so b ad inistere together with the adenylate cyclase activator .

The methods of the present invention also include administering a therapeutically effective amount of a MC4R agonist. The MC4R agonist can foe administered to a ceil or to a patient with Prader-Willi syndrome. The MC4R agonist may be administered to an obese subject. The M 4 agonist can be adnn'nistered orally, intravenously, in iheca y mtranasally, topically, o to the lungs. The MC4 agonist ca include 4 (Setmelanotide), TTP25 , 2 a inothia o e derivatives, M 493. id combinations thereof. The MC4 agonist can be administered in combination with the PDE4 inhibitors and/or adenylate cyclase activators described herein.

The methods of the present invention also include methods wherein the administration results in one or more of the following improvements in the patient: decreases or ameliorates hyperphagia; increases FCSK1 levels; increases PC level and/or activity; decreases circulating proinsulin t insulin ratio, thus increasing insulin secretion; decreases circulating proghrelin t ghre in ratio; decreases circulating PO C to ACT ratio; amelioration of hypothyroidism, decreases circulating ratio of pro-oxytocin to oxytocin, thus increasing oxytocin production in the brain and increases al ha~MSH production in the brain; decreases circulating raiio of pro-BDNF to BDNF (increase brain levels of BDNF); and mcreases the ratio of prohormone: hormone (decreases pro-mature hormone); wherein the symptom, levels, or ratios are in reference to the patient's disease symptom, levels, or ratios.

In certain embodiments, the methods pr ide for treating Prader-W Syndrome (PWS) comprising administering a phosphodiesterase 4 inhibitor (POE4i) io a subject in need thereof, thereby alleviating, eliminating or preventing one or more symptoms of PWS.

In certain embodiments, administering the P E4i upregulates cyclic adenosine monophosphate (cAMP) concentrations or activity in the subject.

In certain embodiments, PWS is characterized by decreased expression of N L 2.

n additional embodiments, decreased expression of N 2 results in. decreased expression of PCSKI .

In certain embodiments, increasing concentrations or activity of cAMP upregulates expression sk

n additional embodiments, the PDE4i is a selective PDE4I. In additional embodiments, the PDE4i i a non-selective PD 4i

In certain embod me s, the selective PDE4i is selected from AN2728, apremilast, cilomilast, diazepam, ibudi s teolin mesembrenone- p e!a ast, ro i ast, rolipram, E60 5 GS 35 2 and M 9 2.. a certain embodiments, he non-selective FDE4i selected from methylated xanthines and derivatives thereof, caffeine, an in phy iin , S-isoboiyi-i-methy!xanihine, paraxanthme, pentoxifylline, theobromine, and theophylline.

In yet additional embodiments, the one or more symptoms include hyperphagia, reduced metabolic rate, obesity, hypogonadism, hyp a re na s , decreased growth hormone production, poor muscle tone, sleep disorders, gastrointestinal disorders, reduced stamina, reduced ability to focus, impaired cognition, behavioral disorders, anxiety, growth failure, reduced conversion of immature hormones to mature and active forms, and diabetes i e !itus and diabetes insipidus.

In certain embodiments, the method further comprises administering one or more additional therapeutic agents effective for treating or alleviating one or more symptoms of S.

In certain embodiments, the immature hormones comprise one or more of insulin, ghreiin, GHRH, alpha-MSH, oxytocin, orexits, BDNF, vasopressin, NPY, AGRP, an gonadotropins, AC'TH.

n certain embodiments, th one or more additional therapeutic agents effective at treating or alleviating PWS include insulin, a insulin receptor agonist, ghreiin, ghreiin receptor agonist, GHRH., a GHRH receptor agonist, alpha-MSH, an alpha M receptor agonist, oxytocin, an oxytocin receptor agonist, r xin, an orexin receptor agonist, BDNF, a BDNF receptor agonist, vasopressin, a vasopressin receptor agonist, NPY, an NPY receptor agonist, AGRP, an AGRP receptor agonist, gonadotropin, gonadotropin receptor against, or combinations thereo BRIEF DESCRIPTION OF THE DRA WINGS

Figure J. is a model showing how deficiencies in hlh2 and PC. may drive the major neuroendocrine phenotypes of PWS. A deficiency i prohormone processing owing to deficits in PC and Nhlh2 production ay explain many of the major neuroendocrine phenotypes of PWS. It is hypothesized that paternal ioss of SNORDI1 may be sufficient to cause deficiencies Nhlh2 and PCI, in tur causing ctional defects i prohormone processing. Arrows/lines mat are dashed indicate theoretical connections. Arrows/lines that are solid indicate pathways that have been investigated.

Figure 2 is a schematic showing the rationale for treatment of PWS utilizing age ts that increase cellular cAMP levels in order to increase level and/or activity of cellular PC and increase prohormone processing.

Figures 3A-P are graphs showing downregulation of PC in PWS models is associated with impaired prohormone processing; PCSKI transcript levels can be increased in unaffected control by treatment with rs o in.

Figure 4 is a schematic showing the therapeutic rationale or coadministration of MC4R agonists and/or AgRP inhibitors in combination with Forskoiin and/or Theophylline in individuals with PWS a d possibly other types of obesity; including common obesity.

Figures A- are graphs showing that th application of Forskoiin, a AC agonist, elevates PCSKJ/PcskI transcript levels in primary mouse neurons, iPSC-derived neurons, and primary mouse pancreatic islets.

Figure A~F are graphs showing that application of phosphodiesterase inhibitors to iPSC-derived neurons increases PCSKI transcript levels and prohormone processing. F g 6A: Theophylline increases PCSKI transcript levels in D34 IPSC-derived hypothalamic ARC neurons ( 2 A line) at 10 M concentration. Figs. B~€ : Rofiu i as increases PCSKI transcript levels in iPSC-derived neurons at Day 40 of differentiation (1043D3 line) at 1 mM concentration. Figs, D-E Combination treatment with ofl um ast ( 0 nM) and Forskoiin ( 1 µΜ) increase PCSKI transcript levels and increase POMC processing to ACTH at lower concentrations tha either agent alone, suggesting an additive or possibly synergistic effect. Fig. F: MK 52 applied at uM in combination with t Μ Forskoiin increases PCSKI transcript levels "-2-fold in iPSC-derived neurons ( 43D3 line). Figures are graphs showing that a treatment with 0952 increases hypothalamic k in wild type mice. Fig. 7A is a graph showing that body weights of all mice used were comparable. Fig. 7B is a graph showing that MK.0952 administered by oral gavage in % methyl cellulose at a ose of 10 mg g body weight increased hypothalamic Pcskl levels by about 25%. Treatment with Forskolm at 25 mg/kg did not result in increased hypothalamic Pcskl levels. Combination treatment with M 952 (10 mg/kg) and Forskolm (25 mg/kg) also resulted in a 25% increase in hypothalamic Pcskl transcript levels, likely due largely to the effects of M 952.

Figures 8A-C are schematics, tables and grapiis showing aspects of the clinical trial design, DETAILED DESCRIPTION

The present disclosure provides fo ethod to regulate PC (prohormone corrvertase

1) levels in vitro or in vivo. The methods can be used to upregniate (increase expression) or increase PCI levels and/or activity. Also encompassed by the present disclosure are methods to treat Prader-Willi syndrome (PWS.) and other forms of obesity. The methods may comprise the ste of administering a therapeutically effective amount of a PDE4 inhibitor and/or a adenylate cyclase activator it is also expected that these methods will be useful for treating patients with Schaaf-Yang Syndrome and Autism Spectrum Disorder (Fabienne Schaller Francoise Wairin. Rachel tu y Anriick Massacrier Pierre Szepetowski Francoise

Museatelli; Hum Moi Ge et (2010) 1.9 (24): 4895-4905. DO : ; Green Fei D, Modahl C, Feinstein C, Waterhouse , Morris M. Oxytocin and autistic disorder; alterations i peptide forms. Biol Psychiatry. 200! Oct 15;50(8}:609- ,

Theoretical mechanisms to increase cellular prohormone convertase 1 levels/activity include but are not limited to: ( ) upregulation at the transcript level by engaging endogenous promoters, (2) directly increasing enzymatic activit of PCI, (3) increasing rates of translation- of PCSKI to PCI, (4) decre asing degradation of PCI o , on possible approach is by decreasing levels (via aatisense o igo "genetic knockdown," traditional small molecule inhibition, or oilier) of the endogenous inhibitor of PC , ProSAAS, (5) decreasing degradation (mi A targeted on-se se mediated decay, putative mRNA methylation levels) of PCSKI transcript, thereby increasing translation, (6) PC itself is processed fr o a 92 a zymogen to a 66 Da mature , thus increasing; levels of preproPCI processing could also have therapeutic utility, and (7) delivery o additional PCSKf cDNA to the cell by gene therapy methods, (8) delivery of SN RD 6 R As by gene therapy methods, and (9 direct delivery of the PC enzyme into the circulation and or tissues with enzyme replacement therapies.

The present .findings suggest that the major neuroendocrine features of PWS are likely due to functional PC deficiency. See Figure 1. As the gene encoding PC , PCSKI, is intact in PWS, increasing the levels of P expression and/or activit in PWS patients wil correct this functional PC deficiency. Pharmacologically, this increase in PC levels can be achieved b ad ii rat on of agents thai increase cyclic adenosine monophosphate (cAMP) levels or block cAMP degradation.

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of cyclic AMP and cyclic GMP, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signaling pathways and, consequently, a myriad of biological responses in health and disease. Maurice el a . Advances in targeting cyclic nucleotide phosphodiesterases Nat. Rev. Drug. Discov. (4); -3 (2014). PD 4 so orrns are highly expressed i ceils tha regulate immuiioinilami¾atory responses and tissue remodeling. d. Inhibition of PDE4 results in an increase in cAMP levels in the cell A iarge number of PDE4 inhibitors are available. on- ifl ti ng of D 4 inhibitors Theophylline, o r niJast, Apremilast b di!as G8 35627 M 52, X 3 sob i i-1-methyixant r e), Mese re one Rolipram. Piclamilast, Lirteolin, Dr ave ne, AN2728, ilo ila t, .Diazepam, L e l and E6 5. Other phosphodiesterase inhibitors include, methylated xanthines and derivatives (such as caffeine, a nophylhne, paraxanthtne, pentoxifylline, theobromine, and theophylline).

T e levels of cAMP also he increased usin age t which activate aden at cyclase. Non it g examples of adenylate cyclase activators include: Forskoim, FD , PD2, D3, FD4, FD5 ( 477), and FD6.

DE4 inhibitors and adenylate cyclase activators can be referred to alone or in combination as therape i agents.

Table 1A: Selected PDE4 Inhibitors GSK356278 Selective Good suspended: GS Phase 1 2 Huntington's Disease MK0952 selective yes suspended: Merck Phase 2 2007 Alzheimer's Disease;

See: Fleckman PR W ters C, P ckae s 1. Phosphodiesterase inhibitors as a targei for cognition enhancement in aging and Alzheimer's disease: a translaiional overview. Curr Pharm D s. 201 S;21{3):3 17-31. Review. Pu Med PM D: 2 59073. Gallant M, et a . Discovery of M -0952, a selective PDE4 inhibitor for the treatment of long-term memory loss and mild cognitive impairment Bioorg Med C e Lett. 2010 Nov i5;20(22):6387-93^ doi: i j.bme .2010.09.087. Epub 2010 Sep j 2 1. PubMed PMID: 2093341 .

B X nonselective n a laboratory use only

Table B: useful as P E inhibitors (PPEi) GEBR~32a 1AVE 8 A BAY 60-7550 Rolipram GRC-4039 Anagrelide GEBR~7b Revamilast Cilostazol Sela¾inpulvilins DG Milrinone Se aginp ivili s L MEM 14 4 O!prinone GSK2 066 Mesopram Parogrelll CbJorbiprara S 36 Pm benda FFPM [ Z 7 37 ibiidilastrotTumiiast Cilo i st | MEM- Ro 20- 24 P la ilast IME -10 € DP840 BCS-15 MEM-19T? Toflrailasi ZL-ft - ! 27 OgkraiSasi NIS-62949 IAV- Teto ast CHF6001 [ AV-4 Lirimifast 4-(8-(3-Fluorophenyl)- 1,7- j nap yr din-6 l)irattscyctohexa«ecarboxy lie Acid Eyevinal Sildenafil 4 5 > ~teira ydro~lH~i 2- diayepin-7-one derivatives Initial Tadala i PDE-310 KC-404 Vardenafsi RPL554 etas Udenafil L-454,560 iM - 6 AvanalB

Table 2 Selected Adenylate Cyclase Activators

Abbreviations

ACT : adrenocorticotropic hormone.

AgRP; Agouti-related protein; a protein also produced in the arcuate nucleus and is an inverse agonist at MC4R. ProAgRP is processed to AgRP by PC . cA P cyclic adenosine monophosphate

G G ret n (the "hunger hormone", also known as e omore in (INN), is a peptide hormone produced by enteroeudocriue ce ls in the fundus of th stomach which functions as a neuropeptide in the central .nervous system. proGHRH: progrowth hormone-releasing hormone. G : Growth hormone-releasing hormone GHR ) also known as somatoliberin or by several other names in its endogenous forms and as soma ore in (INN) in its pharmaceutical form, s a releasing hormone of growih hormone (GH). It is a 44-amino acid peptide hormone produced n the arcuate nucleus of the hypothalamus.

PCI:: Proproiem convertase a so known as prohormone convertase 1 prohormone convertase 3 proprotein convertase 3, neuroendocrine convertase 1, o neuroendocrine convertase 3, and often abbreviated as .PC 1/3 is an enzyme that i humans is encoded fay the PCSK gene. PC and PC2, the protein products of the PCS and PCSK2 genes, differentially cleave marry neuroendocrine or endocrine hor ones. including, proopiomelanocortin, promsniin, and progi ago .

PC2: Proprotein convertase 2 (PC2) a so known as prohormone convertase 2 or neuroendocrine convertase 2 (NEC2) is a serine protease and proprotein convertase PC2, like proprotein convertase I (PCI), is a en .y e responsible for the first step in the maturation of any neuroendocrine peptides from their precursors such as the conversion of pro ns lin to insulin intermediates. To generate the bioacrive form of insulin and many other peptides), a second step involving the removal of C-lerminal basic residues i required; this step is mediated by carboxypeptidases E and/or D . PC2 plays only a minor role in the first step of insulin biosynthesis, but a greater role in the first step of glucagon biosynthesis compared to PC . PC2 binds to the neuroendocrine protein named B2 and if th s protein is not present, p.roPC2 cannot become en y atica y active. 7B2 accomplishes this by preventing the aggregation of proPC2 to inactivatable forms. Tire C-termraal domain of 7.82 also inhibits PC2 activity until it is cleaved into smaller inactive forms. Thus, 7B2 is both an activator and an inhibitor of PC2. In humans, proprotein convertase is encoded by the PCSK2 gene t is related to the bacterial enzyme s i isi and altogether there are 9 diffe rent btilisin- ke genes in mammals: furin, PACE4, PC4, PCS/6, C7/ , P S 9, and S i Sf P.

PCSKI: the gene encoding PC .

P S 2: the gene encoding PC2.

POMC: Proopiomelanocortin (POMC) s precursor polypeptide with 2 amino acid residues. POMC is synthesized in the pituitary from the 285-amino-acid-long polypeptide precursor pre-pro-opiomelanoeortin (pre-POMC), by the removal of 44-amino-acid-iong signal peptide sequence during translation. PDE4; phosphodiesterase 4 .

WS Prader Willi Syndrome.

SNORD 6 : SNORDi 16 (also known as H - 5) is a non-coding RNA (ncR A molecule which functions he modification of other small nuclear RNAs (s As . This type of modifying RNA is usually located in the nucleolus of the eukaryoiic ce l which is a major site o f s RN A. biogenesis. It is known as a small nucleolar RNA (snoRNA) and also often referred to as a guide RNA. SNORDi 16 belongs to -the O X) box class of snoRNAs which contain the conserved sequence motifs known as he C ( A GA) and the D box (CUGA). Most of the members of the box C/D family function in directing site-specific 2'-0- e ylation of substrate RNAs. In the human genome, there are 29 ta dem y repeated copies of SNORDI 16, in ihe PWS region of chromosome 15. in addition, other non-coding RNA species are endocing from the SNORDi 6 locus, including the long noncoding RNA, 6 , fi v n ncRNAs, and o spa neR s. SNORDI 16 s a orphan non-coding RN locus tha lacks clearly defined targets. Mouse models lacking paternal Snor 16 show similar symptoms to human PWS including hyperphagia and growth defi ciency

DPI devices/inhalers: dry powder inhalers; typically hand-held.

M D devices: metered-dose inhalers; typically hand-held. aMSH; is an endogenous iigand of the melanocoriiu 4 receptor.

MC2R: ra e!anoco i 2 receptor.

MC4R: melanocoriiu 4 receptor

WT: wi type

Definitions The term "pharmaceutically acceptable carrier", as used herein means a phar!Maeeutically-acceptable material, composition or vehicle, -such as a liquid o solid filler, diluent, exeipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent. The diluent or carrier ingredients should not be such as to diminish the therapeutic effects of the active compound(s).

The term "composition" as used herein means a product which results from the mixing or combining of more than one element or ingredient

"Treating" or "treatment" of a state, disorder or condition includes: 1) preventing or.delaying the appearance of clinical symptoms of the state, disorder, or condition developing in person who may be afflicted with or predisposed to the state, disorder or condition bu does not yet experience or display clinical symptoms of the state, disorder or condition; or

2) inhibiting the state, disorder or condition, i.e., arresting, reducing o delaying the development of the disease or a relapse thereof in case of maintenance treatment) or at least one clinical symptom, sign, or test, thereof; or

(3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms or signs.

The benefit t a subject to he treated is eithe statistically significant o at least percepti e t the patient or to the physician.

"Patient" or "subject" refers to mammals and includes hu an and veteri ar subjects,

Inhibitors' * and "antagonists," or "activators" and "agonists," refer to inhibitory or activating molecules, respectively, e.g., for the,activation of, e.g., a ligand, receptor, faetor, a gene, cell, tissue or organ. A modulator of, e.g., a gene, a receptor, a ligand, or a cell, is a molecule that alters an activity of the gene, receptor, ligand, or cell, where activity can be activated, inhibited, or altered in its regulatory properties. The modulator ay act alone, or may use a cofactor, e.g., a protein, metal ion, or small molecule. Inhibitors are compounds tha decrease, block, prevent, delay activation, inactivate, desensitize, or down regulate, e.g., a gene, protein, ligartd receptor, or cell. Activators are compounds that increase, activate, facilitate, enhance activation, sensitize, or up regulate, e.g., a gene, protein, ligand, receptor, or cell. An inhibitor may also be defined as a compound that reduces, blocks, or inactivates a constitutive activity. A "agonist" is a compound that interacts with a target to cause or promote an increase in the activation of the target. An "antagonist" is a compound that opposes the actions of an agonist An antagonist prevents, -reduces, inhibits, or neutralises the activity of an agonist. An antagonist ca also prevent, inhibit, or reduce constitutive activity of a target, e.g., a target receptor, even where there is no identified agonist.

To examine the exte t of inhibition, for example, samples or y comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activator or inhibitor a d are compared to control samples without Use inhibitor. Control samples, i.e., samples not treated with antagonist, are assigned a relative activity value of 100%. Inhibition is achieved when the activity value relative to the control s about 9 ¾ or less, typically 85% or less, typically 80% or less, most typically 75% of less, generally 70% orless, more generally 65% or ess, most generally 60% or less, typically 55% or less, l 50% or less usually 45% or less, most visually 40% or less, -preferably 35% or less, more preferably 30% or less, still more preferably 25% or less, and preferably less than 25%. Activation is achieved the activity value relative to the control is about 0%, generally at least 0%. ore generally at least 0%, ore generally at least 0%, often at least 0%, more often at least 2-fold, most often at least 2.5 , usually a least 5- ! more usually at least 10-fold, preferably at least 20-fold, more preferably at least 40-fold, and most preferably over 40-fold higher.

The dosage of the therapeutic formulation w il vary widely, depending upon the nature of the disease, the patient's medical history, the frequency of administration, the .manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, sem -weekly, etc., t tnain ai a effective dosage level In some eases, oral administration will require a higher dose tha f administered intravenously. n some eases, topical administration will include application several times a day, as needed, for a number o days or weeks in order to provide an effective topical dose.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sesame oil an the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not li ited to one or more of a binder (for compressed pills}, a g ida t, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W . Martin. Therapeutic compositions described herein may be administered by an method known in the art, including, without limitation, intranasal, oral, transdermal, ocular, intraperifoneaL inhalation, intravenous, 1CV intracisterrsal injection or infusion, subcutaneous, implant, vaginal, sublingual urethral (e.g., urethral suppository } subcutaneous, intramuscular, intravenous, rectal, sub-lingual, mucosal, ophthalmic, spinal, intrathecal, intra-artieular, ra-arteria , sub-araehinoid, bronchial or lymphatic administration. Topical formulation may b in the form of gel., ointment cream, aerosol, etc.; intranasal formulation can be delivered as a spray or i a drop; transdermal fo tula n may b administered via transdermal patch or iontorphoresis; or, inhalation formulations can be delivered using a nebulizer or similar device. Compositions ca also take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosois, or any oilier appropriate compositions. To prepare such pharmaceutical compositions, one or more PDE4 inhibitors and/or one or more adenylate cyclase activators, and or one or more MC4R agonists may be raked together with a pharmaceutical acceptable carrier, adjuvant and/or exeipient, according to conventional pharmaceutical compounding techniques, Pharmaceutically acceptable carriers that can be used in the present compositions encompass any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and vari ou types of wetting agents. The compositions can additionally contain solid pharmaceutical excipients such as starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate.. glycerol moiiostearate, sodium chloride, dried skim mi k and the like. Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and. various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil etc. Liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose, a d glycols. For examples of carriers, stabilizers and adjuvants, see Remington 's har ac utic 1 S jences, edited by E . W. Martin

(Mack Publishing Company, th ed. « 1990). The compositions also can include stabilizers and preservatives. As used herein, the ter a "therapeutically effective amount" is an amount sufficient to treat a specified disorder or disease or alternatively to obtain a pharmacological response treating a disorder or disease in vitro or in vivo in a mammal such as human o non-human patient. Methods of determining the most effective means and dosage of administration can van' with the composition used for therapy, the purpose of the therapy, the target cell being treated, and. the subject being treated. Treatment dosages generally may be titrated to optimize safety an efficacy. Single or multiple administrations can be carried out wit the dose level and pattern being selected b the -treating physician. Suitable dosage formulations and methods o f administering the agents can be readily determined by those of skill in the art. For example, the composition is administered at about 0.01 g kg to about 200 mg/kg, about 0.1 mg/kg to about 100 mg kg, or about 0.5 mg kg to about 5 mg kg. When the compounds described herein are co-administered with another agent or therapy, e effective amount may b less than, equal to or greater when either agent s used alone. Transdermal ra lations may be prepared by incorporating the active agent in a thixotropie or gelatinous carrier suc as a celluiosic medium, e.g., methyl cellulose or hydroxyethyl cellulose, with the resulting formulation tben being packed in a transdermal device adapted to be secured in dermal contact with the skin of a wearer. If the composition is the form of a gel, the composition may be rubbed onto a membrane of the patient, for example, ihe skin, preferably intact, clean, and dry skin, of the shoulder or upper arm and or the upper torso, an maintained thereon for a period of time sufficient for delivery of the PDE4 inhibitor and/or the adenylate cyclase activator to th blood serum of the patient. The composition of the present invention in gel form may be contained in tube, a sachet, or a metered pump. Such a tube or sachet may contain one unit dose, or ore than one unit dose, of the composition, A metered pump may be capable of dispensing one metered dose of the composition. This invention also provides the compositions as described above for intranasal administration. As such, the compositions ca further comprise a permeation enhancer. Southall et at 2000. The PDE4 inhibitor and/or the adenylate cyclase activator may be administered intranasal!}' in a liquid form such as a solution, an emulsion, a suspension, drops, or in a solid form such as a powder, gel, or ointment. Devices to deliver intranasal medications ar well known in the art. Nasal drug delivery ca be carried out. using devices including, but no limited to, intranasal inhalers, intranasal spray devices, atomizers, nasal spray bottles, unit dose containers, pumps, droppers, squeeze bottles, nebulizers, metered dose inhalers (M ), pressurized dose inhalers, insufflators, and bi-directional devices. The nasa delivery device ca be metered to administer an accurate effective dosage amount to the nasal cavity. The nasal delivery device can b for single unit delivery o multiple unit delivery n a specific example., the Via ase Electronic Atomizer from rve Technology (Bethel!, Washington) can be used i this invention (http: w w k rveteeh .e m) The compounds of the present invention may also be delivered through a tube, a catheter, a syringe, a paekiail, a pledget, a nasal tampon or by submucosal infusion . U.S. Patent Publication os. 20090326275, 2 0 02 T8 4, 2009028 22 and 200903 7377, The PDE4 inhibitor and/or the adenylate cyclase activator can be formulated as aerosols using standard procedures. The PDE4 inhibitor and/or the adenylate cyclase activator may be formulated with or without solvents, and formulated with or without earners. The formulation may be a solution, or may be a n aqueous emulsion with one or more surfactants. For example, an aerosol spray ay be generated from pressurized container with a suitable propellant such as, c orodi oro ethane, trich orofl uoro et a e dic orotetra oroet ane hydrocarbons, compressed air, nitrogen, carbon dioxide, or other suitable gas The dosage unit ca be determined by providing a valve to deliver metered amount. Pomp spray dispensers ca dispense a metered dose or a dose having a specific particle or dropiet size. As used herein, the term "aerosol" refers to a suspension of fine solid particles or liquid solution droplets in a gas. Specifically, aerosol includes a gas-borne suspension of droplets o f a PDE4 inhibitor and/or the adenylate cyclase activator as may be produced in a y suitable device, such as an MD , a nebulizer, or a mist sprayer. Aerosol a so includes a dry powder composition of the composition of the instant invention suspended in air or other carrier gas Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313. Raebnrn et a ( 992) Pharmacol. Toxicol Methods 27:143-159. The PDE4 inhibitor and/or the adenylate cyclase activator ay be delivered to the nasal cavity as a powder in a form such a microspheres delivered by a nasal insufflator. The PDE4 inhibitor and or the adenylate cyclase activator may be absorbed to a solid surface for example, a carrier. The powder or microspheres ma be administered in a dry, air- dispensable form. The powder or microspheres may be stored in container of the insufflator. Alternatively, the powder or microspheres may be filled into a capsule, such as a gelatin capsule, or other single dose unit adapted for nasa administration. The pharmaceutical composition ca be delivered to the nasal cavity by direct placement of the composition in the nasal cavity, for example, in the form of a gel. an ointment, a nasal emulsion, a lotion, a cream, a nasal tampon, a dropper, or a bioadhesive strip. In certain embodiments, it can be desirable to prolong the residence time of the pharmaceutical composition in the Basa cavity, for example, to enhance absorption. Thus, the pharmaceutical composition can optionally be formulated with a bioadhesive polymer, a gum (e.g., xanthan gum), chitosan (e.g., highly purified cationic polysaccharide), pectin (or any carbohydrate that thickens like a gel or emulsifies when applied to nasal mucosa), a microsphere e.g. starch, albumin, dextran, c cl dextr ) gelatin, a liposome, arba er, polyvinyl alcohol, alginate, acacia, chii san and/or cellulose (e.g., methyl o propyl; hydroxyl or earboxy; carboxymetliyl or hydroxy ipropyi). The composition containing the PDE4 inhibitor and/or the adenylate cyclase activator can be administered by oral inhalation into the respiratory tract, i.e., the lungs. Typical delivery- systems for inhalabie agents include nebulizer inhalers, dry powder inhalers (DPI), a d metered-dose inhalers (MDl). Nebulizer devices produce a stream of h gh velocity a r that causes a therapeutic agent i the form of liquid to spray a a mist. The therapeutic age t is formulated i a liquid form such as solution or a suspension of particles of suitable ske. In one embodiment the particles are micronized. The te m cronk ed is defined as having about 90% or more of the particles with a diameter of less than about ra . Suitable nebulizer devices are provided commercially for example, by PARI GmbH (Stamberg, Germany). Other nebulizer devices include Respimat (Boehringer Inge hem ) a those disclosed in, for example, U.S. Patent Nos. 7,568,480 6,123,068, a d WO 97/ 12687 The PDE4 inhibitor and/or the adenylate cyclase activator can be formulated for use in a nebulizer device as an aqueous solution or as a liquid suspension. DPI devices typically administer a therapeutic agent i the form of a free flowing powder that can e dispersed in a patient's air-stream during inspiration. DPI devices which use an external energy source may also be used in the present invention n order to achieve a free flowing powder, the therapeutic agent can be formulated with suitable excipient (e.g., lactose). A dry powder formulation can be made, for example, by combining dry lactose having a particle size between about 1 µ η and 0 µιτι with micronked particles of the present compounds dry blending. Alternatively, the present compounds can be formulated without excipieots. The formulation is loaded i o a dry powder dispenser, or into inhalation cartridges or capsules for use with a dry powder delivery device. Examples of DPI devices provided commercially include Diskhaler (GlaxoSt ith me, Research Triangle Park, .C.) (see, e.g., U.S. Patent No. 5,035,237); Diskus (GlaxoS it e) (see, e.g., U.S. Patent No 6,378, ; Turbuhaler (AstraZeneca, Wilmington, De .) (see, e.g., U.S. Patent No. 4,524,769); a d o aha er (GlaxoSmithK!ine) (see, e.g., U.S. Patent No. 4,353,365). Further examples of suitable DPI devices are described in U.S. Patent Nos. 5,415,162, 5,239,993, and ,7 5,8.10 and references therein MD devices typically discharge a measured amount o hera e tie agent using compressed propei a t gas. Formulations for MDI administration include a solution or suspension of active ingredient in a liquefied propellant. Examples of propellants include hydrofluoroalkiaues (HFA), such as , ,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3- heptailuoro-n-propane, (HFA 227), and chlorofluorocarbons, such as CCI3F. Additional components of HFA formulations for MDI administration include co-solvents, such as ethan l pe ta e, water; and surfactants, such as soxbiian trioleate, oleic acid, ieei in a d glycerin. (See, for example, U.S. Patent Ho. 5,225,183, HP 7 7987, and WO 92/22286) The formulation is loaded into a aerosol canister, which forms a portion of a MDI device. Examples of MDI devices developed specifically for use with HFA propellants are provided in U.S. Pate t Nos. 6,006,745 and 6,143,227. For examples of processes of preparing suitable formulations and devices suitable for inhalation dosing see U.S. Patent Nos. 6,268,533, 5,983,956, 5,874,063, and 6,221,398, and WO 99/53901, WO 00/61 108, WO 99/55319 and WO 00/30614. The P E4 inhibitor may b encapsulated in liposomes or microcapsules for delivery' via inhalation. A liposome is a vesicle composed of a lipid b layer membrane and an aqueous interior. The lipid membrane may be ade of phospholipids, examples of which include phosphatidylcholine s ch as lecithin and lysolecithin; acidic phospholipids such as phosphatidylserine an phosphatidylglycerol; and sphiugophosphoiipids such as phosphatidylethanolamine and sphingomyelin. Alternatively, cholesterol may be added. A microcapsule s a particle coated with a coating material. For example, the coating material may consist of a mixture of a film-forming polymer, a hydrophobic plasticizer, a surface activating agent or/and a lubricant nitrogen-containing polymer. U.S. Patent Nos. 6, ,176 and 7,563,768. The PDE4 inhibitor and/or the adenylate cyclase activator can b given alone or in combination with other drags for the treatment of the above diseases for a short or prolonged period of time, e.g., 1, 2, 3, 4, 5, , 7, 8, 9, 1 , 20, 30, 40, 50 or 60 days or 1, 2, , 5, , 7, , % 10, 1 or 1 months or continuously over the lifetime of the patient The present compositions can be administered to a mammal preferably a human patient. Mammals include but are not limited to, mice, rats, rabbit, simians, bovines, ovine, porcine, canines, feline, farm animals, sport animals, pe s, equine, and primates. The following are non- limiting examples.

EXAMPLE Manipulation of PC expression and activity i iPSC-dedved hypothalamic neurons and β- e s Cyclase activation by Forskolin and/or inhibition of cAMP ca abo ism by inhibition of phosphodiesterase (Theophylline, 1BMX) will increase PC I levels in in vitro models of PWS with consequential increases in prohormone processing. The identification of an apparent l i-tiss deficiency in C : i in vivo and in vitro models of PWS enables rational therapeutic targeting that may alleviate major neuroendocrine s ptoms of PWS ( g. 1). The promoter region of the PCSKl gene contains two cyclic adenosine monophosphate (cAMP)-response elements (Co kr ght et a . 2003; Udupi et a . 1998). Agents tha increase cellular levels of cAMP increase PCSKl R A and increase the secretion of prohormones processed by P (Fig. 2)( d pi 1998). Forskolin and Theophylline are two FD -approved drags with generally safe treatment profiles in pediatric populations. Forskolin binds to adenylate cyclase close to its catalytic domain through hydrophobic interactions and hydrogen bonding (Tang and Hurley 98, Tes er e i 1999). Forskolin binding causes adenylate cyclase conformation to change to its active form, thus increasing AC activity and increasing cellular cAMP levels (Onda et a 2001). Theophylline and other phosphodiesterase 4 (PDE4) inhibitors, such as 0952, increase cellular cAMP levels by blocking its degradation. Non-limiting examples of PDE4 inhibitors include Theophylline, 0952. as well as the other PD inhibitors in Tables A-B . Non-limiting examples of adenylate cyclase (AC) activators include Forskolin and the activators in Table 2 Agents that can be used in the present method also include agents that can modify G protein activity, such as G protein activators or inhibitors, as well as G protein coupled receptor agonists. and PCSKl are downregulated in PWS microdeletion an large deletion iPSC -derived neurons, as shown by RNA sequencing (Figs. 3A 3 . Fig 3A : N 2 is downregulated in PWS .microdeletion and large deletion iPSC-derived neurons compared to unaffected controls. Fig. S : NH 2 protein is downregulated >9 n PWS microdeletion and large deletion iPSC-derived neurons compared to unaffected controls. Figs. 3C-D: PCSKl transcript and its protein product, PC , are downregulated >55 and > %, respectively in PWS microdeletion and large deletion IPSC-derived neurons compared to unaffected controls. Figs. 3 E-F: M ce in which only the paternal copy of S or !Ϊ 16 has been deleted (rest of PWS region s intact) display >40 downreguiaiion of PC I and PC2 protein n isolated islets. Fig. 3G Proinsuiin is dependent on PC fo its proper processing. There s a functional impairment in proinsuiin processing in Sn r l & :S 'i mice compared to WT littermates at 3 minutes following glucose injection. Fig 3 : There is 60% increase in the ratio of proinsuiin to insulin in the plasma of individuals with PWS compared to age and M -matched controls at fasting, indicating a defect in proinsuiin to insulin processing. The effect is less ha that seen n a patient with a .PC mutation., consistent with the reduction of PC 1 in PWS models. Fig. 3 : Proghrelin is also processed by PCI; proghrelin processing is impaired in stomach lysa es fr om Sn rd " mice compared to WT iiter ates. Storaach lysates rom PCI null mice are included as a positive control for impaired proghrelin processing. Fig. 3J; ProGHRH processing may also be impaired in hypothalamic lysates ro Sn r l m ce compared t WT attenuates, p 0. 6. Impaired proGHRH processing is associated with lo circulating G a d dwarfism in PCI null mice. S ' l ~ m also display low GH and severe ranting . Fig 3 ; Preliminary data suggests that treatment of unaf ected control hypothalamic iPSC-derived neurons with Forskolin (FS ) may increase transcript levels of PCSKl in a dose-dependent manner. POMC transcript levels may not be affected. Fig. 3L: Treatment of unaffected control iPSC-derived

β ce ls with. Forskolin increases PCSKl transcript levels. SNORDl 6 and INS transcript levels may be minimally affected. Fig 3M; Transcript levels of P s are decreased 41% in Snordll6p-/m* hypothalamic at .lasting; there is no difference in Pcskl levels at refeeding. Fig. 3N: Transcript levels of Nhih2 are decreased at both fasting and after refeeding in Sn rd 6p-/ hypothalamic compared to WT littermaies. Figs. 30-P: Agrp and Npy transcript levels are increased in Sn rd 6p- - hypothalamic at refeeding compared to WT. Follow-up, independent experiments confirmed these changes by QPCR (gene expression) and Western blotting {protein level) (Figs. 3 B 3D). Individuals with PWS exhibi decreased fasting insulin levels as compared to age and BM matched controls. t was hypothesized that this may be due to Impaired proinsuiin processing. The present dat illustrates that in a mouse model of PWS. in which only the paternal copy of n rd 6 is deleted, PC and PC2 protein levels are decreased in isolated islets and are associated with a functional impairment in the processing of proinsuiin i o insulin (F gs 3E-3G). Proinsuiin processing is also impaired (p :0.089) in plasma from human PWS patients compared to age, BMI-matched controls at fasting (Fig, 3 ). Plasma from a fasted patient harboring a PC mutation was included as a positive control for impaired proinsulin processing. The hyperghrelinemia of PWS patients is a unique phenotype that may be associated with impaired processing of proghrelin o mature ghrel . indeed, the present results illustrate that proghrelin to .mature ghreli processing was impaired in storaach lysates of Snordl * ¾ mice compared to WT littermaies (Fig. 3 ). Stomach lysates from PC n il mice were included as a positive control for impaired proghrelin processing. Like individuals with PWS, -patients- with PCI mutation have decreased circulating OH levels. Mice null for PC have severe r nti g a d decreased circulating GH associated with impaired pro R to GHRH processmg. We found that Sn r mice, which are also ru ted and have low circulating GH, trend towards impaired processing of proG H to GHRH in hypothalamic lysates (Fig. 3J).

A.s outlined in Fig, 2 the identification of a decrease in PC and impaired prohormone processing in PWS suggests a i ed molecular theory which may account for many of the neuroendocrine features of the disease. Thus, agents that increase PC activity and thereby increase prohormone processing, represent rational targeted therapy for the major neuroendocrine features of PWS. Forskolin is know to increase cellular Pcskl levels and can increase prohormone processing. Forskolin was applied to non-PWS iPSC-derived neurons β-cells and the results illustrate that PCSK1 transcript levels increased compared to untreated ceils (Figs. 3 -L). Studies in PWS-derived neurons and β-cel!s will be conducted. The response of PCSK1 transcript levels, PC protein levels, and relevant prohormone processing levels wil be tested in i viiro and n vivo model systems. We will treat unaffected control and hypothalamic iPSC-derived neurons with graded levels of Forskolin, Theophylline, and Forskolin ÷ Theophylline and measure PC transcript and protein, POMC transcript and protein, as wel as protein levels of processed products of POMC including: M SH, β-endorphia, and ACTH. These peptides will be q a ti a e in whole cell lysates as well as the levels secreted i to the cellular medium. The cells will be treated with different concentrations of Forskolin and Theophylline in order to determine whether PC levels ca be increased in a dose-dependent manner and in order to identify an optimal dosage range to increase PC levels and POMC processing. Other P .B4 and adenylate cyclase inhibitors will be tested in these assays as well (see, Tables A-B an 2). Batch RNA sequencing and/or single e ! RNA sequencing will be performed to identify other transcripts that are most affected by the pharmacological treatments. T is approach is expected to predict off-target effects upo In vivo treatment. Single ce l RNA sequencing will be especially -informative regarding P CS J transcript increases .following treatment i POMC-expressing neurons. Other adenylate cyclase activators and PDE4 inhibitors (Tables A- , 2) will be tested n iPSC-derived hypothalamic neurons following the same study protocol as described above. We w l l differentiate iPSC from unaffected control and PWS (large and nun u deletion) to iPSC-derived β-c . We wil treat the iPSC-derived -c s with Fors ol i Theophylline, and Forskoiin Theophylline and measure levels of PCI at the tra sc pt and protein level. We will also measure INS transcript levels as well as protein levels of proinsulin, insulin, and c~pept.de from whole cel lysates as well as the concentrations of the proteins secreted by the -eelSs into the media. These cells may be transplanted into node mice to enable their maturation; these cells can be tested in vivo for insulin processing; excised cells will be tested as described. We wil also use hitman isolated islets from non- diabetic, non-obese individuals (available to us through the National Pancreatic Donors Registry) to test the effects of Forskoiin, Theophylline, and Forskoiin - Theophylline on PCI levels in folly mature human pancreatic islets.

EXAMPLE 2 ' Confirmatory molecular physiology of PC metabolism in > Pet d Pel " mice. Increasing cA P levels by adenylate cyclase (AC) activation and concurrent PDE inhibition wi l increase PCS levels n ex vivo and in vivo models of PWS, and may consequently increase prohormone processing in the Snordl f i mouse model of PWS. Because mice with a paternal deletion of S r 1 (a mouse model of PWS) have impaired processing of proinsulin to insulin associated with reductions in PC transcript and protein, islets wi l be isolated from w ld type (WT) and $nor I.l i ',m* mice and the responses of these cells to Forskoiin, Theophylline, and Forskoiin + Theophylline will be analyzed. The same manipulations and measurements will be performed as for iPSC-derived β-celis. If proinsulin processing can be rescued in isolated islets from Snordl ™ mice as compared to WT littermates, then investigations of proinsulin processing rescue in Snordl 16 ~ i mice treated with Forskoiin, Theophylline, and Forskoiin Theophylline, in vivo will be conducted.

The present results illustrate thai Snordl '"* ' mice have reductions n proinsulin processing to insulin that are associated with reduced PC a d PC2 content n the islets (Figs. 3E-G), Furthermore, proghre i processing is also impaired in the stomachs of Snordl m ice as wel as the processing of proGH to GHRH in the hypothalami of Snord "" ' mice compared to WT littermates (Figs. 3i-J). Furthermore, the ratio of proksuiin to insulin is elevated in fasted individuals with PWS compared to age and BM matched controls, suggesting an impairment in the processing of proinsulin to insulin (Fig. 3 ). isolated islets from Pel null and heterozygous mice -will be included as a control for impaired pr i s tin processing a wel as a predicted negative response to pharmacological treatment. Peripheral levels of glucose, pro s iin insulin, and c peptk e wil be measured at fasting, and , 30, 60, and 120 minutes following intraperitoneal glucose injection. Optimal duration of pharmacological treatment in Snord and WT rake prior to peripheral measures of proinsulin processing will be established empirically. P'cl m l a d heterozygous mice wi be included as a control for impaired proinsulin processing in vivo experiments as well. The initial time periods for testing will be 3 days. 1 week, and month. Several methods of drug delivery will also be tested, Assays that can distinguish between proghrelin and mature g rel , and can thus be used to measure proghrelin processing in the circulation will be developed for both human and mouse. Proghrelin processing following the in vivo pharmacological treatments described above in Snordl , PC null, PC heterozygous, and WT animals will. be measured. The hypothalami of Snordl I WT, and Pel nu l and Pel heterozygous mice treated with Forskolin, Theophylline, and Fors o in + Theophylline, and measure protein levels of PC , POMC, c MSH β-endorphin, and ACTH will be analyzed in order to assess whether >? vivo treatment can affect levels of PC an POMC processing. Because Snordl " animals are runted and do not develop obesity, the main model by which POMC processing wil be assessed is the iPSC-derived human neurons, n which ore extreme downregulations H and PC are observed. However, PC 1 and POMC responses to these pharmacologic agents may also be analyzed in primary neurons from young WT POMC -OFF mice. P MC-expressing neurons can be specifically isolated using mice in which POMC neurons express OFF, We will knock down Pcskl as a control for impaired POMC processing. We may also try to knock down specific isofbrms of Snordl 16 n vitro using si NA- or 2-0-methyi-modified anti-sense o o-based approaches; siRNA are small, double-stranded interfering RNAs that are commonly used to knock down cytoplasmic RNAs, while 2- -met y -modified anti-sense oligos are use to knock down snoRMAs which typically are found in the nucleolus (Liang et a . 20 ). We will then measure PC levels and POMC processing levels and investigate whether pharmacological treatment can increase levels of PCI and increase POMC processing in the primary mouse neurons i which Snordl 16 has been knocked down relative to WT. Additionally, mice with conditional hypomorphic alleles of S RD will be obtained or created. Adult animals with such alleles will have Snordl 16 acutely reduced in specific hypothalamic nuclei (e.g. the arcuate .nucleus) b introduction of suitable ere- expressing constructs, including those driven by specific promote rs eg. For POMC. This approach will circumvent the somatic developmental effects (stunting) of Snor ' hypomorphism in a k e Effect of cyclase activator and/or phosphodiesterase inhibitor - 50% or greater increase in relevant prohormone processing in at least one model tested: PWS iPSC-derived neurons, PWS iPSC-derived p-celis, Snordl f isolated islets, Snordl circulating prohormones, or Snor 16-knockdown primary neurons. These phenoiypes would be accompanied by increases in relevant transcripts and/or proteins in the affected cells. Cyclase activation

Forskolin was applied to various cellular models and the results illustrate that it robustly and reliably increases PCSK! transcript levels, PC protein levels, and functionally increases prohormone processing. P S i/Pcs transcript ievels increased between 2-3 fold in iPSC-derived neurons and primary mouse neurons exposed to 10 uM Forskolin (Figs. 5A, C D).

Cyclic AMP concentrations increased about .5 Foki in iPS de ive hypothalamic ARC neurons exposed to it) µ.Μ Forskolin, supporting the inference that application of Forskolin increases PCSKI transcript ievels by raising cellular cAMP levels which in turn activate PCSKFs cAMP-reaponse element promoter. Fig, 5A is graph showing primary forebraiii neurons were isolated from gestational day 19.5 (El 9.5) embryos of wild type ice and cultured for 2 hours. Subsequently, cells were exposed to either 10 µΜ Forskolin or its vehicle, dimethyl sulfoxide (DMSO) for 20 hours. PcskJ transcript increased ~2.5-fbk1 n primary neurons exposed to Forskolin. Fig, SB is a graph showing unaffected control hypothalamic arcuate-like (ARC) neurons (Hes kx2 1 ESC line) at. da 3? D of differentiation were treated with 10 µΜ Forskolin or vehicle for thirty minutes. Cyclic adenosine monophosphate (cAMP) levels were increased about 8. -fold in ceils exposed to Forskolin. Fig, 5C is a graph showing exposure of unaffected contr ol hypothalamic iPSC- derived neurons (line 1023 ) a day 30 of differentiation to grade concentrations of Forskolin elicits a dose-dependent response in PCSKI transcript levels. Fig. 3D is a graph showing exposure of iPSC-derived neurons (line 1043D3) to 0 µΜ Forskolin for multiple time intervals finds that PCSKI is not significan tly unregulated after only hour of exposure, but is significantly unregulated by 4 and hours of exposure. Four hours of exposure yielded the maximal increase in upregulation, about 2.5-fold, of the time points tested. Figs. E-F are graphs showing of unaffected control ( 2 A) iPSC-derived ypot ala ic ARC neurons at day 30 of differentiation with graded concentration of forskolm identifies a dose-dependent mcrease in POMC processing to both ^endorphin (PEP) and a-melanocyte stimulating hormone ( SH). Figs. 5G- are graphs showing treatment of isolated islets from adult (S- 2 week old) T mice with multiple concentrations of Forskolin shows upregu atio of PCI, but not PC2, protein levels at 25 and 50 µΜ Forskolin concentrations, respectively.

Treatment with forskolin not on y elevated transcript levels but also had functional consequences in that POMC processing to both ^endorphin and MSH were increased (Figs,

5E-F). Furthermore, application of Forskolin to isolated islets from wild type mice resulted in. 3-fold increase in PCI protein levels (Fig. 5 G). PC2 protein levels were unaffected (Fig, SB). Taken together, these results show that P levels increase in response to Forskolin n three separate model systems; iPSC-derived neurons, primary neurons, and isolated islets. Furthermore, Forskolm-itiduced elevation of PCi is functionally consequential, resulting in increased levels of prohormone processing.

Phosphodiesterase inhibition

PDF inhibitors have also been tested in iPSC-derived neurons and found that inhibition of phosphodiesterase can also increase transcription P SK . However, the effect s ze of PDF inhibition on PCSK! transcript levels is less than that induced by AC agonism with forskolin. Theophylline (10 mM and Rofiumilast ( mM> both increase PCSK1 transcription as single agents, while M 0 52 has thus far only been found to increase PCSK1 transcription in combination with Forskolin in vitro (Figs, 6A-C, F). Combination treatment with Forskolin and Rofiumilast demonstrates that these agents ca work together in an additive, possibly synergistic, manner inducing and increased PCSKi transcription at lower concentrations ( 1 µΜ Forskolin, 0 n Rofiumilast) than when either agent is given alone (Fig. 60 ). Again, increased PCSKI transcription due to combination treatment with Forskolin and Rofiumilast also increases prohormone processing of POMC to ACT!! (Fig. 6E) Specifically, tests with graded concentrations of Forskolm in isolated mouse islets showed a 3-fold upregulatiou of PC! protein at 25 µΜ and 50 µΜ concentrations. No change in PC2 protein levels were observed in response to Forskolin application. We also found that µ,Μ Forskolm applied t primary mouse neurons isolated from E 9.5 ic increased P s transcript levels ~2-fold. M S2

The phenoiype most limiting to WS patients r perphagia which is ost .likely mediated by processes occurring i the central nervous system, particularly the hypothalamus. Thus agents tha aim to ameliorate hyperphagia must be able to penetrate the blood brai barrier. 0 52 is an mt n sica!ly potent { ¾) - 0,6 nM) ir -p netrani PDE4 inhibitor with limited whole blood activity (lC$o ~ 555 nM) (M Gallant et a 2010). As described herein, Μ 52 is the lead PDF inhibition candidate at present

A preliminary in vivo test of M 0 52 was performed in wild type- mice. A single administration of 0952 at 10 g kg body weight results in a 25% increase in hypothalamic PcskI transcript levels (Fig. 7A). Administration of Forskolin at 25 mg kg did not resuit in increased hypothalamic Pcskl transcript ieveis. Co-administration of MK0952 at mg/kg and Forskolin at 25 mg/kg again induced an -25% Increase n hypothalamic Pcskl levels, suggesting that this increase was due primarily to the actions of M 0952 (Wig 7B). We will also analyze circulating cAMP levels, cortical pro D /B F, cerebellar Pcskl , gastric Pcsk , and gastric proghrel /g re n, circulating proinsulm and insulin concentrations (and their ratios), and finally pulmonary Pcsk l transcript and cAMP ieveis from these animals as well.

The first in vivo study with 0952 administered by ora -gavage while forskolio was administered intraperitoneaily one time to 4 hoar fasted wild type mice was completed.. Hypothalamic transcript levels of Pcskl were unregulated -25% following administration of either 0 rag/kg M.K0952 as a single agent or both . mg kg Μ Θ952 .and 25 mg kg orskoJi . However, administration of 25 mg/kg Forskolin only did not result in an upregulation of hypothalamic Pcskl, suggesting the Forskolin at tins dose does cannot access the hypothalamus in sufficient quantities to affect Pcskl transcription. This also suggests that the increase in hypothalamic Pcskl following administration of both 25 mg/kg Forskolin and 10 mg/kg M 0952 was mainly due to the actions of M 0952 in the hypothalamus. This difference likely reflects greater CNS penetrance of the M 9S2, not the general relevance of a cyclase activator to therapy of PWS. No change in th ratio of circulating proinsulininsulin was detected following administration M 0952 or Forskolin. Because the processing o proinsulin to insulin is already quite efficient in WT animals, it is in hindsight unlikely that i would further increase at fasting. However, these 'baseline' data are still valuable for assessing proinsulin t insulin processing under the setting of an intraperitoneal glucose tolerance test at 3 mg/ glucose in both WT and S "i m ce Additionally, samples were collected for measurement of ·circulating cAMP levels. cortical. p BDNF/BD F, cerebellar Pc k , gastric P k , an gastric p oghreiin/g m, and finally pulmonary s J transcript and cAMP levels,

EXAMPLE 3

Clinical study of in patients with Prad r Willi (PWS) The preliminary design of the proposed clinical study for individuals with PWS is based on the hypothesis that the expression of proconvertase 1 (P ) in decreased in the neurons of individuals with PWS (Burnett et ah 20 ). Experimental in vitro and in viv exposure to adenylate cyclase agonists and PDE4 inhibitors causes up-regu at n of PC expression and aciivity in human ste cell-derived and rodent rebra neurons, and human fibroblasts. t is anticipated thai administration of these drugs will increase the conversion of implicated pro-hormones to active hormones The clinical study will address and illustrate the efficacy of he drug i enhancing the activity of PC as follows:

1. To illustrate the effects of the candidate therapeutic agents on the behavioral and th endocrine phenotypes of PWS. 2 To monitor the clinical safety profile of such agents. Study design: The clinical study will utilize a cross-over study design (Cleophas et a 2006, Wellek and Blettner 20 , and Louis et ai 1984). This design provides the power to assess the effect of treatment accounting for the variability between subjects in a small. cohort (Fig. 8A). The. washout period etween the two treatment arras w ill mitigate carryover effects; the short duration of the study minimizes the "time-effects" (effects on the change in disease process over time).

Inclusion criteria*: . Genetically proven diagnosis of S 2. Age > 18 years *Recombinant growth hormone therapy is permissible.

I. Severe psychiatric disorder 2. Uncooperative to take the medication 3. Systemic illness, e.g. serious gastrointestinal illness like inflammatory bowel disease, cardiac disease, especially rhythm disturbances, diagnosis of diabetes, hepatic or renal disease or failure. 4 Anemia defined as hemoglobin < 10 g /d 5. Patients on drugs that have potential interaction with the target drug, e.g. FDE4 inhibitors interact with anti-seizure medications, ci et i e, omeprazole, antibiotics etc Many of these drugs alter hepatic n activit and could interfere with the metabolism of a PDE4 inhibitor. A complete list of exclusion drugs wi l be based on the pharmacokinetic and pha aeodynan properties of the identified therapeutic agent.

Recruitment: The recruitment of the subjects wi l b facilitated by partnership with the PWS Foundation (FPWR and PWSA), patient support groups and the clinicians caring for children with PWS. Phone screening wiil identify potentially eligible subjects who wi l be invited for the screening visit

The study will last for 4-6 weeks and consist of the following visits; r visit: At tins visit, a complete review of medical records, medications and physical examination will be performed along with screening iab measurements (from Fig. B, sa e as safety profile aside from the drug level). Subjects will be provided a week of placebo for the run-in period to assess compliance. This will be a short outpatie visit hoars). All other study visits will be 6 hours long shor in-patient stay, 2. Baseli visi t an t3 from Fig. ) Subjects who successfully complete the run-in period will be invited to participate in the study. eligible subjects will be randomized to either the AP group, or the PA group (Fig.SA). Subjects will be advised to fast for > 8 hours for the visit. Physical profiling will include height, weight, body fat measurement, vital signs, resting energy expenditure and a complete physical examination. A complete

pituitary profile that includes ACTH, Cortisol, F SH L estrogen/testosterone, TS fr e T4, G , IGF-1, 1GFBP3 will be performed. The subjects w ill undergo a mixed meal tolerance test (MMT with standardized meal and blood measurements will be obtained at 0,30,60,90,1 0 and 0 minutes from an indwelling V catheter F g. SB). The primary guardians will complete questionnaires relating to hyperphagia (Dykens or modified Dykens) and behavioral assessment will be performed using the Oxytocin Study Questionnaire (25-28). I addition to this, they will complete a foo frequency questionnaire on 3 separate days to include at least one weekend. The visit is expected t last 6-8 hours. Study medication for 1 week will be dispensed with caregiver i st c io . 3 ll - p visit ( and 4 from Fig. 1) : The subject wil return for a follow-up visit n week after the initiation of the study medication. The measurements mentioned above wil be repeated al this visit, a d medication count wil be obtained, along with structured questionnaire for the assessment of toxicity. Each of these visits will be 6-8 hours. A washout period of 1- weeks will be allowed prior to the 2 phase of the study. Outcome measures:

1. Hormonal profile n response to a standard meal: Base on the effect of PC on converssoo of prohormones {such a proinsuiin to insulin etc.), it is anticipated- that administration of the drug will cause increase in the ratio of prohormone: hormone (e.g. proinsuiin: insulin) (Burnett e al. 20 7). This will be tested by hormonal response to

a standard MMTT. MMTT is performed b administration of a liquid meal (6ec/kg of Boost or equivalent to a maximum of 360 cc) followed by periodic measurement of insulin, proinsuiin, POMC prohormone, ACTH, AgRP, proglucagon, glucagon, GLPl, oxytocin (and propeptide), ghrelin, proghrelm, free fatty acids, and glucose. MMTT has been validated in clinical studies of subjects with PWS (P. Gumus Balikciogiu e al 20 5). Relati o values btaiue is anticipated that there w l be a absolute increase io insulin release, decrease in proinsuiin release and a n increase i insidin/promsulin ratio. A l in 25% range. It is also expected that glucose concentrations will be decreased by . 5-2 and ffa as well. In plasma obtained prior to the M.MT, it is anticipated tha POMC will be increased and AgRP reduced by -25%. Oxytocin should be increased by -2 % as well it is also anticipated that proghreim/ghrelin ratio will be reduced. Spinal fluid may also be examined/studied in these subjects as well, but would not be evaluated in relationship to a ea The following components may be assayed: pome prohormone, beta endorphin, alpha msh, AgRP and oxytocin, anticipating that the drugs would reduce pome prohormone, an increase beta endorphin, alpha msh and oxytocin, and reduc AgRP in comparison to untreated ub ect

2. .Pharm me mic profile: Metaboiomic profiling provides an additional opportimity to understand the effects of the drag on the metabolic phenotype. Metaboiomic profile of the study subjects before and after treatme with the drug wit be performed io identify b mar ers for a) response to treatment in the pathways of interest, v insulin metabolism pathwa and others, b) to identify individual differences in treatment, by identification of pathways selectively up- or down- regulated in different individuals, and c) identify the profiles of side-effects or toxicity using a pathway based analysis that may not be obvious by the standard study of established larger molecular profiling ( . Kaddurah-Daouk, R. Weinshilbourn, 2 ; . . Beger etal. 2 6). 3. Changes in per gia related behavior: The Dykens (and modified Dyke s) questionnaire assesses the behavior, severity and drive for hunger. In addition to these outcomes, the Oxytocin Behavior Questionnaire will assess social and emotional behavior related with eating. These outcomes wi l be supplemented with the analysis of the food frequency questionnaires. It is expected that treatment will also improve behavior and/or emotional state. e size: :A pilot sample si of 6 subjects wil be recruited for this study. The power of this sample size t detect outcomes of interest will depend on the eflect size ascertained by in viva hormonal or pharmacometabolomic profiling in animal models. As reflected in Fig.

8C an effect size o -1.47 is required to detect a significant change in a cohort of 6 subjects. The effect size is calcuiated as the difference in the eans of the observation with the placebo as compared to .the active drug divided by the standard deviation of the change. As each subject serves as his/her ow control, th crossover study design limits the variability and allows reaching power in ¼ of the subjects for a similar parallel ar study design.

Additional Study information;

1. Based on the prior studies in children with PWS and the need to achieve high effect size, the standard mixed eal has been selected for the study. 2. The study wil be conducted in the outpatient facility of the Irving institute for Clinical and Translational Research. 3, An RB protocol for the study will be prepared directed to the appropriate PDE inhibitor and/or cyclase activator. 4 The etabolo ic profiling - if obtained in this preliminary study - will be performed in the Hormone and Metabolite Core of the Diabetes and Endocrinology Research Center. EXAMPLE 4 Methods of Treating Prader-Willi s ndrome - Co i ation therapy of endogenous exogenous MC4 agoaism. Individuals with PWS will be treated using agents that increase endogenous levels of processed hormones by virtue of increasing PC production through raising levels of cellular AMP production and/or blocking its degradation.

In the arcuate nucleus, POMC is processed to cMS by praconvertase 1 (PCI) (S.L.

Wardlaw 20 1 ) . «M is an d l gand of the eSano ot i 4 receptor (MC4 Humans and mice with inactivating mutations in POMC, PCSKl (gene produc CSKJ is PCI), or MC4R are hyperphagic and obese (C, Vaisse et ai. 998). Mutations in MC are the most common single gene cause of obesity in humans (R.J. Loos et ai. 2008). AgRP is also produced n the arcuate nucleus and is an inverse agonist at C4R ProAgRP is processed to AgRP by PC (S.L. Wardlaw 20 ) .

It is possible tha increases i P production ay increase the production of both MS and AgR P, w ic have opposite effe cts at MC4 (Fig, 4). The use of s all molecule or peptide-based MC4R agonists could help to ensure that he extracellular pools of agents tha agonize MC4R are in excess of those tha antagonize MC4R (Fig. 4, Table 3). This would be expected to push signaling at the MC4R towards anorexigenic responses (Fig, 4). Agents thai bind to AgRP and block its effects at MC4R could also be a useful strategy i this setting (Table 3) (E.C, Lee and P. A Caipmo 20 ). Compounds that act similarly thai a e not meotioned in Table 3 may also be useful. This strategy may be efficacious not just for treating PWS, but a so other for s of monogenic/syndromic obesity as weil as common obesity.

Table 3: Exampl Compounds that could co-administered with FSK and PDE Inhibitors MS 2 in t iaz i Transtech Phar Alternative derivatives formulations of above TTP25 5 M - 4 3 Merck Small molecule, Robust weight loss in animal on-peptide models bat limited efficacy MC4 agonist for weight loss in s n- genefie obese; possible that individuals with PWS ay ave increased sensitivity to such agents, no observed cardiovascular side effects, oral formulation.

SUMMARY/CONCLUSIONS

Although the gene encoding PC , P K f , is dowmegulaied in ce l based a d animal models of PWS, the gene itself s intact and thus could be subject to pharmacological manipulation. Th present data provides results of ongoing preclinical studies to pharmacologically manipulate cellular levels of PCS P A . In vitro experiments demonstrate that application of Forskolin, an adenylyl cyclase agonist robustly and reliably upregislates PCS expression in human stem cell-derived neurons, mouse primary neurons, and increases PCI protein level in mouse isolated islets. Furthermore-, Forskolm treatment a so increases POMC prohormone processing in ste n cell-derived hypothalamic neurons. Application of PD.E inhibitors Theophylline a d oll u ilast to stem ce l neurons increases PCSKI transcript levels both as single agents m m combination with Forskolin. Combination treatment of o mi ast and Forskolin a so additiveiy increases POMC prohormone processing (to anorexigenic peptides) in stem cell hypothalamic neurons. Treatment of stem cell-derived neurons with both Forskolin and MK.0952 (a class 4 FDE inihibitor) increases PCSKI mRNA. Finally, a single oral dose of 10 rag/kg MK0952 increases hypothalamic Pcskl transcript levels by 25% i wild type mice. Longer applications of MK0 52 in vivo in both wild type and mice for paternal S'n r will be tested next n addition we will collaborate with Andre a an colleagues to measure circulating pro- and processed hormone levels (e.g. proins l n, POMC, pro-oxytocin, proBDNF) in individuals with PWS and matched controls.

Also provided is a protocol for preliminary clinical study of MK.0952 and other candidate compounds in individuals with PWS. The major aims of this clinical study w l be t monitor the clinical safety profile of these agents io PWS subjects as well as to measure behavioral and neuroendocrine endp io s to assess preliminary efficacy.

References: Cors g M. D . et a!. Genome-Wide Analysis o CREB Target Short Article Genes Reveals

A .Core Promoter Requirement for MP Responsiveness Molecular Ce l 11, 101- 08 (2003). tJdupi, V., Townsead, C. M. & Greeley, G . . Stimulation of Prohormone C ertas - mRNA Expression by Second Messenger Signaling Systems. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 246 463-465 (1998). Tang, W .-i & Hurley, J. Catalytic Mechanism and Regulation of Mammalian Adenyiyi Cyclases, Molecular Pharmacology 54 23 1-240 { 998).

Tesmer, . , J. G. l. T c-Meta - o Catalysis in Adenyiyi Cyclase. Science 285, 756-760 (1999). O da, T. et at. Type-specific regulation of adenyiyi cyclase. Selective pharmacological stimulation and inhibition of adenyiyi cyclase soforms hem 276 47785- 47793, do . 074/jbc.M107233200 (2001). Liang, X. H., Vickers, T. A., G o, S. & Crodke, S. T. Efficient and specific knockdown of small non-coding R As in -mammalian cells and in mice. Nucleic Acids Res 39 el 3,

doi:10. 93/nar/gk 1 (20 1). Su a ara R , . & Taussig, R . Isoforms of Mammalian Adenyiyi Cyclase: p e o Signaling. Molecular Interventions 2, 168-184 (2002). S. L. Wardlaw, Hypothalamic proopiornelanocoriiri processing and the regulation of energy balance. Europeanjournal of pharmacology 660, 2 3-2 9 (201 .

C. Yaisse, . Clement, . Gay-Grand, P. Froguel, A frameshift mutation in human MC4R is associated with a dominant form of obesity. Nature Genetics 20, 1 - 4 ( 98). R . J. Loos i αί , Common variants near- C4R are associated with fat mass, weight and risk of obesity. Nat Genet 4», 768-775 (2008). E. C. Lee, P. A . Carpino, Me!anocortin-4 receptor modulators for the treatment of obesity: a patent analysis (2008-2014), Pharmaceutical Patent Analyst 4, 95-10? (2016).

i ia GM, Sass ne C si P. 2001. Cyclic AMP signaling. Celt 14 : 971- 72. L. C. Burnett et αί , Deficiency i prohormone convertase P impairs prohormone processing i Prader-Willi syndrome. J Clin Invest 127, 93-30 (2017), F. D. P. Deborah J. Good, Kathleen A. ahon, Albert F. Pariow, Heiner Westphai, a R. irsch, Hypogonadism and obesity in mice with a targeted deletion of the Nhlh2 gene. Nature Genetics , 397-401 ( 97). D L. Fox, Dissertation, University of Massachusetts Amherst Aim Arbor, ΜΪ (2007). P. Stijnen, . Ramos-Molina, S. O ah y . W M Creemers, PCS 1 mutations and human doc nopathies: from obesity to gas roir esti al disorders. Endocrine Reviews 7, (2016). M. D. Conkright et al. , Genome-Wide Analysis o EB Target Short Article Genes Reveals A Core Promoter Requirement for cAMP Responsi veness Molecular Cell , 1101- 1108 (2003). V d pi, C. M. To ns nd, G. H. Greeley, Stimulation of Prohormone Conver as m A Expression by Second Messenger Signaling Systems. Biochemical and Biophysical Research Communications 246, 63-465 ( 98). W.-J. Tang, J. Hurley, Catalytic Mechanism and Regulation of Mammalian Adenyiyl Cyclases Molecular Pharmacology 54, 231-240 (1998). J J . G . Tesmer e !., v ~M.e ai~ Catalysis in Adenyiyl Cyclase Scienc 285, 756-760 (1999). T. Onda el al., Type-specific regulation of adenyiyl cyclase. Selective pharmacological stimulation and inhibition of adenyiyl cyclase isoforms J Biol Chem 276, 47785- 47793 (2001). M . Gallant et al. Discovery of M -0952, a selective PDE4 inhibitor for the treatment of long-term memory loss and mild cognitive impairment Bioorgamc Medicinal Chemistry Letters 20, 6387-6393 (2 0). Q Zhang, G . J . Bo a . McCiettan, S. Tob t Hypothalamic expression of sno.RNA Soord l is consistent with a lin to the hyperphagia and obesity symptoms of Prader-Willi syndrome. Bit ev Ne ros i 30, 479-485 (2012). Y. Qi e al, Snordl is critical in i regulation of foo intake and body weight Sci Rep 6, 18614 (2016). L. Wang et Differentiation of hypo a amic ike neurons from human phiripoteat stem cells. J Clin Invest 125, (20! 5). V. Grinevich, M. G. Desar enier , B . Chins, M. Tauber, F. Muscatelli, Ontogenesis of oxytocin pathways n the mammalian brain: late maturation and psychosocial disorders. Front e r a! 4 (20 ) . Tauber ., The Use of Oxytocin to Improve Feeding and Social Skills in Infants With Prader-Willi Syndrome Pediatrics 139, (2017). . J . ppens S. . Don e, A. C. Hokken-Koeiega, Promising effects of oxytocin on social and food-related behaviour in young children with Prader-Willi syndrome: a randomized, double-blind, controlled crossover tria . Clin Endocrinol ( x ) 85, 979- 987 (2016). G . Alvarez -Bolado, F. A . Paul, S. Blaess, Sonic hedgeho lineage in the mouse hypothalamus: fro progenitor domains to hypothalamic regions. Neural development 7, (20 ). S. Blaess, N. Szabo R Haddad-Tovolii, X. Zhou, G. Alvarez-Bolado, Sonic hedgehog signaling in the development of the mouse hypothalamus. Front Ne ro nat. 8, 156 (2014). E . azzoni e al. Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity. Nat Neurosci 16, 1219-1227 (2013). P. Arlotia, . Hobert, Homeoiic Transformations of Neuronal Cell identities. Trends Neurosci 38, 7 -762 (2 ) E. S. Deneris, O. Hobert, Maintenance of postmitotic neuronal cell identity. Nat Neurosci 7, 899-907 (2014). T. J. Oeophas, A. H. Zwindemian, T. F. Cleophas, in Statistics Applied to Clinical Trials. (Springer Netherlands, Dordrecht, 2006), pp -228. S. Weliek, M. Bletiner, On the Proper Use of the Crossover Design in Clinical Trials: Part. 1 of a Series Evaluation of Scientific Publications. D rzt l t International \ 2 6-2 (2 2), T. A. Lotus , P. W. avor , J. C. L Bai!ar , M . Polansky Crossover and Se !f Con led Designs in Clinical Research. New England Journal of ed ci , 4- (1984). E, M, Dykens, M, A . Maxwell, E Pantino, R. Kossler, E, Roof, Assessment of Hyperphagia in Prader-Willi Syndrome. Obesity 1 - 26 (2007), S. R. Crawford e al., The International Development of The Modified Hyperphagia Questionnaire. Value n Heath IS, A 76 J . M . MD, D, D . MD, A . Chen, T. E. Hughes, D . D. Kim, paper presented at the Obesity Week 2014, Boston, MA, 2014. R. I . ppen , S. . Donze, A. C. S. Hokkea-Koeiega, Promising effects of oxytocia on social and food-related behaviour in young children with Prader-Willi syndrome: a randomized, doable-blind, controlled crossover trial. Clinical Endocrinology 85, 979- 987 (20 16}.

P. Gu us Baiikciogiu et al. f Macroautrient Regulation of Ghrelin and Peptide YY in Pediatric Obesity an Prader-Willi Syndrome. The Journal of Clinical Endocrinology & Metabolism 00 , 3822-383 (2015). R. Kaddurah-Daouk, R. Weins lbo m, N. on behalf of the Phar aco e abolo ics Research, Metabolomic Signatures for Dru Response P eno pes; P a aco eta olo ics Enables Precision Medicine Clinical Pharmacology Therapeutics 98, 71-75 (2015). R. D. Beger et al., Metaboloroics enables precision medicine: "A White Paper, n Perspective". Metaholomics , 149 (20 ).

The scope of the present invention is not limited by what as been specifically shown and described hereinabove. Numerous references, including patents and various publications, are cited and discussed i the description of this invention. The citation a d discussion of such references s provided merely to cla fy the description of the present invention and is not an admission that any reference i prior art. to the invention described herein. A H references cited and discussed in tins specification are incorporated herein by reference in their entirety. Variations, modifications and other implementations of what is described herein wi occur to those of ordinary skill m the art without departing from the spirit and scope of the ra vention. While certain embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from the spirit and scope of the invention. The matter set forth in the foregoing description and accompanying drawings is offered by way of illustration only and not a a limitation. The aclual scope of the invention is intended to be defined in the following claims. WHAT IS CLAIMED IS:

. A method for regulating prohormone convertase comprising, administering a therapeutically effective amount of a phosphodiesterase 4 inhibitor (PDE4 inhibitor or PDE4i).

2. A method for preg a i expression of pr»horinoae convertase comprising, administering a therapeutically effective amount of a P 4 inhibitor.

Th method of clai wherein the PDE4 inhibitor is administered in m m to a eel

4. The method of claim wherein the P E inhibitor is administered to a patient with

Prader-Wi i syndrome.

5. The method of claim , whereto the PDE inhibitor is administered orally.

6 The method of claim 4, wherein the PDE4 inhibitor is administered intravenously or subcutaneonsly.

7 The method of claim 4, wherein the PDE4 inhibitor is administered intrathecal!}'.

8. The method of claim 4, wherein the PDE4 inhibitor is administered topically.

.10. The method of claim 4, wherein th PDE4 inhibitor is administered intranasaily .

The method of claim 4, wherein the PDE4 inhibitor is administered in th lungs.

. The method of claim wherein the PDE4 inhibitor is selected from the group consisting of theophylline, roflimiiasf apremilast, ibdalast, GS 35 278, M . 952, IBMX, and combinations thereof. 3. A method for regulating convertase comprising administering a therapeutically effective amount of an adenylate cyclase activator.

14. The method of claim 13, wherein the adenylate cyclas activator is administered in vitro to a ceil

5. The method of ciaim 13 , wherein the adenylate cyclase activator is administered to a patient with Prader- il i syndrome.

16. The method of claim S3, wherein the adenylate cyclase activator is administered orally, intravenously, susbcuianeously, intrathecal! or in ra asaily

. The method of clai 3 wherein the adeny late cyclase activator is administered topically.

. The method of claim 13, wherein the adenylate cyciase activator is administered in the kings

. The method of clai 13, wherein the adenylate cyclase activator i selected from group consisting of Forskoltu, FD F , FD3, FD FD5 NK 4 7), FD6, and combinations t ereo

20. A method for regulating prohormone convettase comprising, administering a t erape ica ly effective amount of a P E4 inhibitor and an adenylate cyciase activator.

. The method of clai , wherein the PDE4 inhibitor and the adenylate cyciase activator are administered to a patient with Prader-Willi syndrome.

22. The method of claim 1, wherein the PDE4 inhibitor is administered to an obese subject.

23. The method of claim 3, wherein the adenylate cyclase activator is administered to an obese subject:. 24. The of claim 20, wherein the PDE4 inhibitor and the adenylate cyclase activator are administered to an obese subject.

25. A method for preg lat g expression of prohormone c nvertas comprising administering a therapeutically effective amount of an MC4R agonist,

26. The method of claim 25, wherein the MC4R agonist is administered in vitro to a cell.

27. The method of claim 25, wherein the MC4R agonist i admini stered to a patient w th Prader-Willi syndrome.

28. The method of claim. 25, wherein the MC4R. agonist is administered t an obes subject.

29. The method of claim 25, wherein the MC4R agonist is administered orally.

30 The method of claim 25, wherein the MC4R agonist is administered intravenously or u cntar ons y .

3 ί . The metho of clai 25, wherein the C4R agonist is administered iniratheca!ly.

32. The method of claim 25, wherein the M 4R agonist is administered topically.

33. The method of claim 25, wherein the C4R agonist is administered infranasaliy .

34 . The method of claim. 25, wherein the MC4 agonist is administered i the Jungs.

33. The method of claim 25, w e ein the 4R agonist is selected from, the grou

consisting of M-4 3 (Setmeianotide), TTP2 5 2~a in i zoie derivatives, K. 0493. and combinations thereof

36, The method of any one of claims 1 2, 3 , or 20, further comprising administering a therapeutically effective amount of an MC4R agonist. 37. The of claim 3 wherein the C4R agonist is admini stered in vitro o ce .

38 The method of claim , wherein C agonist is administered to & pati ent with Prader-Willi syndrome.

39. The method of claim wherein the M 4 agonist is administered to an obese subject.

40. The method of claim 36, wherein the C4 agonist is selected from the group consisting of M-4 3 (Setrnelanotide), ΤΪ Ρ2 5, 2 a ino hia o e derivatives, MK 493, a d combinations thereof.

41. The method of any one of claims 4, 15, 21, 27, or 28, wherein the administration results in one or more of the following improvements in the patient: decreases or ameliorates hyperpliagia; increases Peskl levels; increases PCI level and/or activity; decreases circulating proms li to insulin ratio; decreases circulating pr ghre in to ghrelin ratio; decreases circulating POMC to ACTH ratio; amelioration of hypothyroidism, decreases circulating ratio of pro-oxy tocio to oxytocin; decreases circulating ratio of pro-bdnf to bd&f; and increases the ratio of prohormone: hormone; wherein the symptom, levels, or ratios are in reference to the patient's disease symptom, levels, or ratios.

42. A method for treating Prader-Willi Syndrome (PWS) comprising administering a phosphodiesterase 4 inhibitor (PDE4i) to a subject in need thereof, thereby alleviating, eliminating or preventing one or more symptoms of PWS.

43. The method of claim 42, wherein administering the PDE4i preg laies cyclic adenosine monophosphate (cAMP) concentrations or activity in the subject,

44 The method of claim 43, wherein PWS is characterized by decreased expression of Nh 2.

45. The method of claim 44, wherein decreased expression of Nh!¾2 results in. decreased expression of Pcsk . 46. The method of claim 43, wherein increasing concentrations or activity of AM P lipreguiates expression of Pc i .

47. The method of claim 43 wherein the PDE4i a selective PDE4i

48. The method of claim 43, wherein the PDE4i is a non-selective PD E4i

49. The method of claim 47, wherein the selective PDE4i is selected from AN apr mi ast cilomiiast, diazepam, budi ast l eo!in, mesembreoone, piciamiiast, rofiumilast, rolipram, E6005, GSK356278 and M 9 2

50. The method of claim 48, wherein die non-seieclive PDB4i selected from methylated xanthines and derivatives thereof, caffeine, am ophyli ne 3 isob tyl e hy xanth ne, paraxanthine, pentoxifylline, theobroinine, a d theophylline.

. The method of claim 42, wherein the one or more symptoms include !iyperphagia, reduced metabolic rate, obesity, hypogonadism, decreased growth hormone production, poor muscle tone, sleep disorders, gastrointestinal disorders, reduced stamina, reduced ability to focus, impaired cognition, behavioral disorders, anxiety, growth failure, reduced conversion of immature hormones to mature and active forms, and diabetes.

52 The method of claim 42, wherein th method further comprises administering one or mor additional therapeutic agents effective for treating or alleviating one o more symptoms PWS.

53. The method of claim 51, wherein the immature hormones- comprise one or more of insulin, g re m, GHRii, alpha-MSH, oxytocin, orexin, BDNF, vasopressin, NPY, AGRP, and gonadotropin.

54. The method of claim 52, wherein the one or ore additional therapeutic agents effective at treating or alleviating PWS include insulin, an insulin receptor agonist, ghrelm, a. ghrelift receptor agonist, GHRH, a GHRII receptor agonist, alpha-MSH, an alpha-MSH receptor agonist, oxytocin, an oxytocin receptor agonist orexin an orexi receptor agonist, BDNF. BDNF receptor agonist, vasopressin, a vasopressin receptor n , NP , a NPY receptor agonist, AGRP, an AGRP receptor agonist, goaad s rop , a gonadotropin receptor against, or combinations thereof.

INTERNATIONAL SEARCH REPORT International application No.

PCT/US 17/35655

A . CLASSIFICATION O F SUBJECT MATTER IPC(8) - A61 K 31/221 , A23L 1/302, A61 K 31/1 98 (201 7.01 ) CPC - A61 K 36/53, A61 K 36/1 85, A61 K 31/403, A61 K 31/21 6

According to International Patent Classification (IPC) or to both national classification and IPC

B . FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

See Search History Document

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

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) See Search History Document

C . DOCUMENTS CONSIDERED T O E RELEVANT

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

Piro et al. "Chronic Exposure to GLP-1 Increases GLP-1 Synthesis and Release in a Pancreatic 13-14, 16-19, 23 Alpha Cell Line (a-TC1 ): Evidence of a Direct Effect of GLP-1 on Pancreatic Alpha Cells" PLOS One. February 2014, vol 9, pg. 1-14; pg. 1, right col, para 2 , pg. 4 , right col, para 9 , pg. 7 , right 1-8, 10-12, 15, 20-22, 24, col, para 4 , pg. 12, left col, para 2 (36-40)/(1, 2 , 13, 20), 41/(4, 15, 21), 43-50

Jensterle et al. "Short term monotherapy with GLP-1 receptor agonist liraglutide or PDE 4 42, 51-54 inhibitor roflumilast is superior to metformin in weight loss in obese PCOS women: a pilot randomized study" Journal of Ovarian Research. 0 2 June 2015 (02.06.2015) vol 8 , pg. 1-8; pg. 1-8, 10-12, 20-22, 24, (36 2 , left col, para 2 , 5 , pg. 6 , left col, para 2 -40)/(1-2, 20), 41/(4, 21), 43-50

Gabreels et al. "Attenuation of the Polypeptide 7B2, Prohormone Convertase PC2, and 4-8, 10-1 1, 15, 2 1, 41/(4, Vasopressin in the Hypothalamus of Some Prader-Willi Patients: Indications for a Processing 15, 21) Defect" Journal of Clinical Endocrinology and Metabolism. 0 1 February 1998 (01 .02.1998) vol 83, pg. 591-599; pg. 591 , para 1

Rhythm "Rhythm Presents Positive Data from Phase 1b Study of Setmelanotide for the 25-35, (36-40)/(1, 2 , 13, Treatment of Genetic Obesity" 06 November 2015 (06.1 1.2015) ; pg. 1, para 1-3

Chen et al. "Monogenic disorders of obesity and body fat distribution" Journal of Lipid Research. 25-35, (36-40)/(1 , 2 , 13, October 1999, vol 40, pg. 1735-1746; pg. 1739, left col, para 1, pg. 1741, figure 4 20), 41/(27, 28)

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

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

Date of the actual completion of the international search Date of mailing of the international search report 0 3 August 2017 0 S EP 2017

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

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

PCT/US 17/35655

C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

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

Wankhade et al. "Melanocortin 4 receptor is a transcriptional target of nescient helix-loop-helix- 45 2" Molecular and Cellular Endocrinology. 20 July 201 1 (20.07.201 1) vol 341 , pg. 39-47; pg. 40, left col, para 2

Miller et al. "Necdin, a Prader-Willi syndrome candidate gene, regulates gonadotropin-releasing 44 hormone neurons during development" Human Molecular Genetics. 17 October 2008 (17.10.2008) vol 18, pg. 248-260; pg. 249, left col, para 1

Ramos-Molina et al. "PCSK1 Variants and Human Obesity" Progress in Molecular Biology and 46 Translational Science. 29 January 2016 (29.01 .2016) vol 140, pg. 47-74; abstract

Yang et al. "Effect of Caffeine on Erectile Function via Up-Regulating Cavernous Cyclic 48, 50 Guanosine Monophosphate in Diabetic Rats" Journal of Andrology, 02 January 2013 (02.01.2013) vol 29, pg. 586-591 ; pg. 586, right col, para 2

X, P Burnett et al. "Deficiency in prohormone convertase PC1 impairs prohormone processing in 1-8, 10-54 Prader-Willi syndrome" The Journal of Clinical Investigation. January 2017, vol 127, pg. 293- 305; entire document

Form PCT/ISA/210 (continuation of second sheet) (January 201 5)