(19) &   

(11) EP 2 377 530 A2

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 19.10.2011 Bulletin 2011/42 A61K 31/40 (2006.01) A61K 45/06 (2006.01) A61K 31/4015 (2006.01) A61K 31/403 (2006.01) (2006.01) (2006.01) (21) Application number: 10013573.0 A61K 31/416 A61K 31/4166 A61K 31/4184 (2006.01) A61K 31/505 (2006.01) (2006.01) (2006.01) (22) Date of filing: 20.10.2006 A61P 25/00 A61K 31/401

(84) Designated Contracting States: • Lorrain, Kym, I. AT BE BG CH CY CZ DE DK EE ES FI FR GB GR San Diego HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI California 92124 (US) SK TR • Pires, Jammieson, C. Chula Vista (30) Priority: 21.10.2005 US 729366 P CA 91911 (US) 21.03.2006 US 784605 P • Treuner, Kai 17.07.2006 US 807594 P San Diego California 92122 (US) (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: (74) Representative: Roques, Sarah Elizabeth 06826395.3 / 1 940 389 J.A. Kemp & Co. 14 South Square (71) Applicant: Braincells, Inc. Gray’s Inn San Diego, CA 92121 (US) London WC1R 5JJ (GB)

(72) Inventors: Remarks: • Barlow, Carrolee This application was filed on 12-10-2010 as a Del Mar divisional application to the application mentioned California 92014 (US) under INID code 62. • Carter, Todd, A. San Diego California 92129 (US)

(54) Modulation of neurogenesis by PDE inhibition

(57) The instant disclosure describes methods for methods based on use of a PDE agent, optionally in com- treating diseases and conditions of the central and pe- bination with one or more other neurogenic agents, to ripheral nervous system by stimulating or increasing neu- stimulate or activate the formation of new nerve cells. rogenesis. The disclosure includes compositions and EP 2 377 530 A2

Printed by Jouve, 75001 PARIS (FR) 1 EP 2 377 530 A2 2

Description ical and pharmacological properties of the encoded PDEs (e.g., specificity for cAMP and/or cGMP, response RELATED APPLICATIONS to modulatory compounds). [0005] PDE families that specifically/preferentially hy- [0001] This application claims benefit of priority from 5 drolyze cAMP include PDE4, PDE7, and PDE8, whereas U.S. Provisional Patent Applications 60/729,366, filed families that specifically/preferentially hydrolyze cGMP October 21, 2005, 60/784,605, filed March 21, 2006, and include PDE5, PDE6, and PDE9. The PDE1, PDE2, 60/807,594, filed July 17, 2006. All three of these appli- PDE3, PDE10, and PDE11 families show substantial ac- cations are hereby incorporated by reference as if fully tivity agonist both cAMP and cGMP. Many PDE gene set forth. 10 families comprise multiple genes, which give rise to dis- tinct isozymes. For example, the PDE3, PDE6, PDE7, FIELD OF THE DISCLOSURE and PDE8 families each comprise at least two genes (3A, 3B; 6A, 6B; 7A, 7B; 8A, 8B), while the PDE1 family com- [0002] The instant disclosure relates to methods for prises at least three genes (1A, 1B, 1C), and the PDE4 treating diseases and conditions of the central and pe- 15 family comprises at least four genes (4A, 4B, 4C, 4D). In ripheral nervous system by stimulating or increasing neu- addition, the majority of PDE gene transcripts are subject rogenesis via inhibition of cyclic nucleotide phosphodi- to alternative splicing, giving rise to multiple isozymes esterase ("PDE") activity, optionally in combination with within each family. PDE isozymes are differentially ex- another neurogenic agent. The disclosure includes meth- pressed in various tissues, cell types, and subcellular ods based on the application of an PDE inhibitor and20 locations, and numerous PDE isozymes have been de- another neurogenic agent to stimulate or activate the for- tected throughout the CNS. mation of new nerve cells. [0006] Citation of the above documents is not intended as an admission that any of the foregoing is pertinent BACKGROUND OF THE DISCLOSURE prior art. All statements as to the date or representation 25 as to the contents of these documents is based on the [0003] Neurogenesis is a vital process in the brains of information available to the applicant and does not con- animals and humans, whereby new nerve cells are con- stitute any admission as to the correctness of the dates tinuously generated throughout the life span of the or- or contents of these documents. ganism. The newly born cells are able to differentiate into functional cells of the central nervous system and inte- 30 BRIEF SUMMARY OF THE DISCLOSURE grate into existing neural circuits in the brain. Neurogen- esis is known to persist throughout adulthood in two re- [0007] Disclosed herein are compositions and meth- gions of the mammalian brain: the subventricular zone ods for the prophylaxis and treatment of diseases, con- (SVZ) of the lateral ventricles and the dentate gyrus of ditions and injuries of the central and peripheral nervous the hippocampus. In these regions, multipotent neural 35 systems by stimulating or increasing neurogenesis. As- progenitor cells (NPCs) continue to divide and give rise pects of the methods, and activities of the compositions, to new functional neurons and glial cells (for review Gage include increasing or potentiating neurogenesis in cases 2000). It has been shown that a variety of factors can of a disease, disorder, or condition of the nervous system. stimulate adult hippocampal neurogenesis, e.g., adrena- Embodiments of the disclosure include methods of treat- lectomy, voluntary exercise, enriched environment, hip- 40 ing a neurodegenerative disorder, neurological trauma pocampus dependent learning and anti-depressants including brain or central nervous system trauma and/or (Yehuda 1989, van Praag 1999, Brown J 2003, Gould recovery therefrom, depression, anxiety, psychosis, 1999, Malberg 2000, Santarelli 2003). Other factors, learning and memory disorders, and ischemia of the cen- such as adrenal hormones, stress, age and drugs of tral and/or peripheral nervous systems. In other embod- abuse negatively influence neurogenesis (Cameron45 iments, the disclosed methods are used to improve cog- 1994, McEwen 1999, Kuhn 1996, Eisch 2004). nitive outcomes and mood disorders. [0004] Cyclic adenosine monophosphate (cAMP) and [0008] In one aspect, methods of modulating, such as cyclic guanosine monophosphate (cGMP) are ubiquitous by stimulating or increasing, neurogenesis are disclosed. second messengers that mediate a wide range of proc- The neurogenesis may be at the level of a cell or tissue. esses in mammalian cells, including vision, olfaction,50 The cell or tissue may be present in an animal subject or platelet aggregation, aldosterone synthesis, insulin se- a human being, or alternatively be in an in vitro or ex vivo cretion, T cell activation, and smooth muscle relaxation. setting. In some embodiments, neurogenesis is stimulat- Cyclic nucleotide phosphodiesterases ("PDEs") regulate ed or increased in a neural cell or tissue, such as that of intracellular levels of cAMP and cGMP by catalyzing their the central or peripheral nervous system of an animal or hydrolysis to the corresponding nucleotide 5’-monophos- 55 human being. In cases of an animal or human, the meth- phates. Over 20 PDE genes have been cloned, encoding ods may be practiced in connection with one or more 11 gene families (PDE1- PDE11), which are classified ac- diseases, disorders, or conditions of the nervous system cording to sequence homology, as well as the biochem- as present in the animal or human subject. Thus, em-

2 3 EP 2 377 530 A2 4 bodiments disclosed herein include methods of treating the method may be used to improve, maintain, or stabilize a disease, disorder, or condition by administering at least mood in a subject or patient Of course the method may one neurogenesis modulating agent having inhibitory ac- be optionally combined with any other therapy or condi- tivity against a cyclic nucleotide phosphodiesterase tion used in the treatment of a mood disorder. ("PDE"), hereinafter referred to as a "PDE agent". A PDE 5 [0014] In a third aspect, the disclosed methods include agent may be formulated or used alone, or in combination identifying a patient suffering from one or more diseases, with one or more additional neurogenic agents, such as disorders, or conditions, or a symptom thereof, and ad- another PDE agent or a non-PDE agent. ministering to the patient a PDE agent, optionally in com- [0009] The disclosure thus includes a method of using bination with one or more other neurogenic agents, as a chemical entity as a PDE agent to increase neurogen- 10 described herein. In some embodiments, a method in- esis. In some embodiments, a chemical entity used as cluding identification of a subject as in need of an in- an agent is a therapeutically or pharmaceutically accept- crease in neurogenesis, and administering to the subject able reversible PDE inhibitor. Alternatively, an accepta- a PDE agent, optionally in combination with one or more ble irreversible PDE inhibitor may also be used in some other neurogenic agents is disclosed herein. In other em- embodiments of the disclosure. Additional embodiments 15 bodiments, the subject is a patient, such as a human comprise an inhibitor that is a tertiary amine which cross- patient es the blood brain barrier. [0015] Another aspect of the disclosure describes a [0010] While a PDE agent may be considered a "direct" method including administering a PDE agent, optionally agent in that it has direct activity against a PDE by inter- incombination with one or more other neurogenicagents, actions therewith, the disclosure includes a PDE agent 20 to a subject exhibiting the effects of insufficient amounts that may be considered an "indirect" agent in that it does of, or inadequate levels of, neurogenesis. In some em- not directly interact with a PDE. Thus, an indirect agent bodiments, the subject may be one that has been sub- acts on a PDE indirectly, or via production, generation, jected to an agent that decreases or inhibits neurogen- stability, or retention of an intermediate agent which di- esis. Non-limiting examples of an inhibitor of neurogen- rectly interacts with a PDE. 25 esis include opioid receptor agonists, such as a mu re- [0011] Embodiments of the disclosure include a com- ceptor subtype agonist like morphine. In other cases, the bination of a PDE agent and one or more other neuro- need for additional neurogenesis is that detectable as a genic agents disclosed herein or known to the skilled reduction in cognitive function, such as that due to age- person. An additional neurogenic agent as described related cognitive decline, Alzheimer’s Disease, epilepsy, herein may be a direct PDE agent, an indirect PDE agent, 30 or a condition associated with epilepsy as non-limiting or a neurogenic agent that does not act, directly or indi- examples. rectly, through a PDE. Thus in some embodiments, an [0016] In a related manner, a method may include ad- additional neurogenic agent is one that acts, directly or ministering a PDE agent, optionally in combination with indirectly, through a mechanism other than a PDE. An one or more other neurogenic agents, to a subject or additional neurogenic agent as described herein may be 35 person that will be subjected to an agent that decreases one which acts through a known receptor or one which or inhibits neurogenesis. Non-limiting embodiments in- is known for the treatment of a disease or condition. The clude those where the subject or person is about to be disclosure further includes a composition comprising a administered morphine or another opioid receptor ago- combination of a PDE agent with one or more other neu- nist, like another opiate, and so about to be subject to a rogenic agents. 40 decrease or inhibition of neurogenesis. Non-limiting ex- [0012] In a second aspect, the disclosure includes a amples include administering a PDE agent, optionally in method of lessening and/or reducing a decline or de- combination with one or more other neurogenic agents, crease of cognitive function in a subject or patient In some to a subject before, simultaneously with, or after the sub- cases, the method may be applied to maintain and/or ject is administered morphine or other opiate in connec- stabilize cognitive function in the subject or patient. The 45 tion with a surgical procedure. method may comprise administering a PDE agent, op- [0017] In a fifth aspect, the disclosure includes meth- tionally in combination with one or more other neurogenic ods for preparing a population of neural stem cells suit- agents, to a subject or patient in an amount effective to able for transplantation, comprising culturing a popula- lessen or reduce a decline or decrease of cognitive func- tion of neural stem cells (NSCs) in vitro, and contacting tion. 50 the cultured neural stem cells with a PDE agent, option- [0013] In an additional aspect, the disclosure includes ally in combination with one or more other neurogenic a method of treating mood disorders with use of a PDE agents. In some embodiments, the stem cells are pre- agent, optionally in combination with one or more other pared and then transferred to a recipient host animal or neurogenic agents. In some embodiments, the method human. Non-limiting examples of preparation include 1) may be used to moderate or alleviate a mood disorder 55 contact with a PDE agent, optionally in combination with in a subject or patient. Non-limiting examples include a one or more other neurogenic agents, until the cells have subject or patient having, or diagnosed with, a disease undergone neurogenesis, such as that which is detect- or condition as described herein. In other embodiments, able by visual inspection or cell counting, or 2) contact

3 5 EP 2 377 530 A2 6 with a PDE agent, optionally in combination with one or nation, the compounds were combined at equal con- more other neurogenic agents, until the cells have been centrations at each point (for example, the first point sufficiently stimulated or induced toward or into neuro- in the combined curve consisted of a test of 0.01 PM genesis. The cells prepared in such a non-limiting man- captopril and 0.01 PM enoximone). Data is present- ner may be transplanted to a subject, optionally with si- 5 ed as the percentage of the neuronal positive control, multaneous, nearly simultaneous, or subsequent admin- with basal media values subtracted. When used istration of another neurogenic agent to the subject. alone, EC50 was observed at a captopril concentra- While the neural stem cells may be in the form of an in tion of 3.8 PM or an enoximone concentration of 6.8 vitro culture or cell line, in other embodiments, the cells PM in test cells. When used in combination, EC50 may be part of a tissue which is subsequently transplant- 10 was observed at a combination of captopril and ed into a subject. enoximone at concentrations of 1.1 PM each. [0018] In yet another aspect; the disclosure includes FIG. 3 is a dose-response curve analogous to FIG. methods of modulating, such as by stimulating or increas- 2 and showing the effect of the neurogenic agents ing, neurogenesis in a subject by administering a PDE serotonin and enoximone (PDE-3 inhibitor) in com- agent, optionally in combination with one or more other 15 bination on neuronal differentiation compared to the neurogenic agents. In some embodiments, the neuro- effect of either agent alone. When used alone, EC 50 genesis occurs in combination with the stimulation of an- was observed at a serotonin concentration of 7.4 giogenesis which provides new cells with access to the PPM or an enoximone concentration of 6.8P M in circulatory system. test cells. When used in combination, EC 50 was ob- [0019] The details of additional embodiments are set 20 served at a combination of serotonin and enoximone forth in the accompanying drawings and the description at concentrations of 0.74 PM each. below. Other features, objects, and advantages of the FIG. 4 is a dose-response curve analogous to FIG. embodiments will be apparent from the drawings and de- 2 and 3 which shows the effect of the neurogenic tailed description, and from the claims. agents serotonin and rolipram (PDE-4 inhibitor) in 25 combination on neuronal differentiation compared to BRIEF DESCRIPTION OF THE DRAWING the effect of either agent alone. When used alone, EC50 was observed at a serotonin concentration of [0020] 7.4 PM or a rolipram concentration of 2.3 PM in test cells. When used in combination, EC50 was ob- FIG. 1 is a dose-response curve showing the effect 30 served at a combination of serotonin and rolipram at of the neurogenic agents captopril (angiotensin con- concentrations of 0.58 PM each. vertingenzyme, orACE, inhibitor)and ibudilast(PDE FIG. 5 is a dose-response curve analogous to FIG. inhibitor) in combination on neuronal differentiation 2 to 4 and showing the effect of the neurogenic compared to the effect of either agent alone. When agents buspirone (5- HT1a receptor agonist) and rol- run independently, ibudilast was tested in a concen- 35 ipram (PDE-4 inhibitor) in combination on neuronal tration response curve (CRC) ranging from 0.01 PM differentiation compared to the effect of either agent to 10.0 PM and captopril was tested in a CRC ranging alone. When used alone, EC50 was observed at a from 0.01 PM to 31.6 PM. In combination, ibudilast buspirone concentration of 9.4 PM or a rolipram con- was tested in a CRC ranging from 0.003 PM to 10.0 centration of 2.3 PM in test cells. When used in com- 40 PM and captopril was added at a concentration 3.16- bination, EC50 was observed at a combination of fold higher at each point (for example, the first point buspirone and rolipram at concentrations of 0.74 PM in the combined curve reflects a combination of each. 0.003 PM ibudilast and 0.01 PM captopril). Data is FIG. 6 is a dose-response curve analogous to that presented as the percentage of the neuronal positive of FIG. 1 and showing the effect of the neurogenic control, with basal media values subtracted. When 45 agents ibudilast (PDE inhibitor) and candesartan used alone, EC50 was observed at a captopril con- (angiotensin II AT1 receptor antagonist) in combina- centration of 3.8 PM or an ibudilast concentration of tion on neuronal differentiation compared to the ef- 6.2 PM in test cells. When used in combination, EC 50 fect of either agent alone. When used alone, EC50 was observed in a combination of captopril at a con- was observed at a candesartan concentration of 2.2 centration of 0.15 PM and ibudilast at a concentration 50 PM or an ibudilast concentration of 6.2P M in test of 0.05 PM cells. When used in combination, EC50 was ob- FIG. 2 is a dose-response curve showing the effect served at the combination of candesartan at a con- of the neurogenic agents captopril (ACE inhibitor) centration of 0.35 PM and ibudilast at a concentration and enoximone (PDE-3 inhibitor) in combination on of 0.11 PM. neuronal differentiation compared to the effect of ei- 55 ther agent alone. When run independently, each DEFINITIONS compound was tested in a concentration response curve ranging from 0.01 PM to 31.6 PM. In combi- [0021] "Neurogenesis" is defined herein as prolifera-

4 7 EP 2 377 530 A2 8 tion, differentiation, migration and/or survival of a neural cells can produce progeny that are capable of differenti- cell in vivo or in vitro. In some embodiments, the neural ating into more than one cell type. cell is an adult, fetal, or embryonic neural stem cell or [0026] The terms "animal" or "animal subject" refers to population of cells. The cells may be located in the central a non-human mammal, such as a primate, canine, or nervous system or elsewhere in an animal or human be- 5 feline. In other embodiments, the terms refer to an animal ing. The cells may also be in a tissue, such as neural that is domesticated (e.g. livestock) or otherwise subject tissue. In some embodiments, the neural cell is an adult, to human care and/or maintenance (e.g. zoo animals and fetal, or embryonic progenitor cell or population of cells, other animals for exhibition). In other non- limiting exam- or a population of cells comprising a mixture of stem cells ples, the terms refer to ruminants or carnivores, such as and progenitor cells. Neural cells include all brain stem 10 dogs, cats, birds, horses, cattle, sheep, goats, marine cells, all brain progenitor cells, and all brain precursor animals and mammals, penguins, deer, elk, and foxes. cells. Neurogenesis includes neurogenesis as it occurs [0027] The term "PDE agent" as used herein includes during normal development, as well as neural regener- a neurogenic agent, as defmed herein, that elicits an ob- ation that occurs following disease, damage or therapeu- servable response upon contacting a PDE, including one tic intervention, such as by the treatment describe herein. 15 or more of the known isozymes. "PDE agents" useful in [0022] A "neurogenic agent" is defined as a chemical the methods described herein include compounds or agent or reagent that can promote, stimulate, or other- agents that, under certain conditions, may act as modu- wise increase the amount or degree or nature of neuro- lators or inhibitors of PDE activity (able to modulator or genesis in vivo or ex vivo or in vitro relative to the amount, inhibit one or more characteristic activities of a PDE, for degree, or nature of neurogenesis in the absence of the 20 example, by competitively or non-competitively binding agent or reagent. In some embodiments, treatment with to the PDE, a ligand of PDE, and/or a downstream sig- a neurogenic agent increases neurogenesis if it promotes naling molecule). neurogenesis by at least about 5%, at least about 10%, [0028] The term "PDE inhibitor" refers generally to a at least about 25%, at least about 50%, at least about neurogenic agent, as disclosed herein, that decreases 100%, at least about 500%, or more in comparison to the 25 PDE activity relative to PDE activity in the absence of the amount, degree, and/or nature of neurogenesis in the agent. In some embodiments, PDE activity is reduced by absence of the agent, under the conditions of the method at least about 50%, or at least about 75%, or at least used to detect or determine neurogenesis. As described about 90%. In further embodiments, PDE activity is re- herein, a PDE agent that promotes, stimulates, or other- duced by at least about 95%, or by at least about 99%. wise increases the amount or degree or nature of neu- 30 PDE inhibitors useful in methods described herein may rogenesis is a neurogenic agent. act as competitive inhibitors, non-competitive inhibitors, [0023] The term "astrogenic" is defined in relation to allosteric inhibitors, and/or any mechanism that inhibits "astrogenesis" which refers to the activation, prolifera- a target PDE activity. tion, differentiation, migration and/or survival of an astro- [0029] In various embodiments, a PDE agent may act cytic cell in vivo or in vitro. Non-limiting examples of as- 35 directly against a PDE, or indirectly in connection with a trocytic cells include astrocytes, activated microglial co-factor, substrate, or other molecule. For example, cells, astrocyte precursors and potentiated cells, and as- some PDE isozymes are subject to allosteric regulation trocyte progenitor and derived cells. In some embodi- by endogenous activators and/or inhibitors, wherein ments, the astrocyte is an adult, fetal, or embryonic as- binding of an allosteric regulator modulates enzymatic trocyte or population of astrocytes. The astrocytes may 40 activity.Examples of PDEs subject to allosteric regulation be located in the central nervous system or elsewhere in include PDE1, which is allosterically activated by Ca2+/ an animal or human being. The astrocytes may also be calmodulin, and PDE2 and PDE5, which are allosterically in a tissue, such as neural tissue. In some embodiments, activated by cGMP. Allosteric regulators often modulate the astrocyte is an adult, fetal, or embryonic progenitor the susceptibility of PDEs to inhibition with particular in- cell or population of cells, or a population of cells com- 45 hibitors. For example, binding of cGMP to the allosteric prising a mixture of stem and/or progenitor cells, that site of PDE5 enhances binding of PDE5 inhibitors, such is/are capable of developing into astrocytes. Astrogene- as sildenafil. Thus, in some embodiments, a PDE agent sis includes the proliferation and/or differentiation of as- is administered in conjunction with an allosteric regulator trocytes as it occurs during normal development, as well of the target PDE, or an agent that modulates the activity as astrogenesis that occurs following disease, damage 50 and/or levels of an endogenous allosteric regulator of the or therapeutic intervention. target PDE (e.g., calcium- channel modulators, cyclic nu- [0024] The term "stem cell" (or neural stem cell (NSC)), cleotide cyclase activators). Methods for detecting allos- as used herein, refers to an undifferentiated cell that is teric binding to PDEs are described, e.g., in Weeks et capable of self-renewal and differentiation into neurons, al., Methods Mol Biol. 2005;307:239-62. In some embod- astrocytes, and/or oligodendrocytes. 55 iments, a PDE agent may modulate the activity of a PDE [0025] The term "progenitor cell" (e.g., neural progen- in response to another compound or treatment modality. itor cell), as used herein, refers to a cell derived from a [0030] In other embodiments, the PDE agent(s) used stem cell that is not itself a stem cell. Some progenitor in the methods described herein has "selective" activity

5 9 EP 2 377 530 A2 10 under certain conditions against one or more PDE iso- decreasing the degradation of, or increasing the stability zymes with respect to the degree and/or nature of activity of, an intermediate agent. against one or more other PDE isozymes. In certain em- [0034] In some cases, a PDE agent, optionally in com- bodiments, selective activity of one or more PDE agents bination with one or more other neurogenic agents, re- results in enhanced efficacy, fewer side effects, lower 5 sults in improved efficacy, fewer side effects, lower ef- effective dosages, less frequent dosing, or other desira- fective dosages, less frequent dosing, and/or other de- ble attributes. sirable effects relative to use of the neurogenesis mod- [0031] The terms "cGMP-specific PDE" and "cAMP- ulating agents individually (such as at higher doses), due, specific PDE" refer to PDEs that specifically and/or pref- e.g., to synergistic activities and/or the targeting of mol- erentially hydrolyze cGMP or cAMP, respectively. In10 ecules and/or activities that are differentially expressed some embodiments, a PDE preferentially or specifically in particular tissues and/or cell-types. hydrolyzes a particular cyclic nucleotide if the K m for the [0035] The term "neurogenic combination of a PDE non-preferred substrate nucleotide is 2-fold, 5-fold, 10- agent with one or more other neurogenic agents" refers fold, 20-fold, 50-fold, or greater than the Km for the pre- to a combination of neurogenesis modulating agents. In ferred substrate. For example, PDE4, which is selective 15 some embodiments, administering a neurogenic, or neu- for cAMP, has an approximately 1000-fold greater K m for romodulating, combination according to methods provid- cGMP than cAMP, whereas PDE5, which is selective for ed herein modulates neurogenesis in a target tissue cGMP, has an approximately 100-fold greater Km for and/or cell-type by at least about 50%, at least about cAMP than cGMP. In some embodiments, a PDE pref- 75%, or at least about 90% or more in comparison to the erentially or specifically hydrolyzes a particular cyclic nu- 20 absence of the combination. In further embodiments, cleotide if the V max for the preferred substrate nucleotide neurogenesis is modulated by at least about 95% or by is 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or greater than at least about 99% or more. the Vmax for the non-preferred substrate. For example, [0036] A neuromodulating combination may be used PDE3, which preferentially hydrolyzes cAMP, has a sub- to inhibit a neural cell’s proliferation, division, or progress 25 stantially similar Km for cAMP and cGMP, but has an through the cell cycle. Alternatively, a neuromodulating approximately 5-fold greater V max for cAMP. In some em- combination may be used to stimulate survival and/or bodiments, a PDE specifically/preferentially hydrolyzes differentiation in a neural cell. As an additional alterna- cAMP or cGMP due to other and/or additional factors, tive, a neuromodulating combination may be used to in- such as the localization of the PDE in the cell, the inter- hibit, reduce, or prevent astrocyte activation and/or as- action of the PDE with endogenous regulators, etc. The 30 trogenesis or astrocyte differentiation. term "dual-specificity PDE" refers generally to a PDE ca- [0037] "IC50" refers to the concentration of a PDE in- pable of hydrolyzing both cAMP and cGMP under phys- hibitor that reduces the activity of the PDE to half maximal iologically relevant conditions. Generally, PDE1, PDE2, level. IC50 values, as described herein, can be deter- PDE10, and PDE1 are dual-specificity PDEs, PDE3, mined using in vitro assays (e.g.., cell-free assays) or PDE4, and PDE8 are cAMP-specific PDEs, and PDE5, 35 cell-based assays. Without being bound by theory, and PDE6, and PDE9 are cGMP-specific PDEs. The sub- offered to improve the understanding of the disclosure, strate-specificities of PDEs may vary according to a cell-free assays generally detect compounds that exert number of factors, such as the conditions under which their effect directly on a PDE activity and/or required co- they are determined, species differences, tissue- specific factors, whereas cell-based assays detect compounds or disease-specific isoforms/splice variants, and the like. 40 that exert effects directly and/or indirectly. Assays for de- Thus, the above definitions are not intended to be uni- termining and quantifying inhibitory activity against vari- versally applicable. ous PDE activities are known in the art and, are de- [0032] In some embodiments, the PDE agent (s) used scribed, e.g., in 6,348,602, 5,932,465, US20030190672, inthe methods described herein are substantiallyinactive US20020115176, US20040018542, US20050009062, with respect to other receptors (i.e., non- PDE), such as 45 Loughney et al., J. Biol. Chem., 271, pp. 796-806 (1996), muscarinic receptors, 5-HT receptors, dopamine recep- Thompson et al., Biochemistry 10: 311-316 (1971), Kin- tors, epinephrine receptors, histamine receptors, gluta- caid et al, J Biol Chem., 259 (8):5158-66 (1984), Davis et mate receptors, and the like. However, in other embod- al., Biochim. Biophys. Acta 797, 354-362 (1984) and Kin- iments, PDE agent(s) are active against one or more ad- caid et al, Methods Enzymol., 159:457-70 (1988), all of ditional receptor subtypes. 50 which are herein incorporated by reference. PDE activity [0033] In additional embodiments, a PDE agent as can be assayed in vivo, for example as described in Rich used herein includes a neurogenesis modulating agent, et al., J. Gen. Physiology, 118(1); 63-78 (2001), herein as defined herein, that elicits an observable neurogenic incorporated by reference. response by producing, generating, stabilizing, or in- [0038] PDE inhibitors used in methods described here- 55 creasing the retention of an intermediate agent which, in preferably have IC 50 values with respect to one or more when contacted with a PDE, results in the neurogenic target PDE activities residing in the CNS of less than response. As used herein, "increasing the retention of" about 10 PM, or less than about 1 PM, or less than about or variants of that phrase or the term "retention" refer to 0.1 PM. In some embodiments, the PDE inhibitor has an

6 11 EP 2 377 530 A2 12

IC50 of less than about 50 nM, or less than about 10 nM, embodiments, one or more agents in a combination in- or less than about 1 nM. In some embodiments, admin- dividually exhibits selective activity in one or more or- istration of a PDE inhibitor according to methods de- gans, tissues, and/or cell types relative to another organ, scribed herein reduces PDE activity within a target tissue tissue, and/or cell type. For example, in some embodi- by at least about 50%, or at least about 75%, or at least 5 ments, an agent in a combination selectively modulates about 90%. In further embodiments, PDE activity is re- neurogenesis in a neurogenic region of the brain, such duced by at least about 95% or by at least about 99%. as the hippocampus (e.g., the dentate gyrus), the sub- In some embodiments, the PDE inhibitor has the desired ventricular zone, and/or the olfactory bulb. activity at a concentration that is lower than the concen- [0042] In other embodiments, modulation by a combi- tration of the inhibitor that is required to produce another, 10 nation of agents is in a region containing neural cells unrelated biological effect. In some exemplary embodi- affected by disease or injury, region containing neural ments, the concentration of the inhibitor required for PDE cells associated with disease effects or processes, or inhibitory activity is at least about 2- fold lower, or at least region containing neural cells affect other event injurious about 5-fold lower, or at least about 10-fold lower, or at to neural cells. Non-limiting examples of such events in- least about 20- fold lower than the concentration required 15 clude stroke or radiation therapy of the region. In addi- to produce an unrelated biological effect. tional embodiments, a neuromodulating combination [0039] Isozyme-selectivity can be measured as the ra- substantially modulates two or more physiological activ- tio of IC50 for a target PDE:IC50 for a non-target PDE. ities or target molecules, while being substantially inac- Methods for determining isozyme-selectivity are known tive against one or more other molecules and/or activi- in the art, and are described, e.g., in Weeks et al., Int J 20 ties. Impot Res. 2005 Jan-Feb;17(1):5-9 and Gupta et al., [0043] The term "cognitive function" refers to mental Methods Find Exp Clin Pharmacol. 2005 Mar;27(2): processes of an animal or human subject relating to in- 101-18. In some embodiments, a "isozyme-selective" formation gathering and/or processing; the understand- PDE agent has an isozyme selectivity that is less than ing, reasoning, and/or application of information and/or about 1:2, or less than about 1:5, or less than about 1: 25 ideas; the abstraction or specification of ideas and/or in- 10, or less than about 1:50. In further embodiments, the formation; acts of creativity, problem- solving, and possi- isozyme-selective activity of a PDE agent used in meth- bly intuition; and mental processes such as learning, per- ods described herein results in improved efficacy, fewer ception, and/or awareness of ideas and/or information. side effects, lower effective dosages, less frequent dos- The mental processes are distinct from those of beliefs, ing, and/or other desirable attributes relative to non-se- 30 desires, and the like. In some embodiments, cognitive lective inhibitors, due, e.g., to targeting of tissue and/or function may be assessed, and thus optionally defined, cell-specific PDE isozymes. via one or more tests or assays for cognitive function. [0040] "IC50" and "EC50" values also refer to concen- Non-limiting examples of a test or assay for cognitive trations of an agent, in a combination of a PDE agent function include CANTAB (see for example Fray et al. with one or more other neurogenic agents, that reduce 35 "CANTAB battery: proposed utility in neurotoxicology." and promote, respectively, neurogenesis or another Neurotoxicol Teratol. 1996; 18 (4):499-504), Stroop Test, physiological activity (e.g., the activity of a receptor) to a Trail Making, Wechsler Digit Span, or the CogState com- half-maximal level. IC 5o and EC 50 values can be assayed puterized cognitive test (see also Dehaene et al. "Re- in a variety of environments, including cell-free environ- ward-dependent learning in neuronal networks for plan- ments, cellular environments (e.g., cell culture assays), 40 ning and decision making." Prog Brain Res. 2000;126: multicellular environments (e.g., in tissues or other mul- 217-29; Iverson et al. "Interpreting change on the WAIS- ticellular structures), and/or in vivo. In some embodi- III/WMS-III in clinical samples." Arch Clin Neuropsychol. ments, one or more neurogenesis modulating agents in 2001;16(2):183-91; and Weaver et al. "Mild memory im- a combination or method disclosed herein individually pairment in healthy older adults is distinct from normal 45 have IC50 or EC50 values of less than about 10 PM, less aging." Brain Cogn. 2006;60(2):146-55). than about 1 PM, or less than about 0.1 PM or lower. In other embodiments, an agent in a combination has an DETAILED DESCRIPTION OF MODES OF PRACTIC- IC50 of less than about 50 nM, less than about 10 nM, or ING THE DISCLOSURE less than about 1 nM or lower. [0041] In some embodiments, selectivity of one or50 General more agents, in a combination of a PDE agent with one or more other neurogenic agents, is individually meas- [0044] Methods described herein can be used to treat ured as the ratio of the IC50 or EC50 value for a desired any disease or condition for which it is beneficial to pro- effect (e.g., modulation of neurogenesis) relative to the mote or otherwise stimulate or increase neurogenesis. 55 IC50 or EC50 value for an undesired effect In some em- One focus of the methods described herein is to achieve bodiments, a "selective" agent in a combination has a a therapeutic result by stimulating or increasing neuro- selectivity of less than about 1:2, less than about 1:10, genesis via modulation of PDE activity. Thus, certain less than about 1:50, or less than about 1:100. In some methods described herein can be used to treat any dis-

7 13 EP 2 377 530 A2 14 ease or condition susceptible to treatment by increasing cell with a PDE agent, the result may be an increase in neurogenesis. neurodifferentiation. The method may be used to poten- [0045] In some embodiments, a disclosed method is tiate a neural cell for proliferation, and thus neurogenesis, applied to modulating neurogenesis in vivo, in vitro, or via the one or more other agents used with the PDE agent ex vivo. In in vivo embodiments, the cells may be present 5 in combination. Thus the disclosure includes a method in a tissue or organ of a subject animal or human being. of maintaining, stabilizing, stimulating, or increasing neu- Non-limiting examples of cells include those capable of rodifferentiation in a cell or tissue by use of a PDE agent, neurogenesis, such as to result, whether by differentia- optionally in combination with one or more other neuro- tion or by a combination of differentiation and prolifera- genic agents that also increase neurodifferentiation. The tion, in differentiated neural cells. As described herein, 10 method may comprise contacting a cell or tissue with a neurogenesis includes the differentiation of neural cells PDE agent, optionally in combination with one or more along different potential lineages. In some embodiments, other neurogenic agents, to maintain, stabilize stimulate, the differentiation of neural stem or progenitor cells is or increase neurodifferentiation in the cell or tissue. along a neuronal cell lineage to produce neurons. In other [0050] The disclosure also includes a method compris- embodiments, the differentiation is along both neuronal 15 ing contacting the cell or tissue with a PDE agent in com- and glial cell lineages. In additional embodiments, the bination with one or more other neurogenic agents where disclosure further includes differentiation along a neuro- the combination stimulates or increases proliferation or nal cell lineage to the exclusion of one or more cell types cell division in a neural cell. The increase in neuroprolif- in a glial cell lineage. Non-limiting examples of glial cell eration may be due to the one or more other neurogenic types include oligodendrocytes and radial glial cells, as 20 agents and/or to the PDE agent. In some cases, a method well as astrocytes, which have been reported as being comprising such a combination may be used to produce of an "astroglial lineage". Therefore, embodiments of the neurogenesis (in this case both neurodifferentiation disclosure include differentiation along a neuronal cell and/or proliferation) in a population of neural cells. In lineage to the exclusion of one or more cell types selected some embodiments, the cell or tissue is in an animal sub- from oligodendrocytes, radial glial cells, and astrocytes. 25 ject or a human patient as described herein. Non-limiting [0046] In applications to an animal or human being, examples include a human patient treated with chemo- the disclosure includes a method of bringing cells into therapy and/or radiation, or other therapy or condition contact with a PDE agent, optionally in combination with which is detrimental to cognitive function; or a human one or more other neurogenic agents, in effective patient diagnosed as having epilepsy, a condition asso- amounts to result in an increase in neurogenesis in com- 30 ciated with epilepsy, or seizures associated with epilep- parison to the absence of the agent or combination. A sy. non-limiting example is in the administration of the agent [0051] Administration of a PDE agent, optionally in or combination to the animal or human being. Such con- combination with one or more other neurogenic agents, tacting or administration may also be described as ex- may be before, after, or concurrent with, another agent, ogenously supplying the combination to a cell or tissue. 35 condition, or therapy. In some embodiments, the overall [0047] Embodiments of the disclosure include a meth- combination may be of a PDE agent, optionally in com- od to treat, or lessen the level of, a decline or impairment bination with one or more other neurogenic agents. of cognitive function. Also included is a method to treat a mood disorder. In additional embodiments, a disease Uses of a PDE Agent or condition treated with a disclosed method is associat- 40 ed with pain and/or addiction, but in contrast to known [0052] Embodiments of a first aspect of the disclosure methods, the disclosed treatments are substantially me- include a method of modulating neurogenesis by con- diated by increasing neurogenesis. As a further non- lim- tacting one or more neural cells with a PDE agent, op- iting example, a method described herein may involve tionally in combination with one or more other neurogenic increasing neurogenesis ex vivo, such that a composition 45 agents. The amount of a PDE agent, or a combination containing neural stem cells; neural progenitor cells, thereof with one or more other neurogenic agents, may and/or differentiated neural cells can subsequently be be selected to be effective to produce an improvement administered to an individual to treat a disease or condi- in a treated subject, or detectable neurogenesis in vitro. tion. In some embodiments, the amount is one that also min- [0048] In further embodiments, methods described 50 imizes clinical side effects seen with administration of the herein allow treatment of diseases characterized by pain, inhibitor to a subj ect. addiction, and/or depression by directly replenishing, re- [0053] Without being bound by theory, and offered to placing, and/or supplementing neurons and/or glial cells. improve the understanding of the disclosure, phosphodi- In further embodiments, methods described herein en- esterase inhibition is believed to promote neurogenesis hance the growth and/or survival of existing neural cells, 55 by targeting second messenger systems downstream of and/or slow or reverse the loss of such cells in a neuro- neurotransmitters and other signaling molecules. Cyclic degenerative condition. adenosine monophosphate (cAMP) and cyclic guanos- [0049] Where a method comprises contacting a neural ine monophosphate (cGMP) are both examples of such

8 15 EP 2 377 530 A2 16 second messengers, and inhibition of PDEs may prolong such a subject or patient. Non-limiting examples of the cAMP and cGMP signals and may increase signaling method include those comprising administering a PDE through neurogenic signal transduction pathways agent, or a combination thereof with one or more other neurogenic agents, to a subject or patient that is under Cognitive Function 5 treatment with a therapy and/or condition that results in a mood disorder. The administration may be with any [0054] In other embodiments, and if compared to a re- combination and/or amount that is effective to produce duced level of cognitive function, a method of the inven- an improvement in the mood disorder. tion may be for enhancing or improving the reduced cog- [0058] Representative and non-limiting mood disor- nitive function in a subject or patient. The method may 10 ders are described herein. Non-limiting examples of comprise administering a PDE agent, optionally in com- mood disorders include depression, anxiety, hypomania, bination with one or more other neurogenic agents, to a panic attacks, excessive elation, seasonal mood (or af- subject or patient to enhance, or improve a decline or fective) disorder, schizophrenia and other psychoses, lis- decrease of cognitive function due to a therapy and/or sencephaly syndrome, anxiety syndromes, anxiety dis- condition that reduces cognitive function. Other methods 15 orders, phobias, stress and related syndromes, aggres- of the disclosure include treatment to affect or maintain sion, non-senile dementia, post-pain depression, and the cognitive function of a subject or patient In some em- combinations thereof. bodiments, the maintenance or stabilization of cognitive function may be at a level, or thereabouts, present in a Identification of Subjects and Patients subject or patient in the absence of a therapy and/or con- 20 dition that reduces cognitive function. In alternative em- [0059] The disclosure includes methods comprising bodiments, the maintenance or stabilization may be at a identification of an individual suffering from one or more level, or thereabouts, present in a subject or patient as disease, disorders, or conditions, or a symptom thereof, a result of a therapy and/or condition that reduces cog- and administering to the subject or patient a PDE agent, nitive function. 25 optionally in combination with one or more other neuro- [0055] In further embodiments, and if compared to a genic agents, as described herein. The identification of reducedlevel of cognitive function due to a therapy and/or a subject or patient as having one or more diseases, dis- condition that reduces cognitive function, a method of orders or conditions, or a symptom thereof, may be made the invention may be for enhancing or improving the re- by a skilled practitioner using any appropriate means duced cognitive function in a subject or patient. The meth- 30 known in the field. The disclosure also includes identifi- od may comprise administering a PDE agent, or a com- cation or diagnosis of a subject or patient as having one bination thereof with one or more other neurogenic or more diseases, disorders or conditions, or a symptom agents, to a subject or patient to enhance or improve a thereof, which is suitably or beneficially treated or ad- decline or decrease of cognitive function due to the ther- dressed by increasing neurogenesis in the subject or pa- apy or condition. The administering may be in combina- 35 tient. tion with the therapy or condition. [0060] The subsequent administration of a PDE agent, [0056] These methods optionally include assessing or alone or in combination as described herein may be measuring cognitive function of the subject or patient be- based on, or as directed by, the identification or diagnosis fore, during, and/or after administration of the treatment of a subject or patient as in need of one or more effects to detect or determine the effect thereof on cognitive func- 40 provided by a PDE agent or a combination. Non- limiting tion. So in one embodiment, a methods may comprise i) examples of an effect include neurogenic activity and/or treating a subject or patient that has been previously as- potentiation of neurogenesis. sessed for cognitive function and ii) reassessing cogni- [0061] In some embodiments, identification of a patient tive function in the subject or patient during or after the in need of neurogenesis modulation comprises identify- course of treatment. The assessment may measure cog- 45 ing a patient who has or will be exposed to a factor or nitive function for comparison to a control or standard condition known to inhibit neurogenesis, including but value (or range) in subjects or patients in the absence of not limited to, stress, aging, sleep deprivation, hormonal a PDE agent, or a combination thereof with one or more changes (e.g., those associated with puberty, pregnan- other neurogenic agents. This may be used to assess cy, or aging (e.g., menopause), lack of exercise, lack of the efficacy of the PDE agent, alone or in a combination, 50 environmental stimuli (e.g., social isolation), diabetes in alleviating the reduction in cognitive function. and drugs of abuse (e.g., alcohol, especially chronic use; opiates and opioids; psychostimulants). In some cases, Mood disorders the patient hasbeen identified as non-responsive totreat- ment with primary medications for the condition(s) tar- [0057] In other embodiments, a disclosed method may 55 geted for treatment (e.g., non-responsive to antidepres- be used to moderate or alleviate a mood disorder in a sants for the treatment of depression), and a PDE agent, subject or patient as described herein. Thus the disclo- optionally in combination with one or more other neuro- sure includes a method of treating a mood disorder in genic agents, is administered in a method for enhancing

9 17 EP 2 377 530 A2 18 the responsiveness of the patient to a co- existing or pre- ple, in some embodiments, the patient suffers from existing treatment regimen. chronic pain and is prescribed one or more opiate/opioid [0062] In other embodiments, the method or treatment medications; and/or suffers from ADD, ADHD, or a relat- comprisesadministering a combination of a primary med- ed disorder, and is prescribed a psychostimulant, such ication or therapy for the condition(s) targeted for treat- 5 as ritalin, dexedrine, adderall, or a similar medication ment and a PDE agent, optionally in combination with which inhibits neurogenesis. Without being bound by any one or more other neurogenic agents. For example, in particular theory, and offered to improve understanding the treatment of depression or related neuropsychiatric of the invention, it is believed that such medications can disorders, a combination may be administered in con- exert a modulatory effect on neurogenesis, leading to junction with, or in addition to, electroconvulsive shock 10 depression, anxiety and other mood disorders, as well treatment, a monoamine oxidase modulator, and/or a se- as deficits in cognition, learning, and memory. Thus, in lective reuptake modulators of serotonin and/or nore- some preferred embodiments, a PDE agent, optionally pinephrine. incombination with one or more other neurogenicagents, [0063] In additional embodiments, the patient in need is administered to a patient who is currently or has re- of neurogenesis modulation suffers from premenstrual 15 cently been prescribed a medication that exerts a mod- syndrome, post-partum depression, or pregnancy-relat- ulatory effect on neurogenesis, in order to treat depres- ed fatigue and/or depression, and the treatment compris- sion, anxiety, and/or other mood disorders, and/or to im- es administering a therapeutically effective amount of a prove cognition. PDE agent, optionally in combination with one or more [0066] In additional embodiments, the patient suffers other neurogenic agents. Without being bound by any 20 from chronic fatigue syndrome; a sleep disorder; lack of particular theory, and offered to improve understanding exercise (e.g., elderly, infirm, or physically handicapped of the invention, it is believed that levels of steroid hor- patients); and/or lack of environmental stimuli (e.g., so- mones, such as estrogen, are increased during the men- cial isolation); and the treatment comprises administering strual cycle during and following pregnancy, and that a therapeutically effective amount of a PDE agent, op- such hormones can exert a modulatory effect on neuro- 25 tionally in combination with one or more other neurogenic genesis. agents. [0064] In some embodiments, the patient is a user of [0067] In more embodiments, the patient is an individ- a recreational drug including but not limited to alcohol, ual having, or who is likely to develop, a disorder relating amphetamines, PCP, cocaine, and opiates. Without be- to neural degeneration, neural damage and/or neural de- ing bound by any particular theory, and offered to improve 30 myelination. understanding of the invention, it is believed that some [0068] In further embodiments, a subject or patient in- drugs of abuse have a modulatory effect on neurogene- cludes human beings and animals in assays for behavior sis, which is associated with depression, anxiety and oth- linked to neurogenesis. Exemplary human and animal er mood disorders, as well as deficits in cognition, learn- assays are known to the skilled person in the field. ing, and memory. Moreover, mood disorders are causa- 35 [0069] In yet additional embodiments, identifying a pa- tive/risk factors for substance abuse, and substance tient in need of neurogenesis modulation comprises se- abuse is a common behavioral symptom (e.g., self med- lecting a population or sub-population of patients, or an icating) of mood disorders. Thus, substance abuse and individual patient, that is more amenable to treatment mood disorders may reinforce each other, rendering pa- and/or less susceptible to side effects than other patients tients suffering from both conditions non-responsive to 40 having the same disease or condition. In some embodi- treatment. Thus, in some embodiments, a PDE agent, ments, identifying a patient amenable to treatment with optionally in combination with one or more other neuro- a PDE agent, optionally in combination with one or more genic agents, to treat patients suffering from substance other neurogenic agents, comprises identifying a patient abuse and/or mood disorders. In additional embodi- who has been exposed to a factor known to enhance ments, the PDE agent, optionally in combination with one 45 neurogenesis, including but not limited to, exercise, hor- or more other neurogenic agents, can used in combina- mones or other endogenous factors, and drugs taken as tion with one or more additional agents selected from an part of a pre-existing treatment regimen. In some em- antidepressant, an antipsychotic, a mood stabilizer, or bodiments, a sub-population of patients is identified as any other agent known to treat one or more symptoms being more amenable to neurogenesis modulation with exhibited by the patient, In some embodiments, a PDE 50 a PDE agent, optionally in combination with one or more agent exerts a synergistic effect with the one or more other neurogenic agents, by taking a cell or tissue sample additional agents in the treatment of substance abuse from prospective patients, isolating and culturing neural and/or mood disorders in patients suffering from both cells from the sample, and determining the effect of the conditions. combination on the degree or nature of neurogenesis of [0065] In further embodiments, the patient is on a co- 55 the cells, thereby allowing selection of patients for which existing and/or pre-existing treatment regimen involving the therapeutic agent has a substantial effect on neuro- administration of one or more prescription medications genesis. Advantageously, the selection of a patient or having a modulatory effect on neurogenesis. For exam- population of patients in need of or amenable to treatment

10 19 EP 2 377 530 A2 20 with a PDE agent, optionally in combination with one or more other neurogenic agents, as described herein. Non- more other neurogenic agents, of the disclosure allows limiting examples include cases involving an opioid re- more effective treatment of the disease or condition tar- ceptor agonist, which decreases or inhibits neurogene- geted for treatment than known methods using the same sis, and drug addiction, drug rehabilitation, and/or pre- or similar compounds. 5 vention of relapse into addiction. In some embodiments, [0070] In some embodiments, the patient has suffered the opioid receptor agonist is morphine, opium or another a CNS insult, such as a CNS lesion, a seizure (e.g., elec- opiate. troconvulsive seizure treatment; epileptic seizures), ra- [0074] In further embodiments, the disclosure includes diation, chemotherapy and/or stroke or other ischemic methods to treat a cell, tissue, or subject which is exhib- injury. Without being bound by any particular theory, and 10 iting decreased neurogenesis or increased neurodegen- offered to improve understanding of the invention, it is eration. In some cases, the cell, tissue, or subject is, or believed that some CNS insults/injuries leads to in- has been, subjected to, or contacted with, an agent that creased proliferation of neural stem cells, but that the decreases or inhibits neurogenesis. One non-limiting ex- resulting neural cells form aberrant connections which ample is a human subject that has been administered can lead to impaired CNS function and/or diseases, such 15 morphine or other agent which decreases or inhibits neu- as temporal lobe epilepsy. In other embodiments, a PDE rogenesis. Non-limiting examples of other agents include agent, optionally in combination with one or more other opiates and opioid receptor agonists, such as mu recep- neurogenic agents, is administered to a patient who has tor subtype agonists, that inhibit or decrease neurogen- suffered, or is at risk of suffering, a CNS insult or injury esis. to stimulate neurogenesis. Advantageously, stimulation 20 [0075] Thus in additional embodiments, the methods of the differentiation of neural stem cells with a PDE may be used to treat subjects having, or diagnosed with, agent, optionally in combination with one or more other depression or other withdrawal symptoms from morphine neurogenic agents, activates signaling pathways neces- or other agents which decrease or inhibit neurogenesis. sary for progenitor cells to effectively migrate and incor- This is distinct from the treatment of subjects having, or porate into existing neural networks or to block inappro- 25 diagnosed with, depression independent of an opiate, priate proliferation. such as that of a psychiatric nature, as disclosed herein. In further embodiments, the methods may be used to Opiate or Opioid Based Analgesic treat a subject with one or more chemical addictions or dependencies, such as with morphine or other opiates, [0071] Additionally, the disclosed methods provide for 30 where the addiction or dependency is ameliorated or al- the application of a PDE agent, optionally in combination leviated by an increase in neurogenesis. with one or more other neurogenic agents, to treat a sub- ject or patient for a condition due to the anti-neurogenic Transplantation effects of an opiate or opioid based analgesic. In some embodiments, the administration of an opiate or opioid 35 [0076] In other embodiments, methods described based analgesic, such as an opiate like morphine or other herein involve modulating neurogenesis in vitro or ex vivo opioid receptor agonist, to a subject or patient results in with a PDE agent, optionally in combination with one or a decrease in, or inhibition of, neurogenesis. The admin- more other neurogenic agents, such that a composition istration of a PDE agent, optionally in combination with containing neural stem cells, neural progenitor cells, one or more other neurogenic agents, with an opiate or 40 and/or differentiated neural cells can subsequently be opioid based analgesic would reduce the anti-neurogenic administered to an individual to treat a disease or condi- effect. One non- limiting example is administration of such tion. In some embodiments, the method of treatment a combination with an opioid receptor agonist after sur- comprises the steps of contacting a neural stem cell or gery (such as for the treating post-operative pain). progenitor cell with a PDE agent, optionally in combina- [0072] So the disclosed embodiments include a meth- 45 tion with one or more other neurogenic agents, to mod- od of treating post operative pain in a subject or patient ulate neurogenesis, and transplanting the cells into a pa- by combining administration of an opiate or opioid based tient in need of treatment. Methods for transplanting stem analgesic with a PDE agent, optionally in combination and progenitor cells are known in the art, and are de- with one or more other neurogenic agents. The analgesic scribed, e.g., in U.S. Patent Nos. 5,928,947; 5,817,773; may have been administered before, simultaneously50 and 5,800,539,and PCTPublication Nos. WO01/176507 with, or after the combination. In some cases, the anal- and WO 01/170243, all of which are incorporated herein gesic or opioid receptor agonist is morphine or another by reference in their entirety. In some embodiments, opiate. methods described herein allow treatment of diseases [0073] Other disclosed embodiments include a meth- or conditions by directly replenishing, replacing, and/or od to treat or prevent decreases in, or inhibition of, neu- 55 supplementing damaged or dysfunctional neurons. In rogenesis in other cases involving use of an opioid re- further embodiments, methods described herein en- ceptor agonist. The methods comprise the administration hance the growth and/or survival of existing neural cells, of a PDE agent, optionally in combination with one or and/or slow or reverse the loss of such cells in a neuro-

11 21 EP 2 377 530 A2 22 degenerative or other condition. agent and one or more other neurogenic agents, the PDE [0077] In alternative embodiments, the method of agent may be administered separately from the one or treatmentcomprises identifying, generating, and/or prop- more other agents, such that the one or more other agating neural cells in vitro or ex vivo in contact with a agents administered before or after administration of an PDE agent, optionally in combination with one or more 5 angiogenic factor or condition. other neurogenic agents, and transplanting the cells into a subject. In another embodiment, the method of treat- Additional Diseases and Conditions ment comprises the steps of contacting a neural stem cell of progenitor cell with a PDE agent, optionally in com- [0081] As described herein, the disclosed embodi- bination with one or more other neurogenic agents, to 10 ments include methods of treating diseases, disorders, stimulate neurogenesis or neurodifferentiation, and and conditions of the central and/or peripheral nervous transplanting the cells into a patient in need of treatment. systems (CNS and PNS, respectively) by administering Also disclosed are methods for preparing a population a PDE agent, optionally in combination with one or more of neural stem cells suitable for transplantation, compris- other neurogenic agents. As used herein, "treating" in- ing culturing a population of neural stem cells (NSCs) in 15 cludes prevention, amelioration, alleviation, and/or elim- vitro, and contacting the cultured neural stem cells with ination of the disease, disorder, or condition being treated a PDE agent, optionally in combination with one or more or one or more symptoms of the disease, disorder, or other neurogenic agents, as described herein. The dis- condition being treated, as well as improvement in the closure further includes methods of treating the diseases, overall well being of a patient, as measured by objective disorders, and conditions described herein by transplant- 20 and/or subjective criteria. In some embodiments, treating ing such treated cells into a subject or patient. is used for reversing, attenuating, minimizing, suppress- ing, or halting undesirable or deleterious effects of, or Neurogenesis with Angiogenesis effects from the progression of, a disease, disorder, or condition of the central and/or peripheral nervous sys- [0078] In additional embodiments, the disclosure in- 25 tems. In other embodiments, the method of treating may cludes a method of stimulating or increasing neurogen- be advantageously used in cases where additional neu- esis in a subject or patient with stimulation of angiogen- rogenesis would replace, replenish, or increase the num- esis in the subject or patient. The co- stimulation may be bers of cells lost due to injury or disease as non-limiting used to provide the differentiating and/or proliferating examples. cells with increased access to the circulatory system. The 30 [0082] The amount of a PDE agent, optionally in com- neurogenesis is produced by modulation of PDE activity, bination with one or more other neurogenic agents may such as with a PDE agent, optionally in combination with be any that results in a measurable relief of a disease one or more other neurogenic agents, as described here- condition like those described herein. As a non-limiting in. An increase in angiogenesis may be mediated by a example, an improvement in the Hamilton depression means known to the skilled person, including adminis- 35 scale (HAM-D) score for depression may be used to de- tration of an angiogenic factor or treatment with an ang- termine (such as quantitatively) or detect (such as qual- iogenic therapy. Non-limiting examples of angiogenic itatively) a measurable level of improvement in the de- factors or conditions include vascular endothelial growth pression of a subject. factor (VEGF), angiopoietin-1 or -2, erythropoietin, exer- [0083] Non-limiting examples of symptoms that may cise, or a combination thereof. 40 be treated with the methods described herein include ab- [0079] So in some embodiments, the disclosure in- normal behavior, abnormal movement, hyperactivity, cludes a method comprising administering i) a PDE hallucinations, acute delusions, combativeness, hostility, agent, optionally in combination with one or more other negativism, withdrawal, seclusion, memory defects, sen- neurogenic agents, and ii) one or more angiogenic fac- sory defects, cognitive defects, and tension. Non- limiting tors to a subject or patient. In other embodiments, the 45 examples of abnormal behavior include irritability, poor disclosure includes a method comprising administering impulse control, distractibility, and aggressiveness. Out- i) a PDE agent, optionally in combination with one or more comes from treatment with the disclosed methods in- other neurogenic agents, to a subject or patient with ii) clude improvements in cognitive function or capability in treating said subject or patient with one or more ang- comparison to the absence of treatment. iogenic conditions. The subject or patient may be any as 50 [0084] Additional examples of diseases and conditions described herein. treatable by the methods described herein include, but [0080] The co-treatment of a subject or patient in- are not limited to, neurodegenerative disorders and neu- cludes simultaneous treatment or sequential treatment ral disease, such as dementias (e.g., senile dementia, as non-limiting examples. In cases of sequential treat- memory disturbances/memory loss, dementias caused ment, the administration of a PDE agent, optionally with 55 by neurodegenerative disorders (e.g., Alzheimer’s, Par- one or more other neurogenic agents, may be before or kinson’s disease, Parkinson’s disorders, Huntington’s after the administration of an angiogenic factor or condi- disease (Huntington’s Chorea), Lou Gehrig’s disease, tion. Of course in the case of a combination of a PDE multiple sclerosis, Pick’s disease, Parkinsonism demen-

12 23 EP 2 377 530 A2 24 tia syndrome), progressive subcortical gliosis, progres- brain injury, brain surgery, trauma related brain injury, sive supranuclear palsy, thalmic degeneration syn- trauma related to spinal cord injury, brain injury related drome, hereditary aphasia, amyotrophic lateral sclerosis, to cancer treatment, spinal cord injury related to cancer Shy-Drager syndrome, and Lewy body disease; vascular treatment, brain injury related to infection, brain injury conditions (e.g., infarcts, hemorrhage, cardiac disor- 5 related to inflammation, spinal cord injury related to in- ders); mixed vascular and Alzheimer’s; bacterial menin- fection, spinal cord injury related to inflammation, brain gitis; Creutzfeld-Jacob Disease; and Cushing’s disease. injury related to environmental toxin, and spinal cord in- [0085] The disclosed embodiments also provide for jury related to environmental toxin. the treatment of a nervous system disorder related to [0089] Non-limiting examples of nervous system dis- neural damage, cellular degeneration, a psychiatric con- 10 orders related to other neurologically related conditions dition, cellular (neurological) trauma and/or injury (e.g., include learning disorders, memory disorders, age- asso- subdural hematoma or traumatic brain injury), toxic ciated memory impairment (AAMI) or age-related mem- chemicals (e.g., heavy metals, alcohol, some medica- ory loss, autism, learning or attention deficit disorders tions), CNS hypoxia, or other neurologically related con- (ADD or attention deficit hyperactivity disorder, ADHD), ditions. In practice, the disclosed compositions and meth- 15 narcolepsy, sleep disorders and sleep deprivation (e.g., ods may be applied to a subject or patient afflicted with, insomnia, chronic fatigue syndrome), cognitive disor- or diagnosed with, one or more central or peripheral nerv- ders, epilepsy, injury related to epilepsy, and temporal ous system disorders in any combination. Diagnosis may lobe epilepsy. be performed by a skilled person in the applicable fields [0090] Other non-limiting examples of diseases and using known and routine methodologies which identify 20 conditions treatable by the methods described herein in- and/or distinguish these nervous system disorders from clude, but are not limited to, hormonal changes (e.g., other conditions. depression and other mood disorders associated with [0086] Non-limiting examples of nervous system dis- puberty, pregnancy, or aging (e.g., menopause)); and orders related to cellular degeneration include neurode- lack of exercise (e.g., depression or other mental disor- generative disorders, neural stem cell disorders, neural 25 ders in elderly, paralyzed, or physically handicapped pa- progenitor cell disorders, degenerative diseases of the tients); infections (e.g., HIV); genetic abnormalities retina, and ischemic disorders. In some embodiments, (down syndrome); metabolic abnormalities (e.g., vitamin an ischemic disorder comprises an insufficiency, or lack, B12 or folate deficiency); hydrocephalus; memory loss of oxygen or angiogenesis, and non-limiting example in- separate from dementia, including mild cognitive impair- clude spinal ischemia, ischemic stroke, cerebral infarc- 30 ment (MCI), age-related cognitive decline, and memory tion, multi-infarct dementia. While these conditions may loss resulting from the use of general anesthetics, chem- be present individually in a subject or patient, the dis- otherapy, radiation treatment, post-surgical trauma, or closedmethods also providefor the treatment of a subject therapeutic intervention; and diseases of the of the pe- or patient afflicted with, or diagnosed with, more than one ripheral nervous system (PNS), including but not limited of these conditions in any combination. 35 to, PNS neuropathies (e.g., vascular neuropathies, dia- [0087] Non-limiting embodiments of nervous system betic neuropathies, amyloid neuropathies, and the like), disorders related to a psychiatric condition include neu- neuralgias, neoplasms, myelin relaxed diseases, etc. ropsychiatric disorders and affective disorders. As used [0091] Other conditions that can be beneficially treated herein, an affective disorder refers to a disorder of mood by increasing neurogenesis are known in the art (see such as, but not limited to, depression, post-traumatic 40 e.g., U.S. Publication Nos. 20020106731, 2005/0009742 stress disorder (PTSD), hypomania, panic attacks, ex- and 2005/0009847, 20050032702, 2005/0031538, cessive elation, bipolar depression, bipolar disorder 2005/0004046, 2004/0254152, 2004/0229291, and (manic-depression), and seasonal mood (or affective) 2004/0185429, herein incorporated by reference in their disorder. Other non-limiting embodiments include schiz- entirety). ophrenia and other psychoses, lissencephaly syndrome, 45 anxiety syndromes, anxiety disorders, phobias, stress PDE Agents and related syndromes (e.g., panic disorder, phobias, adjustment disorders, migraines), cognitive function dis- [0092] A PDE agent of the disclosure is a ligand which orders, aggression, drug and alcohol abuse, drug addic- modulates activity of one or more PDE isozymes. In some tion, and drug-induced neurological damage, obsessive 50 cases, the ligand may bind or interact with a PDE as an compulsive behavior syndromes, borderline personality inhibitor. In other cases, the agent may modulate activity disorder, non-senile dementia, post-pain depression, indirectly as described herein. In some embodiments, post-partum depression, and cerebral palsy. the agent is an inhibitor of one or more isozymes. In ad- [0088] Examples of nervous system disorders related ditional embodiments, the agent is a blocker or inhibitor to cellular or tissue trauma and/or injury include, but are 55 of PDE activity. not limited to, neurological traumas and injuries, surgery [0093] A PDE agent useful in a method described here- related trauma and/or injury, retinal injury and trauma, in includes an agent that modulates PDE activity at the injury related to epilepsy, cord injury, spinal cord injury, enzyme level (e.g., by binding directly to PDE), at the

13 25 EP 2 377 530 A2 26 transcriptional and/or translational level (e.g., by prevent- includes use of a PDE ligand, alone or in combination ing PDE gene expression), and/or by other modes (e.g., with another agent, wherein the ligand affects the activity by binding to a substrate or co- factor of PDE, or by mod- of more than one PDE isozyme. Stated differently, the ulating the activity of an agent that directly or indirectly ligand may be one which is non- selective in its PDE mod- modulates PDE activity). For example, in some embod- 5 ulating effect(s). Non-limiting examples of such a ligand iments, a PDE agent is a compound that modulates the include ibudilast and dipyridamole. In some cases, the activity of an endogenous PDE inhibitor. The PDE agent non-selective ligand is one which exhibits PDE modulat- can be any, including, but not limited to, a chemical com- ing activity, such as inhibitory activity, at PDE5 and one pound, a protein or polypeptide, a peptidomimetic, or an or more other PDE isozymes. In other cases, the non- antisense molecule or ribozyme. A number of structurally 10 selective ligand exhibits PDE modulating activity at diverse molecules with PDE inhibitory activity are known PDE11 and one or more other PDE isozymes. Non-se- in the art. Structures, synthetic processes, safety profiles, lective ligands that modulate or inhibit two of more PDE biological activity data, methods for determining biolog- isozymes selected from PDE5, PDE6, PDE8, PDE10, ical activity, pharmaceutical preparations, and methods and PDE11, or from PDE1a, PDE2, PDE3, PDE4, PDE5, of administration for a PDE agent useful in a method de- 15 and PDE11, are included within the scope of the disclo- scribed herein are described in the instant text and in the sure. cited references, all of which are herein incorporated by [0098] In other embodiments, the PDE inhibitor inhibits reference in their entirety. a cAMP-specific PDE. Examples of cAMP specific PDE [0094] A PDE ligand for use in embodiments of the inhibitors useful in the methods described herein include disclosure includes a non-selective inhibitor, such as 20 pyrrolidinones, such as the compounds disclosed in ibudilast or MN-166, or dipyridamole (persantine); a 5,665,754, US20040152754 and US20040023945; PDE1 inhibitor, such as vinpocetine; a PDE3 inhibitor, quinazolineones, such as the compounds disclosed in such as enoximone, milrinone, pimobendan, flose- 6,747,035, 6,828,315, WO 97/49702 and WO 97/42174; quinan, levosimendan, vesnarinone, olprinone, amri- xanthine derivatives; phenylpyridines, such as the com- none, inamnirone, anagrelide, cilostazol, or imazodan; a 25 pounds disclosed in 6,410,547, 6,090,817, and WO PDE4 inhibitor, such as cilomilast, roflumilast, rolipram, 97/22585; diazepine derivatives, such as the compounds MEM 1414, MEM 1971, NIK 616, GK 07294A, 256066, disclosed in WO 97/36905; oxime derivatives, such as GW 842470, ONO 6126, PLX369, HT- 0712, IPL 455903, the compounds disclosed in 5,693,659 and WO IC 485, or NVP-ABE171; or a PDE5 inhibitor, such as 96/00215; naphthyridines, such as the compounds de- revatio, cialis (tadalafil), levitra (vardenafil), DA-8159, 30 scribed in 5,817,670, 6,740,662, 6,136,821, 6,331,548, dapoxetine, avanafil (TA-1790), SCH-466132, or ABT- 6,297,248, 6,541,480, 6,642,250, 6,900,205, Trifilieff et 670. al., Pharmacology, 301(1): 241-248 (2002) and Her- [0095] In some embodiments, a combination of a PDE sperger et al., J Med Chem., 43(4):675-82 (2000); ben- ligand and another agent is used in the disclosed com- zofurans, such as the compounds disclosed in positions, formulations, and methods. One non-limiting 35 5,902,824, 6,211,203, 6,514,996, 6,716,987, 6,376,535, example is with a combination of a PDE inhibitor, such 6,080,782, 6,054,475, EP 819688, EP685479, and Per- as ibudilast, that is not limited to a particular PDE subtype rier et al., Bioorg. Med. Chem. Lett. 9:323-326 (1999); with an ACE inhibitor, an angiotensin receptor antago- phenanthridines, such as those disclosed in 6,191,138, nist, an HMG- CoA reductase inhibitor, or a vitamin. Non- 6,121,279, and 6,127,378; benzoxazoles, such as those limiting examples of an ACE inhibitor for use in such a 40 disclosed in 6,166,041 and 6,376,485; purine deriva- combination include captopril, enalapril, randolapril, or tives, such as the compounds disclosed in 6,228,859; perindopril, while non-limiting examples of an angi- benzamides, such as the compounds described in otensin receptor antagonist include candesartan or tel- 5,981,527, 5,712,298, WO95/01338, WO 97/48697 and misartan. A non- limiting example of an HMG- CoA reduct- Ashton et al., J. Med Chem 37: 1696-1703 (1994); sub- ase inhibitor that may be used in such a combination is 45 stituted phenyl compounds, such as the compounds dis- atorvastatin, while a non-limiting example of a vitamin is closed in 6,297,264, 5,866,593,65 5,859,034, 6,245,774, folic acid. 6,197,792, 6,080,790, 6,077,854, 5,962,483, 5,674,880, [0096] In additional embodiments, a combination of 5,786,354, 5,739,144, 5,776,958, 5,798,373, 5,891,896, the disclosure contains a PDE3 inhibitor, such as enoxi- 5,849,770, 5,550,137, 5,340,827, 5,780,478, 5,780,477, mone, or a PDE4 inhibitor, such as rolipram and a second 50 5,633,257, and WO 95/35283; and substituted biphenyl agent. Non-limiting examples of the second agent in the compounds, such as those disclosed in 5,877,190; combination include an ACE inhibitor, an SSRI, or an quinilinones, such as the compounds described in agonist of a 5HT1a receptor. Non-limiting examples of 6,800,625 and WO 98/14432. an ACE inhibitor include captopril and the other inhibitors [0099] Additional examples of cAMP-specific PDE in- described herein,while non-limiting examples ofa 5HT1a 55 hibitors useful in methods provided herein include com- receptor agonist include busipirone and the other ago- pounds disclosed in 6,818,651, 6,737,436, 6,613,778, nists described herein. 6,617,357, 6,146,876, 6,838,559, 6,884,800, 6,716,987, [0097] In yet additional embodiments, the disclosure 6,514,996, 6,376,535, 6,740,655, 6,559,168, 6,069,151,

14 27 EP 2 377 530 A2 28

6,365,585, 6,313,116, 6,245,774, 6,011,037, 6,127,363, PD-190036; PDB-093; PLX650; PLX369; PLX371; 6,303,789, 6,316,472, 6,348,602, 6,331,543, 6,333,354, PLX788; PLX939; Ro-20-1724; RPR-132294; RPR- 5,491,147, 5,608,070, 5,622,977, 5,580,888, 6,680,336, 117658A; RPR-114597; RPR-122818; RPR-132703; 6,569,890, 6,569,885, 6,500,856, 6,486,186, 6,458,787, RS-17597; RS-25344; RS-14203; SCA 40; Sch- 351591; 6,455,562, 6,444,671, 6,423,710, 6,376,489, 6,372,777, 5 SDZ-ISQ-844; SDZ-MKS-492; SKF 94120; SKF-95654; 6,362,213, 6,313,156, 6,294,561, 6,258,843, 6,258,833, SKF-107806; SKF 96231; T- 440; T-2585; WAY-126120; 6,121,279, 6,043,263, RE38,624, 6,297,257, 6,251,923, WAY-122331; WAY-127093B; WIN-63291; WIN- 62582; 6,613,794, 6,407,108, 6,107,295, 6,103,718, 6,479,494, V-11294A; VMX 554; VMX 565; XT- 044; XT-611; Y-590; 6,602,890, 6,545,158, 6,545,025, 6,498,160, 6,743,802, YM-58897; YM-976; ZK-62711; methyl 3-[6-(2H-3,4,5,6- 6,787,554, 6,828,333, 6,869,945, 6,894,041, 6,924,292, 10 tetrahydropyran-2-yloxy)-2-(3-thienylcarbonyl)benzo[b] 6,949,573, 6,953,810, 6,156,753, 5,972,927, 5,962,492, furan-3-yl]propanoate; 4-[4-methoxy-3-(5-phenylpenty- 5,814,651, 5,723,460, 5,716,967, 5,686,434, 5,502,072, loxy)phenyl]-2-methylbenzoic acid; methyl 3- {2-[(4-chlo- 5,116,837, 5,091,431; 4,670,434; 4,490,371; 5,710,160, rophenyl) carbonyl]- 6- hydroxybenzo [b] furan- 3- yl} pro- 5,710,170, 6,384,236, 3,941,785, US20050119225, panoate; (R*,R*)-()-methyl 3-acetyl-4-[3-(cyclopenty- US20050026913, US20050059686, US20040138279, 15 loxy)-4-methoxyphenyl]-3-methyl-1-pyrrolidinecarboxy- US20050222138, US20040214843, US20040106631, lat; or 4-(3-bromophenyl)-1-ethyl-7-methylhydropyridino US 20030045557, US 20020198198, US20030162802, [2,3-b]pyridin-2-one. US20030092908, US 20030104974, US20030100571, [0101] Examples of PDE3 inhibitors include dihydro- 20030092721, US20050148604, WO 99/65880, WO quinolinone compounds such as cilostamide, cilostazol, 00/26201, WO 98/06704, WO 00/59890, W09907704, 20 vesnarinone, and OPC 3911; imidazolones such as WO9422852, WO 98/20007, WO 02/096423, WO piroximone and enoximone; bipyridines such as milri- 98/18796, WO 98/02440, WO 02/096463, WO 97/44337, none, amrinone and olprinone; imidazolines such as im- WO 97/44036, WO 97/44322, EP 0763534, Aoki et al., azodan and 5-methyl-imazodan; pyridazinones such as J Pharmacol Exp Ther., 295(1):255-60 (2000), Del Piaz indolidan, and LY181512; ibudilast, isomazole, motapi- et al., Eur. J. Med. Chem., 35; 463-480 (2000), and Bar- 25 zone, phthalazinol, trequinsin, lixazinone (RS 82856), Y- nette et al., Pharmacol. Rev. Commun. 8: 65-73 (1997). 590, SKF 94120, quazinone, ICI 153,110, bemorandan [0100] In some embodiments, the cAMP- specific PDE (RWJ 22867), siguazodan (SK&F 94836), adibendan inhibitor is Cilomilast (SB-207499); Filaminast; Tibene- (BM 14,478), Pimobendan (UD-CG 115, MCI-154), Sa- last (LY-186655); Ibudilast; Piclamilast (RP 73401); Dox- terinone (BDF 8634), NSP-153, zardaverine, quinazo- ofylline; Cipamfylline (HEP-688); atizoram (CP-80633); 30 lines, benzafentrine, sulmazole (ARL 115), ORG 9935, theophylline; isobutylmethylxanthine; Mesopram (ZK- CI-930, SKF-95654, SDZ-MKS-492, 349-U-85, EMD- 117137); Zardaverine; vinpocetine; Rolipram (ZK- 53998, EMD-57033, NSP-306, NSP-307, Revizinone, 62711); Arofylline (LAS-31025); roflumilast (BY-217); NM-702, WIN-62582, ATZ-1993, WIN-63291, ZK- Pumafentrin (BY-343); Denbufylline; EHNA; milrinone; 62711, PLX650; PLX369; PLX788; PLX939; anagrelide, Siguazodan; Zaprinast; Tolafentrine; Isbufylline; IBMX; 35 carbazeran, ampizone, emoradan, and compounds dis- 1C-485; dyphylline; verolylline; bamifylline; pentoxyfil- closed in 6,156,753. line; enprofilline; lirimilast (BAY 19-8004); filaminast [0102] Examples of PDE4 inhibitors include pyrrolidi- (WAY- PDA-641); benafentrine; trequinsin; nitroqua- nones, such as the compounds disclosed in 5,665,754, zone; cilostamide; vesnarinone; piroximone; enoximone; U520040152754 and US20040023945; quinazolin- amrinone; olprinone; imazodan and 5- methyl-imazodan; 40 eones, such as the compounds disclosed in 6,747,035, indolidan; anagrelide; carbazeran; ampizone; emo- 6,828,315, WO 97/49702 and WO 97/42174; xanthine radan; motapizone; phthalazinol; lixazinone (RS 82856); derivatives; phenylpyridines, such as the compounds quazinone; bemorandan (RWJ 22867); adibendan (BM disclosed in 6,410,547, 6,090,817, and WO 97/22585; 14,478); Pimobendan (MCI-154); Saterinone (BDF diazepine derivatives, such as the compounds disclosed 8634); Tetomilast (OPC-6535); benzafentrine; sulma- 45 in WO 97/36905; oxime derivatives, such as the com- zole (ARL 115); Revizinone; 349- U-85; AH-21-132; ATZ- pounds disclosed in 5,693,659 and WO 96/00215; naph- 1993; AWD-12-343; AWD-12-281; AWD-12-232; BRL thyridines, such as the compounds described in 50481; CC-7085; CDC-801; CDC-998; CDP-840; CH- 5,817,670, 6,740,662, 6,136,821, 6,331,548,6,297,248, 422; CH-673; CH-928; CH-3697; CH-3442; CH-2874; 6,541,480, 6,642,250, 6,900,205, Trifilieff et al., Pharma- CH-4139; Chiroscience 245412; CI-930; CI-1018; CI- 50 cology, 301(1): 241-248 (2002) and Hersperger et al., J 1044; CI-1118; CP-353164; CP-77059; CP- 146523; CP- Med Chem, 43(4):675-82 (2000); benzofurans, such as 293321; CP-220629; CT-2450; CT-2820; CT-3883; CT- the compounds disclosed in 5,902,824, 6,211,203, 5210; D-4418; D-22888; E-4021; EMD 54622; EMD- 6,514,996, 6,716,987, 6,376,535, 6,080,782, 6,054,475, 53998; EMD-57033; GF-248; GW-3600; IC-485; ICI EP 819688, EP685479, and Perrier et al., Bioorg. Med. 63197; ICI 153,110; IPL-4088; KF-19514; KW-4490; L- 55 Chem. Lett. 9: 323-326 (1999); phenanthridines, such as 787258; L-826141;L-791943; LY181512; NCS- 613; NM- those disclosed in 6,191,138, 6,121,279, and 6,127,378; 702; NSP-153; NSP-306; NSP-307; Org-30029; Org- benzoxazoles, such as those disclosed in 6,166,041 and 20241; Org-9731; ORG 9935; PD-168787; PD-190749; 6,376,485; purine derivatives, such as the compounds

15 29 EP 2 377 530 A2 30 disclosed in 6,228,859; benzamides, such as the com- 4490; L-787258; L-826141; L-791943; NCS-613; Org- pounds described in 5,981,527, 5,712,298,30029; Org-20241; Org-9731; PD-168787; PD-190749; WO95/01338, WO 97/48697 and Ashton et al., J. Med PD-190036; PDB-093; PLX650; PLX369; PLX371; Chem 37: 1696-1703 (1994); substituted phenyl com- PLX788; PIX939; Ro-20-1724; RPR-132294; RPR- pounds, such as the compounds disclosed in 6,297,264, 5 117658A; RPR-114597; RPR-122818; RPR-132703; 5,866,593,65 5,859,034, 6,245,774, 6,197,792,RS-17597; RS-25344; RS-14203; SCA 40; Sch- 351591; 6,080,790, 6,077,854, 5,962,483, 5,674,880, 5,786,354, SDZ-ISQ-844; SKF-107806; SKF 96231; T-440; T- 2585; 5,739,144, 5,776,958, 5,798,373, 5,891,896, 5,849,770, WAY-126120; WAY-122331; WAY-127093B; V- 5,550,137, 5,340,827, 5,780,478, 5,780,477, 5,633,257, 11294A;VMX 554; VMX 565; XT-044; XT-611; YM- and WO 95/35283; and substituted biphenyl compounds, 10 58897; YM-976; methyl 3-[6-(2H-3,4,5,6-tetrahydro- such as those disclosed in 5,877,190; quinilinones, such pyran-2-yloxy)-2-(3-thienylcarbonyl)benzo[b]furan-3-yl] as the compounds described in 6,800,625 and WO propanoate; 4-[4-methoxy-3-(5-phenylpentyloxy)phe- 98/14432. nyl]-2-methylbenzoic acid; methyl 3- {2-[(4-chlorophenyl) [0103] Additional examples of PDE4 inhibitors useful carbonyl]-6-hydroxybenzo[b]furan-3-yl}propanoate; (R*, in methods provided herein include compounds dis-15 R*)-()-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-meth- closed in 6,716,987, 6,514,996,6,376,535, 6,740,655, oxyphenyl]-3-methyl-l-pyrrolidinecarboxylat; or 4-(3- 6,559,168, 6,069,151,6,365,585,6,313,116,6,245,774, bromophenyl)-1-ethyl-7-methylhydropyridino[2,3-b]pyri- 6,011,037, 6,127,363, 6,303,789, 6,316,472, 6,348,602, din-2-one. 6,331,543, 6,333,354, 5,491,147, 5,608,070, 5,622,977, [0105] Examples of PDE7 inhibitors useful in methods 5,580,888, 6,680,336, 6,569,890, 6,569,885, 6,500,856, 20 described herein include BRL 50481; PLX369; PLX788; 6,486,186, 6,458,787, 6,455,562, 6,444,671, 6,423,710, and compounds described in 6,818,651; 6,737,436, 6,376,489, 6,372,777, 6,362,213, 6,313,156, 6,294,561, 6,613,778, 6,617,357; 6,146,876, 6,838,559, 6,884,800, 6,258,843, 6,258,833, 6,121,279, 6,043,263, RE38,624, US20050059686; US20040138279; US20050222138; 6,297,257, 6,251,923, 6,613,794, 6,407,108, 6,107,295, US20040214843; US20040106631; US 20030045557; 6,103,718, 6,479,494, 6,602,890, 6,545,158, 6,545,025, 25 US 20020198198; US20030162802, US20030092908, 6,498,160, 6,743,802, 6,787,554, 6,828,333, 6,869,945, US 20030104974; US20030100571; 20030092721; and 6,894,041, 6,924,292, 6,949,573, 6,953,810, 5,972,927, US20050148604. 5,962,492, 5,814,651, 5,723,460, 5,716,967, 5,686,434, 5,502,072, 5,116,837, 5,091,431; 4,670,434; 4,490,371; Examples of inhibitors of PDE8 include dipyridamole. 5,710,160, 5,710,170, 6,384,236,3,941,785,30 US20050119225, US20050026913, WO 99/65880, WO [0106] In some embodiments, the PDE inhibitor inhib- 00/26201, WO 98/06704, WO 00/59890, WO9907704, its a cGMP-specific PDE. Examples of cGMP specific WO9422852, WO 98/20007, WO 02/096423, WO PDE inhibitors useful in the methods described herein 98/18796, WO 98/02440, WO 02/096463, WO 97/44337, include pyrimidine and pyrimidinone derivatives, such as WO 97/44036, WO 97/44322, EP 0763534, Aoki et al., 35 the compounds described in 6,677,335, 6,458,951, J Pharmacol Exp Ther., 295(1):255-60 (2000), Del Piaz 6,251,904, 6,787,548, 5,294,612, 5,250,534, 6,469,012, et al., Eur. J. Med. Chem., 35; 463-480 (2000), and Bar- WO 94/28902, WO96/16657, EP0702555, and Eddahibi, nette et al., Pharmacol. Rev. Commun. 8: 65-73 (1997). Br. J. Pharmacol., 125 (4): 681-688 (1988); griseolic acid [0104] In some embodiments, the PDE4 inhibitor is derivatives, such as the compounds disclosed in Cilomilast (SB-207499); Filaminast; Tibenelast (LY-40 4,460,765; 1-arylnaphthalene lignans, such as those de- 186655); Ibudilast; Piclamilast (RP 73401); Doxofylline; scribed in Ukita, J. Med. Chem. 42 (7): 1293-1305 (1999); Cipamfylline (HEP-688); atizoram (CP- 80633); theophyl- quinazoline derivatives, such as 4-[[3’,4’-(methylenedi- line; isobutylmethylxanthine; Mesopram (ZK-117137); oxy)benzyl]amino]-6 methoxyquinazoline) and com- Zardaverine; vinpocetine; Rolipram (ZK-62711); Arofyl- pounds described in 3,932,407, 4,146,718, and line(LAS- 31025); roflumilast (BY- 217); Pumafentrin(BY- 45 RE31,617; pyrroloquinolones and pyrrolopyridinones, 343); Denbufylline; EHNA; milrinone; Siguazodan; Zap- such as those described in 6,686,349, 6,635,638, rinast; Tolafentrine; Isbufylline; IBMX; 1C-485; dyphyl- 6,818,646, US20050113402; carboline derivatives, such line; verolylline; bamifylline; pentoxyfilline; enprofilline; the compounds described in 6,492,358, 6,462,047, lirimilast (BAY 19-8004); filaminast (WAY- PDA-641); 6,821,975, 6,306,870, 6,117,881, 6,043,252, 3,819,631, benafentrine; trequinsin; nitroquazone; Tetomilast50 US20030166641, WO 97/43287, Daugan et al., J Med (OPC-6535); AH-21-132; AWD-12-343; AWD-12-281; Chem., 46(21):4533-42 (2003), and Daugan et al., J Med AWD-12-232; CC-7085; CDC-801; CDC-998; CDP-840; Chem., 9;46(21):4525-32 (2003); imidazo derivatives, CH-422; CH-673; CH-928; CH-3697; CH-3442; CH- such as the compounds disclosed in 6,130,333, 2874; CH-4139; Chiroscience 245412; CI-1018; CI- 6,566,360, 6,362,178, 6,582,351, US20050070541, and 1044; CI-1118; CP-353164; CP-77059; CP- 146523; CP- 55 US20040067945; and compounds described in 293321; CP-220629; CT-2450; CT-2820; CT-3883; CT- 6,825,197, 5,719,283, 6,943,166, 5,981,527, 6,576,644, 5210; D-4418; D-22888; E-4021; END 54622; GF-248; 5,859,009, 6,943,253, 6,864,253, 5,869,516, 5,488,055, GW-3600; IC-485; ICI 63197; IPL-4088; KF- 19514; KW- 6,140,329, 5,859,006, 6,143,777, WO 96/16644, WO

16 31 EP 2 377 530 A2 32

01/19802, WO 96/26940, Dunn, Org. Proc. Res. Dev., 1081-92 (2004); UK- 235,187 and related compounds de- 9: 88-97 (2005), and Bi et al., Bioorg Med Chem Lett., scribed in EP 579496; and compounds described in 11(18):2461-4 (2001). 6,930,114, 4,861,891, US20020132754 [0107] Examples of PDE5 inhibitors useful in methods US20040138249, US20040249148, US2004010663 1, described herein include pyrimidine and pyrimidinone 5 WO 951997, and Maw et al., Bioorg Med Chem Lett. derivatives, such as the compounds described in 2003 Apr 17;13(8):1425-8. 6,677,335, 6,458,951, 6,251,904, 6,787,548, 5,294,612, [0113] Examples of PDE1 inhibitors include IBMX; vin- 5,250,534, 6,469,012, WO 94/28902, WO96/16657, pocetine; MMPX; KS-505a; SCH-51866; W-7; PLX650; EP0702555, and Eddahibi, Br. J. Pharmacol., 125(4): PLX371; PLX788; Phenothiazines; and compounds de- 681-688 (1988); griseolic acid derivatives, such as the 10 scribed in 4,861,891. compounds disclosed in 4,460,765; 1-arylnaphthalene [0114] Examples of PDE2 inhibitors include EHNA; lignans, such as those described in Ukita, J. Med. Chem. PLX650; PLX369; PLX788; PLX 939; Bay 60-7550 and 42(7): 1293-1305 (1999); quinazoline derivatives, such related compounds described in Boess et al., Neurop- as 4-[[3’,4’-(methylenedioxy)benzyl]amino]-6-methoxy- harmacology,47(7): 1081-92 (2004); andcompounds de- quinazoline) and compounds described in 3,932,407, 15 scribed in US20020132754. 4,146,718, and RE31,617; pyrroloquinolones and pyrrol- [0115] Examples of PDE10 inhibitors include sildena- opyridinones, such as those described in 6,686,349, fil; SCH-51866; papaverine; Zaprinast; Dipyridamole; 6,635,638, 6,818,646, US20050113402; carboline deriv- E4021; Vinpocetine; EHNA; Milrinone; Rolipram; atives, such the compounds described in 6,492,358, PLX107; and compounds described in 6,930,114, 6,462,047, 6,821,975, 6,306,870, 6,117,881, 6,043,252, 20 US20040138249, US20040249148. 3,819,631, US20030166641, WO 97/43287, Daugan et [0116] Examples of PDE11 inhibitors include IC-351 al., J Med Chem., 46(21):4533-42 (2003), and Daugan and related compounds described in WO 9519978; et al., J Med Chem., 9;46(21):4525-32 (2003); imidazo E4021 and related compounds described in WO derivatives, such as the compounds disclosed in9307124;UK- 235,187 andrelated compoundsdescribed 6,130,333, 6,566,360, 6,362,178, 6,582,351,25 in EP 579496; PLX788; Zaprinast; Dipyridamole; and US20050070541, and US20040067945; and com- compounds described in U20040106631 and Maw et al., pounds described in 6,825,197, 6,943,166, 5,981,527, Bioorg Med Chem Lett. 2003 Apr 17;13(8):1425-8. 6,576,644, 5,859,009, 6,943,253, 6,864,253, 5,869,516, [0117] In some embodiments, the PDE inhibitor is a 5,488,055, 6,140,329, 5,859,006, 6,143,777, WO compound described in 5,091,431, 5,081,242, 96/16644, WO 01/19802, WO 96/26940, Dunn, Org.30 5,066,653, 5,010,086, 4,971,972, 4,963,561, 4,943,573, Proc. Res. Dev., 9: 88-97 (2005), and Bi et al., Bioorg 4,906,628, 4,861,891, 4,775,674, 4,766,118, 4,761,416, Med Chem Lett., 11(18):2461-4 (2001). 4,739,056, 4,721,784, 4,701,459, 4,670,434, 4,663,320, [0108] In some embodiments, the PDE5 inhibitor is 4,642,345, 4,593,029, 4,564,619, 4,490,371, 4,489,078, zaprinast; MY-5445; dipyridamole; vinpocetine; 4,404,380, 4,370,328, 4,366,156, 4,298,734,4,289,772, FR229934; methyl- 1- 3-isobutyl-8-(methylamino)xan- 35 RE30,511, 4,188,391, 4,123,534, 4,107,309, 4,107,307, thine; furazlocillin; Sch-51866; E4021; GF-196960; IC- 4,096,257, 4,093,617, 4,051,236, or 4,036,840. 351; T- 1032; sildenafil; tadalafil; vardenafil; DMPPO; RX- [0118] A PDEagent as describedherein includesphar- RA-69; KT-734; SKF-96231; ER-21355; BF/GP-385; maceutically acceptable salts, derivatives, prodrugs, me- NM-702; PLX650; PLX134; PLX369; PLX788; or vesna- tabolites, stereoisomer, or other variant of the agent. For rinone. 40 example, in some embodiments, the PDE inhibitor is rof- [0109] In some embodiments, the PDE5 inhibitor is lumilastN-oxide, which is the primary metabolite of roflu- sildenafil or a related compound disclosed in 5,346,901, milast in humans and has been shown to have enhanced 5,250,534, or 6,469,012; tadalafil or a related compound efficacy in vivo. In some embodiments, a PDE inhibitor disclosed in 5,859,006, 6,140,329, 6,821,975, oris chemically modified to reduce side effects, toxicity, sol- 6,943,166; or vardenafil or a related compound disclosed 45 ubility, and/or other characteristics. For example, in some in 6,362,178. embodiments, the PDE inhibitor is benzylated in order to [0110] Examples of PDE6 inhibitors useful in methods reduce emetogenic effects, as described, e.g., in described herein include dipyridamole and zaprinast. 6,555,572, herein incorporated by reference. Methods [0111] Examples of PDE9 inhibitors useful in methods for preparing and administering salts, derivatives, prod- described herein include SCH-51866; IBMX; and BAY 50 rugs, and metabolites of various compounds are well 73-6691. known in the art. [0112] In some embodiments, the PDE inhibitor inhib- [0119] Compounds described herein that contain a its dual-specificity PDE. Examples of dual-specificity chiral center include all possible stereoisomers of the PDE inhibitors useful in the methods described herein compound, including compositions comprising the ra- include the cAMP-specific and cGMP-specific PDE in- 55 cemic mixture of the two enantiomers, as well as com- hibitors describe herein; MMPX; KS-505a; W-7; Pheno- positions comprising each enantiomer individually, sub- thiazines; Bay 60-7550 and related compounds de- stantially free of the other enantiomer. Thus, for example, scribed in Boess et al., Neuropharmacology, (7): 47 contemplated herein is a composition comprising the S

17 33 EP 2 377 530 A2 34 enantiomer substantially free of the R enantiomer, or the "target selective"activity under certain conditions,where- R enantiomer substantially free of the S enantiomer. If in the PDE inhibitor is substantially inactive against non- the named compound comprises more than one chiral PDE molecular targets, such as (i) CNS receptors, in- center, the scope of the present disclosure also includes cluding but not limited to, glutamate receptors, GABA compositions comprising mixtures of varying proportions 5 receptors, opioid receptors (e.g., mu, delta, and kappa between the diastereomers, as well as compositions opioid receptors), muscarinic receptors (e.g., m1- m5 re- comprising one or more diastereomers substantially free ceptors), histaminergic receptors, phencyclidine recep- of one or more of the other diastereomers. By "substan- tors, dopamine receptors, alpha and beta-adrenocep- tially free" it is meant that the composition comprises less tors, sigma receptors (type-1 and type- 2), and 5HT-1 and than 25%, 15%, 10%, 8%, 5%, 3%, or less than 1% of 10 5-HT-2 receptors; (ii) kinases, including but not limited the minor enantiomer or diastereomer(s). Methods for to, Mitogen-activated protein kinase, PKA, PKB, PKC, synthesizing, isolating, preparing, and administering var- CK-2; c-Met, JAK, SYK, KDR, FLT-3, c-Kit, Aurora ki- ious stereoisomers are known in the art. nase, CDK kinases (e.g., CDK4/cyclin D, CDK2/cyclin E, [0120] In some embodiments, compositions compris- CDK2/cyclin A, CDK1/cyclin B), and TAK- 1; (iii) ion chan- ing one or more stereoisomers substantially free from 15 nels (e.g., calcium, chloride, potassium, and the like) one or more other stereoisomers provide enhanced af- and/or (iv) enzymes, including but not limited to, adenyl finity, potency, selectivity and/or therapeutic efficacy rel- or guanyl cyclases, HDACs, and the like. In other em- ative to compositions comprising a greater proportion of bodiments, a PDE agent used in the methods described the minor stereoisomer (s). For example, in some embod- herein is substantially inactive with respect to other re- iments, a PDE agent is the (-)-enantiomer of rolipram, 20 ceptors, such as muscarinic receptors, nicotinic recep- which has a higher pharmacological potency than the tors, dopamine receptors, and opioid receptors as non- (+)-enantiomer, due e.g., to a higher affinity for PDEs limiting examples. (e.g., PDE4) and/or stereospecific metabolism, with the [0125] In some embodiments, a PDE inhibitor exhibits (+)-enantiomer exhibiting a faster clearance rate than the both isozyme and target selectivity. In further embodi- (-)-enantiomer. In some embodiments, a PDE agent is a 25 ments, isozyme and/or target selectivity is achieved by variant of a known PDE inhibitor that has enhanced af- administering a PDE inhibitor at a dosage and in a man- finity, potency, selectivity and/or therapeutic efficacy. ner that produces a concentration of the PDE inhibitor in [0121] In some embodiments, a PDE agent used in a the target organ or tissue that is therapeutically effective method described herein have similar activity against against one or more PDE isozymes, while being sub- several PDE isozymes. Examples of non- selective inhib- 30 therapeutic at other PDE isozymes and/or targets. Ad- itors include, e.g., theophylline, theobromine, dipyrida- vantageously, the isozyme and/or target selectivity of a mole, IBMX, pentoxifylline, ibudilast, and papaverine. PDE inhibitor results in enhanced efficacy, fewer side [0122] In other embodiments, a PDE agent may exhibit effects, lower effective dosages, less frequent dosing, "isozyme-selective" activity. For example, in various em- and other desirable attributes relative to non-selective bodiments, a PDE agent is active against one or more 35 modulators. PDE families and substantially inactive against one or [0126] In some embodiments, the PDE inhibitor exhib- more other PDE families. In some embodiments, a PDE its dual-selectivity, being substantially more active agent exhibits isozyme-selective activity against one or against two PDE isozymes relative to other PDE iso- more PDE families that are active in the CNS, including zymes. For example, in some embodiments, the PDE but not limited to, PDE1, PDE2, PDE3, PDE4 (e.g.,40 inhibitor is a dual PDE4/PDE7 inhibitor, such as the com- PDE4A, PDE4B, PDE4D), PDE5, PDE7, PDE8, (e.g., pounds described in US20030104974; a dual PDE8B), PDE9, PDE10 and/or Pre11. In further embod- PDE3/PDE4 inhibitor, such as zardaverine, tolafentrine, iments, a PDE agent exhibits isozyme-selectivity within benafentrine, trequinsine, Org-30029, L-686398, SDZ- a PDE family. For example, the PDE4 inhibitor cilomilast ISQ-844, Org-20241, EMD-54622, or a compound de- is approximately 10-fold more selective for PDE4D, than 45 scribed in 5,521,187, or 6,306,869; or a dual PDE1/PDE4 for PDE4A, PDE4B, and PDE4C. inhibitor, such as KF19514 (5-phenyl-3-(3-pyridyl)me- [0123] In additional embodiments, PDE inhibitors ex- thyl-3H-imidazo[4,5-c][1,8]naphthyridin-4 (5H)-one) hibit isozyme-selective activity against one or more PDEs [0127] In some embodiments, the PDE inhibitor used residing in a neurogenic region of the brain, such as the in a combination or method disclosed herein is caffeine dentate gyrus, the subventricular zone, and/or the olfac- 50 or other ingested compound such as a vitamin like folic tory bulb. For example, PDE inhibitors are active against acid. In some embodiments, the caffeine or folic acid is PDE1A, PDE1B, PDE1C, PDE2A, and/or PDE5A, which administered in a formulation comprising a PDE agent. are expressed in the subventricular zone, hippocampus In other embodiments, the caffeine or folic acid is admin- and olfactory bulb, as well as throughout the brain. Other istered simultaneously with a PDE agent. In alternative isozymes expressed at high levels in the brain include, 55 embodiments, the caffeine is administered in a formula- but are not limited to, PDE4A, PDE4B, PDE4D, PDE9A, tion, dosage, or concentration lower or higher than that and PDE10A. of a caffeinated beverage such as coffee, tea, or soft [0124] In some embodiments, PDE inhibitors have drinks. Similarly, folic acid may be administered in a for-

18 35 EP 2 377 530 A2 36 mulation, dosage, or concentration lower or higher than is achieved by administering a combination of at least that of a nutritional supplement containing folic acid. In one PDE inhibitor, and at least one PDE transcriptional/ further embodiments, the caffeine or folic acid is admin- translational inhibitor. istered by a non-oral means, including, but not limited to, [0131] As described herein, a PDE agent, optionally in parenteral (e.g., intravenous, intradermal, subcutane- 5 combination with one or more other neurogenic agents, ous, inhalation), transdermal (topical), transmucosal, is administered to an animal or human subject to result rectal, or intranasal (including, but not limited to, inhala- in neurogenesis. A combination may thus be used to treat tion of aerosol suspensions for delivery of compositions a disease, disorder, or condition of the disclosure. to the nasal mucosa, trachea and bronchioli) administra- [0132] Methods for assessing the nature and/or de- tion. The disclosure includes embodiments with the ex- 10 gree of neurogenesis in vivo and in vitro, for detecting plicit exclusion of caffeine, folic acid, or another one or changes in the nature and/or degree of neurogenesis, more of the described agents for use in combination with for identifying neurogenesis modulating agents, for iso- a PDE agent. lating and culturing neural stem cells, and for preparing [0128] In further alternative embodiments, the caffeine neural stem cells for transplantation or other purposes is in an isolated form, such as that which is separated 15 are disclosed, for example, in U.S. Provisional Applica- from one or more molecules or macromolecules normally tion No. 60/697,905, and U.S. Publication Nos. found with caffeine before use in a combination or method 2005/0009742 and 2005/0009847, 20050032702, as disclosed herein. In other embodiments, the caffeine 2005/0031538, 2005/0004046, 2004/0254152, is completely or partially purified from one or more mol- 2004/0229291, and 2004/0185429, all of which are here- ecules or macromolecules normally found with the caf- 20 in incorporated by reference in their entirety. feine. Exemplary cases of molecules or macromolecules [0133] Selection of a PDE agent, or additional agent found with caffeine include a plant or plant part, an animal of a combination, may be readily determined by evaluat- or animal part, and a food or beverage product. ing their potency in relation to neurogenesis and their [0129] In some embodiments, a PDE agent is a novel target selectivity with routine methods as described here- PDE inhibitor identified using structure-activity relation- 25 in and as known to the skilled person. The agent (s) may ships and teachings in the art and described, e.g., in Kel- then be evaluated for their toxicity (if any), pharmacoki- ler et al., Chem Pharm Bull (Tokyo). 2001 Aug;49(8): netics (such as absorption, metabolism, distribution and 1009-17; Jin et al., J Biol Chem 267: 18929-18939 degradation/elimination) by use of with recognized (1992), Pillai et al., Proc Natl Acad Sci USA 90:standard pharmaceutical techniques. Embodiments of 11970-11974 (1993), Atienza et al., J. Biol. Chem., 274: 30 the disclosure include use of agent (s) that are potent and 4839-4847 (1997), Xu et al., Science (Wash DC) 288: selective, and have either an acceptable level of toxicity 1822-1825 (2000), Boyle et al., Bioorg Med Chem Lett., or no significant toxic effect, at the therapeutic dose. Ad- 15(9):2365-9 (2005); Lee et al., FEBS Lett., 530(1-3): ditional selections may be made based on bioavailability 53-8 (2002); Maw et al., Bioorg Med Chem Lett. 2003 of the agent following oral administration. Apr 17; 13(8): 1425-8; and Richter et al., Cell Signal., 13 35 (3): 159-67 (2001), each of which are herein incorporated Formulations and Doses by reference in their entirety. [0130] In other embodiments, a PDE agent is a mole- [0134] In some embodiments of the disclosure, a PDE cule or composition that inhibits the expression of a target agent, optionally in combination with one or more other PDE, such as an antisense nucleotide (e.g., siRNA) that 40 neurogenic agents, is in the form of a composition that specifically hybridizes with the cellular mRNA and/or ge- includes at least one pharmaceutically acceptable excip- nomic DNA corresponding to the gene(s) of the target ient. As used herein, the term "pharmaceutically accept- PDE so as to inhibit their transcription and/or translation, able excipient" includes any excipient known in the field or a ribozyme that specifically cleaves the mRNA of a as suitable for pharmaceutical application. Suitable phar- target PDE. Antisense nucleotides and ribozymes can 45 maceutical excipients and formulations are known in the be delivered directly to cells, or indirectly via an expres- art and are described, for example, in Remington’s Phar- sion vector which produces the nucleotide when tran- maceutical Sciences (19th ed.) (Genarro, ed. (1995) scribed in the cell. Methods for designing and adminis- Mack Publishing Co., Easton, Pa.). Preferably, pharma- tering antisense oligonucleotides and ribozymes are ceutical carriers are chosen based upon the intended known in the art, and are described, e.g., in Mautino et 50 mode of administration of a PDE agent, optionally in com- al., Hum Gene Ther 13:1027-37 (2002) and Pachori et bination with one or more other neurogenic agents. The al., Hypertension 39:969-75 (2002), herein incorporated pharmaceutically acceptable carrier may include, for ex- by reference. Examples of antisense compositions ample, disintegrants, binders, lubricants, glidants, emol- against PDEs include, e.g., the anti- PDE4 compositions lients, humectants, thickeners, silicones, flavoring disclosed in US20030045490 and WO 00/40714, and 55 agents, and water. the anti-PDE1 and anti-PDE4 compositions disclosed in [0135] A PDE agent, optionally in combination with one 5,885,834, all of which are herein incorporated by refer- or more other neurogenic agents, may be incorporated ence. In some embodiments, neurogenesis modulation with excipients and administered in the form of ingestible

19 37 EP 2 377 530 A2 38 tablets, buccal tablets, troches, capsules, elixirs, suspen- to about 2 mg/kg/day, about 2 to about 20 mg/kg/day, or sions, syrups, wafers, or any other form known in the about 20 to about 200 mg/kg/day. However, as under- pharmaceutical arts. The pharmaceutical compositions stood by those skilled in the art, the exact dosage of a may also be formulated in a sustained release form. Sus- PDE agent, optionally in combination with one or more tained release compositions, enteric coatings, and the 5 other neurogenic agents, used to treat a particular con- like are known in the art. Alternatively, the compositions dition will vary in practice due to a wide variety of factors. may be a quick release formulation. Accordingly, dosage guidelines provided herein are not [0136] The amount of a combination of a PDE agent, limiting as the range of actual dosages, but rather provide or a combination thereof with one or more other neuro- guidance to skilled practitioners in selecting dosages genic agents, may be an amount that also potentiates or 10 useful in the empirical determination of dosages for indi- sensitizes, such as by activating or inducing cells to dif- vidual patients. Advantageously, methods described ferentiate, a population of neural cells for neurogenesis. herein allow treatment of one or more conditions with The degree of potentiation or sensitization for neurogen- reductions in side effects, dosage levels, dosage fre- esis may be determined with use of the combination in quency, treatment duration, safety, tolerability, and/or any appropriate neurogenesis assay, including, but not 15 other factors. So where suitable dosages for a PDE agent limited to, a neuronal differentiation assay described to modulate a PDE activity are known to a skilled person, herein. In some embodiments, the amount of a combi- the disclosure includes the use of about 75%, about 50%, nation of a PDE agent, optionally in combination with one about 33%, about 25%, about 20%, about 15%, about or more other neurogenic agents, is based on the highest 10%, about 5%, about 2.5%, about 1%, about 0.5%, amount of one agent in a combination, which amount 20 about 0.25%, about 0.2%, about 0.1%, about 0.05%, produces no detectable neuroproliferation in vitro but yet about 0.025%, about 0.02%, about 0.01%, or less than produces neurogenesis, or a measurable shift in efficacy the known dosage. inpromoting neurogenesis in vitro,when used in the com- [0139] In some embodiments, an effective, neurogen- bination. esis modulating amount is an amount that achieves a [0137] As disclosed herein, an effective amount of a 25 concentration within the target tissue, using the particular PDE agent, optionally in combination with one or more mode of administration, at or above the IC50 for activity other neurogenic agents, in the described methods is an of a PDE agent. In some embodiments, the PDE agent amount sufficient, when used as described herein, to is administered in a manner and dosage that gives a peak stimulate or increase neurogenesis in the subject target- concentration of about 1, 1.5, 2, 2.5, 5, 10, 20 or more 30 ed for treatment when compared to the absence of the times the IC50 concentration. IC50 values and bioavaila- combination. An effective amount of a PDE agent alone bility data for various PDE agent are known in the art, or in combination may vary based on a number of factors, and are described, e.g., in the references cited herein. including but not limited to, the activity of the active com- [0140] Infurther embodiments, an effective, neurogen- pounds, the physiological characteristics of the subject, esis modulating amount is a dose that lies within a range 35 the nature of the condition to be treated, and the route of circulating concentrations that includes the ED50 (the and/or method of administration. General dosage ranges pharmacologically effective dose in 50% of subjects) with of certain compounds are provided herein and in the cited little or no toxicity. references based on animal models of CNS diseases [0141] In some embodiments, an effective, neurogen- and conditions. Various conversion factors, formulas, esis modulating amount is an amount that achieves a and methods for determining human dose equivalents of 40 peak concentration within the target tissue, using the par- animal dosages are known in the art, and are described, ticular mode of administration, at or above the IC50 or e.g., in Freireich et al., Cancer Chemother Repts 50(4): EC50 concentration for the modulation of neurogenesis. 219 (1966), Monro et al., Toxicology Pathology, 23: In various embodiments, a PDE agent is administered in 187-98 (1995), Boxenbaum and Dilea, J.Clin.Pharmacol. a manner and dosage that gives a peak concentration of 45 35: 957-966 (1995), and Voisin et al., Reg. Toxicol. Phar- about 1, 1.5, 2, 2.5, 5, 10, 20 or more times the IC50 or macol., 12(2): 107-116 (1990), which are herein incor- EC50 concentration for the modulation of neurogenesis. porated by reference. In some embodiments, the IC50 or EC50 concentration [0138] The disclosed methods typically involve the ad- for the modulation of neurogenesis is substantially lower ministration of a PDE agent, optionally in combination than the IC50 concentration for activity of a PDE agent, with one or more other neurogenic agents, in a dosage 50 allowing treatment of conditions for which it is beneficial range of from about 0.001 ng/kg/day to about 200 to modulate neurogenesis with lower dosage levels, dos- mg/kg/day. Other non-limiting dosages include from age frequencies, and/or treatment durations relative to about 0.001 to about 0.01 ng/kg/day, about 0.01 to about known therapies. IC50 and EC50 values for the modula- 0.1 ng/kg/day, about 0.1 to about 1 ng/kg/day, about 1 tion of neurogenesis can be determined using methods to about 10 ng/kg/day, about 10 to about 100 ng/kg/day, 55 described in U.S. Provisional Application No. 60/697,905 about 100 ng/kg/day to about 1P g/kg/day, about 1 to to Barlow et al., filed July 8, 2005, incorporated by refer- about 2 Pg/kg/day, about 2 Pg/kg/day to about 0.02 ence, or by other methods known in the art. mg/kg/day, about 0.02 to about 0.2 mg/kg/day, about 0.2 [0142] In some embodiments IC 50 or EC 50 concentra-

20 39 EP 2 377 530 A2 40 tion for the modulation of neurogenesis is substantially or in a combination disclosed herein, may be in the form lower than the IC50 or EC50 concentration for activity of of a pharmaceutical composition. a PDE agent at non-PDE targets, such as other kinases, [0147] In some embodiments, an effective, neurogen- receptors, or signaling molecules. IC 50 and EC50 values esis modulating amount of a combination of a PDE agent, for PDE agents at various kinases and other molecules 5 optionally in combination with one or more other neuro- are known in the art, and can be readily determined using genic agents, is an amount of a PDE agent (or of each established methods. agent in a combination) that achieves a concentration [0143] In other embodiments, the amount of a PDE within the target tissue, using the particular mode of ad- agent used in vivo may be about 50%, about 45%, about ministration, at or above the IC50 or EC50 for activity of 40%, about 35%, about 30%, about 25%, about 20%, 10 target molecule or physiological process. In some cases, about 18%, about 16%, about 14%, about 12%, about a PDE agent, optionally in combination with one or more 10%, about 8%, about 6%, about 4%, about 2%, or about other neurogenic agents, is administered in a manner 1%or less than the maximum tolerateddose for a subject, and dosage that gives a peak concentration of about 1, including where one or more other neurogenic agents is about 1.5, about 2, about 2.5, about 5, about 10, about 15 used in combination with the PDE agent. This is readily 20 or more times the IC50 or EC50 concentration of the determined for each PDE agent that has been in clinical PDE agent (or each agent in the combination). IC 50 and use or testing, such as in humans. EC50 values and bioavailability data for a PDE agent and [0144] Alternatively, the amount of a PDE agent, op- other agent(s) described herein are known in the art, and tionally in combination with one or more other neurogenic are described, e.g., in the references cited herein or can agents, may be an amount selected to be effective to 20 be readily determined using established methods. In ad- produce an improvement in a treated subject based on dition, methods for determining the concentration of a detectable neurogenesis in vitro as described above. In free compound in plasma and extracellular fluids in the some embodiments, such as in the case of a known PDE CNS, as well pharmacokinetic properties, are known in agent, the amount is one that minimizes clinical side ef- the art, and are described, e.g., in de Lange et al., AAPS fects seen with administration of the agent to a subject. 25 Journal, 7(3): 532-543 (2005). In some embodiments, a The amount of an agent used in vivo may be about 50%, PDE agent, optionally in combination with one or more about 45%, about 40%, about 35%, about 30%, about other neurogenic agents, described herein is adminis- 25%, about 20%, about 18%, about 16%, about 14%, tered, as a combination or separate agents used togeth- about 12%, about 10%, about 8%, about 6%, about 4%, er, at a frequency of at least about once daily, or about about 2%, or about 1% or less of the maximum tolerated 30 twice daily, or about three or more times daily, and for a dose in terms of acceptable side effects for a subject. duration of at least about 3 days, about 5 days, about 7 This is readily determined for each PDE agent or other days, about 10 days, about 14 days, or about 21 days, agent(s) of a combination disclosed herein as well as or about 4 weeks, or about 2 months, or about 4 months, those that have been in clinical use or testing, such as or about 6 months, or about 8 months, or about 10 in humans. 35 months, or about 1 year, or about 2 years, or about 4 [0145] In other embodiments, the amount of an addi- years, or about 6 years or longer. tional neurogenic sensitizing agent in a combination with [0148] In other embodiments, an effective, neurogen- a PDE agent of the disclosure is the highest amount esis modulating amount is a dose that produces a con- which produces no detectable neurogenesis in vitro, in- centration of a PDE agent (or each agent in a combina- cluding in animal (or non-human) models for behavior 40 tion) in an organ, tissue, cell, and/or other region of in- linked to neurogenesis, but yet produces neurogenesis, terest that includes the ED50 (the pharmacologically ef- or a measurable shift in efficacy in promoting neurogen- fective dose in 50% of subjects) with little or no toxicity. esis in the in vitro assay, when used in combination with IC50 and EC 50 values for the modulation of neurogenesis a PDE agent. Embodiments include amounts which pro- can be determined using methods described in U.S. Pro- duce about 1%, about 2%, about 4%, about 6%, about 45 visional Application No. 60/697,905 to Barlow et al., filed 8%, about 10%, about 12%, about 14%, about 16%, July 8, 2005, incorporated by reference, or by other meth- about 18%, about 20%, about 25%, about 30%, about ods known in the art. In some embodiments, the IC 50 or 35%, or about 40% or more of the neurogenesis seen EC50 concentration for the modulation of neurogenesis with the amount that produces the highest level of neu- is substantially lower than the IC 50 or EC 50 concentration rogenesis in an in vitro assay. 50 for activity of a PDE agent and/or other agent(s) at non- [0146] As described herein, the amount of a PDE targeted molecules and/or physiological processes. agent, optionally in combination with one or more other [0149] In some methods described herein, the appli- neurogenic agents, may be any that is effective to pro- cation of a PDE agent in combination with one or more duce neurogenesis, optionally with reduced or minimized other neurogenic agents may allow effective treatment amounts of astrogenesis. In some embodiments, the55 with substantially fewer and/or less severe side effects amount may be the lowest needed to produce a desired, compared to existing treatments. In some embodiments, or minimum, level of detectable neurogenesis or benefi- combination therapy with a PDE agent and one or more cial effect. Of course the administered PDE agent, alone additional neurogenic agents allows the combination to

21 41 EP 2 377 530 A2 42 be administered at dosages that would be sub- therapeu- or a PDE5 inhibitor, such as revatio, cialis (tadalafil), lev- tic when administered individually or when compared to itra (vardenafil), DA-8159, dapoxetine, avanafil (TA- other treatments. In other embodiments, each agent in 1790), SCH- 466132, or ABT- 670 in combination with one a combination of agents may be present in an amount or more other neurogenic agents. In other embodiments, that results in fewer and/or less severe side effects than 5 a combination of two or more of the above agents, is that which occurs with a larger amount. Thus the com- used in combination with one or more other neurogenic bined effect of the neurogenic agents will provide a de- agents. sired neurogenic activity while exhibiting fewer and/or [0153] In some embodiments, methods of treatment less severe side effects overall. Non-limiting examples disclosed herein comprise the step of administering to a of side effects which may be reduced, in number and/or 10 mammal a PDE agent, optionally in combination with one severity, include, but are not limited to, sweating, di- or more other neurogenic agents, for a time and at a arrhea, flushing, hypotension, bradycardia, bronchocon- concentration sufficient to treat the condition targeted for striction, urinary bladder contraction, nausea, vomiting, treatment. The disclosed methods can be applied to in- parkinsonism, and increased mortality risk. In further em- dividuals having, or who are likely to develop, disorders bodiments, methods described herein allow treatment of 15 relating to neural degeneration, neural damage and/or certain conditions for which treatment with the same or neural demyelination. similar compounds is ineffective using known methods [0154] Depending on the desired clinical result, the dis- due, for example, to dose-limiting side effects, toxicity, closed agents or pharmaceutical compositions are ad- and/or other factors. ministered by any means suitable for achieving a desired [0150] In some embodiments, the disclosure includes 20 effect. Various delivery methods are known in the art and a combination, of a PDE agent and a second agent, can be used to deliver an agent to a subject or to NSCs where one or both of the agents is used to treat hyper- or progenitor cells within a tissue of interest. The delivery tension, such as in a case where one or both agents has method will depend on factors such as the tissue of in- hypotensive effects. The disclosure includes a composi- terest, the nature of the compound (e.g., its stability and tion or formulation of such a combination in doses that 25 ability to cross the blood-brain barrier), and the duration reduces such hypotensive effects. Non-limiting exam- of the experiment or treatment, among other factors. For ples of such doses include amounts that are lower than example, an osmotic minipump can be implanted into a those used to treat hypertension, or lower than those neurogenic region, such as the lateral ventricle. Alterna- which are sufficient to treat hypertension, in an animal tively, compounds can be administered by direct injection subject or human patient. In some cases, the dose of one 30 into the cerebrospinal fluid of the brain or spinal column, or both agents may be from about one-thirtieth to about or into the eye. Compounds can also be administered one-half of an amount used to treat hypertension or an into the periphery (such as by intravenous or subcutane- amount sufficient to treat hypertension. In other cases, ous injection, or oral delivery), and subsequently cross the dose of one or both agents may be less than the the blood-brain barrier. about one-thirtieth amount. 35 [0155] In some embodiments, the disclosed agents or [0151] In other embodiments, the dose of one or both pharmaceutical compositions are administered in a man- agents is an amount which produces an acceptable level ner that allows them to contact the subventricular zone of hypotension in a normotensive subject or patient. The (SVZ) of the lateral ventricles and/or the dentate gyrus amount may be one which is insufficient to produce a of the hippocampus. The delivery or targeting of a PDE detectable or measurable reduction in hypertension in a 40 agent, optionally in combination with one or more other hypertensive subject or patient. neurogenic agents, to a neurogenic region, such as the dentate gyrus or the subventicular zone, may enhances Routes of Administration efficacy and reduces side effects compared to known methods involving administration with the same or similar [0152] As described, the methods of the disclosure 45 compounds. Examples of routes of administration in- comprise contacting a cell with a PDE agent, optionally clude parenteral, e.g., intravenous, intradermal, subcu- incombination with one or more other neurogenicagents, taneous, oral (e.g., inhalation), transdermal (topical), or administering such an agent or combination to a sub- transmucosal, and rectal administration. Intranasal ad- ject, to result in neurogenesis. Some embodiments com- ministration generally includes, but is not limited to, in- prise the use of one PDE agent, such as ibudilast or MN- 50 halation of aerosol suspensions for delivery of composi- 166, or dipyridamole (persantine); a PDE1 inhibitor, such tions to the nasal mucosa, trachea and bronchioli. as vinpocetine; a PDE3 inhibitor, such as enoximone, [0156] In other embodiments, a combination of a PDE milrinone, pimobendan, flosequinan, levosimendan, ve- agent, optionally in combination with one or more other snarinone, olprinone, amrinone, inamnirone, anagrelide, neurogenic agents, is administered so as to either pass cilostazol, or imazodan; a PDE4 inhibitor, such as cilo- 55 through or by-pass the blood-brain barrier. Methods for milast, roflumilast, rolipram, MEM 1414, MEM 1971, NIK allowing factors to pass through the blood-brain barrier 616, GK 07294A, 256066, GW 842470, ONO 6126, are known in the art, and include minimizing the size of PLX369, HT-0712, IPL 455903, IC 485, or NVP-ABE171; the factor, providing hydrophobic factors which facilitate

22 43 EP 2 377 530 A2 44 passage, and conjugation to a carrier molecule that has ine maleate (CAS RN: 61718-82-9) and Luvox®, parox- substantial permeability across the blood brain barrier. etine (Paxil®; described, e.g., in U.S. Pat. 3,912,743 and In some instances, an agent or combination of agents 4,007,196), or sertraline (Zoloft®; described, e.g., in U.S. can be administered by a surgical procedure implanting Pat. 4,536,518), or alaproclate; the compound nefazo- a catheter coupled to a pump device. The pump device 5 done (Serozone®; described, e.g., in U.S. Pat. can also be implanted or be extracorporally positioned. 4,338,317). As would be recognized by a skilled person, Administration of a PDE agent, optionally in combination the effects of these agents is reflected by the effects of with one or more other neurogenic agents, can be in in- serotonin. Additional non-limiting examples of such termittent pulses or as a continuous infusion. Devices for agents include a selective norepinephrine reuptake in- injection to discrete areas of the brain are known in the 10 hibitor (SNRI) such as reboxetine (Edronax®), atomox- art. In certain embodiments, the combination is adminis- etine (Strattera®), milnacipran (described, e.g., in U.S. tered locally to the ventricle of the brain, substantia nigra, Pat. 4,478,836), sibutramine or its primary amine metab- striatum, locus ceruleous, nucleus basalis Meynert, pe- olite (BTS 54 505), amoxapine, or maprotiline; a selective dunculopontine nucleus, cerebral cortex, and/or spinal serotonin & norepinephrine reuptake inhibitor (SSNRI) cord by, e.g., injection. Methods, compositions, and de- 15 such as venlafaxine (Effexor; described, e.g., in U.S. Pat. vices for delivering therapeutics, including therapeutics 4,761,501), and its reported metabolite desvenlafaxine, for the treatment of diseases and conditions of the CNS or duloxetine (Cymbalta; described, e.g., in U.S. Pat. and PNS, are known in the art. 4,956,388); a serotonin, noradrenaline, and a dopamine [0157] In some embodiments, a PDE agent and/or oth- "triple uptake inhibitor", such as er agent(s) in a combination is modified to facilitate cross- 20 DOV 102,677 (see Popik et al. "Pharmacological Profile ing of the gut epithelium. For example, in some embod- of the "Triple" Monoamine Neurotransmitter Uptake In- iments, a PDE agent or other agent(s) is a prodrug that hibitor, DOV 102,677." Cell Mol Neurobiol. 2006 Apr 25; is actively transported across the intestinal epithelium Epub ahead of print), and metabolized into the active agent in systemic circu- DOV 216,303 (see Beer et al. "DOV 216,303, lation and/or in the CNS. 25 a "triple" reuptake inhibitor: safety, tolerability, and phar- [0158] In other embodiments, a PDE agent and/or oth- macokinetic profile." J Clin Pharmacol. 2004 (12): 44 er agent(s) of a combination is conjugated to a targeting 1360-7), domain to form a chimeric therapeutic, where the target- DOV 21,947 ((+)-1-(3,4-dichlorophenyl)-3-azabicyc- ing domain facilitates passage of the blood- brain barrier lo-(3.1.0)hexane hydrochloride), see Skolnick et al. "Anti- (as described above) and/or binds one or more molecular 30 depressant-like actions of DOV 21,947: targets in the CNS. In some embodiments, the targeting a "triple" reuptake inhibitor." Eur J Pharmacol. 2003 461 domain binds a target that is differentially expressed or (2-3):99-104), displayed on, or in close proximity to, tissues, organs, NS-2330 or tesofensine (CAS RN 402856-42-2), or NS and/or cells of interest. In some cases, the target is pref- 2359 (CAS RN 843660-54-8); erentially distributed in a neurogenic region of the brain, 35 and agents like dehydroepiandrosterone (DHEA), and such as the dentate gyrus and/or the SVZ. For example, DHEA sulfate (DHEAS), 122,721 CP- (CAS RN in some embodiments, a PDE agent and/or other agent 145742-28-5). (s) of a combination is conjugated or complexed with the [0160] Additional non-limiting examples of such fatty acid docosahexaenoic acid (DHA), which is readily agents include a tricyclic compound such as clomi- transported across the blood brain barrier and imported 40 pramine, dosulepin or dothiepin, lofepramine (described, into cells of the CNS. e.g., in 4,172,074), trimipramine, protriptyline, amitriptyl- ine, desipramine(described, e.g., in U.S. Pat. 3,454,554), Representative Conditions doxepin, imipramine, or nortriptyline; a psychostimulant such as dextroamphetamine and methylphenidate; an [0159] The disclosure includes methods for treating 45 MAO inhibitor such as selegiline (Emsam®); an ampa- depression and other neurological diseases and condi- kine such as CX516 (or Ampalex, CAS RN: tions. In some embodiments, a method may comprise 154235-83-3), CX546 (or 1-(1,4-benzodioxan-6-ylcarb- use of a combination of a PDE agent and one or more onyl)piperidine), and CX614 (CAS RN 191744-13-5) agents reported as anti-depressant agents. Thus a meth- from Cortex Pharmaceuticals; a V1b antagonist such as od may comprise treatment with a PDE agent and one 50 SSR149415 ((2S,4R)-1-[5-Chloro-1-[(2,4-dimethoxy- or more reported anti- depressant agents as known to the phenyl)sulfonyl]-3-(2-methoxy-phenyl)-2-oxo-2,3-dihy- skilled person. Non-limiting examples of such agents in- dro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2-pyrrolidine clude an SSRI (selective serotonine reuptake inhibitor), carboxamide), such as fluoxetine (Prozac®; described, e.g., in U.S. Pat. [1-(beta-mercapto-beta,beta-cyclopentamethylenepro- 4,314,081 and 4,194,009), citalopram (Celexa; de-55 pionic acid), 2-O-ethyltyrosine, 4-valine] arginine vaso- scribed, e.g., in U.S. Pat. 4,136,193), escitalopram (Lex- pressin (d(CH2)5[Tyr(Et2)]VAVP (WK 1-1), apro; described, e.g., in U.S. Pat. 4,136,193), fluvoxam- 9-desglycine[1-(beta-mercapto-beta,beta- cyclopen- ine (described, e.g., in U.S. Pat. 4,085,225) or fluvoxam- tamethylenepropionic acid), 2-O-ethyltyrosine, 4-valine]

23 45 EP 2 377 530 A2 46 arginine vasopressin desGly9d(CH2)5 [Tyr(Et2)]-VAVP (PRX-00023) for the treatment of anxiety and depres- (WK 3-6), or sion." J Med Chem. 2006 49(11):3116-35), Vestipitant 9-desglycine [1-(beta-mercapto-beta,beta- cyclopen- (or GW597599, CAS RN 334476-46-9), OPC-14523 or tamethylenepropionic acid),2-D-(O-ethyl)tyrosine, 4- VPI-013 (see Bermack et al. "Effects of the potential anti- valine ] arginine vasopressin des Gly9d(CH2)5[D-Tyr 5 depressant OPC-14523 [1-[3-[4-(3-chlorophenyl)-1-pip- (Et2)]VAVP (AO 3-21); a corticotropin-releasing factor erazinyl] propyl]- 5- methoxy- 3,4- dihydro- 2- quinolinone (CRF) R antagonist such as CP-154,526 (structure dis- monomethanesulfonate] a combined sigma and 5- HT1A closed in Schulz et al. "CP-154,526: a potent and selec- ligand: modulation of neuronal activity in the dorsal raphe tive nonpeptide antagonist of corticotropin releasing fac- nucleus." J Pharmacol Exp Ther. 2004 310(2):578-83), tor receptors." Proc Natl Acad Sci USA. 1996 93(19): 10 Casopitant or GW679769 (CAS RN 852393-14-7), Elza- 10477-82), NBI 30775 (also known as R121919 or 2,5- sonan or CP-448,187 (CAS RN 361343-19-3), dimethyl- 3-(6- dimethyl- 4- methylpyridin- 3- yl)- 7- dipro- GW823296 (see published U.S. Patent Application pylaminopyrazolo[1,5-a]pyrimidine), astressin (CAS RN US2005/0119248), Delucemine or NPS 1506 (CAS RN 170809-51-5), or a photoactivatable analog thereof as 186495-49-8), or Ocinaplon (CAS RN 96604-21-6). described in Bonk et al. "Novel high- affinity photoactivat- 15 [0163] Yet additional non-limiting examples of such able antagonists of corticotropin-releasing factor (CRF)" agents include CX717 from Cortex Pharmaceuticals, Eur. J. Biochem. 267:3017-3024 (2000), or AAG561 TGBA01AD (a serotonin reuptake inhibitor, 5-HT2 ago- (from Novartis); a melanin concentrating hormone (MCH) nist, 5-HT1A agonist, and 5-HT1D agonist) from Fabre- antagonist such as 3,5-dimethoxy-N-(1-(naphthalen-2- Kramer Pharmaceuticals, Inc., ORG 4420 (an NaSSA ylmethyl)piperidin-4-yl)benzamide or (R)-3,5-dimeth-20 (noradrenergic/specific serotonergic antidepressant) oxy-N-(1-(naphthalen-2-ylmethyl)-pyrrolidin-3-yl)benza- from Organon, CP-316,311 (a CRF1 antagonist) from mide (see Kim et al. "Identification of substituted 4-ami- Pfizer, BMS-562086 (a CRF1 antagonist) from Bristol- nopiperidines and 3-aminopyrrolidines as potent MCH- Myers Squibb, GW876008 (a CRF1 antagonist) from R1 antagonists for the treatment of obesity." Bioorg Med Neurocrine/GlaxoSmithKline, ONO-2333Ms (a CRF1 Chem Lett. 2006 Jul 29; [Epub ahead of print] for both), 25 antagonist) from Ono Pharmaceutical Co., Ltd., JNJ- or any MCH antagonist disclosed in U.S. Patent19567470 or TS-041 (a CRF1 antagonist) from Janssen 7,045,636 or published U.S. Patent Application(Johnson & Johnson) and Taisho, SSR 125543 or SSR US2005/0171098. 126374 (a CRF1 antagonist) from Sanofi-Aventis, Lu [0161] Further non-limiting examples of such agents AA21004 and Lu AA24530 (both from H. Lundbeck A/S), include a tetracyclic compound such as mirtazapine (de- 30 SEP-225289 from Sepracor Inc., ND7001 (a PDE2 in- scribed, e.g., in U.S. Pat. 4,062,848; see CAS RN hibitor) from Neuro3d, SSR 411298 or SSR 101010 (a 61337-67-5; also known as Remeron, or CAS RN fatty acid amide hydrolase, or FAAH, inhibitor) from Sa- 85650-52-8), mianserin (described, e.g., in U.S. Pat. nofi-Aventis, 163090 (a mixed serotonin receptor inhibi- 3,534,041), or setiptiline. tor) from GlaxoSmithKline, SSR 241586 (an NK2 and [0162] Further non-limiting examples of such agents 35 NK3 receptor antagonist) from Sanofi-Aventis, SAR include agomelatine (CAS RN 138112-76-2), pindolol 102279 (an NK2 receptor antagonist) from Sanofi-Avent- (CAS RN 13523-86-9), antalarmin (CAS is, RN YKP581 from SK Pharmaceuticals (Johnson & John- 157284-96-3), mifepristone (CAS RN 84371-65-3), ne- son), R1576 (a GPCR modulator) from Roche, or mifitide (CAS RN 173240-15-8) or nemifitide ditriflutate ND1251 (a PDE4 inhibitor) from Neuro3d. (CAS RN 204992-09-6), YKP- 10A or R228060 (CAS RN 40 [0164] In other embodiments, a method may comprise 561069-23-6), trazodone (CAS RN 19794-93-5), bupro- use of a combination of a PDE agent and one or more pion (CAS RN 34841-39-9 or 34911-55-2) or bupropion agents reported as anti-psychotic agents. Non-limiting hydrochloride (or Wellbutrin, CAS RN 31677-93-7) and examples of areported anti-psychotic agent as amember its reported metabolite radafaxine (CAS of RN a combination include olanzapine, quetiapine (Sero- 192374-14-4), NS2359 (CAS RN 843660-54-8), Org45 quel), clozapine (CAS RN 5786-21-0) or its metabolite 34517 (CAS RN 189035-07-2), Org 34850 (CAS RN ACP-104 (N-desmethylclozapine or norclozapine, CAS 162607-84-3), vilazodone (CAS RN 163521-12-8), CP- RN 6104-71-8), reserpine, aripiprazole, risperidone, 122,721 (CAS RN 145742-28-5), gepirone (CAS RN ziprasidone, sertindole, trazodone, paliperidone (CAS 83928-76-1), SR58611 (see Mizuno et al. "The stimula- RN 144598-75-4), mifepristone (CAS RN 84371-65-3), tion of beta(3)-adrenoceptor causes phosphorylation of 50 bifeprunox or DU-127090 (CAS RN 350992-10-8), extracellular signal-regulated kinases 1 and 2 through a asenapine or ORG 5222 (CAS RN 65576-45-6), iloperi- G(s)- but not G(i)-dependent pathway in 3T3-L1 adi- done (CAS RN 133454-47-4), ocaperidone (CAS RN pocytes." Eur J Pharmacol. 2000 404 (1-2):63-8), saredu- 129029-23-8), SLV 308 (CAS RN 269718-83-4), licar- tant or SR 48968 (CAS RN 142001-63-6), PRX-00023 bazepine or GP 47779 (CAS RN 29331-92-8), Org 34517 (N- {3-[4-(4- cyclohexylmethanesulfonylaminobutyl) pip- 55 (CAS RN 189035-07-2), ORG 34850 (CAS RN erazin-1-yl]phenyl}acetamide, see Becker et al. "An in- 162607-84-3), Org 24448 (CAS RN 211735-76-1), lur- tegrated in silico 3D model-driven discovery of a novel, asidone (CAS RN 367514-87-2), blonanserin or lonasen potent, and selective amidosulfonamide S-HT1A agonist (CAS RN 132810-10-7), Talnetant or SB-223412 (CAS

24 47 EP 2 377 530 A2 48

RN 174636-32-9), secretin (CAS RN 1393-25-5) or hu- includevarious diet pills that are commercially or clinically man secretin (CAS RN 108153-74-8) which are endog- available. In some embodiments, the reported agent is enous pancreatic hormones, ABT 089 (CAS RNorlistat (CAS RN 96829-58-2), sibutramine (CAS RN 161417-03-4), SSR 504734 (see compound 13 in Hash- 106650-56-0) or sibutramine hydrochloride (CAS RN imoto "Glycine Transporter Inhibitors as Therapeutic5 84485-00-7), phetermine (CAS RN 122-09-8) or pheter- Agents for Schizophrenia." Recent Patents on CNS Drug mine hydrochloride (CAS RN 1197-21-3), diethylpropion Discovery, 2006 1:43-53), MEM 3454 (see Mazurov et or amfepramone (CAS RN 90-84-6) or diethylpropion hy- al. "Selective alpha7 nicotinic acetylcholine receptor lig- drochloride, benzphetamine (CAS RN 156-08-1) or ben- ands." Curr Med Chem. 2006 13(13):1567-84), a phos- zphetamine hydrochloride, phendimetrazine (CAS RN phodiesterase 10A (PDE10A) inhibitor such as papaver- 10 634-03-7 or 21784-30-5) or phendimetrazine hydrochlo- ine (CAS RN 58-74-2) or papaverine hydrochloride (CAS ride (CAS RN 17140-98-6) or phendimetrazine tartrate, RN 61-25-6), paliperidone (CAS RN 144598-75-4), trif- rimonabant (CAS RN 168273-06-1), bupropion hydro- luoperazine (CAS RN 117-89-5), or trifluoperazine hy- chloride (CAS RN: 31677-93-7), topiramate (CAS RN drochloride (CAS RN 440-17-5). 97240-79-4), zonisamide (CAS RN 68291-97-4), or [0165] Additional non-limiting examples of such15 APD-356 (CAS RN 846589-98-8). agents include trifluoperazine, fluphenazine, chlorpro- [0169] In other non-limiting embodiments, the agent mazine, perphenazine, thioridazine, haloperidol, loxap- may be fenfluramine or Pondimin (CAS RN 458-24-2), ine, mesoridazine, molindone, pimoxide, or thiothixene, dexfenfluramine or Redux (CAS RN 3239-44-9), or lev- SSR 146977 (see Emonds-Alt et al. "Biochemical and ofenfluramine (CAS RN 37577-24-5); or a combination pharmacological activities of SSR 146977, a new potent 20 thereof or a combination with phentermine. Non-limiting nonpeptide tachykinin NK3 receptor antagonist." Can J examples include a combination of fenfluramine and Physiol Pharmacol. 2002 80(5):482-8), SSR181507 ((3- phentermine (or "fen-phen") and of dexfenfluramine and exo)-8-benzoyl-N-[[(2 s)7-chloro-2,3-dihydro-1,4-ben- phentermine (or "dexfen-phen"). zodioxin- 1- yl] methyl]- 8- azabicyclo [3.2.1] octane- 3- [0170] The combination therapy may be of one of the methanamine monohydrochloride), or SLV313 (1-(2,3- 25 above with a PDE agent as described herein to improve dihydro-benzo[1,4]dioxin-5-yl)-4-[5-(4-fluorophenyl)-py- the condition of the subject or patient. Non-limiting ex- ridin-3-ylmethyl]-piperazine). amples of combination therapy include the use of lower [0166] Further non-limiting examples of such agents dosages of the above additional agents, or combinations include Lu-35-138 (a D4/5-HT antagonist) from Lund- thereof, which reduce side effects of the agent or com- beck, AVE 1625 (a CB1 antagonist) from Sanofi-Aventis, 30 bination when used alone. For example, an anti-depres- SLV 310,313 (a 5-HT2A antagonist) from Solvay, SSR sant agent like fluoxetine or paroxetine or sertraline may 181507 (a D2/5-HT2 antagonist) from Sanofi-Aventis, be administered at a reduced or limited dose, optionally GW07034 (a 5-HT6 antagonist) or GW773812 (a D2, 5- also reduced in frequency of administration, in combina- HT antagonist) from GlaxoSmithKline, YKP 1538 from tion with a PDE agent. SK Pharmaceuticals, SSR 125047 (a sigma receptor an- 35 [0171] Similarly, a combination of fenfluramine and tagonist) from Sanofi-Aventis, MEM1003 (a L- type calci- phentermine, or phentermine and dexfenfluramine, may um channel modulator) from Memory Pharmaceuticals, be administered at a reduced or limited dose, optionally JNJ-17305600 (a GLYT1 inhibitor) from Johnson & John- also reduced in frequency of administration, in combina- son, XY 2401 (a glycine site specific NMDA modulator) tion with a PDE agent. The reduced dose or frequency from Xytis, PNU 170413 from Pfizer, RGH- 188 (a D2, D3 40 may be that which reduces or eliminates the side effects antagonist) from Forrest, SSR 180711 (an alpha7 nico- of the combination. tinic acetylcholine receptor partial agonist) or SSR [0172] In light of the positive recitation (above and be- 103800 (a GLYT1 (Type 1 glycine transporter) inhibitor) low) of combinations with alternative agents to treat con- or SSR 241586 (a NK3 antagonist) from Sanofi-Aventis. ditions disclosed herein, the disclosure includes embod- [0167] In other disclosed embodiments, a reported an- 45 iments with the explicit exclusion of one or more of the ti-psychotic agent may be one used in treating schizo- alternative agents or one or more types of alternative phrenia. Non-limiting examples of a reported anti-schiz- agents. As would be recognized by the skilled person, a ophrenia agent as a member of a combination with a description of the whole of a plurality of alternative agents PDE agent include molindone hydrochloride (MOBAN®) (or classes of agents) necessarily includes and describes and TC-1827 (see Bohme et al. "In vitro and in vivo char- 50 subsets of the possible alternatives, such as the part re- acterization of TC-1827, a novel brain α4β2 nicotinic re- maining with the exclusion of one or more of the alterna- ceptor agonist with pro-cognitive activity." Drug Devel- tives or exclusion of one or more classes. opment Research 2004 62(1):26-40). [0168] In some embodiments, a method may comprise Representative Combinations use of a combination of a PDE agent and one or more 55 agents reported for treating weight gain, metabolic syn- [0173] As indicated herein, the disclosure includes drome, or obesity, and/or to induce weight loss or prevent combination therapy, where a PDE agent in combination weight gain. Non- limiting examples of the reported agent with one or more other neurogenic agents is used to pro-

25 49 EP 2 377 530 A2 50 duce neurogenesis. When administered as a combina- receptor subtypes, identified as OP 1, OP 2, and OP 3 (also tion, the therapeutic compounds can be formulated as know as delta, or δ, kappa, or κ, and mu, or P, respec- separate compositions that are administered at the same tively). Thus an opioid that antagonizes any two, or all time or sequentially at different times, or the therapeutic three, of these subtypes, or an inverse agonist that is compounds can be given as a single composition. The 5 specific or selective for any two or all three of these sub- methods of the disclosure are not limited in the sequence types, may be used as the neurogenic agent in the prac- of administration. tice. Alternatively, an antagonist or inverse agonist may [0174] Instead, the disclosure includes methods be specific or selective for one of the three subtypes, wherein treatment with a PDE agent and another neuro- such as the kappa subtype as a non-limiting example. genic agent occurs over a period of more than about 48 10 [0177] Non-limiting examples of reported opioid antag- hours, more than about 72 hours, more than about 96 onists include naltrindol, naloxone,naloxene, naltrexone, hours, more than about 120 hours, more than about 144 JDTic (Registry Number 785835-79-2; also known as 3- hours, more than about 7 days, more than about 9 days, isoquinolinecarboxamide, 1,2,3,4-tetrahydro-7-hydroxy- more than about 11 days, more than about 14 days, more N-[(1S)-1-[[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl- than about 21 days, more than about 28 days, more than 15 1- piperidinyl] methyl]- 2- methylpropyl]-dihydrochloride, about 35 days, more than about 42 days, more than about (3R)-(9CI)), nor-binaltorphimine, and buprenorphine. In 49 days, more than about 56 days, more than about 63 some embodiments, a reported selective kappa opioid days, more than about 70 days, more than about 77 days, receptor antagonist compound, as described in US more than about 12 weeks, more than about 16 weeks, 20020132828, U.S. Patent 6,559,159, and/or WO more than about 20 weeks, or more than about 24 weeks 20 2002/053533, may be used. All three of these documents or more. In some embodiments, treatment by adminis- are herein incorporated by reference in their entireties tering a PDE agent, occurs at least about 12 hours, such as if fully set forth. Further non- limiting examples of such as at least about 24, or at least about 36 hours, before reported antagonists is a compound disclosed in U.S. administration of another neurogenic agent. Following Patent 6,900,228 (herein incorporated by reference in its administration of a PDE agent, further administrations 25 entirety), arodyn (Ac[Phe(1,2,3),Arg(4),d-Ala(8)]Dyn may be of only the other neurogenic agent in some em- A-(1-11)NH(2), as described in Bennett, et al. (2002) J. bodiments of the disclosure. In other embodiments, fur- Med. Chem. 45:5617-5619), and an active analog of ar- ther administrations may be of only the PDE agent. odyn as described in Bennett e al. (2005) J Pept Res. 65 [0175] In some cases, combination therapy with a PDE (3):322-32, alvimopan. agent and one or more additional agents results in a en- 30 [0178] In some embodiments, the neurogenic agent hanced efficacy, safety, therapeutic index, and/or toler- used in the methods described herein has "selective" ac- ability, and/or reduced side effects (frequency, severity, tivity (such as in the case of an antagonist or inverse or other aspects), dosage levels, dosage frequency, agonist) under certain conditions against one or more and/or treatment duration. Examples of compounds use- opioid receptor subtypes with respect to the degree ful in combinations described herein are provided above 35 and/or nature of activity against one or more other opioid and below. Structures, synthetic processes, safety pro- receptor subtypes. For example, in some embodiments, files, biological activity data, methods for determining bi- the neurogenic agent has an antagonist effect against ological activity, pharmaceutical preparations, and meth- one or more subtypes, and a much weaker effect or sub- ods of administration relating to the compounds are stantially no effect against other subtypes. As another known in the art and/or provided in the cited references, 40 example, an additional neurogenic agent used in the all of which are herein incorporated by reference in their methods described herein may act as an agonist at one entirety. Dosages of compounds administered in combi- or more opioid receptor subtypes and as antagonist at nation with a PDE agent can be, e.g., a dosage within one or more other opioid receptor subtypes. In some em- the range of pharmacological dosages established in hu- bodiments, a neurogenic agent has activity against kap- mans, or a dosage that is a fraction of the established 45 pa opioid receptors, while having substantially lesser ac- human dosage, e.g., 70%, 50%, 30%, 10%, or less than tivity against one or both of the delta and mu receptor the established human dosage. subtypes. In other embodiments, a neurogenic agent has [0176] In some embodiments, the neurogenic agent activity against two opioid receptor subtypes, such as the combined with a PDE agent may be a reported opioid or kappa and delta subtypes. As non-limiting examples, the non-opioid (acts independently of an opioid receptor) 50 agents naloxone and naltrexone have nonselective an- agent. In some embodiments, the neurogenic agent is tagonist activities against more than one opioid receptor one reported as antagonizing one or more opioid recep- subtypes. In certain embodiments, selective activity of tors or as an inverse agonist of at least one opioid recep- one or more opioid antagonists results in enhanced effi- tor. A opioid receptor antagonist or inverse agonist may cacy, fewer side effects, lower effective dosages, less be specific or selective (or alternatively non-specific or 55 frequent dosing, or other desirable attributes. non-selective) for opioid receptor subtypes. So an antag- [0179] An opioid receptor antagonist is an agent able onist may be non-specific or non-selective such that it to inhibit one or more characteristic responses of an opi- antagonizes more than one of the three known opioid oid receptor or receptor subtype. As a non-limiting ex-

26 51 EP 2 377 530 A2 52 ample, an antagonist may competitively or non- compet- Pharmacol Ther. 2002 93(2-3):225-32); or a substituted itively bind to an opioid receptor, an agonist or partial isoquinolinesulfonamide compound as disclosed in U.S. agonist (or other ligand) of a receptor, and/or a down- Patent 6,906,061. stream signaling molecule to inhibit a receptor’s function. [0184] Furthermore, the neurogenic agent in combina- [0180] An inverse agonist able to block or inhibit a con- 5 tion with a PDE agent may be a reported GSK- 3 inhibitor stitutive activity of an opioid receptor may also be used. or modulator. In some non- limiting embodiments, the re- An inverse agonist may competitively or non-competi- ported GSK3-beta modulator is a paullone, such as al- tively bind to an opioid receptor and/or a downstream sterpaullone, kenpaullone (9-bromo-7,12-dihydroindolo signaling molecule to inhibit a receptor’s function. Non- [3,2-d][1]benzazepin-6(5H)-one), gwennpaullone (see limiting examples of inverse agonists for use in the dis- 10 Knockaert et al. "Intracellular Targets of Paullones. Iden- closed methods include ICI-174864 ( N,N-diallyl-Tyr-Aib- tification following affinity purification on immobilized in- Aib-Phe-Leu), RTI-5989-1, RTI-5989-23, and RTI- hibitor." J Biol Chem. 2002 277(28):25493-501), azak- 5989-25 (see Zaki et al. J. Pharmacol. Exp. Therap. 298 enpaullone (see Kunick et al. "1- Azakenpaullone is a se- (3): 1015-1020, 2001). lective inhibitor of glycogen synthase kinase-3 beta." [0181] Additional embodiments of the disclosure in- 15 Bioorg Med Chem Lett. 2004 14(2):413-6), or the com- clude a combination of a PDE agent with an additional pounds described in U.S. Publication No. 20030181439; agent such as acetylcholine or a reported modulator of International Publication No. WO 01/60374; Leost et al., an androgen receptor. Non- limiting examples include the Eur. J. Biochem. 267:5983-5994 (2000); Kunick et al., J androgen receptor agonists ehydroepiandrosterone Med Chem.; 47 (1): 22-36 (2004); or Shultz et al., J. Med. (DHEA) and DHEA sulfate (DHEAS). 20 Chem. 42:2909-2919 (1999); an anticonvulsant, such as [0182] Alternatively, the neurogenic agent in combina- lithium or a derivative thereof (e.g., a compound de- tion with a PDE agent may be an enzymatic inhibitor, scribed in U.S. Patent Nos. 1,873,732; 3,814,812; and such as a reported inhibitor of HMG CoA reductase. Non- 4,301,176); valproic acid or a derivative thereof (e.g., val- limiting examples of such inhibitors include atorvastatin proate, or a compound described in Werstuck et al., (CAS RN 134523-00-5), cerivastatin (CAS 25 RNBioorg Med Chem Lett., 14(22): 5465-7 (2004)); lamot- 145599-86-6), crilvastatin (CAS RN 120551-59-9), fluv- rigine; SL/76002 (Progabide), Gabapentin; tiagabine; or astatin (CAS RN 93957-54-1) and fluvastatin sodium vigabatrin; a maleimide or a related compound, such as (CAS RN 93957-55-2), simvastatin (CAS Ro RN 31-8220, SB-216763, SB-410111, SB-495052, or 79902-63-9), lovastatin (CAS RN 75330-75-5), pravas- SB-415286, or a compound described, e.g., in U.S. Pat. tatin (CAS RN 81093-37-0) or pravastatin sodium, rosu- 30 No. 6,719,520; U.S. Publication No. 20040010031; In- vastatin (CAS RN 287714-41-4), and simvastatin (CAS ternational Publication Nos. WO-2004072062; WO- RN 79902-63-9). Formulations containing one or more 03082859; WO-03104222; WO-03103663, WO- of such inhibitors may also be used in a combination. 03095452, WO-2005000836; WO 0021927; WO- Non-limiting examples include formulations comprising 03076398; WO-00021927; WO-00038675; or WO- lovastatin such as Advicor (an extended-release, niacin 35 03076442; or Coghlan et al., Chemistry & Biology 7: 793 containing formulation) or Altocor (an extended release (2000); a pyridine or pyrimidine derivative, or a related formulation); and formulations comprising simvastatin compound (such as 5-iodotubercidin, GI 179186X, GW such as Vytorin (combination of simvastatin and784752X and GW 784775X, and compounds described, ezetimibe). e.g., in U.S. Pat. Nos. 6489344; 6417185; and 6153618; [0183] In other non-limiting embodiments, the neuro- 40 U.S. Publication Nos. 20050171094; and 20030130289; genic agent in combination with a PDE agent may be a European Patent Nos. EP- 01454908, EP-01454910,EP- reported Rho kinase inhibitor. Non-limiting examples of 01295884, EP-01295885; and EP -01460076; EP- such an inhibitor include fasudil (CAS RN 103745-39-7); 01454900; International Publication Nos. WO 01/70683; fasudil hydrochloride (CAS RN 105628-07-7); the me- WO 01/70729; WO 01/70728; WO 01/70727; WO tabolite of fasudil, which is hydroxyfasudil 45 (see01/70726; WO 01/70725; WO-00218385; WO- Shimokawa et al. "Rho-kinase-mediated pathway induc- 00218386; WO-03072579; WO-03072580; WO- es enhanced myosin light chain phosphorylations in a 03027115; WO-03027116; WO-2004078760; WO- swine model of coronary artery spasm." Cardiovasc Res. 2005037800, WO-2004026881, WO-03076437, WO- 1999 43:1029-1039), Y 27632 (CAS RN 138381-45-0); 03029223; WO-2004098607; WO-2005026155; WO- a fasudil analog thereof such as (S)-Hexahydro-1-(4- 50 2005026159; WO-2005025567; WO-03070730 ; WO- ethenylisoquinoline- 5- sulfonyl)- 2- methyl- 1H- 1,4- di- 03070729; WO-2005019218; WO-2005019219; WO- azepine, (S)-hexahydro-4-glycyl-2-methyl-1-(4-methyl- 2004013140; WO-2004080977; WO-2004026229, WO- isoquinoline-5-sulfonyl)-1H-1,4-diazepine, or (S)-(+)-2- 2004022561; WO-03080616; WO-03080609; WO- methyl- 1-[(4- methyl- 5- isoquinoline) sulfonyl]-homopi- 03051847; WO-2004009602; WO-2004009596; WO- perazine (also known as H-1152P; see Sasaki et al. "The 55 2004009597; WO-03045949; WO-03068773; WO- novel and specific Rho- kinase inhibitor (S)-(+)- 2-methyl- 03080617; WO 99/65897; WO 00/18758; WO0307073; 1-[(4- methyl- 5- isoquinoline) sulfonyl]-homopiperazine WO-00220495; WO-2004043953, WO-2004056368, as a probing molecule for Rho- kinase-involved pathway." WO-2005012298, WO-2005012262, WO-2005042525,

27 53 EP 2 377 530 A2 54

WO-2005005438, WO-2004009562, WO-03037877; 3R)-1-aminocyclopentane-1,3-dicarboxylic acid WO-03037869; WO-03037891; WO-05012307; WO- (ACPD), a broad spectrum mGlu agonist having substan- 05012304 and WO 98/16528; and in Massillon et al., Bi- tial activity at Group I and II receptors; (ii) (-)-2-thia-4- ochem J 299:123-8 (1994)); a pyrazine derivative, such aminobicyclo-hexane-4,6-dicarboxylate (LY389795), as Aloisine A (7-n-Butyl-6-(4-hydroxyphenyl)[5H]pyrrolo 5 which is described in Monn et al., J. Med. Chem., 42 (6): [2,3-b]pyrazine) or a compound described in Internation- 1027-40 (1999); (iii) compounds described in US App. al Publication Nos. WO-00144206; WO014424 or WO- No. 20040102521 and Pellicciari et al., J. Med. Chem., 2005035532; a thiadiazole or thiazole, such as TDZD-8 39, 2259-2269 (1996); and (iv) the Group II-specific mod- (Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione); OT- ulators described below. DZT (4-Dibenzyl-5-oxothiadiazolidine-3-thione); or a re- 10 [0186] Non-limiting examples of reported Group II an- lated compound described, e.g., in U.S. Patent Nos. tagonists include: (i) phenylglycine analogues, such as 6645990 or 6762179; U.S. Publication (RS)-alpha- No. methyl-4-sulphonophenylglycine (MSPG), 20010039275; International Publication Nos. WO (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG), 01/56567, WO-03011843, WO-03004478, or WO- and (RS)-alpha-methyl-4-tetrazolylphenylglycine (MT- 03089419; or Mettey, Y., et al., J. Med. Chem. 46, 222 15 PG), described in Jane et al., Neuropharmacology 34: (2003); TWS119 or a related compound, such as a com- 851-856 (1995); (ii) LY366457, which is described in pound described in Ding et al., Proc Natl Acad Sci USA., O’Neill et al., Neuropharmacol., 45 (5): 565-74 (2003); (iii) 100(13): 7632-7 (2003); an indole derivative, such as a compounds described in US App Nos. 20050049243, compound described in International Publication Nos. 20050119345 and 20030157647; and (iv) the Group II- WO-03053330, WO-03053444, WO-03055877, WO-20 specific modulators described below. 03055492, WO-03082853, or WO-2005027823; a pyra- [0187] In some non-limiting embodiments, the report- zine or pyrazole derivative, such as a compound de- ed Group II modulator is a Group II- selective modulator, scribed in U.S. Patent Nos. 6727251, 6696452, 6664247, capable of modulating mGlu2 and/or mGlu3 under con- 666073, 6656939, 6653301, 6653300, 6638926,ditions where it is substantially inactive at other mGlu 6613776, or 6610677; or International Publication Nos. 25 subtypes (of Groups I and III). Examples of Group II- WO-2005002552, WO-2005002576, or WO-selective modulators include compounds described in 2005012256; a compound described in U.S. Pat. Nos. Monn, et al., J. Med. Chem., 40, 528-537 (1997); Sch- 6719520; 6,498,176; 6,800,632; or 6,872,737; U.S. Pub- oepp, et al., Neuropharmacol., 36, 1-11 (1997) (e.g., 1S, lication Nos. 20050137201; 20050176713;2S, SR, 6S- 2- aminobicyclohexane- 2,6- dicarboxylate); 20050004125; 20040010031; 20030105075;30 and Schoepp, Neurochem. Int., 24, 439 (1994). 20030008866; 20010044436; 20040138273; or[0188] Non-limiting examples of reported Group II- se- 20040214928; International Publication Nos. WO lective agonists include (i) (+)-2-aminobicyclohexane- 99/21859; WO-00210158; WO-05051919; WO-2,6-dicarboxylic acid (LY354740), which is described in 00232896; WO-2004046117; WO-2004106343; WO- Johnson et al., Drug Metab. Disposition, 30(1): 27-33 00210141; WO-00218346; WO 00/21927; 35 WO (2002) and Bond et al., NeuroReport 8: 1463-1466 01/81345; WO 01/74771; WO 05/028475; WO 01/09106; (1997),and issystemically active afteroral administration WO 00/21927; WO01/41768; WO 00/17184; WO (e.g., Grillon et al., Psychopharmacol. (Berl), 168: 04/037791; WO-04065370; WO 01/37819; WO446-454 (2003)); (ii) (-)-2-Oxa-4-aminobicyclohexane- 01/42224; WO 01/85685; WO 04/072063; WO-4,6-dicarboxylic acid (LY379268), which is described in 2004085439; WO-2005000303; WO-2005000304; or 40 Monn et al., J. Med. Chem. 42: 1027-1040 (1999) and WO 99/47522; or Naerum, L., et al., Bioorg. Med. Chem. US Pat. No. 5,688,826. LY379268 is readily permeable Lett. 12, 1525 (2002); CP-79049, GI 179186X, GW across the blood-brain barrier, and has EC50 values in 784752X, GW 784775X, AZD-1080, AR-014418, SN- the low nanomolar range (e.g., below about 10 nM, or 8914, SN-3728, OTDZT, Aloisine A, TWS119,below about 5 nM) against human mGlu 2 and mGlu3 re- CHIR98023, CHIR99021, CHIR98014, CHIR98023, 5- 45 ceptors in vitro; (iii) (2R,4R)-4-aminopyrrolidine-2,4-di- iodotubercidin, Ro 31-8220, SB-216763, SB-410111, carboxylate ((2R,4R)-APDC), which is described in Monn SB-495052, SB-415286, alsterpaullone, kenpaullone, et al., J. Med. Chem. 39: 2990 (1996) and Schoepp et gwennpaullone, LY294002, wortmannin, sildenafil, al., Neuropharmacology, 38: 1431 (1999); (iv) (1S,3S)- CT98014, CT-99025, flavoperidol, or L803-mts. 1-aminocyclopentane-1,3-dicarboxylic acid ((1S, [0185] In yet further embodiments, the neurogenic50 3S)-ACPD), described in Schoepp, Neurochem. Int., 24: agent used in combination with a PDE agent may be a 439 (1994); (v) (2R, 4R)-4-aminopyrrolidine-2,4-dicarbo- reported glutamate modulator or metabotropic glutamate xylic acid ((2R,4R)-APDC), described in Howson and (mGlu) receptor modulator. In some embodiments, the Jane, British Journal of Pharmacology, 139, 147-155 reported mGlu receptor modulator is a Group II modula- (2003); (vi) (25,1’S,2’S)-2-(carboxycyclopropyl)-glycine tor, having activity against one or more Group II receptors 55 (L-CCG-I), described in Brabet et al., Neuropharmacol- (mGlu2 and/or mGlu3). Embodiments include those ogy 37: 1043-1051 (1998); (vii) (2S,2’R,3R)-2-(2’,3’-di- where the Group II modulator is a Group II agonist. Non- carboxycyclopropyl)glycine (DCG-IV), described in Ha- limiting examples of Group II agonists include: (i) (1S, yashi et al., Nature, 366, 687-690 (1993); (viii) 1S, 2S,5R,

28 55 EP 2 377 530 A2 56

6S-2-aminobicyclohexane-2,6-dicarboxylate, described non-limiting examples of subtype-selective modulators in Monn, et al., J. Med. Chem., 40, 528 (1997) and Sch- include allosteric mGlu receptor modulators (mGlu 2 and oepp, et al., Neuropharmacol., 36, 1 (1997); and (vii) mGlu3) and NAAG-related compounds (mGlu 3), such as compounds described in US App. No. 20040002478; US those described below. Pat. Nos. 6,204,292, 6,333,428, 5,750,566 5 and[0192] In other non-limiting embodiments, a reported 6,498,180; and Bond et al., Neuroreport 8: 1463-1466 Group II modulator is a compound with activity at Group (1997). I and/or Group III receptors, in addition to Group II recep- [0189] Non-limiting examples of reported Group II- se- tors, while having selectivity with respect to one or more lective antagonists useful in methods provided herein in- mGlu receptor subtypes. Non-limiting examples of such clude the competitive antagonist (2S)-2-amino-2-(1S, 10 compounds include: (i) (2S,3S,4S)-2-(carboxycyclopro- 2S-2-carboxycycloprop-1-yl)-3-(xanth-9-yl) propanoic pyl)glycine (L-CCG-1) (Group I/Group II agonist), which acid (LY341495), which is described, e.g., in Kingston et is described in Nicoletti et al., Trends Neurosci. 19: al., Neuropharmacology 37: 1-12 (1998) and Monn et al., 267-271 (1996), Nakagawa, et al., Eur. J. Pharmacol. J Med Chem 42: 1027-1040 (1999). LY341495 is readily 184, 205 (1990), Hayashi, et al., Br. J. Pharmacol., 107, 15 permeably across the blood-brain barrier, and has IC50 539 (1992), and Schoepp et al., J. Neurochem., 63., page values in the low nanomolar range (e.g., below about 10 769-772 (1994); (ii) (S)-4-carboxy-3-hydroxyphenylgly- nM, or below about 5 nM) against cloned human mGlu 2 cine (4C3HPG) (Group II agonist/ Group I competitive an- and mGlu3 receptors. LY341495 has a high degree of tagonist); (iii) gamma-carboxy-L-glutamic acid (GLA) selectivity for Group II receptors relative to Group I and (Group II antagonist/ Group III partial agonist/ antagonist); Group III receptors at low concentrations (e.g., nanomo- 20 (iv) (2S,2’R,3’R)-2-(2,3-dicarboxycyclopropyl)glycine lar range), whereas at higher concentrations (e.g., above (DCG-IV) (Group II agonist/Group III antagonist), which 1PM), LY341495 also has antagonist activity against is described in Ohfune et al, Bioorg. Med. Chem, Lett., mGlu7 and mGlu8, in addition to mGlu2/3. LY341495 is 3: 15 (1993); (v) (RS)-a-methyl-4-carboxyphenylglycine substantially inactive against KA, AMPA, and NMDA iGlu (MCPG) (Group I/ Group II competitive antagonist), which receptors. 25 is described in Eaton et al., Eur. J. Pharmacol., 244: 195 [0190] Additional non-limiting examples of reported (1993), Collingridge and Watkins, TiPS, 15: 333 (1994), Group II-selective antagonists include the following com- and Joly et al., J. Neurosci., 15: 3970 (1995); and (vi) the pounds, indicated by chemical name and/or described in Group II/III modulators described in US Pat Nos. the cited references: (i) α-methyl-L-(carboxycyclopropyl) 5,916,920, 5,688,826, 5,945,417, 5,958,960, 6,143,783, glycine (CCG); (ii) (2S,3S,4S)-2-methyl-2-(carboxycy- 30 6,268,507, 6,284,785. clopropyl) glycine (MCCG); (iii) (1R,2R,3R,5R,6R)-2- [0193] In some non-limiting embodiments, the report- amino-3-(3,4-dichlorobenzyloxy)-6 fluorobicyclohex- ed mGlu receptor modulator comprises (S)-MCPG (the ane-2,6-dicarboxylic acid (MGS0039), which is de- active isomer of the Group I/Group II competitive antag- scribed in Nakazato et al., J. Med. Chem.,(18): 47 onist (RS)-MCPG) substantially free from (R)-MCPG. 4570-87 (2004); (iv) an n- hexyl, n-heptyl, n-octyl, 5-meth- 35 (S)-MCPG is described, e.g., in Sekiyama et al., Br. J. ylbutyl, or 6- methylpentyl ester prodrug of MGS0039; (v) Pharmacol., 117: 1493 (1996) and Collingridge and Wat- MGS0210 (3-(3,4-dichlorobenzyloxy)-2-amino-6-fluoro- kins, TiPS, 15: 333 (1994). bicyclohexane-2,6-dicarboxylic acid n-heptyl ester); (vi) [0194] Additional non-limiting examples of reported (RS)-1-amino-5-phosphonoindan-1-carboxylic acid mGlu modulators useful in methods disclosed herein in- (APICA), which is described in Ma et al., Bioorg. Med. 40 clude compounds described in US Pat Nos. 6,956,049, Chem. Lett., 7: 1195 (1997); (vii) (2S)-ethylglutamic acid 6,825,211, 5,473,077, 5,912,248,6,054,448, and (EGLU), which is described in Thomas et al., Br. J. Phar- 5,500,420; US App Nos. 20040077599, 20040147482, macol. 117: 70P (1996); (viii) (2S,1’S, 2’S,3’R)-2-(2’-car- 20040102521, 20030199533 and 20050234048; and Intl boxy-3’-phenylcyclopropyl)glycine (PCCG-IV); and (ix) Pub/App Nos. WO 97/19049, WO 98/00391, and compounds described in US Pat No. 6,107,342 and US 45 EP0870760. App No. 20040006114. APICA has an IC50 value of ap- [0195] In some non-limiting embodiments, the report- proximately 30 PM against mGluR 2 and mGluR 3, with no ed mGlu receptor modulator is a prodrug, metabolite, or appreciable activity against Group I or Group III receptors other derivative of N- Acetylaspartylglutamate (NAAG), a at sub-mM concentrations. peptide neurotransmitter in the mammalian CNS that is 50 [0191] In some non-limiting embodiments, a reported a highly selective agonist for mGluR3 receptors, as de- Group II-selective modulator is a subtype-selective mod- scribed in Wroblewska et al., J. Neurochem., (1): 69 ulator, capable of modulating the activity of mGlu 2 under 174-181 (1997). In other embodiments, the mGlu mod- conditions in which it is substantially inactive at mGlu3 ulator is a compound that modulates the levels of endog- (mGlu2-selective), or vice versa (mGlu 3-selective). Non- enous NAAG, such as an inhibitor of the enzyme N- limiting examples of subtype-selective modulators in- 55 acetylated-alpha-linked-acidic dipeptidase (NAALA- clude compounds described in US Pat Nos. 6,376,532 Dase), which catalyzes the hydrolysis of NAAG to N- (mGlu2-selective agonists) and US App acetyl- No. aspartate and glutamate. Examples of NAALA- 20040002478 (mGlu3-selective agonists). Additional Dase inhibitors include 2-PMPA (2-(phosphonomethyl)

29 57 EP 2 377 530 A2 58 pentanedioic acid), which is described in Slusher et al., 40,20050026963,20050020585,and 20040259917. Nat. Med., 5(12): 1396-402 (1999); and compounds de- [0199] Non-limiting examples of compounds reported scribed in J. Med. Chem. 39: 619 (1996), US Pub. No. to modulate Group III receptors include (i) the Group III- 20040002478, and US Pat Nos. 6,313,159, 6,479,470, selective agonists (L)-2-amino-4-phosphonobutyric acid and 6,528,499. In some embodiments, the mGlu modu- 5 (L-AP4), described in Knopfel et al., J. Med Chem., 38, lator is the mGlu3-selective antagonist, beta-NAAG. 1417-1426 (1995); and 2-Amino- (S)- 2-methyl-4- [0196] Additional non-limiting examples of reported phosphonobutanoic acid; (ii) the Group III-selective an- glutamate modulators include memantine (CAS RN tagonists (RS)-α-Cyclopropyl-4-phosphonophenylgly- 19982-08-2), memantine hydrochloride (CAS RN cine; (RS)-α-Methylserine-O-phosphate (MSOP); and 41100-52-1), and riluzole (CAS RN 1744-22-5). 10 compounds described in US App. No. 20030109504; and [0197] In some non-limiting embodiments, a reported (iii) (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxyl- Group II modulator is administered in combination with ic acid (ACPT-I). one or more additional compounds reported as active [0200] In additional embodiments, the neurogenic against a Group I and/or a Group III mGlu receptor. For agent used in combination with a PDE agent may be a example, in some cases, methods comprise modulating 15 reported AMPA modulator. Non-limiting examples in- the activity of at least one Group I receptor and at least clude CX-516 or ampalex (CAS RN 154235-83-3), Org- one Group II mGlu receptor (e.g., with a compound de- 24448 (CAS RN 211735-76-1), LY451395 (2-pro- scribed herein). Examples of compounds useful in mod- panesulfonamide, N-[(2R)-2-[4’-[2-[methylsulfonyl)ami- ulating the activity of Group I receptors include Group I- no]ethyl][1,1’-biphenyl]-4-yl]propyl]-), LY-450108 (see selective agonists, such as (i) trans- azetidine-2,4,-dicar- 20 Jhee et al. "Multiple-dose plasma pharmacokinetic and boxylic acid (tADA), which is described in Kozikowski et safety study of LY450108 and LY451395 (AMPA recep- al., J. Med. Chem., 36: 2706 (1993) and Manahan- tor potentiators) and their concentration in cerebrospinal Vaughan et al., Neuroscience 72: 999 (1996); (ii) (RS)- fluid in healthy human subjects." J Clin Pharmacol. 2006 3,5-Dihydroxyphenylglycine (DHPG), which is described 46(4):424-32), and CX717. Additional examples of re- in Ito et al., NeuroReport 3: 1013 (1992); or a composition 25 ported antagonists include irampanel (CAS RN comprising (S)-DHPG substantially free of (R)-DHPG, as 206260-33-5) and E-2007. described, e.g., in Baker et al., Bioorg.Med.Chem.Lett. [0201] Further non-limiting examples of reported AM- 5: 223 (1995); (iii) (RS)-3-Hydroxyphenylglycine, which PA receptor antagonists for use in combinations include is described in Birse et al., Neuroscience 52: 481 (1993); YM90K (CAS RN 154164-30-4), YM872 or Zonampanel or a composition comprising (S)- 3-Hydroxyphenylgly- 30 (CAS RN 210245-80-0), NBQX (or 2,3-Dioxo-6-nitro-7- cine substantially free of (R)- 3-Hydroxyphenylglycine, sulfamoylbenzo(f)quinoxaline; CAS RN 118876-58-7), as described, e.g., in Hayashi et al., J.Neurosci. 14: 3370 PNQX (1,4,7,8,9,10-hexahydro-9-methyl-6-nitropyrido (1994); (iv)and (S)-Homoquisqualate, which is described [3, 4-f]quinoxaline-2,3-dione), and ZK200775 ([1,2,3,4- in Porter et al., Br. J. Pharmacol., 106: 509 (1992). tetrahydro-7-morpholinyl-2,3-dioxo-6-(fluoromethyl)qui- [0198] Additional non-limiting examples of reported 35 noxalin-1-yl] methylphosphonate). Group I modulators include (i) Group I agonists, such as [0202] In additional embodiments, a neurogenic agent (RS)-3,5-dihydroxyphenylglycine, described in Brabet et used in combination with a PDE agent may be a reported al., Neuropharmacology, 34, 895-903, 1995; and com- muscarinic agent. Non-limiting examples of a reported pounds described in US Pat Nos. 6,399,641 and muscarinic agent include a muscarinic agonist such as 6,589,978, and US Pub No. 20030212066; (ii) Group I 40 milameline (CI-979), or a structurally or functionally re- antagonists, such as (S)-4-Carboxy-3-hydroxyphenylg- lated compound disclosed in U.S. Patent Nos. 4,786,648, lycine; 7-(Hydroxyimino)cyclopropa-β-chromen-1α- car- 5,362,860, 5,424,301, 5,650,174, 4,710,508, 5,314,901, boxylate ethyl ester; (RS)-1-Aminoindan-1,5-dicarboxy- 5,356,914, or 5,356,912; or xanomeline, or a structurally lic acid (AIDA); Methyl- 2- 6 (phenylethynyl)pyridine or functionally related compound disclosed in U.S. Patent (MPEP); 2-Methyl-6-(2-phenylethenyl)pyridine (SIB- 45 Nos. 5,041,455, 5,043,345, or 5,260,314. 1893); 6-Methyl-2-(phenylazo)-3-pyridinol (SIB-1757); [0203] Other non-limiting examples include a mus- (Sα-Amino-4-carboxy-2-methylbenzeneacetic acid; and carinic agent such as alvameline (LU 25-109), or a func- compounds described in US Pat Nos. 6,586,422, tionally or structurally compound disclosed in U.S. Pat. 5,783,575, 5,843,988, 5,536,721, 6,429,207, 5,696,148, Nos. 6,297,262, 4,866,077, RE36,374, 4,925,858, PCT and 6,218,385, and US Pub Nos. 20030109504,50 Publication No. WO 97/17074, or in Moltzen et al., J Med 20030013715, 20050154027, 20050004130, 2005- Chem. 1994 Nov 25;37(24):4085-99; 2,8-dimethyl-3- 0209273, 20050197361, and 20040082592; (iii)methylene-1-oxa-8-azaspiro[4.5]decane (YM-796) or mGlu5-selective agonists, such as (RS)-2-Chloro-5-hy- YM-954, or a functionally or structurally related com- droxyphenylglycine (CHPG); and (iv) mGlu5-selective pound disclosed in U.S. Patent Nos. 4,940,795, antagonists, such as 2-methyl-6-(phenylethynyl)-pyrid- 55 RE34,653, 4,996,210, 5,041,549, 5,403,931, or ine (MPEP); and compounds described in US Pat No. 5,412,096, or in Wanibuchi et al., Eur. J. Pharmacol., 6,660,753; and US Pub Nos. 20030195139,187, 479-486 (1990); cevimeline (AF102B), or a func- 20040229917,20050153986,20050085514,200500653 tionally or structurally compound disclosed in U.S. Pat.

30 59 EP 2 377 530 A2 60

Nos. 4,855,290, 5,340,821, 5,580,880 (American Home capable of inhibiting, reducing, or otherwise modulating Products), or 4,981,858 (optical isomers of AF102B); the deacetylation of histones mediated by a histone sabcomeline (SB 202026), or a functionally or structurally deacetylase. Non-limiting examples of a reported HDAC related compound described in U.S. Patent Nos.inhibitor include a short-chain fatty acid, such as butyric 5,278,170, RE35,593, 6,468,560, 5,773,619, 5,808,075, 5 acid, phenylbutyrate (PB), 4-phenylbutyrate (4-PBA), 5,545,740, 5,534,522, or 6,596,869, U.S. Patent Publi- pivaloyloxymethyl butyrate (Pivanex, AN- 9), isovalerate, cation Nos. 2002/0127271, 2003/0129246, 2002/0- valerate, valproate, valproic acid, propionate, butyra- 150618, 2001/0018074, 2003/0157169, or 2001/0- mide, isobutyramide, phenylacetate, 3-bromopropion- 003588, Bromidge et al., J Med Chem. 19;40(26): ate, or tributyrin; a compound bearing a hydroxyamic acid 4265-80 (1997), or Harries et al., British J. Pharm., 124, 10 group, such as suberoylanlide hydroxamic acid (SAHA), 409-415 (1998); talsaclidine (WAL 2014 FU), or a func- trichostatin A (TSA), trichostatin C (TSC), salicylhy- tionally or structurally compound disclosed in U.S. Patent droxamic acid, oxamflatin, suberic bishydroxamic acid Nos. 5,451,587, 5,286,864, 5,508,405, 5,451,587, (SBHA), m-carboxy-cinnamic acid bishydroxamic acid 5,286,864, 5,508,405, or 5,137,895, or in Pharmacol. (CBHA), pyroxamide (CAS RN 382180-17-8), diethyl Toxicol., 78, 59-68 (1996); or a 1- methyl-1,2,5,6-tetrahy- 15 bis-(pentamethylene-N,N-dimethylcarboxamide) malo- dropyridyl-1,2,5-thiadiazole derivative, such as tetra(eth- nate (EMBA), azelaic bishydroxamic acid (ABHA), azela- yleneglycol)(4-methoxy-1,2,5-thiadiazol-3-yl)[3-(1-me- ic-1-hydroxamate-9-anilide (AAHA), 6-(3-Chloropheny- thyl-1,2,5,6-tetrahydropyrid-3-yl)-1,2,5-thiadiazol-4-yl] lureido) carpoic hydroxamic acid, or A-161906; a cyclic ether, or a compound that is functionally or structurally tetrapeptide, such as Depsipeptide (FK228), FR225497, related to a 1-methyl-1,2,5,6-tetrahydropyridyl-1,2,5-thi- 20 trapoxin A, apicidin, chlamydocin, or HC-toxin; a benza- adiazole derivative as provided by Cao et al. ("Synthesis mide, such as MS-275; depudecin, a sulfonamide anilide and biological characterization of 1-methyl-1,2,5,6-tet- (e.g., diallyl sulfide), BL1521, curcumin (diferuloylmeth- rahydropyridyl-1,2,5 thiadiazole derivatives as muscarin- ane), CI-994 (N-acetyldinaline), spiruchostatin A, Scrip- ic agonists for the treatment of neurological disorders." taid, carbamazepine (CBZ), or a related compound; a J. Med. Chem. 46(20):4273-4286, 2003). 25 compound comprising a cyclic tetrapeptide group and a [0204] Yet additional non-limiting examples include hydroxamic acid group (examples of such compounds besipiridine, SR-46559, L-689,660, S-9977-2, AF-102, are described in U.S. Patent Nos. 6,833,384 and thiopilocarpine, or an analog of clozapine, such as a phar- 6,552,065); a compound comprising a benzamide group maceutically acceptable salt, ester, amide, or prodrug and a hydroxamic acid group (examples of such com- form thereof, or a diaryl[a,d]cycloheptene, such as an 30 pounds are described in Ryu et al., Cancer Lett. 2005 amino substituted form thereof, or N-desmethylclozap- Jul 9 (epub), Plumb et al., Mol Cancer Ther., 2(8):721-8 ine, which has been reported to be a metabolite of cloz- (2003), Ragno et al., J Med Chem., 47 (6):1351-9 (2004), apine, or an analog or related compound disclosed in US Mai et al., J Med Chem., 47(5):1098-109 (2004), Mai et 2005/0192268 or WO 05/63254. al., J Med Chem., 46 (4):512-24 (2003), Mai et al., J Med [0205] In other embodiments, the muscarinic agent is 35 Chem., 45(9):1778-84 (2002), Massa et al., J Med an m1 receptor agonist selected from 55-LH-3B, 55-LH- Chem., 44(13):2069-72 (2001), Mai et al., J Med Chem., 25A, 55-LH-30B, 55-LH-4-1A, 40-LH-67, 55-LH- 15A, 55- 48(9):3344-53 (2005), and Mai et al., J Med Chem., 46 LH-16B, 55-LH-11C, 55-LH-31A, 55-LH-46, 55-LH-47, (23):4826-9 (2003)); a compound described in U.S. Pat- 55-LH-4-3A, or a compound that is functionally or struc- ent Nos. 6,897,220, 6,888,027, 5,369,108, 6,541,661, turally related to one or more of these agonists disclosed 40 6,720,445, 6,562,995, 6,777,217, or 6,387,673, or U.S. in US 2005/0130961 or WO 04/087158. Patent Publication Nos. 20050171347, 20050165016, [0206] In additional embodiments, the muscarinic 20050159470, 20050143385, 20050137234, 2005- agent is a benzimidazolidinone derivative, or a function- 0137232, 20050119250, 20050113373, 20050107445, ally or structurally compound disclosed in U.S. Patent 20050107384, 20050096468, 20050085515,2005- 6,951,849, US 2003/0100545, WO 04/089942, or WO 45 0032831,20050014839,20040266769,20040254220,2 03/028650; a spiroazacyclic compound, or a functionally 0040229889, 20040198830, 20040142953, 2004- or structurally related related compound like 1-oxa-3,8- 0106599, 20040092598, 20040077726, 20040077698, diaza-spiro[4,5]decan-2-one or a compound disclosed in 20040053960, 20030187027, 20020177594, 2002- U.S. Patent 6,911,452 or WO 03/057698; or a tetrahyd- 0161045, 20020119996, 20020115826, 20020103192, roquinoline analog, or a functionally or structurally com- 50 or 20020065282; FK228, AN- 9, MS-275, CI-994, SAHA, pound disclosed in US 2003/0176418, G2M- US777, PXD-101, LBH-589, MGCD-0103, MK0683, 2005/0209226, or WO 03/057672. sodium phenylbutyrate, CRA-024781, and derivatives, [0207] In yet additional embodiments, the neurogenic salts, metabolites, prodrugs, and stereoisomers thereof; agent in combination with a PDE agent is a reported and a molecule that inhibits the transcription and/or trans- HDAC inhibitor. The term "HDAC" refers to any one of a 55 lation of one or more HDACs. family of enzymes that remove acetyl groups from the [0208] Additional non-limiting examples include a re- epsilon-amino groups of lysine residues at the N-termi- ported HDac inhibitor selected from ONO- 2506 or arun- nus of a histone. An HDAC inhibitor refers to compounds dic acid (CAS RN 185517-21-9); MGCD0103 (see Gelm-

31 61 EP 2 377 530 A2 62 on et al. "Phase I trials of the oral histone deacetylase 6,218,547; 6,399,604; 6,646,124; 6,515,140; 6,451,809; (HDAC) inhibitor MGCD0103 given either daily or 3x 6,448,259; 6,448,246; 6,423,711; 6,414,147; 6,399,604; weekly for 14 days every 3 weeks in patients (pts) with 6,380,209; 6,353,109; 6,297,256; 6,297,252; 6,268,496; advanced solid tumors." Journal of Clinical Oncology, 6,211,365; 6,166,203; 6,177,569; 6,194,427; 6,156,898; 2005 ASCO Annual Meeting Proceedings. 23 (16S, June 5 6,143,760; 6,127,395; 6,103,903; 6,103,731; 6,723,735; 1 Supplement), 2005: 3147 and Kalita et al. "Pharmaco- 6,479,506; 6,476,030; 6,337,331; 6,730,676; 6,730,681; dynamic effect of MGCD0103, an oral isotype-selective 6,828,322; 6,872,720; 6,699,859; 6,696,444; 6,617,326; histone deacetylase (HDAC) inhibitor, on HDAC enzyme 6,608,062; 6,579,875; 6,541,484; 6,500,828; 6,355,798; inhibition and histone acetylation induction in Phase I 6,333,336; 6,319,924; 6,303,605; 6,303,597; 6,291,460; clinical trials in patients (pts) with advanced solid tumors 10 6,255,305; 6,133,255; 6,872,731; 6,900,215; 6,642,229; or non-Hodgkin’s lymphoma (NHL)" Journal of Clinical 6,593,325; 6,914,060; 6,914,063; 6,914,065; 6,936,608; Oncology, 2005 ASCO Annual Meeting Proceedings. 23 6,534,505; 6,426,343; 6,313,125 ; 6,310,203; (16S, Part I of II, June 1 Supplement), 2005: 9631), a 6,200,975; 6,071,909; 5,922,724; 6,096,887; 6,080,873; reported thiophenyl derivative of benzamide HDac inhib- 6,013,799; 5,936,095; 5,925,770; 5,910,590; 5,908,932; itor as presented at the 97th American Association for 15 5,849,927; 5,840,888; 5,817,813; 5,804,686; 5,792,766; Cancer Research (AACR) Annual Meeting in Washing- 5,750,702; 5,744,603; 5,744,602; 5,723,462; 5,696,260; ton, DC. in a poster titled "Enhanced Isotype-Selectivity 5,693,801; 5,677,309; 5,668,283; 5,637,725; 5,637,724; and Antiproliferative Activity of Thiophenyl Derivatives of 5,625,063; 5,610,299; 5,608,079; 5,606,059; 5,604,235; BenzamideHDAC Inhibitors In Human Cancer Cells," 5,585,490; 5,510,480; 5,484,944; 5,473,073; 5,463,054; (abstract #4725), and a reported HDac inhibitor as de- 20 5,451,585; 5,426,186; 5,367,077; 5,328,912 5,326,868; scribed in U.S. Patent 6,541,661; SAHA or Vorinostat 5,312,822; 5,306;819; 5,286,860; 5,266,698; 5,243,049; (CAS RN 149647-78-9); PXD101 or PXD 101 or PX 5,216,159; 5,212,310; 5,185,446; 5,185,446; 5,182,290; 105684 (CAS RN 414864-00-9), CI- 994 or Tacedinaline 5,130,430; 5,095,015; 20050014939; 20040171633; (CAS RN 112522-64-2), 275 MS- (CAS RN 20050165048; 20050165023; 20040259818; and 209783-80-2), or an inhibitor reported25 20040192692. in WO2005/108367. [0211] In some embodiments, the GABA-A modulator [0209] In other embodiments, the neurogenic agent in is a subunit-selective modulator. Non-limiting examples combination with a PDE agent is a reported GABA mod- of GABA-A modulator having specificity for the alpha1 ulator which modulates GABA receptor activity at the re- subunit include alpidem and zolpidem. Non-limiting ex- ceptor level (e.g., by binding directly to GABA receptors), 30 amples of GABA-A modulator having specificity for the at the transcriptional and/or translational level (e.g., by alpha2 and/or alpha3 subunits include compounds de- preventing GABA receptor gene expression), and/or by scribed in 6,730,681; 6,828,322; 6,872,720; 6,699,859; other modes (e.g., by binding to a ligand or effector of a 6,696,444; 6,617,326; 6,608,062; 6,579,875; 6,541,484; GABA receptor, or by modulating the activity of an agent 6,500,828; 6,355,798; 6,333,336; 6,319,924; 6,303,605; that directly or indirectly modulates GABA receptor ac- 35 6,303,597; 6,291,460; 6,255,305; 6,133,255; 6,900,215; tivity). Non-limiting examples of GABA-A receptor mod- 6,642,229; 6,593,325; and 6,914,063. Non-limiting ex- ulators useful in methods described herein include tria- amples of GABA-A modulator having specificity for the zolophthalazine derivatives, such as those disclosed in alpha2, alpha3 and/or alpha5 subunits include com- WO 99/25353, and WO/98/04560; tricyclic pyrazolo-py- pounds described in 6,730,676 and 6,936,608. Non- lim- ridazinone analogues, such as those disclosed in WO 40 iting examples of GABA- A modulators having specificity 99/00391; fenamates, such as those disclosed in for the alpha5 subunit include compounds described in 5,637,617; triazolo-pyridazine derivatives, such as those 6,534,505; 6,426,343; 6,313,125 ; 6,310,203; 6,200,975 disclosed in WO 99/37649, WO 99/37648, and WO and 6,399,604. Additional non-limiting subunit selective 99/37644; pyrazolopyridine derivatives, such as those GABA-A modulators include CL218,872 and related disclosed in WO 99/48892; nicotinic derivatives, such as 45 compounds disclosed in Squires et al., Pharmacol. Bio- those disclosed in WO 99/43661 and 5,723,462; musci- chem. Behav., 10: 825 (1979); and beta- carboline-3-car- mol, thiomuscimol, and compounds disclosed inboxylic acid esters described in Nielsen et al., Nature, 3,242,190; baclofen and compounds disclosed in 286: 606 (1980). 3,471,548; phaclofen; quisqualamine; ZAPA; zaleplon; [0212] In some embodiments, the GABA-A receptor THIP; imidazole-4-acetic acid (IMA); (+)-bicuculline; gab- 50 modulator is a reported allosteric modulator. In various alinoleamide; isoguvicaine; 3-aminopropane sulphonic embodiments, allosteric modulators modulate one or acid; piperidine-4-sulphonic acid; 4,5,6,7-tetrahy- more aspects of the activity of GABA at the target GABA dro-[5,4-c]-pyridin-3-ol;SR 95531; RU5315; CGP 55845; receptor, such as potency, maximal effect, affinity, and/or CGP 35348; FG 8094; SCH 50911; NG2-73; NGD-96-3; responsiveness to other GABA modulators. In some em- pricrotoxin and other bicyclophosphates disclosed in55 bodiments, allosteric modulators potentiate the effect of Bowery et al., Br. J. Pharmacol., 57; 435 (1976). GABA (e.g., positive allosteric modulators), and/or re- [0210] Additional non-limiting examples of GABA-A duce the effect of GABA (e.g., inverse agonists). Non- modulators include compounds described in 6,503,925; limiting examples of benzodiazepine GABA-A modula-

32 63 EP 2 377 530 A2 64 tors include aiprazolam, bentazepam, bretazenil, bro- beta-carboline-3-carboxylic acid, 6-methoxy-1,2,3,4-tet- mazepam, brotizolam, cannazepam, chlordiazepoxide, rahydro-beta-carboline, N-BOC-L-1,2,3,4-tetrahydro-b- clobazam, clonazepam, cinolazepam, clotiazepam, eta-carboline-3-carboxylic acid, tryptoline, pinoline, cloxazolam, clozapin, delorazepam, diazepam, diben- methoxyharmalan, tetrahydro-beta-carboline (THBC), 1- zepin, dipotassium chlorazepat, divaplon, estazolam, 5 methyl-THBC, 6-methoxy-THBC, 6-hydroxy-THBC, 6- ethyl-loflazepat, etizolam, fludiazepam, flumazenil, fluni- methoxyharmalan, norharman, 3,4-dihydro-beta-carbo- trazepam, flurazepamI 1HCl, flutoprazepam,line, and compounds described in Nielsen et al., Nature, halazeparn, haloxazolam, imidazenil, ketazolam, lo- 286: 606 (1980). razepam, loprazolam, lormetazepam, medazepam, met- [0217] In some embodiments, the GABA modulator aclazepam, mexozolam, midazolam-HCl, nabanezil, 10 modulates GABA-B receptor activity. Non-limiting exam- nimetazepam, nitrazepam, nordazepam, oxazepam- ples of reported GABA-B receptor modulators useful in tazepam, oxazolam, pinazepam, prazepam, quazepam, methods described herein include CGP36742; CGP- sarmazenil, suriclone, temazepam, tetrazepam,64213; CGP 56999A; CGP 54433A; CGP 36742; SCH tofisopam, triazolam, zaleplon,zolezepam, zolpidem, zo- 50911; CGP 7930; CGP 13501; baclofen and com- piclone, and zopielon. 15 pounds disclosed in 3,471,548; saclofen; phaclofen; 2- [0213] Additional non-limiting examples of benzodi- hydroxysalofen; SKF 97541; CGP 35348 and related azepine GABA-A modulators include Ro15-4513, compounds described in Olpe, et al, Eur. J. Pharmacol., CL218872, CGS 8216, CGS 9895, PK 9084, U-93631, 187, 27 (1990); phosphinic acid derivatives described in beta-CCM, beta-CCB, beta-CCP, Ro 19-8022, CGS Hills, et al, Br. J. Pharmacol., 102, pp. 5-6 (1991); and 20625, NNC 14-0590, Ru 33-203, 5-amino-1-bromour- 20 compounds described in 4,656,298, 5,929,236, acil, GYKI-52322, FG 8205, Ro 19-4603, 63, ZG- EP0463969, EP 0356128, Kaupmann et al., Nature 368: RWJ46771, SX-3228, and L-655,078; NNC 14-0578, 239 (1997), Karla et al., J Med Chem., 42(11):2053-9 NNC 14-8198, and additional compounds described in (1992), Ansar et al., Therapie. 54(5):651-8 (1999), and Wong et al., Eur J Pharmacol 209: 319-325 (1995); Y- Castelli et al., Eur J Pharmacol., 446(1-3):1-5 (2002). 23684 and additional compounds in Yasumatsu et al., Br 25 [0218] In some embodiments, the GABA modulator J Pharmacol 111: 1170-1178 (1994); and compounds de- modulates GABA-C receptor activity. Non- limiting exam- scribed in U.S. Patent 4,513,135. ples of reported GABA-C receptor modulators useful in [0214] Non-limiting examples of barbiturate or barbi- methodsdescribed herein include cis- aminocrotonic acid turic acid derivative GABA-A modulators include pheno- (CACA); 1,2,5,6-tetrahydropyridine-4-yl methyl phos- barbital, pentobarbital, pentobarbitone, primidone, bar- 30 phinic acid (TPMPA) and related compounds such as bexaclon, dipropyl barbituric acid, eunarcon, hexobarbi- P4MPA, PPA and SEPI; 2-methyl-TACA; (+/-)-TAMP; tal, mephobarbital, methohexital, Na- methohexital, 2,4,6 muscimol and compounds disclosed in 3,242,190; ZA- (1H,3H,5)-pyrimidintrion, secbutabarbital and/or thio- PA; THIP and related analogues, such as aza- THIP; pri- pental. cotroxin; imidazole-4-acetic acid (IMA); and CGP36742. [0215] Non-limiting examples of neurosteroid GABA- 35 [0219] In some embodiments, the GABA modulator A modulators include alphaxalone, allotetrahydrodeoxy- modulates the activity of glutamic acid decarboxylase corticosterone, tetrahydrodeoxycorticosterone, estro- (GAD). gen, progesterone 3-beta-hydroxyandrost-5-en-17-on- [0220] In some embodiments, the GABA modulator 3-sulfate, dehydroepianrosterone, eltanolone, ethi- modulates GABA transaminase (GTA). Non-limiting ex- nylestradiol, 5-pregnen-3-beta-ol-20 on-sulfate, 5a- 40 amples of GTA modulators include the GABA analogue pregnan-3a-ol-20-one (5PG),allopregnanolone, pregna- vigabatrin and compounds disclosed in 3,960,927. nolone, and steroid derivatives and metabolites de- [0221] In some embodiments, the GABA modulator scribed in 5,939,545, 5,925,630, 6,277,838, 6,143,736, modulates the reuptake and/or transport of GABA from RE35,517, 5,925,630, 5,591,733, 5,232,917,extracellular regions. In other embodiments, the GABA 20050176976, WO 96116076, WO 98/05337, WO45 modulator modulates the activity of the GABA transport- 95/21617, WO 94/27608, WO 93/18053, WO 93/05786, ers, GAT-1, GAT-2, GAT-3 and/or BGT-1. Non-limiting WO 93/03732,, WO 91116897, EP01038880, and Han examples of GABA reuptake and/or transport modulators et al., J. Med. Chem., 36, 3956-3967 (1993), Anderson include nipecotic acid and related derivatives, such as et al., J. Med. Chem., 40, 1668-1681 (1997), Hogenkamp CI 966; SKF 89976A; TACA; stiripentol; tiagabine and et al., J. Med. Chem., 40, 61-72 (1997), Upasani et al., 50 GAT-1 inhibitors disclosed in 5,010,090; (R)-1-(4,4- J. Med. Chem., 40, 73-84 (1997), Majewska et al., Sci- diphenyl-3-butenyl)-3-piperidinecarboxylic acid and re- ence 232:1004-1007 (1986), Harrison et al., J. Pharma- lated compounds disclosed in 4,383,999; (R)-1-[4,4-bis col. Exp. Ther. 241:346-353 (1987), Gee et al., Eur. J. (3- methyl- 2- thienyl)- 3- butenyl]- 3- piperidinecarboxylic Pharmacol., 136:419-423 (1987) and Birtran et al., Brain acid and related compounds disclosed in Anderson et Res., 561, 157-161 (1991). 55 al., J. Med. Chem. 36, (1993) 1716-1725; guvacine and [0216] Non-limiting examples of beta-carboline relatedcompounds disclosed in Krogsgaard-Larsen, Mo- GABA-A modulators include abecarnil, 3,4-dihydro-beta- lecular & Cellular Biochemistry 31, 105-121 (1980); GAT- carboline, gedocamil, methyl- 1- 1-vinyl-2,3,4-trihydro- 4 inhibitors disclosed in 6,071,932; and compounds dis-

33 65 EP 2 377 530 A2 66 closed in 6,906,177 and Ali, F. E., et al. J. Med. Chem. 35348, which is described, e.g., in Olpe et al. Eur.J. Phar- 1985, 28, 653-660. Methods for detecting GABA re- macol. 1990,187,27; Hao et al. Neurosci. Lett. 1994, 182, uptake inhibitors are known in the art, and are described, 299; and Froestl et al. Pharmacol. Rev. Comm. e.g., in 6,906,177; 6,225,115; 4,383,999; Ali, F. E., et al. 1996,8,127; the selective GABA-B antagonist CGP J. Med. Chem. 1985, 28, 653-660. 5 46381, which is described, e.g., in Lingenhoehl, Phar- [0222] In some embodiments, the GABA modulator is macol. Comm. 1993, 3, 49; the selective GABA- B antag- the benzodiazepine Clonazepam, which is described, onist CGP 52432, which is described, e.g., in Lanza et e.g., in 3,121,076 and 3,116,203; the benzodiazepine al. Eur. J. Pharmacol. 1993, 237, 191; Froestl et al. Phar- Diazepam, which is described, e.g., in 3,371,085; macol. Rev. Comm. 1996, 8, 127; Bonanno et al. Eur. J. 3,109,843; and 3,136,815; the short-acting diazepam de- 10 Pharmacol. 1998, 362, 143; and Libri et al. Naunyn- rivative Midazolam, which is a described, e.g., in Schmied. Arch. Pharmacol. 1998, 358,168; the selective 4,280,957; the imidazodiazepine Flumazenil, which is GABA-Bantagonist CGP 54626, which is described, e.g., described, e.g., in 4,316,839; the benzodiazepine Lo- in Brugger et al. Eur. J. Pharmacol. 1993,235,153; razepam is described, e.g., in 3,296,249; the benzodi- Froestl et al. Pharmacol. Rev. Comm. 1996, 8, 127; and azepine L-655708, which is described, e.g., in Quirk et 15 Kaupmann et al. Nature 1998,396,683; the selective al. Neuropharmacology 1996, 35, 1331; Sur et al. Mol. GABA-B antagonist CGP 55845, which is a GABA-re- Pharmacol. 1998, 54, 928; and Sur et al. Brain Res. 1999, ceptor antagonist described, e.g., in Davies et al. Neu- 822, 265; the benzodiazepine Gabitril; Zopiclone, which ropharmacology 1993, 32, 1071; Froestl et al. Pharma- binds the benzodiazepine site on GABA-A receptors, and col. Rev. Comm. 1996, 8, 127; and Deisz Neuroscience is disclosed, e.g., in 3,862,149 and 4,220,646.; the20 1999, 93, 1241; the selective GABA-B antagonist GABA-A potentiator Indiplon as described, e.g., in Foster Saclofen, which is described, e.g., in Bowery, TiPS, et al., J Pharmacol Exp Ther., 311(2):547-59 (2004), 1989,10,401; and Kerr et al. Neurosci Lett. 1988;92(1): 4,521,422 and 4,900,836; Zolpidem, described, e.g., in 92-6; the GABA-B antagonist 2-Hydroxysaclofen, which 4,794,185 and EP50563; Zaleplon, described, e.g., in is described, e.g., in Kerr et al. Neurosci. Lett. 1988, 92, 4,626,538; Abecarnil, described, e.g., in Stephens et al., 25 92; and Curtis et al. Neurosci. Lett. 1988, 92, 97; the J Pharmacol Exp Ther., 253(1):334-43 (1990); the GABA-B antagonist SCH 50,911, which is described, GABA-A agonist Isoguvacine, which is described, e.g., e.g., in Carruthers et al., Bioorg Med Chem Lett 8: in Chebib et al., Clin. Exp. Pharmacol. Physiol.3059-3064 (1998); Bolser et al. J. Pharmacol. Exp. Ther. 1999,26,937-940; Leinekugel et al. J. Physiol.1996,274,1393; Hosford et al. J. Pharmacol. Exp. Ther. 1995,487,319-29; and White et al., J. Neurochem. 1983, 30 1996,274, 1399; and Ong et al. Eur. J. Pharmacol. 1998, 40(6), 1701-8; the GABA-A agonist Gaboxadol (THIP), 362, 35; the selective GABA-C antagonist TPMPA, which which is described, e.g., in 4,278,676 and Krogsgaard- is described, e.g., in Schlicker et al., Brain Res. Bull. Larsen, Acta. Chem. Scand. 1977,31,584; the GABA-A 2004, 63(2), 91-7; Murata et al., Bioorg.Med.Chem.Lett. agonist Muscimol, which is described, e.g., in 3,242,190 6: 2073 (1996); and Ragozzino et al., Mol.Pharmacol. and 3,397,209; the inverse GABA-A agonist beta-CCP, 35 50: 1024 (1996); a GABA derivative, such as Pregabalin which is described, e.g., in Nielsen et al., J. Neurochem, [(S)-(+)-3-isobutylgaba] or gabapentin [1-(aminomethyl) 36(1):276-85 (1981); the GABA-A potentiator Riluzole, cyclohexane acetic acid]. Gabapentin is described, e.g., which is described, e.g., in 4,370,338 and EP 50,551; in U.S. Patent 4,024,175; the lipid- soluble GABA agonist the GABA-B agonist and GABA-C antagonist SKF Progabide,which is metabolized in vivo into GABA and/or 97541, which is described, e.g., in Froestl et 40 al.,pharmaceutically active GABA derivatives in vivo. Pro- J.Med.Chem. 38 3297 (1995); Hoskison et al., Neurosci. gabide is described, e.g., in U.S. Patents 4,094,992 and Lett. 2004, 365 (1), 48-53 and Hue et al., J. Insect Physiol. 4,361,583; the GAT1 inhibitor Tiagabine, which is de- 1997,43(12),1125-1131; the GABA-B agonist Baclofen, scribed, e.g., in U.S. Patent 5,010,090 and Andersen et which is described, e.g., in U.S. Patent 3,471,548; the al. J. Med. Chem. 1993,36,1716; the GABA transami- GABA-C agonist cis-4-aminocrotonic acid (CACA), 45 nase inhibitor Valproic Acid (2-propylpentanoic acid or which is described, e.g., in Ulloor et al. J. Neurophysiol. dispropylacetic acid), which is described, e.g., in U.S. 2004, 91(4),1822-31; the GABA- A antagonist Phaclofen, Patent 4,699,927 and Carraz et al., Therapie, which is described, e.g., in Kerr et al. Brain Res. 1987, 1965,20,419; the GABA transaminase inhibitor Viga- 405, 150; Karlsson et al. Eur. J Pharmacol.batrin, which is described, e.g., in U.S. Patent 3,960,927; 1988,148,485; and Hasuo, Gallagher Neurosci. Lett.50 or Topiramate, which is described, e.g., in U.S. Patent 1988,86,77; the GABA-A antagonist SR 95531, which is 4,513,006. described, e.g., in Stell et al. J. Neurosci. 2002,22(10), [0223] Additionally, the neurogenic agent in combina- RC223; Wermuth et al., J.Med.Chem. 30 239 (1987); and tion with a PDE agent may be a neurogenic sensitizing Luddens and Korpi, J.Neurosci. 15: 6957 (1995); the agent that is a reported anti- epileptic agent. Non-limiting GABA-A antagonist Bicuculline, which is a described, 55 examples of such agents include carbamazepine or te- e.g., in Groenewoud, J. Chem. Soc. 1936,199; Olsen et gretol (CAS RN 298-46-4), clonazepam (CAS RN al., Brain Res. 102: 283 (1976) and Haworth et al. Nature 1622-61-3), BPAor 3-(p- Boronophenyl)alanine(CAS RN 1950,165,529; the selective GABA-B antagonist CGP 90580-64-6), gabapentin or neurontin (CAS RN

34 67 EP 2 377 530 A2 68

60142-96-3), phenytoin (CAS RN 57-41-0), topiramate, ported calcium channel antagonist such as amlodipine lamotrigine or lamictal (CAS RN 84057-84-1), phenobar- (CAS RN 88150-42-9) or amlodipine maleate (CAS RN bital (CAS RN 50-06-6), oxcarbazepine (CAS RN 88150-47-4), nifedipine (CAS RN 21829-25-4), MEM- 28721-07-5), primidone (CAS RN 125-33-7), ethosux- 1003 (CAS RN see Rose et al. "Efficacy of MEM 1003, imide (CAS RN 77-67-8), levetiracetam (CAS RN5 a novel calcium channel blocker, in delay and trace eye- 102767-28-2), zonisamide, tiagabine (CAS RNblink conditioning in older rabbits." Neurobiol Aging. 2006 115103-54-3), depakote or divalproex sodium (CAS RN Apr 16; [Epub ahead of print]), isradipine (CAS RN 76584-70-8), Felbamate (Na-channel and NMDA recep- 75695-93-1), felodipine (CAS RN 72509-76-3; 3,5-Pyri- tor antagonist), or pregabalin (CAS RN 148553-50-8). dinedicarboxylic acid, 1,4-dihydro-4-(2,3-dichlorophe- [0224] In further embodiments, the neurogenic sensi- 10 nyl)-2,6-dimethyl-, ethyl methyl ester) or felodipine (CAS tizing agent may be a reported direct or indirect modulator RN 86189-69-7; 3,5-Pyridinedicarboxylic acid, 4-(2,3- of dopamine receptors. Non-limiting examples of such dichlorophenyl)-1,4-dihydro-2,6-dimethyl-, ethyl methyl agents include the indirect dopamine agonists methyl- ester, (+-)-), lemildipine (CAS RN 125729-29-5 or phenidate (CAS RN 113-45-1) or Methylphenidate hy- 94739-29-4), clevidipine (CAS RN 166432-28-6 or drochloride (also known as ritalin CAS RN 298-59-9), 15 167221-71-8), verapamil (CAS RN 52-53-9), ziconotide amphetamine (CAS RN 300-62-9) and methampheta- (CAS RN 107452-89-1), monatepil maleate (CAS RN mine (CAS RN 537-46-2), and the direct dopamine ag- 132046-06-1), manidipine (CAS RN 89226-50-6), Fur- onists sumanirole (CAS RN 179396-43-7), roprinirole nidipine (CAS RN 138661-03-7), Nitrendipine (CAS RN (CAS RN 91374-21-9), and rotigotine (CAS RN39562-70-4), Loperamide (CAS RN 53179-11-6), Amio- 99755-59-6). Additional non- limiting examples include 7- 20 darone (CAS RN 1951-25-3), Bepridil (CAS RN OH-DPAT, quinpirole, haloperidole, or clozapine. 64706-54-3), diltiazem (CAS RN 42399-41-7), Ni- [0225] Additional non-limiting examples include bro- modipine (CAS RN 66085-59-4), Lamotrigine, Cinnar- mocriptine (CAS RN 25614-03-3), adrogolide (CAS RN izine (CAS RN 298-57-7), lacipidine (CAS RN 171752-56-0), pramipexole (CAS RN 104632-26-0), 103890-78-4), nilvadipine (CAS RN 75530-68-6), dota- Ropinirole (CAS RN 91374-21-9), apomorphine (CAS 25 rizine (CAS RN 84625-59-2), cilnidipine (CAS RN RN 58-00-4) or apomorphine hydrochloride (CAS RN 132203-70-4), Oxodipine (CAS RN 90729-41-2), ara- 314-19-2), lisuride (CAS RN 18016-80-3), Sibenadet hy- nidipine (CAS RN 86780-90-7), anipamil (CAS RN drochloride or Viozan (CAS RN 154189-24-9), L-DOPA 83200-10-6), ipenoxazone (CAS RN 104454-71-9), Efo- or Levodopa (CAS RN 59-92-7), Melevodopa (CAS RN nidipine hydrochloride or NZ 105 (CAS RN 111011-53-1) 7101-51-1), etilevodopa (CAS RN 37178-37-3), Talipex- 30 or Efonidipine (CAS RN111011-63-3), temiverine (CAS ole hydrochloride (CAS RN 36085-73-1) or Talipexole RN 173324-94-2), pranidipine (CAS RN 99522-79-9), (CAS RN 101626-70-4), Nolomirole (CAS RNdopropidil (CAS RN 79700-61-1), lercanidipine (CAS RN 90060-42-7), quinelorane (CAS RN 97466-90-5), per- 100427-26-7), terodiline (CAS RN 15793-40-5), fantofar- golide (CAS RN 66104-22-1), fenoldopam (CAS RN one (CAS RN 114432-13-2), azelnidipine (CAS RN 67227-56-9), Carmoxirole (CAS RN 98323-83-2), tergu- 35 123524-52-7), mibefradil (CAS RN 116644-53-2) or ride (CAS RN 37686-84-3), cabergoline (CAS RN mibefradil dihydrochloride (CAS RN 116666-63-8), SB- 81409-90-7), quinagolide (CAS RN 87056-78-8) or 237376 (see Xu et al. "Electrophysiologic effects of SB- quinagolide hydrochloride (CAS RN 94424-50-7), 237376: a new antiarrhythmic compound with dual po- sumanirole, docarpamine (CAS RN 74639-40-0), SLV- tassium and calcium channel blocking action." J Cardio- 308 or (3H)-Benzoxazolone, 2 7-(4-methyl-1-piperazi- 40 vasc Pharmacol. 2003 41(3):414-21), BRL-32872 (CAS nyl)-monohydrochloride (CAS RN 269718-83-4), arip- RN 113241-47-7), S- 2150 (see Ishibashi et al. "Pharma- iprazole (CAS RN 129722-12-9), bifeprunox, lisdexam- codynamics of S- 2150, a simultaneous calcium- blocking fetamine dimesylate (CAS RN 608137-33-3), safinamide and alphal-inhibiting antihypertensive drug, in rats." J (CAS RN 133865-89-1), or Adderall or Amfetamine (CAS Pharm Pharmacol. 2000 52 (3):273-80), nisoldipine (CAS RN 300-62-9). 45 RN 63675-72-9), semotiadil (CAS RN 116476-13-2), pal- [0226] In further embodiments, the neurogenic agent onidipine (CAS RN 96515-73-0) or palonidipine hydro- used in combination with a PDE agent may be a reported chloride (CAS RN 96515-74-1), SL-87.0495 (see U.S. dual sodium and calcium channel modulator. Non-limit- Patent 6,897,305), YM430 (4(((S)-2-hydroxy-3-phenox- ing examples of such agents include safinamide and ypropyl)amino)butyl methyl 2,6- dimethyl-((S)-4-(m-nitro- zonisamide. Additional non-limiting examples include 50 phenyl))-1,4-dihydropyridine-3,5-dicarboxylate), bar- enecadin (CAS RN 259525-01-4), Levosemotiadil (CAS nidipine (CAS RN 104713-75-9), and AM336 or CVID RN 116476-16-5), bisaramil (CAS RN 89194-77-4), SL- (see Adams et al. "Omega-Conotoxin CVID Inhibits a 34.0829 (see U.S. Patent 6,897,305), lifarizine (CAS RN Pharmacologically Distinct Voltage-sensitive Calcium 119514-66-8), JTV-519 (4-[3-(4-benzylpiperidin-1-yl) Channel Associated with Transmitter Release from Pre- propionyl]-7-methoxy-2,3,4,5-tetrahy dro-1,4-benzothi- 55 ganglionic Nerve Terminals" J. Biol. Chem., 278(6): azepine monohydrochloride), and delapril. 4057-4062, 2003). An additional non- limiting example is [0227] In further embodiments, the neurogenic agent NMED-160. in used in combination with a PDE agent may be a re- [0228] In other embodiments, the neurogenic agent

35 69 EP 2 377 530 A2 70 used in combination with a PDE agent may be a reported tasosartan or its metabolite enoltasosartan; omapatrilat modulator of a melatonin receptor. Non-limiting exam- (CAS RN 167305-00-2); or a a combination of nateglinide ples of such modulators include the melatonin receptor and valsartan, amoldipine and benazepril (Lotrel 10-40 agonists melatonin, LY-156735 (CAS RN 118702-11-7), or Lotrel 5-40), or delapril and manidipine (CHF 1521). agomelatine (CAS RN 138112-76-2),6-chloromelatonin 5 [0233] Additionally, the agent used with a PDE agent (CAS RN 63762-74-3), Ramelteon (CAS may RN be a reported 5HT1a receptor agonist (or partial 196597-26-9), 2-Methyl-6,7-dichloromelatonin (CAS RN agonist) such as buspirone (buspar). In some embodi- 104513-29-3), and ML 23 (CAS RN 108929-03-9). ments, a reported 5HTla receptor agonist is an aza- [0229] In yet further embodiments, the neurogenic pirone, such as, but not limited to, tandospirone, gepirone agent in combination with a PDE agent may be a reported 10 and ipsapirone. Non-limiting examples of additional re- modulator of a melanocortin receptor. Non-limiting ex- ported 5HT1a receptor agonists include flesinoxan (CAS amples of such agents include a melanocortin receptor RN 98206-10-1), MDL 72832 hydrochloride, U-92016A, agonists selected from melanotan II (CAS RN(+)-UH 301, F 13714, F 13640, 6- hydroxy-buspirone (see 121062-08-6), PT-141 or Bremelanotide (CAS RN US 2005/0137206), S-6-hydroxy-buspirone (see US 189691-06-3), HP-228 (see Getting et al. "The melano- 15 2003/0022899), R-6-hydroxy-buspirone (see US cortin peptide HP228 displays protective effects in acute 2003/0009851), adatanserin, buspirone- saccharide(see models of inflammation and organ damage." Eur J Phar- WO 00/12067) or 8-hydroxy-2-dipropylaminotetralin (8- macol. 2006 Jan 24), or AP214 from Action Pharma A/S. OHDPAT). [0230] Additional embodiments include a combination [0234] Additional non-limiting examples of reported of a PDE agent and a reported modulator of angiotensin 20 5HT1a receptor agonists include OPC- 14523 (1-[3-[4-(3- II function, such as at an angiotensin II receptor. In some chlorophenyl)-1-piperazinyl] propyl]- 5-methoxy- 3,4- di- embodiments, the neurogenic sensitizing agent used hydro-2[1H]-quinolinone monomethanesulfonate); with a PDE agent may be a reported inhibitor of an an- BMS-181100 or BMY 14802 (CAS RN 105565-56-8); fli- giotensin converting enzyme (ACE). Non-limiting exam- banserin (CAS RN 167933-07-5); repinotan (CAS RN ples of such reported inhibitors include a sulfhydryl- con- 25 144980-29-0); lesopitron (CAS RN 132449-46-8); picloz- taining (or mercapto-containing) agent, such as Alace- otan (CAS RN 182415-09-4); Aripiprazole, Org-13011 pril, captopril (Capoten®), fentiapril, pivopril, pivalopril, (1-(4-trifluoromethyl-2-pyridinyl)-4- [4-[2-oxo-1-pyrrolidi- or zofenopril; a dicarboxylate-containing agent, such as nyl]butyl]piperazine (E)-2-butenedioate); SDZ-MAR-327 enalapril (Vasotec® or Renitec®) or enalaprilat, ramipril (see Christian et al. "Positron emission tomographic (Altace® or Tritace® or Ramace®), quinapril (Accupril®) 30 analysis of central dopamine D1 receptor binding in nor- or quinapril hydrochloride, perindopril (Coversyl®) or mal subjects treated with the atypical neuroleptic, SDZ perindopril erbumine (Aceon®), lisinopril (Lisodur® or MAR 327." Int J Mol Med. 1998 1(1):243-7); MKC-242 Prinivil® or Zestril®); a phosphonate-containing (or phos- ((S)- 5-[3-[(1,4- benzodioxan- 2- ylmethyl) amino] pro- phate-containing) agent, such as fosinopril (Monopril®), poxy]-1,3-benzodioxole HCl); vilazodone; sarizotan fosinoprilat, fosinopril sodium (CAS RN 88889-14-9),35 (CAS RN 177975-08-5); roxindole (CAS RN benazepril (Lotensin®) or benazepril hydrochloride, im- 112192-04-8) or roxindole methanesulfonate (CAS RN idapril or imidapril hydrochloride, moexipril (Univasc®), 119742-13-1); alnespirone (CAS RN 138298-79-0); bro- or trandolapril (Mavik®). In other embodiments, a mod- merguride (CAS RN 83455-48-5); xaliproden (CAS RN ulator is administered in the form of an ester that increas- 135354-02-8); mazapertine succinate (CAS RN es biovavailability upon oral administration with subse- 40 134208-18-7) or mazapertine (CAS RN 134208-17-6); quent conversion into metabolites with greater activity. PRX-00023; F-13640 ((3-chloro-4-fluoro-phenyl)-[4- [0231] Further embodiments include reported angi- fluoro- 4-[[(5- methyl- pyridin- 2- ylmethyl)-amino] methyl] otensin II modulating entities that are naturally occurring, piperidin-1-yl]methanone, fumaric acid salt); eptapirone such as casokinins and lactokinins (breakdown products (CAS RN 179756-85-5); Ziprasidone (CAS RN of casein and whey) which may be administered as such 45 146939-27-7); Sunepitron (see Becker et al. "G protein- to obviate the need for their formation during digestion. coupled receptors: In silico drug discovery in 3D" PNAS Additional non-limiting embodiments of reported angi- 2004 101(31):11304-11309); umespirone (CAS RN otensin receptor antagonists include candesartan (Ata- 107736-98-1); SLV-308; bifeprunox; and zalospirone cand® or Ratacand®, 139481-59-7) or candesartan (CAS RN 114298-18-9). cilexetil; eprosartan (Teveten®) or eprosartan mesylate; 50 [0235] Yet further non-limiting examples include AP- irbesartan (Aprovel® or Karvea® or Avapro®); losartan 521 (partial agonist from AsahiKasei) and Du-123015 (Cozaar® or Hyzaar®); olmesartan (Benicar®, CAS RN (from Solvay). 144689-24-7) or olmesartan medoxomil (CAS RN [0236] Alternatively, the agent used with a PDE agent 144689-63-4); telmisartan (Micardis® or Pritor®); or val- may be a reported 5HT4 receptor agonist (or partial ag- sartan (Diovan®). 55 onist). In some embodiments, a reported 5HT4 receptor [0232] Additional non-limiting examples of a reported agonist or partial agonist is a substituted benzamide, angiotensin modulator that may be used in a combination such as cisapride; individual, or a combination of, cis- include nateglinide or starlix (CAS RN 105816-04-4); apride enantiomers ((+) cisapride and (-) cisapride); mos-

36 71 EP 2 377 530 A2 72 apride; and renzapride as non- limiting examples. In other onists and partial agonists for use in combination with a embodiments, the chemical entity is a benzofuran deriv- PDE agent include metoclopramide (CAS RN 364-62-5), ative, such as prucalopride. Additional embodiments in- 5-methoxytryptamine (CAS RN 608-07-1), RS67506 clude indoles, such as tegaserod, or benzimidazolones. (CAS RN 168986-61-6), 2-[1-(4-piperonyl)piperazinyl] Other non-limiting chemical entities reported as a 5HT4 5 benzothiazole (CAS RN 155106-73-3), RS66331 (see receptor agonist or partial agonist include zacopride Buccafusco et al. "Multiple Central Nervous System Tar- (CAS RN 90182-92-6), 53116 SC- (CAS RN gets for Eliciting Beneficial Effects on Memory and Cog- 141196-99-8) and its racemate SC-49518 (CAS RN nition." (2000) Pharmacology 295(2):438-446), BIMU8 146388-57-0), BIMU1 (CAS RN 127595-43-1), TS-951 (endo-N-8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-de- (CAS RN 174486-39-6), or ML10302 CAS 10 RNhydro-2-oxo-3-(prop-2-yl)-1H-benzimid-azole-1-carbox- 148868-55-7). Additional non-limiting chemical entities amide), or SB 205149 (the n-butyl quaternary analog of include metoclopramide, methoxytryptamine, 5- renzapride). Compounds related to metoclopramide, RS67506, 2-[1-(4-piperonyl)piperazinyl]benzothiazole, such as metoclopramide dihydrochloride (CAS RN RS66331, BIMU8, SB 205149 (the n-butyl quaternary 2576-84-3) or metoclopramide dihydrochloride (CAS RN analog of renzapride), or an indole carbazimidamide as 15 5581-45-3) or metoclopramide hydrochloride (CAS RN described by Buchheit et al. ("The serotonin 5-HT4 re- 7232-21-5 or 54143-57-6) may also be used in a combi- ceptor. 2. Structure-activity studies of the indole carbaz- nation or method as described herein. imidamide class of agonists." J Med Chem. (1995) 38 [0238] Additionally, the agent used with a PDE agent (13):2331-8). Yet additional non-limiting examples in- may be a reported 5HT3 receptor antagonist such as clude norcisapride (CAS RN 102671-04-5) which is the 20 azasetron (CAS RN 123039-99-6); Ondansetron (CAS metabolite of cisapride; mosapride citrate; the maleate RN 99614-02-5) or Ondansetron hydrochloride (CAS RN form of tegaserod (CAS RN 189188-57-6); zacopride hy- 99614-01-4); Cilansetron (CAS RN 120635-74-7); Aloxi drochloride (CAS RN 99617-34-2); mezacopride (CAS or Palonosetron Hydrochloride (CAS RN 135729-62-3); RN 89613-77-4); SK-951 ((+-)- 4-amino-N-(2-(1-azabicy- Palenosetron (CAS RN 135729-61-2 or 135729-56-5); clo(3.3.0)octan-5-yl)ethyl)-5-chloro-2,3-dihydro-2-meth- 25 Cisplatin (CAS RN 15663-27-1); Lotronex or Alosetron ylbenzo(b)furan-7-carboxamide hemifumarate); ATI- hydrochloride (CAS RN 122852-69-1); Anzemet or Do- 7505, a cisapride analog from ARYx Therapeutics; SDZ- lasetron mesylate (CAS RN 115956-13-3); zacopride or 216-454, a selective 5HT4 receptor agonist that stimu- R-Zacopride; E-3620 ([3(S)-endo]-4-amino-5-chloro- lates cAMP formation in a concentration dependent man- N-(8-methyl-- 8-azabicyclo[3.2.1-]oct-3-yl-2[(1-methyl- ner (see Markstein et al. "Pharmacological characterisa- 30 2-butynyl)oxy]benzamide) or E-3620 HCl (3(S)-endo-4- tion of 5-HT receptors positively coupled to adenylyl cy- amino-5-chloro-N-(8 methyl-8- azabicyclo [3.2.1] oct- 3- clase in the rat hippocampus." Naunyn Schmiedebergs yl)-2-(1-methyl-2-butinyl)oxy)-benzamide-HCl); YM 060 Arch Pharmacol. (1999) 359(6):454-9);SC-54750,or or Ramosetron hydrochloride (CAS RN 132907-72-3); a Aminomethylazaadamantane; Y-36912, or 4-amino- thieno[2,3-d]pyrimidine derivative antagonist described N-[1-[3-(benzylsulfonyl) propyl] piperidin- 4- ylmethyl]- 5- 35 in U.S. Patent 6,846,823, such as DDP 225 or MCI 225 chloro-2-methoxybenzamide as disclosed by Sonda et (CAS RN 135991-48-9); Marinol or Dronabinol (CAS RN al. ("Synthesis and pharmacological properties of ben- 1972-08-3); or Lac Hydrin or Ammonium lactate (CAS zamide derivatives as selective serotonin 4 receptor ag- RN 515-98-0); Kytril or Granisetron hydrochloride (CAS onists." Bioorg Med Chem. (2004) (10): 12 2737-47); RN 107007-99-8); Bemesetron (CAS RN 40796-97-2); TKS159, or 4-amino-5-chloro-2-methoxy-N-[(2S,4S)-1- 40 Tropisetron (CAS RN 89565-68-4); Zatosetron (CAS RN ethyl-2-hydroxymethyl-4-pyrrolidinyl] benzamide, as re- 123482-22-4); Mirisetron (CAS RN 135905-89-4) or Miri- ported by Haga et al. ("Effect of TKS159, a novel 5- setron maleate (CAS RN 148611-75-0); or renzapride hydroxytryptamine4 agonist, on gastric contractile activ- (CAS RN 112727-80-7). ity in conscious dogs."; RS67333, or 1-(4-amino-5-chlo- [0239] Additionally, the agent used with a PDE agent ro-2-methoxyphenyl)-3-(1-n-butyl-4-piperidinyl)-1-pro- 45 may be a reported 5HT2A/2C receptor antagonist such panone; KDR-5169, or 4-amino-5-chloro-N-[1-(3-fluoro- as Ketanserin (CAS RN 74050-98-9) or ketanserin tar- 4-methoxybenzyl)piperidin-4-yl]-2-(2-hydro xyethoxy) trate; risperidone; olanzapine; adatanserin (CAS RN benzamide hydrochloride dihydrate as reported by 127266-56-2);Ritanserin (CAS RN 87051-43-2); etoperi- Tazawa, et al. (2002) "KDR- 5169, a new gastrointestinal done; nefazodone; deramciclane (CAS RN prokinetic agent, enhances gastric contractile and emp- 50 120444-71-5); Geoden or Ziprasidone hydrochloride tying activities in dogs and rats." Eur J Pharmacol 434 (CASRN 138982-67-9); Zeldox or Ziprasidone or Ziprasi- (3):169-76; SL65.0155, or 5-(8- amino-7-chloro-2,3-dihy- done hydrochloride; EMD 281014 (7-[4-[2-(4-fluoro- phe- dro-1,4-benzodioxin-5-yl)-3-[1-(2-phenyl ethyl)-4-pipe- nyl)-ethyl]-piperazine-1-carbonyl]-1H-indole-3-carboni- ridinyl]-1,3,4-oxadiazol-2(3H)-one monohydrochloride; trileHCl); MDL 100907 or M100907 (CAS RN and 34959, Y- or Amino- 4- 5-chloro-2-methoxy- 55 139290-65-6); Effexor XR (Venlafaxine formulation); N-[1-[5-(1-methylindol-3-ylcarbonylamino)pentyl]piperi- Zomaril or Iloperidone; quetiapine (CAS RN din-4-ylmethyl]benzamide. 111974-69-7) or Quetiapine fumarate (CAS RN [0237] Other non-limiting reported 5HT4 receptor ag- 111974-72-2) or Seroquel; SB 228357 or SB 243213

37 73 EP 2 377 530 A2 74

(see Bromidge et al. "Biarylcarbamoylindolines are novel ferred to herein as "monoamines") or other biogenic and selective 5-HT(2C) receptor inverse agonists: iden- amines, such as trace amines (TAs) as a non-limiting tification of 5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]- 5- example. TAs are endogenous, CNS-active amines that pyridyl]carbamoyl]-6-trifluoromethylindoline (SB-2432- are structurally related to classical biogenic amines (e.g., 13) as a potential antidepressant/ anxiolytic agent." J Med 5 norepinephrine, dopamine (4-(2-aminoethyl)benzene- Chem. 2000 43(6): 1123-34; SB 220453 or Tonabersat 1,2-diol), and/or serotonin (5-hydroxytryptamine (5-HT), (CAS RN 175013-84-0); Sertindole (CAS or RN a metabolite, precursor, prodrug, or analogue thereof. 106516-24-9); Eplivanserin (CAS RN 130579-75-8) or The methods of the disclosure thus include administra- Eplivanserin fumarate (CAS RN 130580-02-8); Lubazo- tion of one or more reported TAs in a combination with done hydrochloride (CAS RN 161178-10-5); Cyprohep- 10 a PDE agent. Additional CNS-active monoamine recep- tadine (CAS RN 129-03-3); Pizotyline or pizotifen (CAS tor modulators are well known in the art, and are de- RN 15574-96-6); Mesulergine (CAS RN 64795-35-3); Ir- scribed, e.g., in the Merck Index, 12th Ed. (1996). indalone (CAS RN 96478-43-2); MDL 11939 (CAS RN [0244] Certain food products, e.g., chocolates, chees- 107703-78-6); or pruvanserin (CAS RN 443144-26-1). es, and wines, can also provide a significant dietary [0240] Additional non-limiting examples of modulators 15 source of TAs and/or TA-related compounds. Non-limit- include reported 5-HT2C agonists or partial agonists, ing examples of mammalian TAs useful as constitutive such as m-chlorophenylpiperazine; or 5-HT2A receptor factors include, but are not limited to, tryptamine, p- inverse agonists, such as ACP 103 (CAS RN:tyramine, m-tyramine, octopamine, synephrine or β-phe- 868855-07-6), APD125 (from Arena Pharmaceuticals), nylethylamine β ( -PEA). Additional useful TA-related AVE 8488 (from Sanofi- Aventis) or TGWOOAD/AA (from 20 compounds include, but are not limited to, 5- hydroxytryp- Fabre Kramer Pharmaceuticals). tamine, amphetamine, bufotenin, 5-methoxytryptamine, [0241] Additionally, the agent used with a PDE agent dihydromethoxytryptamine, phenylephrine, or a metab- may be a reported 5HT6 receptor antagonist such as SB- olite, precursor, prodrug, or analogue thereof. 357134 (N-(2,5-Dibromo-3-fluorophenyl)-4-methoxy-3- [0245] In some embodiments, the constitutive factor is piperazin-1-ylbenzenesulfonamide); SB-271046 (5- 25 a biogenic amine or a ligand of a trace amine-associated chloro-N-(4-methoxy-3-(piperazin-1-yl)phenyl)-3-meth- receptor (TAAR), and/or an agent that mediates one or ylbenzo[b]thiophene-2-sulfonamide); Ro 04-06790 (N- more biological effects of a TA. TAs have been shown (2, 6-bis(methylamino)pyrimidin-4-yl)-4-aminobenzene- to bind to and activate a number of unique receptors, sulfonamide); Ro 63-0563 (4-amino-N-(2,6 bis-methyl- termed TAARs, whichcomprise a family of G- proteincou- amino-pyridin-4-yl)-benzene sulfonamide); clozapine or 30 pled receptors (TAAR1-TAAR9) with homology to clas- its metabolite N-desmethylclozapine; olanzapine (CAS sical biogenic amine receptors. For example, TAAR1 is RN 132539-06-1); fluperlapine (CAS RN 67121-76-0); activated by both tyramine and β-PEA. seroquel (quetiapine or quetiapine fumarate); clomi- [0246] Thus non-limiting embodiments include meth- pramine (CAS RN 303-49-1); amitriptyline (CASods and combination compositions wherein the constitu- RN50-48-6); doxepin (CAS RN 1668-19-5); nortryptyline 35 tive factor is β-PEA, which has been indicated as having (CAS RN 72-69-5); methoxytryptamine 5- (CAS RN a significant neuromodulatory role in the mammalian 608-07-1); bromocryptine (CAS RN 25614-03-3); octo- CNS and is found at relatively high levels in the hippoc- clothepin (CAS RN 13448-22-1); chlorpromazine (CAS ampus (e.g., Taga et al., Biomed Chromatogr.,(3): 3 RN 50-53-3); loxapine (CAS RN 1977-10-2); fluphena- 118-20 (1989)); ametabolite, prodrug, precursor,or other zine (CAS RN 69-23-8); or GSK 742457 (presented by 40 analogue of β-PEA, such as the β-PEA precursor L-phe- David Witty, "Early Optimisation of in vivo Activity: the nylalanine, the β-PEA metabolite β-phenylacetic acid (β- discovery of 5-HT6 Receptor Antagonist 742457" Glax- PAA), or the β-PEA analogues methylphenidate, am- oSmithKline at SCIpharm 2006, International Pharma- phetamine, and related compounds. ceutical Industry Conference in Edinburgh, 16 May [0247] Most TAs and monoamines have a short half- 2006). 45 life (e.g., less than about 30 s) due, e.g., to their rapid [0242] As an additional non-limiting example, the re- extracellular metabolism. Thus embodiments of the dis- ported 5HT6 modulator may be SB- 258585 (4-Iodo-N-[4- closure include use of a monoamine "metabolic modula- methoxy- 3-(4- methyl- piperazin- 1- yl)-phenyl]-benzen tor," which increases the extracellular concentration of esulphonamide); PRX 07034 (from Predix Pharmaceu- one or more monoamines by inhibiting monoamine me- ticals) or a partial agonist, such as 6801 E- (6-chloro- 50 tabolism. In some embodiments, the metabolic modula- N-(3-(2-(dimethylamino) ethyl)- 1H- indol- 5- yl) imidazo tor is an inhibitor of the enzyme monoamine oxidase [2,1-b]thiazole-5-sulfonamide) or E-6837 (5-chloro- (MAO), which catalyzes the extracellular breakdown of N-(3-(2-(dimethylamino) ethyl)- 1H- indol- 5- yl) naphtha- monoamines into inactive species. Isoforms MAO-A lene-2-sulfonamide). and/or MAO-B provide the major pathway for TA metab- [0243] Additionally, the agent used in combination with 55 olism. Thus, in some embodiments, TA levels are regu- a PDE agent may be a reported compound (or "monoam- lated by modulating the activity of MAO-A and/or MAO- ine modulator") that modulates neurotransmission medi- B. For example, in some embodiments, endogenous TA ated by one or more monoamine neurotransmitters (re- levels are increased (and TA signaling is enhanced) by

38 75 EP 2 377 530 A2 76 administering an inhibitor of MAO-A and/or MAO-B, in 3,885,046), or (S,S)-hydroxybupropion (described, e.g., combination with a PDE agent as described herein. in U.S. Pat. 6,342,496); (ii) selective dopamine reuptake [0248] Non-limiting examples of inhibitors of monoam- inhibitors, such as medifoxamine, amineptine (de- ine oxidase (MAO) include reported inhibitors of the scribed, e.g., in U.S. Pat. 3,758,528 and 3,821,249), MAO-A isoform, which preferentially deaminates 5-hy- 5 GBR12909, GBR12783 and GBR13069, described in droxytryptamine (serotonin) (5-HT) and norepinephrine Andersen, Eur J Pharmacol, 166:493-504 (1989); or (iii) (NE), and/or the MAO-B isoform, which preferentially a monoamine "releaser" which stimulates the release of deaminates phenylethylamine (PEA) and benzylamine monoamines, such as biogenic amines from presynaptic (both MAO-A and MAO-B metabolize Dopamine (DA)). sites, e.g., by modulating presynaptic receptors (e.g., au- In various embodiments, MAO inhibitors may be irrevers- 10 toreceptors, heteroreceptors), modulating the packaging ible or reversible (e.g., reversible inhibitors of MAO- A (RI- (e.g., vesicular formation) and/or release (e.g., vesicular MA)), and may have varying potencies against MAO-A fusion and release) of monoamines, and/or otherwise and/or MAO-B (e.g., non-selective dual inhibitors or iso- modulating monoamine release. Advantageously, form-selective inhibitors). Non- limiting examples of MAO monoamine releasers provide a method for increasing inhibitors useful in methods described herein include15 levels of one or more monoamines within the synaptic clorgyline, L-deprenyl, isocarboxazid (Marplan), aya- cleft or other extracellular region independently of the huasca, nialamide, iproniazide, iproclozide, mo-activity of the presynaptic neuron. clobemide (Aurorix), phenelzine (Nardil), tranylcy- [0251] Monoamine releasers useful in combinations promine (Parnate) (the congeneric of phenelzine), provided herein include fenfluramine or p- chloroamphet- toloxatone, levodeprenyl (Selegiline), harmala, RIMAs 20 amine (PCA) or the dopamine, norepinephrine, and se- (e.g., moclobemide, described in Da Prada et al., J Phar- rotonin releasing compound amineptine (described, e.g., macol Exp Ther 248: 400-414 (1989); brofaromine; and in U.S. Pat. 3,758,528 and 3,821,249). befloxatone, described in Curet et al., J Affect Disord 51: [0252] Furthermore, the neurogenic agent in combina- 287-303 (1998)), lazabemide (Ro 19 6327), described in tion with a PDE agent may be a reported neurosteroid. Ann. Neurol., 40(1): 99-107 (1996), and SL25.1131, de- 25 Non-limiting examples of such a neurosteroid include scribed in Aubin et al., J. Pharmacol. Exp. Ther., 310: pregnenolone and allopregnenalone. 1171-1182 (2004). [0253] Alternatively, the neurogenic sensitizing agent [0249] In additional embodiments, the monoamine may be a reported non-steroidal anti-inflammatory drug modulator is an "uptake inhibitor," which increases ex- (NSAID) or an anti-inflammatory mechanism targeting tracellular monoamine levels by inhibiting the transport 30 agent in general. Non-limiting examples of a reported of monoamines away from the synaptic cleft and/or other NSAID include a cyclooxygenase inhibitor, such as in- extracellular regions. In some embodiments, thedomethacin, ibuprofen, celecoxib, cofecoxib, naproxen, monoamine modulator is a monoamine uptake inhibitor, or aspirin. Additional non-limiting examples for use in which may selectively/ preferentially inhibit uptake of one combination with a PDE agent include rofecoxib, melox- or more monoamines relative to one or more other35 icam, piroxicam, valdecoxib, parecoxib, etoricoxib, monoamines. The term "uptake inhibitors" includes com- etodolac, nimesulide, acemetacin, bufexamac, diflunisal, pounds that inhibit the transport of monoamines (e.g., ethenzamide, etofenamate, flobufen, isoxicam, kebu- uptake inhibitors) and/or the binding of monoamine sub- zone, lonazolac, meclofenamic acid, metamizol, mofeb- strates (e.g., uptake blockers) by transporter proteins utazone, niflumic acid, oxyphenbutazone, paracetamol, (e.g., the dopamine transporter (DAT), the NE transporter 40 phenidine, propacetamol, propyphenazone, salicyla- (NET),the 5- HT transporter (SERT), and/or the extraneu- mide, tenoxicam, tiaprofenic acid, oxaprozin,lornoxicam, ronal monoamine transporter (EMT)) and/or other mole- nabumetone, minocycline, benorylate, aloxiprin, sal- cules that mediate the removal of extracellular monoam- salate, flurbiprofen, ketoprofen, fenoprofen, fenbufen, ines. Monoamine uptake inhibitors are generally classi- benoxaprofen, suprofen, piroxicam, meloxicam, di- fied according to their potencies with respect to particular 45 clofenac, ketorolac, fenclofenac, sulindac, tolmetin, xy- monoamines, as described, e.g., in Koe, J. Pharmacol. phenbutazone, phenylbutazone, feprazone, azapropa- Exp. Ther. 199: 649-661 (1976). However, references to zone, flufenamic acid or mefenamic acid. compounds as being active against one or more[0254] In additional embodiments, the neurogenic monoamines are not intended to be exhaustive or inclu- agent in combination with a PDE agent may be a reported sive of the monoamines modulated in vivo, but rather as 50 agent for treating migraines. Non-limiting examples of general guidance for the skilled practitioner in selecting such an agent include a triptan, such as almotriptan or compounds for use in therapeutic methods provided almotriptan malate; naratriptan or naratriptan hydrochlo- herein. ride; rizatriptan or rizatriptan benzoate; sumatriptan or [0250] In embodiments relating to a biogenic amine sumatriptan succinate; zolmatriptan or zolmitriptan, fro- modulator used in a combination or method with a PDE 55 vatriptan or frovatriptan succinate; or eletriptan or elet- agent as disclosed herein, the modulator may be (i) a riptan hydrobromide. Embodiments of the disclosure norepinephrine and dopamine reuptake inhibitor, such may exclude combinations of triptans and an SSRI or as bupropion (described, e.g., in U.S. Pat. 3,819,706 and SNRI that result in life threatening serotonin syndrome.

39 77 EP 2 377 530 A2 78

[0255] Other non-limiting examples include an ergot derivative, such as dihydroergotamine or dihydroergot- amine mesylate, ergotamine or ergotamine tartrate; di- clofenac or diclofenac potassium or diclofenac sodium; flurbiprofen; amitriptyline; nortriptyline; divalproex or di- 5 valproex sodium; propranolol or propranolol hydrochlo- ride; verapamil; methysergide (CAS RN 361-37-5); meto- clopramide; prochlorperazine (CAS RN 58-38-8); aceta- minophen; topiramate; GW274150 ([2-[(1-iminoethyl) amino]ethyl]-L-homocysteine); or ganaxalone (CAS RN 10 [0260] (see Allan et al. "Therapeutic androgen recep- 38398-32-2). tor ligands" Nucl Recept Signal 2003; 1: e009); a phtha- [0256] Additional non-limiting examples include a lamide, such as a modulator as described by Miyachi et COX-2 inhibitor, such as Celecoxib. al. ("Potent novelnonsteroidal androgenantagonists with [0257] In other embodiments, the neurogenic agent in a phthalimide skeleton." Bioorg. Med. Chem. Lett. 1997 combination with a PDE agent may be a reported mod- 15 7:1483-1488); osaterone or osaterone acetate; hydrox- ulator of a nuclear hormone receptor. Nuclear hormone yflutamide; or a non-steroidal antagonist described in receptors are activated via ligand interactions to regulate U.S. Pat. No.6,017,924. gene expression, in some cases as part of cell signaling [0261] Other non-limiting examples of a reported mod- pathways. Non-limiting examples of a reported modula- ulator include a retinoic acid receptor agonist such as all- tor include a dihydrotestosterone agonist such as dihy- 20 trans retinoic acid (Tretinoin); isotretinoin (13- cis-retinoic drotestosterone; a 2-quinolone like LG121071 (4-ethyl- acid); 9-cis retinoic acid; bexarotene; 101 TAC- 1,2,3,4-tetrahydro-6-(trifluoromethyl)-8-pyridono[5,6-g]- (4-[3,5-bis (trimethylsilyl) benzamide] benzoic acid); AC- quinoline); a non-steroidal agonist or partial agonist com- 261066 (see Lund et al. "Discovery of a potent, orally pound described in U.S. Pat. No.6,017,924; LGD2226 available, and isoform-selective retinoic acid beta2 re- (see WO 01/16108, WO 01/16133, WO 01/16139, and 25 ceptor agonist." J Med Chem. 2005 (24):7517-9); 48 Rosen et al. "Novel, non-steroidal, selective androgen LGD1550 ((2E,4E,6E)-3-methyl-7-(3,5-di-ter-butylphen- receptor modulators (SARMs) with anabolic activity in yl)octatrienoic acid); E6060 (E6060 [4-{5-[7-fluoro- 4-(tri- bone and muscle and improved safety profile." J Musc- fluoromethyl)benzo[b]furan-2-yl]-1H-2-pyrrolyl}benzoic uloskelet Neuronal Interact. 2002(3): 222-4); 2 or acid]; agonist 1 or 2 as described by Schapira et al. ("In LGD2941 (from collaboration between Ligand Pharma- 30 silico discovery of novel Retinoic Acid Receptor agonist ceuticals Inc. and TAP Pharmaceutical Products Inc.). structures." BMC Struct Biol. 2001; 1: 1 (published online [0258] Additional non-limiting examples of a reported 2001 June 4) where "Agonist 1 was purchased from Bi- modulator include a selective androgen receptor modu- onet Research (catalog number1G- 433S).Agonist 2 was lator (SARM) such as andarine, ostarine, prostarin, or purchased from Sigma-Aldrich (Sigma Aldrich library of andromustine (all from GTx, Inc.); bicalutamide or a bi- 35 rare chemicals. Catalog number S08503-1"); a synthetic calutamide derivative such as GTx-007 (U.S. Pat. acetylenic retinoic acid, such as AGN 190121 (CAS RN: 6,492,554); or a SARM as described in U.S. Pat. 132032-67-8), AGN 190168 (or Tazarotene or CAS RN 6,492,554. 118292-40-3), or its metabolite AGN 190299 (CAS RN [0259] Further non-limiting examples of a reported 118292-41-4); Etretinate; acitretin; an acetylenic retin- modulator include an androgen receptor antagonist such 40 oate, such as AGN 190073 (CAS 132032-68-9), or AGN as cyproterone, bicalutamide, flutamide, or nilutamide; a 190089 (or 3-Pyridinecarboxylic acid, 6-(4-(2,6,6-trime- 2-quinolone such as LG120907, represented by the fol- thyl-1-cyclohexen-1-yl)-3-buten-1-ynyl)-, ethyl ester or lowing structure CAS RN 116627-73-7). [0262] In further embodiments, the additional agent for 45 use in combination with a PDE agent may be a reported modulator selected from thyroxin, tri-iodothyronine, or levothyroxine. [0263] Alternatively, the additional agent is a vitamin D (1,25-dihydroxyvitamine D3) receptor modulator, such 50 as calcitriol or a compound described in Ma et al. ("Iden- tification and characterization of noncalcemic, tissue- se- lective, nonsecosteroidal vitamin D receptor modula- tors." J Clin Invest 2006 116 (4):892-904) or Molnar et al. or a derivative compound represented by the following ("Vitamin D receptor agonists specifically modulate the structure 55 volume of the ligand- binding pocket." J Biol Chem. 2006 281(15):10516-26) or Milliken et al. ("EB1089, a vitamin D receptor agonist, reduces proliferation and decreases tumor growth rate in a mouse model of hormone-induced

40 79 EP 2 377 530 A2 80 mammary cancer." Cancer Lett. 2005 229 (2):205-15) or CAR receptor, such as 1,4 bis[2-(3,5- dichloropyridyloxy)] Yee et al. ("Vitamin D receptor modulators for inflamma- benzene (TCPOBOP); or a PXR receptor, such as SR- tion and cancer." Mini Rev Med Chem. 2005 5 (8):761-78) 12813 (tetra-ethyl 2-(3,5-di-tert-butyl-4-hydroxyphenyl) or Adachi et al. "Selective activation of vitamin D receptor ethenyl-1, 1-bisphosphonate). by lithocholic acid acetate, a bile acid derivative." J Lipid 5 [0269] In additional embodiments, the agent in combi- Res. 2005 46(1):46-57). nation with a PDE agent is ethyl eicosapentaenoate or [0264] Furthermore, the additional agent may be a re- ethyl-EPA (also known as 5,8,11,14,17- eicosapentaeno- ported cortisol receptor modulator, such as methylpred- ic acid ethyl ester or miraxion, CAS RN 86227-47-6), do- nisolone or its prodrug methylprednisolone suleptanate; cosahexaenoic acid (DHA), or a retinoid acid drug. As PI-1020 (NCX-1020 or budesonide-21-nitrooxymethyl- 10 an additional non-limiting example, the agent may be benzoate); fluticasone furoate; GW-215864; betameth- Omacor, a combination of DHA and EPA, or idebenone asone valerate; beclomethasone; prednisolone; or BVT- (CAS RN 58186-27-9). 3498 (AMG-311). [0270] In further embodiments, a reported nootropic [0265] Alternatively, the additional agent may be a re- compound may be used as an agent in combination with ported aldosterone (or mineralocorticoid) receptor mod- 15 a PDE agent. Non- limiting examples of such a compound ulator, such as Spironolactone or Eplerenone. include Piracetam (Nootropil), Aniracetam, Oxiracetam, [0266] In other embodiments, the additional agent may Pramiracetam, Pyritinol (Enerbol), Ergoloid mesylates be a reported progesterone receptor modulator such as (Hydergine), Galantamine or Galantamine hydrobro- Asoprisnil (CAS RN 199396-76-4); mesoprogestin or mide, Selegiline, Centrophenoxine (Lucidril), Desmo- J1042; J956; medroxyprogesterone acetate (MPA);20 pressin (DDAVP), Nicergoline, Vinpocetine, Picamilon, R5020; tanaproget; trimegestone; progesterone; norg- Vasopressin, Milacemide, FK-960, FK-962, levetira- estomet; melengestrol acetate; mifepristone; onapris- cetam, nefiracetam, or hyperzine A (CAS RN: tone; ZK137316; ZK230211 (see Fuhrmann et al. "Syn- 102518-79-6). thesis and biological activity of a novel, highly potent pro- [0271] Additional non-limiting examples of such a com- gesterone receptor antagonist." J Med Chem. 2000 43 25 pound include anapsos (CAS RN 75919-65-2), nebra- (26):5010-6); or a compound described in Spitz "Proges- cetam (CAS RN 97205-34-0 or 116041-13-5), metri- terone antagonists and progesterone receptor modula- fonate, ensaculin (or CAS RN 155773-59-4 or KA-672) tors: an overview." Steroids 2003 68(10-13):981-93. or ensaculin HCl, Rokan (CAS RN 122933-57-7 or EGb [0267] In further embodiments, the additional agent 761), AC-3933 (5-(3- methoxyphenyl)-3-(5-methyl-1,2,4- may be a reported i) peroxisome proliferator-activated 30 oxadiazol-3-yl)-2-oxo-1,2-dihydro-1,6-naphthyridine) or receptor (PPAR) agonist such as muraglitazar; tesagli- its hydroxylated metabolite SX-5745 (3-(5-hydroxyme- tazar; reglitazar; GW-409544 (see Xu et al. "Structural thyl-1,2,4-oxadiazol-3-yl)-5-(3-methoxyphenyl)-2-oxo- determinants of ligand binding selectivity between the 1,2-dihydro-1,6-naphthyridine), JTP-2942 (CAS RN peroxisome proliferator-activated receptors." Proc Natl 148152-77-6), sabeluzole (CAS RN 104383-17-7), Acad Sci USA. 2001 98(24):13919-24); or DRL 11605 35 ladostigil (CAS RN 209394-27-4), choline alphoscerate (Dr. Reddy’s Laboratories); ii) a peroxisome proliferator- (CAS RN 28319-77-9 or Gliatilin), Dimebon (CAS RN activated receptor alpha agonist like clofibrate; ciprofi- 3613-73-8), tramiprosate (CAS RN 3687-18-1), omigapil brate; fenofibrate; gemfibrozil; DRF-10945 (Dr. Reddy’s (CAS RN 181296-84-4), cebaracetam (CAS RN Laboratories); iii) a peroxisome proliferator- activated re- 113957-09-8), fasoracetam (CAS RN 110958-19-5), PD- ceptor delta agonist such as GW501516 (CAS RN40 151832 (see Jaen et al. "In vitro and in vivo evaluation 317318-70-0); or iv) a peroxisome proliferator-activated of the subtype-selective muscarinic agonist PD 151832." gamma receptor agonist like a hydroxyoctadecadienoic Life Sci. 1995 56(11-12):845-52), Vinconate (CAS RN acid (HODE); a prostaglandin derivative, such as 15- de- 70704-03-9), PYM-50028 PYM-50028 (Cogane) or oxy-Delta12,14-prostaglandin J2; a thiazolidinedione (gl- PYM-50018(Myogane) as described by Harvey ("Natural itazone), such as pioglitazone, troglitazone; rosiglitazone 45 Products in Drug Discovery and Development. 27-28 or rosiglitazone maleate; ciglitazone; Balaglitazone or June 2005, London, UK." IDrugs. 2005 8 (9):719-21), SR- DRF-2593; AMG 131 (from Amgen); or G1262570 (from 46559A (3-[N-(2 diethyl-amino-2-methylpropyl)-6-phe- Glaxo Wellcome). In additional embodiments, a PPAR nyl-5-propyl), dihydroergocristine (CAS RN 17479-19-5), ligand is a PPARγ antagonist such as T0070907 (CAS dabelotine (CAS RN 118976-38-8), zanapezil (CAS RN RN 313516-66-4) or GW9662 (CAS RN 22978-25-2). 50 142852-50-4). [0268] In additional embodiments, the additional agent [0272] Further non-limiting examples include NBI- 113 may be a reported modulator of an "orphan" nuclear hor- (from Neurocrine Biosciences, Inc.), NDD- 094 (from No- mone receptor. Embodiments include a reported modu- vartis), P-58 or P58 (from Pfizer), or SR- 57667 (from Sa- lator of a liver X receptor, such as a compound described nofi-Synthelabo). in U.S. Pat. 6,924,311; a farnesoid X receptor, such as 55 [0273] Moreover, an agent in combination with a PDE GW4064 as described by Maloney et al. ("Identification agent may be a reported modulator of the nicotinic re- of a chemical tool for the orphan nuclear receptor FXR." ceptor. Non-limiting examples of such a modulator in- J Med Chem. 2000 43(16):2971-4); a RXR receptor, a clude nicotine, acetylcholine, carbamylcholine, epibati-

41 81 EP 2 377 530 A2 82 dine, ABT-418 (structurally similar to nicotine, with an cannabichromene (CBC), cannabicyclol (CBL), canna- ixoxazole moiety replacing the pyridyl group of nicotine), bivarol (CBV), tetrahydrocannabivarin (THCV), can- epiboxidine (a structural analogue with elements of both nabidivarin (CBDV), cannabichromevarin (CBCV), can- epibatidine and ABT-418), ABT- 594 (azetidine analogue nabigerovarin (CBGV), or cannabigerol monoethyl ether of epibatidine), lobeline, SSR-591813, represented by 5 (CBGM). the following formula [0278] In yet further embodiments, an agent used in combination with a PDE agent is a reported FAAH (fatty acid amide hydrolase) inhibitor. Non-limiting examples of reported inhibitor agents include URB597 car- (3’- 10 bamoyl- biphenyl- 3- yl- cyclohexylcarbamate); CAY10401 (1-oxazolo[4,5-b]pyridin-2-yl-9-octadecyn-1- one); OL-135 (1-oxo-1[5-(2-pyridyl)-2-yl]-7-phenylhep- tane); anandamide (CAS RN 94421-68-8); AA- 5-HT (see Bisogno et al. "Arachidonoylserotonin and other novel 15 inhibitors of fatty acid amide hydrolase." Biochem Bio- phys Res Commun. 1998 248(3):515-22); 1-Octanesul- or SIB-1508 (altinicline). fonyl fluoride; or O-2142 or another arvanil derivative [0274] In additional embodiments, an agent used in FAAH inhibitor as described by Di Marzo et al. ("A struc- combination with a PDE agent is a reported aromatase ture/activity relationship study on arvanil, an endocan- inhibitor. Reported aromatase inhibitors include, but are 20 nabinoid and vanilloid hybrid." J Pharmacol Exp Ther. not limited to, nonsteroidal or steroidal agents. Non-lim- 2002 300(3):984-91). iting examples of the former, which inhibit aromatase via [0279] Further non-limiting examples include SSR the heme prosthetic group, include anastrozole (Arimi- 411298 (fromSanofi-Aventis), JNJ28614118(from John- dex®), letrozole (Femara®), or vorozole (Rivisor). Non- son & Johnson), or SSR 101010 (from Sanofi-Aventis) limiting examples of steroidal aromatase inhibitors AIs, 25 [0280] In additional embodiments, an agent in combi- which inactivate aromatase, include, but are not limited nation with a PDE agent may be a reported modulator of to, exemestane (Aromasin®), androstenedione, or form- nitric oxide function. One non-limiting example is silde- estane (lentaron). nafil (Viagra®). [0275] Additional non-limiting examples of a reported [0281] In additional embodiments, an agent in combi- aromatase for use in a combination or method as dis- 30 nation with a PDE agent may be a reported modulator of closed herein include aminoglutethimide, 4-androstene- prolactin or a prolactin modulator. 3,6,17-trione (or "6-OXO"), or zoledronic acid or Zometa [0282] In additional embodiments, an agent in combi- (CAS RN 118072-93-8). nation with a PDE agent is a reported anti-viral agent, [0276] Further embodiments include a combination of with ribavirin and amantadine as non-limiting examples. a PDE agent and a reported selective estrogen receptor 35 [0283] In additional embodiments, an agent in combi- modulator (SERM) may be used as described herein. nation with a PDE agent may be a component of a natural Non-limiting examples include tamoxifen, raloxifene, product or a derivative of such a component. In some toremifene, clomifene, bazedoxifene, arzoxifene, or la- embodiments, the component or derivative thereof is in sofoxifene. Additional non-limiting examples include a an isolated form, such as that which is separated from steroid antagonist or partial agonist, such as centchro- 40 one or more molecules or macromolecules normally man, clomiphene, or droloxifene), found with the component or derivative before use in a [0277] In other embodiments, a combination of a PDE combination or method as disclosed herein. In other em- agent and a reported cannabinoid receptor modulator bodiments, the component or derivative is completely or may be used as described herein. Non- limiting examples partially purified from one or more molecules or macro- include synthetic cannabinoids, endogenous cannabi- 45 molecules normally found with the component or deriv- noids, or natural cannabinoids. In some embodiments, ative. Exemplary cases of molecules or macromolecules the reported cannabinoid receptor modulator is rimona- found with a component or derivative as described herein bant (SR141716 or Acomplia), nabilone, levonantradol, include a plant or plant part, an animal or animal part, marinol, or sativex (an extract containing both THC and and a food or beverage product. CBD). Non-limiting examples of endogenous cannabi- 50 [0284] Non-limiting examples such a component in- noids include arachidonyl ethanolamine (anandamide); clude folic acid; a flavinoid, such as as citrus flavonoid; analogs of anandamide, such as docosatetraenyleth- a flavonol, such as Quercetin, Kaempferol, Myricetin, or anolamide or homo-γ-linoenylethanolamide; N-acyl eth- Isorhamnetin; a flavone, such as Luteolin or Apigenin; a anolamine signalling lipids, such as the noncannabimi- flavanone, such as Hesperetin, Naringenin, or Eriodicty- metic palmitoylethanolamine or oleoylethanolamine; or 55 ol; a flavan-3-ol (including a monomeric, dimeric, or pol- 2-arachidonyl glycerol. Non-limiting examples of natural ymeric flavanol), such as (+)-Catechin, (+)-Gallocate- cannabinoids include tetrahydrocannabinol (THC), can- chin, (-)-Epicatechin, (-)-Epigallocatechin, (-)-Epicate- nabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), chin 3-gallate, (-)-Epigallocatechin 3- gallate, Theaflavin,

42 83 EP 2 377 530 A2 84

Theaflavin 3-gallate, Theaflavin 3’-gallate, Theaflavin 4,6,7-trimethylbenzofuranyl) acetic acid); L- carnosine; L- 3,3’ digallate, a Thearubigin, or Proanthocyanidin; an an- Histidine; glycine; flavocoxid (or LIMBREL); baicalin, op- thocyanidin, such as Cyanidin, Delphinidin, Malvidin, tionally with catechin (3,3’,4’,5,7-pentahydroxyflavan Pelargonidin, Peonidin, or Petunidin; an isoflavone, such (2R,3S form)), and/or its stereoisomer; masoprocol (CAS as daidzein, genistein, or glycitein; flavopiridol; a pre- 5 RN 27686-84-6); mesna (CAS RN 19767-45-4); probucol nylated chalcone, such as Xanthohumol; a prenylated (CAS RN 23288-49-5); silibinin (CAS RN 22888-70-6); flavanone, such as Isoxanthohumol; a non-prenylated sorbinil (CAS RN 68367-52-2); spermine; tangeretin chalcone, such as Chalconaringenin; a non-prenylated (CAS RN 481-53-8); butylated hydroxyanisole (BHA); flavanone, such as Naringenin; Resveratrol; or an anti- butylated hydroxytoluene (BHT); propyl gallate (PG); ter- oxidant neutraceutical (such as any present in chocolate, 10 tiary-butyl-hydroquinone (TBHQ); nordihydroguaiaretic like dark chocolate or unprocessed or unrefined choco- acid (CAS RN 500-38-9); astaxanthin (CAS RN late). 472-61-7); or an antioxidant flavonoid. [0285] Additional non-limiting examples include a [0291] Additional non-limiting examples include a vita- component of Gingko biloba, such as a flavo glycoside min, such as vitamin A (Retinol) or C (Ascorbic acid) or or a terpene. In some embodiments, the component is a 15 E (including Tocotrienol and/or Tocopherol); a vitamin flavanoid, such as a flavonol or flavone glycoside, or a cofactors or mineral, such as Coenzyme Q10 (CoQ10), quercetin or kaempferol glycoside, or rutin; or a terpe- Manganese, or Melatonin; a carotenoid terpenoid, such noid, such as ginkgolides A, B, C, or M, or bilobalide. as Lycopene, Lutein, Alpha-carotene, Beta-carotene, [0286] Further non-limiting examples include a com- Zeaxanthin, Astaxanthin, or Canthaxantin; a non- carote- ponent that is a flavanol, or a related oligomer, or 20 a noid terpenoid, such as Eugenol; a flavonoid polyphe- polyphenol as described in US2005/245601AA,nolic (or bioflavonoid); a flavonol, such as Resveratrol, US2002/018807AA, US2003/180406AA, US2002/0868- Pterostilbene (methoxylated analogue of resveratrol), 33AA, US2004/0236123, WO9809533, or WO9945788; Kaempferol, Myricetin, Isorhamnetin, a Proanthocyani- a procyanidin or derivative thereof or polyphenol as de- din, or a tannin; a flavone, such as Quercetin, rutin, Lute- scribed in US2005/171029AA; a procyanidin, optionally 25 olin, Apigenin, or Tangeritin; a flavanone, such as Hes- in combination with L-arginine as described peretin in or its metabolite hesperidin, naringenin or its pre- US2003/104075AA; a low fat cocoa extract as described cursor naringin, or Eriodictyol; a flavan-3-ols (anthocya- in US2005/031762AA; lipophilic bioactive compound nidins), such as Catechin, Gallocatechin, Epicatechin or containing composition as described in a US- gallate form thereof, Epigallocatechin or a gallate form 2002/107292AA; a cocoa extract, such as those contain- 30 thereof, Theaflavin or a gallate form thereof, or a Thearu- ing one or more polyphenols or procyanidins as de- bigin; an isoflavone phytoestrogens, such as Genistein, scribed in US2002/004523AA; an extract of oxidized tea Daidzein, or Glycitein; an anthocyanins, such as Cyani- leaves as described in US Pat. 5,139,802 or 5,130,154; din, Delphinidin, Malvidin, Pelargonidin, Peonidin, or Pe- a food supplement as described in WO 2002/024002. tunidin; a phenolic acid or ester thereof, such as Ellagic [0287] Of course a composition comprising any of the 35 acid, Gallic acid, Salicylic acid, Rosmarinic acid, Cinnam- above components, alone or in combination with a PDE ic acid or a derivative thereof like ferulic acid, Chlorogenic agent as described herein is included within the disclo- acid, Chicoric acid, a Gallotannin, or an Ellagitannin; a sure. nonflavonoid phenolic, such as Curcumin; an anthoxan- [0288] In additional embodiments, an agent in combi- thin, betacyanin, Citric acid, Uric acid, R- α-lipoic acid, or nation with a PDE agent may be a reported calcitonin 40 Silymarin. receptor agonist such as calcitonin or the ’orphan pep- [0292] Further non-limiting examples include 1-(car- tide’ PHM-27 (see Ma et al. "Discovery of novel peptide/ boxymethylthio)tetradecane; 2,2,5,7,8-pentamethyl-1- receptor interactions: identification of PHM-27 as a po- hydroxychroman; 2,2,6,6-tetramethyl-4-piperidinol-N- tent agonist of the human calcitonin receptor." Biochem oxyl; 2,5-di-tert-butylhydroquinone; 2-tert-butylhydroqui- Pharmacol. 2004 67(7):1279-84). A further non-limiting 45 none; 3,4-dihydroxyphenylethanol; 3-hydroxypyridine; example is the agonist from Kemia, Inc. 3-hydroxytamoxifen; 4-coumaric acid; 4-hydroxyanisole; [0289] In an alternative embodiment, the agent may 4-hydroxyphenylethanol; 4-methylcatechol; 5,6,7,8-tet- be a reported modulator of parathyroid hormone activity, rahydrobiopterin; 6,6’-methylenebis(2,2-dimethyl-4-me- such as parathyroid hormone, or a modulator of the par- thanesulfonic acid-1,2-dihydroquinoline); 6-hydroxy- athyroid hormone receptor. 50 2,5,7,8-tetramethylchroman-2-carboxylic acid; me- 6- [0290] In additional embodiments, an agent in combi- thyl-2-ethyl-3-hydroxypyridine; O- 6- palmitoylascorbic nation with a PDE agent may a reported antioxidant, such acid; acetovanillone; acteoside; Actovegin; allicin; allyl as N-acetylcysteine or acetylcysteine; disufenton sodium sulfide; alpha-pentyl-3-(2-quinolinylmethoxy)benzene- (or CAS RN 168021-79-2 or Cerovive); activin (CAS RN methanol; alpha-tocopherol acetate; apolipoprotein A- 104625-48-1); selenium; L-methionine; an alpha, gam- 55 IV; bemethyl; boldine; bucillamine; Calcium Citrate; Can- ma, beta, or delta, or mixed, tocopherol; alpha lipoic acid; thaxanthin; crocetin; diallyl trisulfide; dicarbine; dihydrol- Coenzyme Q; Benzimidazole; benzoic acid; dipyrida- ipoic acid; dimephosphon; ebselen; Efamol; enkephalin- mole; glucosamine; IRFI-016 (2(2,3-dihydro-5-acetoxy- Leu, Ala(2)-Arg(6)-; Ergothioneine; esculetin; essential

43 85 EP 2 377 530 A2 86

303 forte; Ethonium; etofyllinclofibrate; fenozan; glau- nylpropanolamine; or pseudoephedrine. cine; H290-51; histidyl-proline diketopiperazine; hydro- [0297] Further non-limiting embodiments include a re- quinone; hypotaurine; idebenone; indole-3-carbinol; iso- ported modulator of a beta adrenergic receptor such as ascorbic acid; kojic acid, lacidipine, lodoxamide trometh- arbutamine, befunolol, cimaterol, higenamine, isoxsu- amine; mexidol; morin; N’- N, diphenyl-4-phenylenedi- 5 prine, methoxyphenamine, oxyfedrine, ractopamine, tre- amine; N-isopropyl-N-phenyl-4-phenylenediamine; N- toquinol, or TQ-1016 (from TheraQuest Biosciences, monoacetylcystine; nicaraven, nicotinoyl-GABA; niteca- LLC), or a reportedβ 1-adrenergic receptor modulator pone; nitroxyl; nobiletin; oxymethacil; p-tert-butyl cate- such as prenalterol, Ro 363, or xamoterol or a reported chol; phenidone; pramipexol; proanthocyanidin; procya- β1-adrenergic receptor agonist like dobutamine. nidin; prolinedithiocarbamate; Propyl Gallate; purpuro- 10 [0298] Alternatively, the reported modulator may be of gallin; pyrrolidine dithiocarbamicacid; rebamipide; retinol a β2-adrenergic receptor such as levosalbutamol (CAS palmitate; salvin; Selenious Acid; sesamin; sesamol; so- RN 34391-04-3), metaproterenol, MN-221 or KUR- 1246 dium selenate; sodium thiosulfate; theaflavin; thiazolid- ((-)-bis(2-{[(2S)-2-({(2R)-2-hydroxy-2-[4-hydroxy-3-(2- ine-4-carboxylic acid; tirilazad; tocopherylquinone; tocot- hydroxyethyl) phenyl] ethyl}amino)-1,2,3,4-tetrahydro- rienol, alpha; a Tocotrienol; tricyclodecane-9-yl-xantho- 15 naphthalen-7-yl]oxy}-N,N-dimethylacetamide)monosul- genate; turmeric extract; U 74389F; U 74500A; U fate or (2-[[(2S)- bis 2-([(2R)-2-hydroxy-2-[4-hydroxy- 78517F; ubiquinone 9; vanillin; vinpocetine; xylometazo- 3-(2-hydroxyethyl)-phenyl]ethyl]amino)-1,2,3,4-tetrahy- line; zeta Carotene; zilascorb; zinc thionein; or zonisa- dronaphthalen-7-yl]oxy]-N,N-dimethylacetamide) sul- mide. fate or CAS RN 194785-31-4), nylidrin, orciprenaline, pir- [0293] In additional embodiments, an agent in combi- 20 buterol, procaterol, reproterol, ritodrine, salmeterol, sal- nation with a PDE agent may be a reported modulator of meterol xinafoate, terbutaline, tulobuterol, zinterol or bro- a norepinephrine receptor. Non-limiting examples in- moacetylalprenololmenthane, or a reported β2-adrener- clude Atomoxetine (Strattera); a norepinephrine re- gic receptor agonist like albuterol, albuterol sulfate, salb- uptake inhibitor, such as talsupram, tomoxetine,utamol (CAS RN 35763-26-9), clenbuterol, broxaterol, nortriptyline, nisoxetine, reboxetine (described, e.g., in 25 dopexamine, formoterol, formoterol fumarate, iso- U.S. Pat. 4,229,449), or tomoxetine (described, e.g., in etharine, levalbuterol tartrate hydrofluoroalkane, or U.S. Pat. 4,314,081); or a direct agonist, such as a beta mabuterol. adrenergic agonist. [0299] Additional non-limiting embodiments include a [0294] Additional non-limiting examples include an al- reported modulator of a β3-adrenergic receptor such as pha adrenergic agonist such as etilefrine or a reported 30 AJ-9677 or TAK677 ([3-[(2R)-[[(2R)-(3-chlorophenyl)-2- agonist of the α2-adrenergic receptor (or α2 adrenocep- hydroxyethyl]amino]propyl]-1H-indol-7-yloxy]acetic ac- tor) like clonidine (CAS RN 4205-90-7), yohimbine, mir- id), or a reportedβ 3-adrenergic receptor agonist like tazepine, atipamezole, carvedilol; dexmedetomidine or SR58611A (described in Simiand et al., Eur J Pharmacol, dexmedetomidine hydrochloride; ephedrine, epine- 219:193-201 (1992), BRL 26830A, BRL 35135, BRL phrine; etilefrine; lidamidine; tetramethylpyrazine; tizani- 35 37344, CL 316243 or ICI D7114. dine or tizanidine hydrochloride; apraclonidine; bitolterol [0300] Further alternative embodiments include a re- mesylate; brimonidine or brimonidine tartrate; dipivefrin ported nonselective alpha and beta adrenergic receptor (which is converted to epinephrine in vivo); guanabenz; agonist such as epinephrine or ephedrine; a reported guanfacine; methyldopa; alphamethylnoradrenaline; nonselective alpha and beta adrenergic receptor antag- mivazerol; natural ephedrine or D(-)ephedrine; any one 40 onist such as carvedilol; a β1 and β2 adrenergic receptor or any mixture of two, three, or four of the optically active agonist such as isopreoterenol; or a β1 and β2 adrenergic forms of ephedrine; CHF1035 or nolomirole hydrochlo- receptor antagonist such as CGP 12177, fenoterol, or ride (CAS RN 138531-51-8); or lofexidine (CAS RN hexoprenaline. 31036-80-3). [0301] In further embodiments, an agent in combination [0295] Alternative non-limiting examples include an 45 with a PDE agent may be a reported modulator of carbonic adrenergic antagonist such as a reported antagonist of anhydrase. Non-limiting examples of such an agent in- the α2-adrenergic receptor like yohimbine (CAS RN clude acetazolamide, benzenesulfonamide, benzola- 146-48-5) or yohimbine hydrochloride, idazoxan, flupar- mide, brinzolamide, dichlorphenamide, dorzolamide or oxan,mirtazepine, atipamezole, orRX781094 (see Elliott dorzolamide HCl, ethoxzolamide, flurbiprofen, mafenide, et al. "Peripheral pre and postjunctional alpha 2-adren- 50 methazolamide, sezolamide, zonisamide, bendroflume- oceptors in man: studies with RX781094, a selective al- thiazide, benzthiazide, chlorothiazide, cyclothiazide, dan- pha 2 antagonist." J Hypertens Suppl. 1983 1 (2):109-11). sylamide, diazoxide, ethinamate, furosemide, hydrochlo- [0296] Other non-limiting embodiments include a re- rothiazide, hydroflumethiazide, mercuribenzoic acid, ported modulator of an α1-adrenergic receptor such as methyclothiazide, trichloromethazide, amlodipine, cyana- cirazoline; modafinil; ergotamine; metaraminol; methox- 55 mide, or a benzenesulfonamide. Additional non-limitinge amine; midodrine (a prodrug which is metabolized to the examples of such an agent include (4s-Trans)-4-(Ethyl- major metabolite desglymidodrine formed by deglycina- amino)- 5,6- Dihydro- 6- Methyl- 4h- Thieno (2,3- B) Thi- tion of midodrine); oxymetazoline; phenylephrine; phe- opyran-2-Sulfonamide-7,7-Dioxide; (4s-Trans)-4-(Meth-

44 87 EP 2 377 530 A2 88 ylamino)- 5,6- Dihydro- 6- Methyl- 4h- Thieno (2,3- B) Thi- clopamine, jervine, ezetimibe, regadenoson (CAS RN opyran-2-Sulfonamide-7,7-Dioxide; (R)-N-(3-Indol-1-Yl- 313348-27-5, or CVT-3146), a compound described in 2-Methyl-Propyl)-4-Sulfamoyl-Benzamide; (S)-N-(3-In- U.S. Pat. 6,683,192 or identified as described in U.S. Pat. dol-1-Yl-2-MethylPropyl)-4-Sulfamoyl-Benzamide; 1,2,4- 7,060,450, or CUR-61414 or another compound de- Triazole; 1-Methyl-3-Oxo-1,3-Dihydro-Benzo[C]Isothia- 5 scribed in U.S. Pat. 6,552,016. zole-5-Sulfonic Acid Amide; 2,6- Difluorobenzenesulfona- [0304] In other embodiments, an agent in combination mide; 3,5-Difluorobenzenesulfonamide; 3-Mercuri-4- with a PDE agent may be a reported modulator of IMPDH, Aminobenzenesulfonamide; 3-Nitro-4-(2-OxoPyrrolidin- such as mycophenolic acid or mycophenolate mofetil 1- Yl)-Benzenesulfonamide; 4-(Aminosulfonyl)-N-[(2,3,4- (CAS RN 128794-94-5). Trifluorophenyl)Methyl]-Benzamide; 4-(Aminosulfonyl)- 10 [0305] In yet additional embodiments, an agent in com- N-[(2,4,6-Trifluorophenyl)Methyl]-Benzamide; 4-(Amino- bination with a PDE agent may be a reported modulator sulfonyl)-N-[(2,4- Difluorophenyl) Methyl]-Benzamide; of a sigma receptor, including sigma-1 and sigma- 2. Non- 4-(Aminosulfonyl)-N-[(2,5- Difluorophenyl) Methyl]-Ben- limiting examples of such a modulator include an agonist zamide; 4-(Aminosulfonyl)-N-[(3,4,5-Trifluorophenyl)Me- of sigma-1 and/or sigma-2 receptor, such as (+)-penta- thyl]-Benzamide; 4-(Aminosulfonyl)-N-[(4-Fluorophenyl) 15 zocine, SKF 10,047 (N-allylnormetazocine), or 1,3-di-o- Methyl]-Benzamide; 4-(Hydroxymercury)Benzoic Acid; 4- tolylguanidine (DTG). Additional non-limiting examples Flourobenzenesulfonamide; 4-Methylimidazole; 4-Sul- include SPD-473 (from Shire Pharmaceuticals); a mole- fonamide-[1-(4-Aminobutane)]Benzamide; Sulfona- 4- cule with sigma modulatory activity as known in the field mide-[4-(Thiomethylaminobutane)]Benzamide; 5-Aceta- (see e.g., Bowen et al., Pharmaceutica Acta Helvetiae mido-1,3,4-Thiadiazole-2-Sulfonamide; 6-Oxo-8,9,10,20 74: 211-218 (2000)); a guanidine derivative such as 11- Tetrahydro- 7h- Cyclohepta [C] [1] Benzopyran- 3- O- those described in U.S. Pat. Nos. 5,489,709; 6,147,063; Sulfamate; (4-sulfamoyl-phenyl)-thiocarbamic acid O-(2- 5,298,657; 6,087,346; 5,574,070; 5,502,255; 4,709,094; thiophen-3-yl-ethyl) ester; (R)-4-ethylamino-3,4-dihydro- 5,478,863; 5,385,946; 5,312,840; or 5,093,525; 2-(2-methoylethyl)-2H-thieno[3,2-E]-1,2-thiazine-6-sul- WO9014067;an antipsychotic with activity at one ormore fonamide-1,1-dioxide; 3,4-dihydro-4-hydroxy-2-(2-thie- 25 sigma receptors, such as haloperidol, rimcazole, per- nymethyl)-2H-thieno[3,2-E]-1,2-thiazine-6-sulfonamide- phenazine, fluphenazine, (-)-butaclamol, acetophena- 1,1-dioxide; 3,4-dihydro-4-hydroxy-2-(4-methoxyphe- zine, trifluoperazine, molindone, pimozide, thioridazine, nyl)-2H-thieno[3,2-E]-1,2-thiazine-6-sulfonamide-1,1-di- chlorpromazine and triflupromazine, BMY 14802, BMY oxide; N-[(4-methoxyphenyl)methyl]2,5-thiophenedesul- 13980, remoxipride, tiospirone, cinuperone (HR 375), or fonamide; 2-(3-methoxyphenyl)-2H-thieno-[3,2-E]-1,2- 30 WY47384. thiazine-6-sulfinamide-1,1-dioxide; (R)-3,4-didhydro-2- [0306] Additional non-limiting examples include igme- (3-methoxyphenyl)-4-methylamino-2H-thieno[3,2-E]-1,2- sine; BD1008 and related compounds disclosed in U.S. thiazine-6-sulfonamide-1,1-dioxide; (S)-3,4-dihydro-2-(3- Publication No. 20030171347; cis-isomers of U50488 methoxyphenyl)- 4- methylamino- 2H- thieno [3,2- E]- 1,2- and related compounds described in de Costa et al, J. thiazine-6-sulfonamide-1,1-dioxide; 3"4-dihydro-2-(3- 35 Med. Chem., 32(8): 1996-2002 (1989); U101958; methoxyphenyl)-2H-thieno-[3,2-E]-1,2-thiazine-6-sulfon- SKF10,047; apomorphine; OPC- 14523 and related com- amide-1,1-dioxide; [2h-Thieno[3,2-E]-1,2-Thiazine-6- pounds described in Oshiro et al., J Med Chem.; 43(2): Sulfonamide, 2-(3- Hydroxyphenyl)- 3-(4- Morpholinyl)-, 177-89 (2000); arylcyclohexamines such as PCP; 1,1-Dioxide]; [2h-Thieno[3,2-E]-1,2-Thiazine-6-Sulfona- (+)-morphinans such as dextrallorphan; phenylpiperid- mide,2-(3-Methoxyphenyl)-3-(4-Morpholinyl)-, 1,1-Diox- 40 ines such as (+)-3- PPP and OHBQs; neurosteroids such ide]; Aminodi(Ethyloxy)Ethylaminocarbonylbenzenesul- as progesterone and desoxycorticosterone; butryophe- fonamide; N-(2,3,4,5,6-Pentaflouro-Benzyl)-4-Sulfamo- nones; BD614; or PRX-00023. Yet additional non-limiting yl-Benzamide; N-(2,6-Diflouro-Benzyl)-4-Sulfamoyl-Ben- examples include a compound described in U.S. Pat. zamide; N-(2-FLOURO-BENZYL)-4-SULFAMOYL-BEN- Nos. 6,908,914; 6,872,716; 5,169,855; 5,561,135; ZAMIDE; N-(2-Thienylmethyl)-2,5-Thiophenedisulfona- 45 5,395,841; 4,929,734; 5,061,728; 5,731,307; 5,086,054; mide; N-[2-(1H-INDOL-5-YL)-BUTYL]-4-SULFAMOYL- 5,158,947; 5,116,995; 5,149,817; 5,109,002; 5,162,341; BENZAMIDE; N-Benzyl-4-Sulfamoyl-Benzamide; or Sul- 4,956,368; 4,831,031; or 4,957,916; U.S. Publication famic Acid 2,3-O-(1-Methylethylidene)-4,5-O-Sulfonyl- Nos. 20050132429; 20050107432; 20050038011, Beta-Fructopyranose Ester. 20030105079; 20030171355; 20030212094; or [0302] In yet additional embodiments, an agent in com- 50 20040019060; European Patent Nos. EP 503 411; EP bination with a PDE agent may be a reported modulator 362 001-A1; or EP 461 986; International Publication of a catechol-O-methyltransferase (COMT), such as flo- Nos. WO 92/14464; WO 93/09094; WO 92/22554; WO proprione, or a COMT inhibitor, such as tolcapone (CAS 95/15948; WO 92/18127; 91/06297; WO01/02380; RN 134308-13-7), nitecapone (CAS RN 116313-94-1), WO91/18868; or WO 93/00313; or in Russell et al., J or entacapone(CAS RN 116314-67-1 or 130929-57-6). 55 Med Chem.; 35(11): 2025-33 (1992) or Chambers et al., [0303] In yet further embodiments, an agent in combi- J.Med Chem.; 35(11): 2033-9 (1992). nation with a PDE agent may be a reported modulator of [0307] Further non-limiting examples include a sigma- hedgehog pathway or signaling activity such as cy- 1 agonist, such as IPAG (1-(4-iodophenyl)-3-(2-ada-

45 89 EP 2 377 530 A2 90 mantyl)guanidine); pre-084; carbetapentane; 4-IBP; L- ence to the following examples which are provided by 687,384 and related compounds described in Middlem- way of illustration, and are not intended to be limiting of iss et al., Br. J. Pharm., 102: 153 (1991); BD 737 and the disclosed invention, unless specified. related compounds described in Bowen et al., J Pharma- col Exp Ther., 262(1): 32-40 (1992)); OPC-14523 or a 5 EXAMPLES related compound described in Oshiro et al., J Med Chem.; 43(2): 177-89 (2000); a sigma-1 selective ago- Example 1 - Effect of combining captopril and ibudilast nist, such as igmesine; (+)-benzomorphans, such as on neuronal differentiation of human neural stem cells (+)-pentazocine and (+)-ethylketocyclazocine; SA-4503 or a related compound described in U.S. Pat. No.10 [0312] Human neural stem cells (hNSCs) were isolat- 5,736,546 or by Matsuno et al., Eur J Pharmacol., 306 ed and grown in monolayer culture, plated, treated with (1-3): 271-9 (1996); SK&F 10047; or ifenprodil; a sigma- varying concentrations of captopril and/or ibudilast (test 2 agonist, such as haloperidol, (+)-5,8-disubstituted mor- compounds), and stained with TUJ-1 antibody, as de- phan-7-ones, including CB 64D, CB 184, or a related scribed in U.S. Provisional Application No. 60/697,905 compound described in Bowen et al., Eur. J. Parmacol. 15 (incorporated by reference). Mitogen- free test media with 278:257-260 (1995) or Bertha et al., J. Med. Chem. 38: a positive control for neuronal differentiation was used 4776-4785 (1995); or a sigma-2 selective agonist, such along with basal media without growth factors as a neg- as 1-(4-fluorophenyl)-3-[4-[3-(4-fluorophenyl)-8-azabi- ative control. cyclo[3.2.1]oct-2- en- 8-yl]-1-butyl]-1H-indole, Lu 28-179, [0313] Results are shown in Figure 1, which shows Lu 29-253 or a related compound disclosed in U.S. Pat. 20 concentration response curves of neuronal differentia- Nos. 5,665,725 or 6,844,352, U.S. Publication No. tion after background media values are subtracted. The 20050171135, International Patent Publication Nos. WO concentration response curve of the combination of cap- 92/22554 or WO 99/24436, Moltzen et al., J. Med Chem., topril and ibudilast is shown with the concentration re- 26; 38(11): 2009-17 (1995) or Perregaard et al., J Med sponse curves of captropril or ibudilast alone. The data Chem., 26; 38(11): 1998-2008 (1995). 25 is presented as a percent of neuronal positive control. [0308] Alternative non-limiting examples include a sig- The data indicate that the combination of captopril and ma-1 antagonist such as BD-1047 (N(-)[2-(3,4-dichlo- ibudilast resulted in superior promotion of neuronal dif- rophenyl)ethyl]-N-methyl-2-(dimethylamin- ethyl- o) ferentiation than either agent alone. amine), BD-1063 (-) (1[2-(3,4-dichlorophenyl)ethyl]-4- methylpiperazine, rimcazole, haloperidol, BD-1047, BD- 30 Example 2 - Effect ofcombining captopril and enoximone 1063, BMY 14802, DuP 734, NE-100, AC915, or R-(+)- on neuronal differentiation of human neural stem cells 3-PPP. Particular non-limiting examples include fluoxe- tine, fluvoxamine, citalopram, sertaline, clorgyline, imi- [0314] Human neural stem cells (hNSCs) were isolat- pramine, igmesine, opipramol, siramesine, SL 82.0715, ed and grown in monolayer culture, plated, treated with imcazole, DuP 734, BMY 14802, SA 4503, OPC 14523, 35 varying concentrations of captopril and/or enoximone panamasine, or PRX-00023. (test compounds), and stained with TUJ-1 antibody, as [0309] Other non-limiting examples of an agent in com- described in U.S. Provisional Application No. 60/697,905 bination with a PDE agent include acamprosate (CAS (incorporated by reference). RN 77337-76-9); a growth factor, like LIF, EGF, FGF, [0315] Results are shown in Figure 2, which shows bFGF or VEGF as non-limiting examples; octreotide 40 concentration response curves of neuronal differentia- (CAS RN 83150-76-9); an NMDA modulator like keta- tion after background media values are subtracted. The mine, DTG, (+)-pentazocine, DHEA, Lu 28-179concentration response curve of the combination of cap- (1’-[4-[1-(4- fluorophenyl)- 1H- indol- 3- yl]- 1- butyl]-spiro topril and enoximone is shown with the concentration re- [isobenzofuran-1(3H), 4’piperidine]), BD 1008 (CAS RN sponse curves of captropril or enoximone alone.The data 138356-08-8), ACEA1021 (Licostinel or CAS 45 RN is presented as a percent of neuronal positive control. 153504-81-5), GV150526A (Gavestinel or CAS RN The data indicate that the combination of captopril and 153436-22-7), sertraline, clorgyline, or memantine as enoximone resulted in superior promotion of neuronal non-limiting examples; or metformin. differentiation than either agent alone. [0310] Of course a further combination therapy may also be that of a PDE agent, optionally in combination 50 Example 3- Effect of combining serotonin and enoxi- with one or more other neurogenic agents, with a non- mone on neuronal differentiation of human neural stem chemical based therapy. Non-limiting examples include cells the use of psychotherapy for the treatment of many con- ditions described herein, such as the psychiatric condi- [0316] Human neural stem cells (hNSCs) were isolat- tions, as well as behavior modification therapy such as 55 ed and grown in monolayer culture, plated, treated with that use in connection with a weight loss program. varying concentrations of serotonin and/or enoximone [0311] Having now generally described the invention, (test compounds), and stained with TUJ-1 antibody, as the same will be more readily understood through refer- described in Example 2.

46 91 EP 2 377 530 A2 92

[0317] Results are shown in Figure 3, which shows [0323] Results are shown in Figure 6, which shows concentration response curves of neuronal differentia- concentration response curves of neuronal differentia- tion after background media values are subtracted. The tion after background media values are subtracted. The concentration response curve of the combination of se- concentration response curve of the combination of ibud- rotonin and enoximone is shown with the concentration 5 ilast and candesartan is shown with the concentration response curves of serotonin or enoximone alone. The response curves of ibudilast or candesartan alone. The data is presented as a percent of neuronal positive con- data is presented as a percent of neuronal positive con- trol. The data indicate that the combination of serotonin trol. The data indicate that the combination of ibudilast and enoximone resulted in superior promotion of neuro- and candesartan resulted in superior promotion of neu- nal differentiation than either agent alone. 10 ronal differentiation than either agent alone.

Example 4- Effect of combining serotonin and rolipram Example 7 - Determination of Synergy on neuronal differentiation of human neural stem cells [0324] The presence of synergy was determined by [0318] Human neural stem cells (hNSCs) were isolat- 15 use of a combination index (CI). The CI based on the ed and grown in monolayer culture, plated, treated with EC50 as used to determine whether a pair of compounds varying concentrations of serotonin and/or rolipram (test had an additive, synergistic (greater than additive), or compounds), and stained with TUJ-1 antibody, as de- antagonistic effect when run in combination. The CI is a scribed in Examples 2 and 3. quantitative measure of the nature of drug interactions, 20 [0319] Results are shown in Figure 4, which shows comparing the EC50’s of two compounds, when each is concentration response curves of neuronal differentia- assayed alone, to the EC 50 of each compound when as- tion after background media values are subtracted. The sayed in combination. The combination index (CI) is concentration response curve of the combination of se- equal to the following formula: rotonin and rolipram is shown with the concentration re- sponse curves of serotonin or rolipram alone. The data 25 is presented as a percent of neuronal positive control. The data indicate that the combination of serotonin and rolipram resulted in superior promotion of neuronal dif- ferentiation than either agent alone. 30 [0325] where C1 and C2 are the concentrations of a Example 5- Effect of combining buspirone and rolipram first and a second compound, respectively, resulting in on neuronal differentiation of human neural stem cells 50% activity in neuronal differentiation when assayed in combination; and IC1 and IC2 are the concentrations of [0320] Human neural stem cells (hNSCs) were isolat- each compound resulting in 50% activity when assayed ed and grown in monolayer culture, plated, treated with 35 independently. A CI of less than 1 indicates the presence varying concentrations of buspirone and/or rolipram (test of synergy; a CI equal to 1 indicates an additive effect; compounds), and stained with TUJ-1 antibody, as de- and a CI greater than 1 indicates antagonism between scribed in Examples 2 to 4. the two compounds. [0321] Results are shown in Figure 5, which shows [0326] Non-limiting examples of combinations of a concentration response curves of neuronal differentia- 40 PDE inhibitor and an additionalagent asdescribed herein tion after background media values are subtracted. The were observed to result in synergistic activity. The exem- concentration response curve of the combination of bus- plary results, based on FIG. 1 to 6, are shown in the pirone and rolipram is shown with the concentration re- following table. sponse curves of buspirone or rolipram alone. The data is presented as a percent of neuronal positive control. 45 Figure Combo CI The data indicate that the combination of buspirone and Fig. 1 Ibudilast-Captopril 0.05 rolipram resulted in superior promotion of neuronal dif- ferentiation than either agent alone. Fig. 2 Captopril-Enoximone 0.50 Fig. 3 Serotonin-Enoximone 0.22 Example 6- Effect of combining ibudilast and cande- 50 sartan on neuronal differentiation of human neural stem Fig. 4 Serotonin-Rolipram 0.35 cells Fig. 5 Buspirone-Rolipram 0.42

[0322] Human neural stem cells (hNSCs) were isolat- Fig. 6 Ibudilast-Candesartan 0.18 ed and grown in monolayer culture, plated, treated with 55 varying concentrations of ibudilast and/or candesartan [0327] As the CI is less than 1 for each of these com- (test compounds), and stained with TUJ-1 antibody, as binations, the two compounds have a synergistic effect described in Example 1.

47 93 EP 2 377 530 A2 94 in neuronal differentiation. 6. The method of Embodiment 1, wherein said neu- [0328] The above is based on the selection of EC 50 as rogenesis comprises differentiation of neural stem the point of comparison for the two compounds. The com- cells (NSCs) along a neuronal lineage. parison is not limited by the point used, but rather the 7. The method of Embodiment 1, wherein said neu- same comparison may be made at another point, such 5 rogenesis comprises differentiation of neural stem as EC20, EC30, EC40, EC60, EC70, EC80, or any other EC cells (NSCs) along a glial lineage. value above, below, or between any of those points. 8. The method of Embodiment 1, wherein said PDE [0329] All references cited herein, including patents, agent is ibudilast. patent applications, and publications, are hereby incor- 9. The method of Embodiment 1, wherein said PDE porated by reference in their entireties, whether previ- 10 agent is an inhibitor of PDE3 or PDE4. ously specifically incorporated or not. 10. The method of Embodiment 9, wherein said [0330] Having now fully provided the instant disclo- agent is enoximone or rolipram. sure, it will be appreciated by those skilled in the art that 11. The method of Embodiment 2 or 3, wherein said the same can be performed within a wide range of equiv- cell or tissue exhibits decreased neurogenesis or is alent parameters, concentrations, and conditions without 15 subjected to an agent which decreases or inhibits departing from the spirit and scope of the disclosure and neurogenesis. without undue experimentation. 12. The method of Embodiment 11, wherein said [0331] While the disclosure has been described in con- agent which decreases or inhibits neurogenesis is nection with specific embodiments thereof, it will be un- an opioid receptor agonist. derstood that it is capable of further modifications. This 20 13. The method of Embodiment 12, wherein said opi- application is intended to cover any variations, uses, or oid receptor agonist is morphine or another opiate. adaptations of the disclosure following, in general, the 14. The method of Embodiment 2 or 3 wherein said disclosed principles and including such departures from subject or patient has one or more chemical addic- the disclosure as come within known or customary prac- tion or dependency. tice within the art to which the disclosure pertains and as 25 15. A method of treating a nervous system disorder may be applied to the essential features hereinbefore set related to cellular degeneration, a psychiatric condi- forth. tion, cellular trauma and/or injury, or another neuro- logically related condition in a subject or patient, said EMBODIMENTS OF THE INVENTION method comprising 30 administering a PDE agent in combination with one [0332] or more other neurogenic agents, to produce an im- provement in said disorder in said subject or patient. 1. A method of stimulating or increasing neurogen- 16. The method of Embodiment 15, wherein said esis in a cell or tissue, said method comprising nervous system disorder related to cellular degen- contacting said cell or tissue with a PDE agent in 35 eration is selected from a neurodegenerative disor- combination with one or more other neurogenic der, a neural stem cell disorder, a neural progenitor agents, cell disorder, a degenerative disease of the retina, wherein the agent or combination is effective to pro- an ischemic disorder, and combinations thereof. duce neurogenesis in said cell or tissue. 17. The method of Embodiment 15, wherein said 2. The method of Embodiment 1, wherein said cell 40 nervous system disorder related to a psychiatric con- or tissue is in an animal subject or a human patient. dition is selected from a neuropsychiatric disorder, 3. The method of Embodiment 2, wherein said pa- and affective disorder, depression, hypomania, pan- tient has been identified as in need of neurogenesis ic attacks, anxiety, excessive elation, bipolar depres- or has been diagnosed with a disease, condition, or sion, bipolar disorder (manic-depression), seasonal injury of the central or peripheral nervous system. 45 mood (or affective) disorder, schizophrenia and oth- 4. The method of any one of Embodiment 1-3, where- er psychoses, lissencephaly syndrome, anxiety syn- in said one or more other neurogenic agent is an dromes, anxiety disorders, phobias, stress and re- ACE inhibitor, and angiotensin receptor antagonist, lated syndromes, cognitive function disorders, ag- an HMG-CoA reductase inhibitor, an SSRI, an ago- gression, drug and alcohol abuse, obsessive com- nist of a 5HTla receptor, or folic acid. 50 pulsive behaviour syndromes, borderline personality 5. The method of Embodiment 4, wherein said ACE disorder, non-senile dementia, post-pain depres- inhibitor is captopril, enalapril, randolapril, or perin- sion, post-partum depression, cerebral palsy, and dopril; or combinations thereof. said angiotensin receptor antagonist is candesartan 18. The method of Embodiment 15, wherein said or telmisartan; or 55 nervous system disorder related to cellular trauma said HMG-CoA reductase inhibitor is atorvastatin; or and/or injury is selected from neurological traumas said SSRI is fluoroxetine; or and injuries, surgery related trauma and/or injury, said agonist of a 5HT1 a receptor is busipirone. retinal injury and trauma, injury related to epilepsy,

48 95 EP 2 377 530 A2 96

spinal cord injury, brain injury, brain surgery, trauma position produces an improvement in the disorder in related brain injury, trauma related to spinal cord in- the subject or patient; jury, brain injury related to cancer treatment, spinal the nervous system disorder related to a psychiatric cord injury related to cancer treatment, brain injury condition is selected from a neuropsychiatric disor- related to infection, brain injury related to inflamma- 5 der, an affective disorder, depression, hypomania, tion, spinal cord injury related to infection, spinal cord panic attacks, anxiety, excessive elation, bipolar de- injury related to inflammation, brain injury related to pression, bipolar disorder (manic-depression), sea- environmental toxin, spinal cord injury related to en- sonal mood (or affective) disorder, schizophrenia vironmental toxin, and combinations thereof. and other psychoses, lissencephaly syndrome, an 19. The method of Embodiment 15, wherein said 10 anxiety syndrome, an anxiety disorder, a phobia, neurologically related condition is selected form stress and related syndromes, a cognitive function learning disorders, memory disorders, autism, atten- disorder, aggression, drug and alcohol abuse, an ob- tion deficit disorders, narcolepsy, sleep disorders, sessive compulsive behavior syndrome, borderline cognitive disorders, epilepsy, temporal lobe epilep- personality disorder, non- senile dementia, post- pain sy, and combinations thereof. 15 depression, post-partum depression, cerebral palsy, 20. The method of Embodiment 17, wherein said and combinations thereof; psychiatric condition comprises depression. the nervous system disorder related to cellular de- 21. The method of Embodiment 20, wherein said one generation is selected froma neurodegenerativedis- or more other neurogenic agents comprises an anti- order, a neural stem cell disorder, a neural progenitor depressant agent. 20 cell disorder, a degenerative disease of the retina, 22. The method of Embodiment 20, wherein said de- an ischemic disorder, and combinations thereof; pression is due to morphine, alcohol, or drug use by the nervous system disorder related to cellular trau- the subject or patient. ma and/or injury is selected from neurological trau- 23. The method of any one of Embodiments 15-22, mas and injuries, surgery related trauma and/or in- wherein said PDE agent is an inhibitor of PDE3 or 25 jury, retinal injury and trauma, injury related to epi- PDE4. lepsy, spinal cord injury, brain injury, brain surgery, 24. The method of Embodiment 15, wherein said trauma related brain injury, trauma related to spinal PDE agent is ibudilast, enoximone, or rolipram. cord injury, brain injury related to cancer treatment, 25. The method of Embodiment 15, wherein said spinal cord injury related to cancer treatment, brain combination is in a pharmaceutically acceptable for- 30 injury related to infection, brain injury related to in- mulation. flammation, spinal cord injury related to infection, 26. A method of preparing cells or tissue for trans- spinal cord injury related to inflammation, brain injury plantation to a subject of patient, said method com- related to environmental toxin, spinal cord injury re- prising lated to environmental toxin, and combinations contacting said cell or tissue with a PDE agent in 35 thereof; and combination with one or more other neurogenic the neurologically related condition is selected from agents, to stimulate or increase neurogenesis in said a learning disorder, a memory disorder, autism, at- cell or tissue. tention deficit disorder, narcolepsy, a sleep disorder, a cognitive disorder, epilepsy, temporal lobe epilep- 40 sy, and combinations thereof. Claims 3. The composition for use according to claim 1, where- 1. A composition of an PDE agent, in combination with in the subject or patient in need of an increase in a neurogenic agent, for use in stimulating or increas- neurogenesis has a reduction in cognitive function. ing neurogenesis in a cell or tissue in an animal sub- 45 jector a human patient, wherein the subject or patient 4. The composition for use according to claim 3, where- is in need of stimulation of or an increase in neuro- in the reduction in cognitive function is due to age- genesis; wherein the neurogenic agent is an angi- related cognitive decline, Alzheimer’s Disease, epi- otensin modulator, an antidepressant, an anticon- lepsy, or a condition associated with epilepsy. vulsant, a peroxisome proliferator- activated receptor 50 (PPAR) agonist, or a 5HT1a receptor agonist. 5. The composition for use according to claim 2, where- in the psychiatric condition comprises depression, 2. The composition for use according to claim 1 for anxiety, or combinations thereof. treating a nervous system disorder in the patient, wherein the nervous system disorder is related to a 55 6. The composition for use according to claim 2, where- psychiatric condition, cellular degeneration, cellular in the neurologically related condition is a cognitive trauma and/or injury, or another neurologically relat- disorder. ed condition in a subject or patient, wherein the com-

49 97 EP 2 377 530 A2 98

7. The composition for use according to claim 2, where- in the memory disorder is mild cognitive impairment (MCI); age-related cognitive decline; or memory loss resulting from the use of general anesthetics, chem- otherapy, radiation treatment, post-surgical trauma, 5 or therapeutic intervention.

8. A composition comprising a PDE agent and an an- giotensin modulator, an antidepressant, an anticon- vulsant, a peroxisome proliferator- activated receptor 10 (PPAR) agonist, or a 5HT1 a receptor agonist.

9. The composition for use according to any one of claim 1 to claim 7, or the composition of claim 8, wherein the PDE agent is a PDE inhibitor. 15

10. The composition for use or composition according to claim 9, wherein the PDE inhibitor is a non- selec- tive PDE inhibitor, a PDE4 inhibitor, or a PDE5 in- hibitor. 20

11. The composition for use or composition according to claim 10, wherein the non- selective PDE inhibitor is ibudilast or theophylline. 25 12. The composition for use according to claim 1 or claim 2, or the composition of claim 8, wherein the angi- otensin modulator is an angiotensin converting en- zyme (ACE) inhibitor, or an angiotensin receptor an- tagonist (ARAn). 30

13. The composition for use according to claim 1 or claim 2, or the composition of claim 8, wherein the antide- pressant is an SSRI. 35 14. The composition of claim 8, compising ibudilast and captopril, enoximone and captopril, rolipram and buspirone, rolipram and an SSRI, enoximone and an SSRI, or ibudilast and candesartan. 40

45

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

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• US 6043252 A [0106] [0107] • US 4298734 A [0117] • US 3819631 A [0106] [0107] • US 4289772 A [0117] • US 20030166641 A [0106] [0107] • US RE30511 E [0117] • WO 9743287 A [0106] [0107] • US 4188391 A [0117] • US 6130333 A [0106] [0107] • US 4123534 A [0117] • US 6566360 B [0106] [0107] • US 4107309 A [0117] • US 6362178 B [0106] [0107] [0109] • US 4107307 A [0117] • US 6582351 B [0106] [0107] • US 4096257 A [0117] • US 20050070541 A [0106] [0107] • US 4093617 A [0117] • US 20040067945 A [0106] [0107] • US 4051236 A [0117] • US 6825197 B [0106] [0107] • US 4036840 A [0117] • US 5719283 A [0106] • US 6555572 B [0118] • US 6943166 B [0106] [0107] [0109] • US 20030045490 A [0130] • US 6576644 B [0106] [0107] • WO 0040714 A [0130] • US 5859009 A [0106] [0107] • US 697905 P [0132] [0312] [0314] • US 6943253 B [0106] [0107] • US 69790505 P, Barlow [0141] [0148] • US 6864253 B [0106] [0107] • US 4314081 A [0159] [0293] • US 5869516 A [0106] [0107] • US 4194009 A [0159] • US 5488055 A [0106] [0107] • US 4136193 A [0159] • US 6140329 A [0106] [0107] [0109] • US 4085225 A [0159] • US 5859006 A [0106] [0107] [0109] • US 3912743 A [0159] • US 6143777 A [0106] [0107] • US 4007196 A [0159] • WO 9616644 A [0106] [0107] • US 4536518 A [0159] • WO 0119802 A [0106] [0107] • US 4338317 A [0159] • WO 9626940 A [0106] [0107] • US 4478836 A [0159] • US 5346901 A [0109] • US 4761501 A [0159] • GB 235187 A [0112] [0116] • US 4956388 A [0159] • EP 579496 A [0112] [0116] • US 3454554 A [0160] • US 6930114 B [0112] [0115] • US 7045636 B [0160] • US 4861891 A [0112] [0117] • US 20050171098 A [0160] • US 20020132754 A [0112] [0114] • US 4062848 A [0161] • US 20040138249 A [0112] [0115] • US 3534041 A [0161] • US 20040249148 A [0112] [0115] • US 20050119248 A [0162] • WO 951997 A [0112] • US 20020132828 A [0177] • WO 9519978 A [0116] • US 6559159 B [0177] • WO 9307124 A [0116] • WO 2002053533 A [0177] • US 0040106631 B [0116] • US 6900228 B [0177] • US 5081242 A [0117] • US 6906061 B [0183] • US 5066653 A [0117] • US 20030181439 A [0184] • US 5010086 A [0117] • WO 0160374 A [0184] • US 4971972 A [0117] • US 1873732 A [0184] • US 4963561 A [0117] • US 3814812 A [0184] • US 4943573 A [0117] • US 4301176 A [0184] • US 4906628 A [0117] • US 6719520 B [0184] • US 4775674 A [0117] • US 20040010031 A [0184] • US 4766118 A [0117] • WO 2004072062 A [0184] • US 4761416 A [0117] • WO 03082859 A [0184] • US 4739056 A [0117] • WO 03104222 A [0184] • US 4721784 A [0117] • WO 03103663 A [0184] • US 4701459 A [0117] • WO 03095452 A [0184] • US 4663320 A [0117] • WO 2005000836 A [0184] • US 4642345 A [0117] • WO 0021927 A [0184] • US 4593029 A [0117] • WO 03076398 A [0184] • US 4564619 A [0117] • WO 00021927 A [0184] • US 4489078 A [0117] • WO 00038675 A [0184] • US 4404380 A [0117] • WO 03076442 A [0184] • US 4370328 A [0117] • US 6489344 B [0184] • US 4366156 A [0117] • US 6417185 B [0184]

59 EP 2 377 530 A2

• US 6153618 A [0184] • WO 03037877 A [0184] • US 20050171094 A [0184] • WO 03037869 A [0184] • US 20030130289 A [0184] • WO 03037891 A [0184] • EP 01454908 A [0184] • WO 05012307 A [0184] • EP 01454910 A [0184] • WO 05012304 A [0184] • EP 01295884 A [0184] • WO 9816528 A [0184] • EP 01295885 A [0184] • WO 00144206 A [0184] • EP 01460076 A [0184] • WO 014424 A [0184] • EP 01454900 A [0184] • WO 2005035532 A [0184] • WO 0170683 A [0184] • US 6645990 B [0184] • WO 0170729 A [0184] • US 6762179 B [0184] • WO 0170728 A [0184] • US 20010039275 A [0184] • WO 0170727 A [0184] • WO 0156567 A [0184] • WO 0170726 A [0184] • WO 03011843 A [0184] • WO 0170725 A [0184] • WO 03004478 A [0184] • WO 00218385 A [0184] • WO 03089419 A [0184] • WO 00218386 A [0184] • WO 03053330 A [0184] • WO 03072579 A [0184] • WO 03053444 A [0184] • WO 03072580 A [0184] • WO 03055877 A [0184] • WO 03027115 A [0184] • WO 03055492 A [0184] • WO 03027116 A [0184] • WO 03082853 A [0184] • WO 2004078760 A [0184] • WO 2005027823 A [0184] • WO 2005037800 A [0184] • US 6727251 B [0184] • WO 2004026881 A [0184] • US 6696452 B [0184] • WO 03076437 A [0184] • US 6664247 B [0184] • WO 03029223 A [0184] • US 666073 A [0184] • WO 2004098607 A [0184] • US 6656939 B [0184] • WO 2005026155 A [0184] • US 6653301 B [0184] • WO 2005026159 A [0184] • US 6653300 B [0184] • WO 2005025567 A [0184] • US 6638926 B [0184] • WO 03070730 A [0184] • US 6613776 B [0184] • WO 03070729 A [0184] • US 6610677 B [0184] • WO 2005019218 A [0184] • WO 2005002552 A [0184] • WO 2005019219 A [0184] • WO 2005002576 A [0184] • WO 2004013140 A [0184] • WO 2005012256 A [0184] • WO 2004080977 A [0184] • US 6498176 B [0184] • WO 2004026229 A [0184] • US 6800632 B [0184] • WO 2004022561 A [0184] • US 6872737 B [0184] • WO 03080616 A [0184] • US 20050137201 A [0184] • WO 03080609 A [0184] • US 20050176713 A [0184] • WO 03051847 A [0184] • US 20050004125 A [0184] • WO 2004009602 A [0184] • US 20030105075 A [0184] • WO 2004009596 A [0184] • US 20030008866 A [0184] • WO 2004009597 A [0184] • US 20010044436 A [0184] • WO 03045949 A [0184] • US 20040138273 A [0184] • WO 03068773 A [0184] • US 20040214928 A [0184] • WO 03080617 A [0184] • WO 9921859 A [0184] • WO 9965897 A [0184] • WO 00210158 A [0184] • WO 0018758 A [0184] • WO 05051919 A [0184] • WO 0307073 A [0184] • WO 00232896 A [0184] • WO 00220495 A [0184] • WO 2004046117 A [0184] • WO 2004043953 A [0184] • WO 2004106343 A [0184] • WO 2004056368 A [0184] • WO 00210141 A [0184] • WO 2005012298 A [0184] • WO 00218346 A [0184] • WO 2005012262 A [0184] • WO 0181345 A [0184] • WO 2005042525 A [0184] • WO 0174771 A [0184] • WO 2005005438 A [0184] • WO 05028475 A [0184] • WO 2004009562 A [0184] • WO 0109106 A [0184]

60 EP 2 377 530 A2

• WO 0141768 A [0184] • US 20030013715 A [0198] • WO 0017184 A [0184] • US 20050154027 A [0198] • WO 04037791 A [0184] • US 20050004130 A [0198] • WO 04065370 A [0184] • US 20050209273 A [0198] • WO 0137819 A [0184] • US 20050197361 A [0198] • WO 0142224 A [0184] • US 20040082592 A [0198] • WO 0185685 A [0184] • US 6660753 B [0198] • WO 04072063 A [0184] • US 20030195139 A [0198] • WO 2004085439 A [0184] • US 20040229917 A [0198] • WO 2005000303 A [0184] • US 20050153986 A [0198] • WO 2005000304 A [0184] • US 20050085514 A [0198] • WO 9947522 A [0184] • US 20050065340 A [0198] • US 20040102521 A [0185] [0194] • US 20050026963 A [0198] • US 20050049243 A [0186] • US 20050020585 A [0198] • US 20050119345 A [0186] • US 20040259917 A [0198] • US 20030157647 A [0186] • US 4786648 A [0202] • US 5688826 A [0188] [0192] • US 5362860 A [0202] • US 20040002478 A [0188] [0191] [0195] • US 5424301 A [0202] • US 6204292 B [0188] • US 5650174 A [0202] • US 6333428 B [0188] • US 4710508 A [0202] • US 5750566 A [0188] • US 5314901 A [0202] • US 6498180 B [0188] • US 5356914 A [0202] • US 6107342 A [0190] • US 5356912 A [0202] • US 20040006114 A [0190] • US 5041455 A [0202] • US 6376532 B [0191] • US 5043345 A [0202] • US 5916920 A [0192] • US 5260314 A [0202] • US 5945417 A [0192] • US 6297262 B [0203] • US 5958960 A [0192] • US 4866077 A [0203] • US 6143783 A [0192] • US RE36374 E [0203] • US 6268507 B [0192] • US 4925858 A [0203] • US 6284785 B [0192] • WO 9717074 A [0203] • US 6956049 B [0194] • US 4940795 A [0203] • US 6825211 B [0194] • US RE34653 E [0203] • US 5473077 A [0194] • US 4996210 A [0203] • US 5912248 A [0194] • US 5041549 A [0203] • US 6054448 A [0194] • US 5403931 A [0203] • US 5500420 A [0194] • US 5412096 A [0203] • US 20040077599 A [0194] • US 4855290 A [0203] • US 20040147482 A [0194] • US 5340821 A [0203] • US 20030199533 A [0194] • US 5580880 A [0203] • US 20050234048 A [0194] • US 4981858 A [0203] • WO 9719049 A [0194] • US 5278170 A [0203] • WO 9800391 A [0194] • US RE35593 E [0203] • EP 0870760 A [0194] • US 6468560 B [0203] • US 6313159 B [0195] • US 5773619 A [0203] • US 6479470 B [0195] • US 5808075 A [0203] • US 6528499 B [0195] • US 5545740 A [0203] • US 6399641 B [0198] • US 5534522 A [0203] • US 6589978 B [0198] • US 6596869 B [0203] • US 20030212066 A [0198] • US 20020127271 A [0203] • US 6586422 B [0198] • US 20030129246 A [0203] • US 5783575 A [0198] • US 20020150618 A [0203] • US 5843988 A [0198] • US 20010018074 A [0203] • US 5536721 A [0198] • US 20030157169 A [0203] • US 6429207 B [0198] • US 20010003588 A [0203] • US 5696148 A [0198] • US 5451587 A [0203] • US 6218385 B [0198] • US 5286864 A [0203] • US 20030109504 A [0198] [0199] • US 5508405 A [0203]

61 EP 2 377 530 A2

• US 5137895 A [0203] • WO 9900391 A [0209] • US 20050192268 A [0204] • WO 5637617 A [0209] • WO 0563254 A [0204] • WO 9937649 A [0209] • US 20050130961 A [0205] • WO 9937648 A [0209] • WO 04087158 A [0205] • WO 9937644 A [0209] • US 6951849 B [0206] • WO 9948892 A [0209] • US 20030100545 A [0206] • WO 9943661 A [0209] • WO 04089942 A [0206] • WO 5723462 A [0209] • WO 03028650 A [0206] • US 3242190 A [0209] [0222] • US 6911452 B [0206] • US 6503925 B [0210] • WO 03057698 A [0206] • US 6218547 B [0210] • US 20030176418 A [0206] • US 6399604 B [0210] [0211] • US 20050209226 A [0206] • US 6646124 B [0210] • WO 03057672 A [0206] • US 6515140 B [0210] • US 6833384 B [0207] • US 6451809 B [0210] • US 6552065 B [0207] • US 6448259 B [0210] • US 6897220 B [0207] • US 6448246 B [0210] • US 6888027 B [0207] • US 6423711 B [0210] • US 5369108 A [0207] • US 6414147 B [0210] • US 6541661 B [0207] [0208] • US 6380209 B [0210] • US 6720445 B [0207] • US 6353109 B [0210] • US 6562995 B [0207] • US 6297256 B [0210] • US 6777217 B [0207] • US 6297252 B [0210] • US 6387673 B [0207] • US 6268496 B [0210] • US 20050171347 A [0207] • US 6211365 B [0210] • US 20050165016 A [0207] • US 6166203 A [0210] • US 20050159470 A [0207] • US 6177569 B [0210] • US 20050143385 A [0207] • US 6194427 B [0210] • US 20050137234 A [0207] • US 6156898 A [0210] • US 20050137232 A [0207] • US 6143760 A [0210] • US 20050119250 A [0207] • US 6127395 A [0210] • US 20050113373 A [0207] • US 6103903 A [0210] • US 20050107445 A [0207] • US 6103731 A [0210] • US 20050107384 A [0207] • US 6723735 B [0210] • US 20050096468 A [0207] • US 6479506 B [0210] • US 20050085515 A [0207] • US 6476030 B [0210] • US 20050032831 A [0207] • US 6337331 B [0210] • US 20050014839 A [0207] • US 6730676 B [0210] • US 20040266769 A [0207] • US 6730681 B [0210] [0211] • US 20040254220 A [0207] • US 6828322 B [0210] [0211] • US 20040229889 A [0207] • US 6872720 B [0210] [0211] • US 20040198830 A [0207] • US 6699859 B [0210] [0211] • US 20040142953 A [0207] • US 6696444 B [0210] [0211] • US 20040106599 A [0207] • US 6617326 B [0210] [0211] • US 20040092598 A [0207] • US 6608062 B [0210] [0211] • US 20040077726 A [0207] • US 6579875 B [0210] [0211] • US 20040077698 A [0207] • US 6541484 B [0210] [0211] • US 20040053960 A [0207] • US 6500828 B [0210] [0211] • US 20030187027 A [0207] • US 6355798 B [0210] [0211] • US 20020177594 A [0207] • US 6333336 B [0210] [0211] • US 20020161045 A [0207] • US 6319924 B [0210] [0211] • US 20020119996 A [0207] • US 6303605 B [0210] [0211] • US 20020115826 A [0207] • US 6303597 B [0210] [0211] • US 20020103192 A [0207] • US 6291460 B [0210] [0211] • US 20020065282 A [0207] • US 6255305 B [0210] [0211] • WO 2005108367 A [0208] • US 6133255 A [0210] [0211] • WO 9925353 A [0209] • US 6872731 B [0210] • WO 9804560 A [0209] • US 6900215 B [0210] [0211]

62 EP 2 377 530 A2

• US 6642229 B [0210] [0211] • US 5182290 A [0210] • US 6593325 B [0210] [0211] • US 5130430 A [0210] • US 6914060 B [0210] • US 5095015 A [0210] • US 6914063 B [0210] [0211] • US 20050014939 A [0210] • US 6914065 B [0210] • US 20040171633 A [0210] • US 6936608 B [0210] • US 20050165048 A [0210] • US 6534505 B [0210] [0211] • US 20050165023 A [0210] • US 6426343 B [0210] [0211] • US 20040259818 A [0210] • US 6313125 B [0210] [0211] • US 20040192692 A [0210] • US 6310203 B [0210] [0211] • US 4513135 A [0213] • US 6200975 B [0210] [0211] • US 5939545 A [0215] • US 6071909 A [0210] • US 5925630 A [0215] • US 5922724 A [0210] • US 6277838 B [0215] • US 6096887 A [0210] • US 6143736 A [0215] • US 6080873 A [0210] • US RE35517 E [0215] • US 6013799 A [0210] • US 5591733 A [0215] • US 5936095 A [0210] • US 5232917 A [0215] • US 5925770 A [0210] • US 20050176976 A [0215] • US 5910590 A [0210] • WO 96116076 A [0215] • US 5908932 A [0210] • WO 9805337 A [0215] • US 5849927 A [0210] • WO 9521617 A [0215] • US 5840888 A [0210] • WO 9427608 A [0215] • US 5817813 A [0210] • WO 9318053 A [0215] • US 5804686 A [0210] • WO 9305786 A [0215] • US 5792766 A [0210] • WO 9303732 A [0215] • US 5750702 A [0210] • WO 91116897 A [0215] • US 5744603 A [0210] • EP 01038880 A [0215] • US 5744602 A [0210] • US 4656298 A [0217] • US 5723462 A [0210] • US 5929236 A [0217] • US 5696260 A [0210] • EP 0463969 A [0217] • US 5693801 A [0210] • EP 0356128 A [0217] • US 5677309 A [0210] • US 3960927 A [0220] [0222] • US 5668283 A [0210] • US 4383999 A [0221] • US 5637725 A [0210] • US 6071932 A [0221] • US 5637724 A [0210] • US 6906177 B [0221] • US 5625063 A [0210] • US 6225115 B [0221] • US 5610299 A [0210] • US 3121076 A [0222] • US 5608079 A [0210] • US 3116203 A [0222] • US 5606059 A [0210] • US 3371085 A [0222] • US 5604235 A [0210] • US 3109843 A [0222] • US 5585490 A [0210] • US 3136815 A [0222] • US 5510480 A [0210] • US 4280957 A [0222] • US 5484944 A [0210] • US 4316839 A [0222] • US 5473073 A [0210] • US 3296249 A [0222] • US 5463054 A [0210] • US 3862149 A [0222] • US 5451585 A [0210] • US 4220646 A [0222] • US 5426186 A [0210] • US 4521422 A [0222] • US 5367077 A [0210] • US 4900836 A [0222] • US 5328912 A [0210] • US 4794185 A [0222] • US 5326868 A [0210] • EP 50563 A [0222] • US 5312822 A [0210] • US 4626538 A [0222] • US 5306819 A [0210] • US 4278676 A [0222] • US 5286860 A [0210] • US 3397209 A [0222] • US 5266698 A [0210] • US 4370338 A [0222] • US 5243049 A [0210] • EP 50551 A [0222] • US 5216159 A [0210] • US 3471548 A [0222] • US 5212310 A [0210] • US 4024175 A [0222] • US 5185446 A [0210] • US 4094992 A [0222]

63 EP 2 377 530 A2

• US 4361583 A [0222] • US 5478863 A [0305] • US 5010090 A [0222] • US 5385946 A [0305] • US 4699927 A [0222] • US 5312840 A [0305] • US 4513006 A [0222] • US 5093525 A [0305] • US 6897305 B [0226] [0227] • WO 9014067 A [0305] • US 20050137206 A [0233] • US 20030171347 A [0306] • US 20030022899 A [0233] • US 6908914 B [0306] • US 20030009851 A [0233] • US 6872716 B [0306] • WO 0012067 A [0233] • US 5169855 B [0306] • US 6846823 B [0238] • US 5561135 B [0306] • US 3819706 A [0250] • US 5395841 B [0306] • US 3885046 A [0250] • US 4929734 B [0306] • US 6342496 B [0250] • US 5061728 B [0306] • US 3758528 A [0250] [0251] • US 5731307 B [0306] • US 3821249 A [0250] [0251] • US 5086054 B [0306] • US 6017924 A [0257] [0260] • US 5158947 B [0306] • WO 0116108 A [0257] • US 5116995 B [0306] • WO 0116133 A [0257] • US 5149817 B [0306] • WO 0116139 A [0257] • US 5109002 B [0306] • US 6492554 B [0258] • US 5162341 B [0306] • US 6924311 B [0268] • US 4956368 B [0306] • US 2005245601 AA [0286] • US 4831031 B [0306] • US 2002018807 AA [0286] • US 4957916 B [0306] • US 2003180406 AA [0286] • US 20050132429 A [0306] • US 2002086833 AA [0286] • US 20050107432 A [0306] • US 20040236123 A [0286] • US 20050038011 A [0306] • WO 9809533 A [0286] • US 20030105079 A [0306] • WO 9945788 A [0286] • US 20030171355 A [0306] • US 2005171029A A [0286] • US 20030212094 A [0306] • US 2003104075A A [0286] • US 20040019060 A [0306] • US 2005031762 AA [0286] • EP 503411 A [0306] • US 2002107292 AA [0286] • EP 362001 A1 [0306] • US 2002004523A A [0286] • EP 461986 A [0306] • US 5139802 A [0286] • WO 9214464 A [0306] • US 5130154 A [0286] • WO 9309094 A [0306] • WO 2002024002 A [0286] • WO 9222554 A [0306] [0307] • US 4229449 A [0293] • WO 9515948 A [0306] • US 6683192 B [0303] • WO 9218127 A [0306] • US 7060450 B [0303] • WO 9106297 A [0306] • US 6552016 B [0303] • WO 0102380 A [0306] • US 5489709 A [0305] • WO 9118868 A [0306] • US 6147063 A [0305] • WO 9300313 A [0306] • US 5298657 A [0305] • US 5736546 A [0307] • US 6087346 A [0305] • US 5665725 A [0307] • US 5574070 A [0305] • US 6844352 B [0307] • US 5502255 A [0305] • US 20050171135 A [0307] • US 4709094 A [0305] • WO 9924436 A [0307]

Non-patent literature cited in the description

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