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US 20120076770A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0076770 A1 Espina et al. (43) Pub. Date: Mar. 29, 2012

(54) MODULATION OF AUTOPHAGY AND AND Publication Classification SEROTONINFOR TREATMENT OF MULTIPLE MYELOMA RELATED DISEASES (51) Int. Cl. A6II 3/4706 (2006.01) A63L/436 (2006.01) (76) Inventors: Virginia Espina, Rockville, MD A63L/404 (2006.01) (US); Lance Liotta, Bethesda, MD A63L/506 (2006.01) (US); Antonella Chiechi, Matera A63/675 (2006.01) (IT); Alessandra Romano, Catania A 6LX 39/395 (2006.01) (IT); Emanuel Petricoin, A6IP 9/08 (2006.01) Gainesville, VA (US); Amy Van A6IP35/00 (2006.01) Meter, Bristow, VA (US) A6II 3/663 (2006.01) A63L/353 (2006.01) (21) Appl. No.: 13/073,989 A 6LX 3/57 (2006.01) (52) U.S. Cl...... 424/130.1: 514/456; 514/291; (22) Filed: Mar. 28, 2011 514/.414: 514/252.19; 514/266.24: 514/313; Related U.S. Application Data 514/108: 514/94 (63) Continuation-in-part of application No. PCT/US2009/ (57) ABSTRACT 004608, filed on Dec. 8, 2009. THE INVENTION RELATESTO compounds, proteins and methods of treatment therewith. Aspects of embodiments of (60) Provisional application No. 61/318,074, filed on Mar. the invention further relates to compounds and methods of 26, 2010. treatment for bone, bone marrow, and bone tissue. Patent Application Publication Mar. 29, 2012 Sheet 1 of 11 US 2012/0076770 A1

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MODULATION OF AUTOPHAGY AND AND 0012. In another embodiment, a process to modulate bone SEROTONINFOR TREATMENT OF remodeling in a Subject may include administering to a Sub MULTIPLE MYELOMA RELATED DISEASES ject a modulator in an effective amount wherein serotonin levels in a subject are modulated. In a further teach CROSS-REFERENCE TO RELATED ing, a process to modulate bone remodeling produces plasma APPLICATIONS concentrations of platelet derived serotonin. In another aspect 0001. This application claims the benefit of U.S. Provi of an embodiment, a process to modulate bone remodeling in sional Application No. 61/318,074, filed 26 Mar. 2010, a subject may include administering to a subject a medica entitled “Modulation of Autophagy and Serotonin for Treat ment in an effective amount wherein serotonin levels in a ment of Multiple Myeloma Related Diseases.” and is a con Subject are modulated. tinuation-in-part (CIP) of PCT application PCT/US2009/ 0013. In still another embodiment a process for modulat 004608, with an international filing date of 12 Aug. 2009, ing bone dysplasia, treating monoclonal gammopathy of each of which is hereby incorporated by reference in its unknown significance (MGUS), or treating multiple entirety. myeloma may include administering a serotonin modulator to a Subject in an effective amount. Said serotonin modulator BRIEF DESCRIPTION OF THE SEVERAL may include a kinase inhibitor, a selective serotonin VIEWS OF THE DRAWINGS , a heterocyclic , a monoam ine oxidase inhibitor, an antidepressant, an anti-, an 0002 FIG. 1 is an artist's rendering of a physiological antiepileptic, or an antibody. mechanism of Serotonin-based regulation of bone mass as 0014. In an aspect of an embodiment, an article of manu per aspect of an embodiment of the present invention. facture may include at least one vessel containing purified 0003 FIG. 2 depicts three graphs illustrating a relation chloroquine and purified HA14-1, instructions for the use of ship between Serotonin, the angiogenesis switch (HIF-1C), chloroquine and HA14-1 for the treatment monoclonal gam and bone lesions in patients treated with bone breakdown mopathy of unknown significance (MGUS), multiple inhibitors (bisphosphonates) as per aspect of an embodiment myeloma, bone dysplasia and/or abnormalities in bone, the of the present invention. treatment comprising (a) identifying a patient Suspected of 0004 FIG.3 reveals two graphs depicting the serum-Se having said disease, and (b) administering an effective rotonin concentration and the platelet grains-Serotonin con amount of chloroquine and HA14-1 to the patient. centration for healthy, diseased, and disease-free bone tissue 0015. In an aspect of an embodiment, an article of manu as per aspect of an embodiment of the present invention. facture may include a label that indicates the contents of the 0005 FIG. 4 is a multipanel figure depicting a correlation package may be used to treat at least one of multiple mono between Serotonin levels and RANK. DKK1, and cytokines clonal gammopathy of unknown significance (MGUS), mul that are known to regulate bone. tiple myeloma, bone dysplasia and/or abnormalities in bone, 0006 FIG. 5 contains two graphs depicting Serotonin's packaging material, and contained within the packing mate positive correlation with both IL-10 and TNF-C. as per aspect rial purified chloroquine (or other 4-amino quinoline), and at of an embodiment of the present invention. least one of a purified tyrosine kinase inhibitor, a purified 0007 FIG. 6 is a graph comparing the Serotonin, RANK, selective serotonin reuptake inhibitor (SSRI), a purified het lrp6, progest rec S.190, beta-arrestin, and DEPTOR concen erocyclic antidepressant, a purified trations in diseased and healthy bone marrow aspirates as per inhibitor, a purified antidepressant, a purified anti-anxiety aspect of an embodiment of the present invention. compound, a purified anti-epileptic, and a purified antibody. 0016. In an aspect of an embodiment a method of treating DETAILED DESCRIPTION OF EMBODIMENTS bone disease may include administering a serotonin modula 0008 Embodiments relate to methods of treating disease torto a subject. In an aspect of an embodiment, a method may in subjects. Other embodiments relate to articles of manufac include treating wherein said treating includes at least one of ture useful in treating disease, methods of making therapeutic increasing bone density, decreasing bone density, maintain compositions, combinations of therapeutic compositions, ing bone density, or regulating elements associated with bone methods of administering therapeutic compositions, and dos aOW. ages of therapeutic compositions. 0017. An aspect of an embodiment a method of regulating 0009. In an aspect of an embodiment, a process to treat may include altering at least one of preneoplastic differentia myelodysplasia includes methods of treating monoclonal tion of bone marrow cells, neoplastic angiogenesis of bone gammopathy of unknown significance (MGUS), processes to marrow cells, or bone marrow stem cell function. treat multiple myeloma, processes to treat bone dysplasia and 0018. In an aspect of an embodiment a method of regulat processes to treat abnormalities in bone. ing may include wherein serotonin modulator alter at least 0010. In one aspect of an embodiment, a process to treat one of the ratio of serotonin in platelets to plasma, concen monoclonal gammopathy of unknown significance (MGUS) tration of serotonin in the bone marrow, serotonin receptor may include administering to a patientatherapeutically effec activity of cells within the bone marrow, production of sero tive amount of a 4-aminoquinoline compound and a tyrosine tonin by cells associated with the bone, intracellular signaling kinase inhibitor. pathways associated with serotonin. 0011. In one embodiment, a process to modulate bone 0019. In an aspect of an embodiment, a method of treating remodeling in a Subject may include modulating serotonin in bone disease may include administering a serotonin modula a Subject. In an additional embodiment, said process to modu tor to a subject. In a further embodiment the method of treat late bone remodeling may include increasing or decreasing ing bone disease may include wherein said treating comprises bone density. at least one of the following increasing bone density, decreas US 2012/0076770 A1 Mar. 29, 2012 ing bone density, maintaining bone density; and regulating ing the amount of serotonin in said biological sample, com elements associated with bone marrow. paring said amount of serotonin in said known normal sample 0020. A method of claim 2 where said regulating com to the amount of serotonin in said biological sample. In prises altering at least one of the following: preneoplastic another aspect of an embodiment, diagnostic methods differentiation of bone marrow cells, neoplastic angiogenesis include treating a subject based on serotonin level determi of bone marrow cells, or bone marrow stem cell function. nation and or modulating serotonin in the Subject based on 0021. In an aspect of an embodiment, serotonin modulator said diagnosing. may alter at least one of ratio of serotonin in platelets to plasma, concentration of serotonin in the bone marrow, sero 0032. In a still further teaching, diagnostic methods tonin receptor activity of cells within the bone marrow, pro include assays evaluating post translational modification of duction of serotonin by cells associated with the bone, intra signaling proteins, caspase cleavage, poly(ADP-ribose) poly cellular signaling pathways associated with serotonin, merase (PARP) cleavage or dye exclusion/uptake. 0022. In an aspect of an embodiment a serotonin modula 0033 According to another aspect of an embodiment, tor may include a least one of a tyrosine kinase inhibitor, a diagnostic methods include evaluating methods employing at selective serotonin reuptake inhibitor (SSRI), a heterocyclic least one of reverse phase microarray (RPMA), ELISA, flow antidepressant, a monoamine oxidase inhibitor, an antide cytometry, Immunohistochemistry, Immunoassay, high reso pressant, an antianxiety compound, an antiepileptic com lution mass spectroscopy, and Suspension bead array. pound, an antibody 0034. In a further teaching, a method of treating mono 0023. In an aspect of an embodiment, a monoamine oxi clonal gammopathy of unknown significance (MGUS), pre dase inhibitor may include a selective monoamine oxidase malignant bone marrow cells or multiple myeloma in a Sub inhibitor, a inhibitor, a monoamine ject includes treating with an autophagy pathway inhibitor, oxidase B inhibitor or a nonselective monoamine oxidase and at least one of a tyrosine kinase inhibitor, a serotonin inhibitor. modulator, an antidepressant, an anti-anxiety compound, an 0024. In an aspect of an embodiment, a method of treating antiepileptic, a monoamine oxidase inhibitor, an antibody, a bone disease may include administering at least one of an non-chemotherapeutic agent and a bisphosphonate. autophagy inhibitor, a non-chemotherapeutic agent, and 0035. In a further teaching, an autophagy pathway inhibi angiogenesis inhibitor, a bone breakdown inhibitor, an osteo tor Such as 4-amino quinoline may be used. clast or osteoblast activity inhibitor, and an immune signal 0036. In a further aspect of an embodiment, treatment modulator. methods may retard the progression from a pre-disease state 0025. An aspect of an embodiment, the method of treating to multiple myeloma. In a further teaching, a modulator may bone disease may include reducing serotonin from platelet include a lease one of a tyrosine kinase inhibitor, a selective cells, gastrointestinal cells, neural cells, immune cells, bone serotonin reuptake inhibitor (SSRI); a heterocyclic antide marrow microenvironment cells or cancer cells. pressant; a monoamine oxidase inhibitor, an antidepressant; 0026. In an aspect of an embodiment, a “bone breakdown an anti-anxiety compound; an anti-epileptic; and an antibody. inhibitor” is administered in an effective amount to modulate 0037. In a further embodiment, a monoamine oxidase at least one of bone cell activity, stem cell activity, gas inhibitor is a selective monoamine oxidase inhibitor, a trointestinal cell activity, cancer cell activity, platelet cell monoamine oxidase A inhibitor, a monoamine oxidase B activity, and neural cell activity. inhibitor or a nonselective monoamine oxidase inhibitor. 0027. In a further teaching, a method of treating bone disease includes bone diseases Such as brittle bone disease, 0038 An additional aspect of an embodiment may include multiple myeloma, osteogenesis imperfecta, osteolytic bone a method of treating bone disease including treating with at disease, amyloidosis, monoclonal gammopathy, alterations least one of an autophagy inhibitor; a non-chemotherapeutic in bone marrow hematopoetic precursor cells; and myelod agent; an angiogenesis inhibitor, a bone breakdown inhibitor; ysplasia. a osteoclast or osteoblast activity inhibitor; and an immune 0028. In still another aspect, the method of treating bone signal modulator. disease encompasses treating Subject at least one of a chor 0039. In a further aspect of an embodiment, the method of date, mammal, primate, and human. Another embodiment treating bone disease may include reducing serotonin from at may include diagnosing and/or treating myeloma cells, least one of platelet cells, gastrointestinal cells, neural cells, including wherein said cells are inhibited, Suppressed, or immune cells, bone marrow microenvironment cells or can killed to a greater extent as compared to the non-myeloma cer cells. cells. 0040. In a further aspect of an embodiment, administering 0029. Still another aspect of an embodiment includes a a bone breakdown inhibitor may include administering in an method of diagnosing a Subject for a bone disease including at effective amount to modulate at least one of bone cell activity, least the steps of assaying a biological sample of the Subject, stem cell activity, gastrointestinal cell activity, cancer cell determining the amount of serotonin in said biological activity, platelet cell activity, and neural cell activity. In a sample and determining a disease state based on said amount further aspect of an embodiment, a method of treating bone of serotonin. disease may include treating at least one of brittle bone dis 0030 Still another aspect of an embodiment includes ease; multiple myeloma; osteogenesis imperfecta (OI), diagnostic methods wherein said biological sample com osteolytic bone disease, amyloidosis; monoclonal gammopa prises at least one of the bone marrow aspirate, tissue, blood, thy; alterations in bone marrow hematopoetic precursor cells; serum, whole blood, cells; and blood. and myelodysplasia. 0031 Still another aspect of an embodiment includes 0041 According to an aspect of an embodiment, the treat diagnostic methods including at least the steps of determining ment methods may include treating at least one of a chordate, the amount of serotonin in a known normal sample, determin mammal, primate, and human. US 2012/0076770 A1 Mar. 29, 2012

0042. According to embodiments, myeloma cells are antidepressant, a purified anti-anxiety compound, a purified inhibited, Suppressed, or killed to a greater extent as com anti-epileptic, and a purified antibody. pared to the non-myeloma cells. 0056. In a further teaching, a method of treating may 0043. According to embodiments, methods include select include a route of administration wherein said route of the ing a subject in need of treatment. administration may include at least one of intramuscular, 0044 According to embodiments, a method of diagnosing transdermally, transmucossally, rectally, orally, via nasal a Subject for a bone disease includes assaying a biological insufflation, intravenous administration, and via cerebrospi sample of the Subject; determining the amount of serotonin in nal fluid or lumbar injection. said biological sample; and determining a disease State based 0057 According to an additional embodiment, the form of on said amount of serotonin. the medicament may include a lotion, patch, injectable, tab 0045. According to embodiments, a biological sample to let, or nasal spray. be used in the method includes at least one of the following 0058. In an additional embodiment, chloroquine analogs bone marrow aspirate; tissue; blood serum; whole blood; include Chloroquine (CQ), 7-chloro-4-4-(diethylamino)-1- cells; and blood. methylbutylaminoquinoline phosphate (1:2), Chloroquine 0046 According to embodiments, diagnostic methods Phosphate, USP, Qualiquin (Quinine), Plaquenil (hydroxy include determining the amount of serotonin in a known chloroquine), Aralen, Aralen Phosphate, Lariam, or 4-amino normal sample, determining the amount of serotonin in a quinoline compounds. biological sample, and comparing said amount of serotonin in 0059. According to embodiments, tyrosine kinase inhibi said known normal sample to the amount of serotonin in said tors include Azitinib, Bosutinib, Cediranib, Crizotinib, Dam biological sample. According to embodiments, diagnostic nacanthal, Dasatinib, Erlotnib, Gefitinib, Imatinib, Lapatinib, methods further include treating a subject based on said deter Lestaurtinib, Neratinib, Nilotinib, Regorafenib, Ruxolitinb. mination. Semaxanib, Sunitinib, Toceranib, Tofacitinib, Vandetanib, or 0047 According to embodiments, regulating bone remod Vatalnib. eling in the Subject includes modulating serotonin in the 0060 According to embodiments, serotonin modulators Subject based on said diagnosing. include selective serotonin reuptake inhibitors (SSRIs). 0.048. According to embodiments, the assaying step may SSRIs include , Dapoxetine, , Fluox include at least one of assays evaluating post translational etine, , , or . modification of signaling proteins, caspase cleavage, poly 0061 Additional embodiments include antide (ADP-ribose) polymerase (PARP) cleavage or dye exclusion? pressants (TCAS). TCAS include , Butripyline, uptake. , , Dawsey Letha, , 0049 According to embodiments, diagnostic methods , , or . may include at least one or more of evaluating where the 0062. In another embodiment, monoamine oxidase inhibi evaluating includes at least one of reverse phase microarray tors (MAOIs) include selective monoamine oxidase inhibi (RPMA), ELISA, flow cytometry, Immunohistochemistry, tors, non-selective monoamine oxidase inhibitors, monoam Immunoassay, high resolution mass spectroscopy, and Sus ine oxidase-A (MAO-A) inhibitors (, pension bead array. Resveratrol, Berberine, Coptisine, , , , , or ), monoamine oxi 0050. According to embodiments, a method of treating dase-B (MAO-B), inhibitors (, , Rasa monoclonal gammopathy of unknown significance (MGUS), giline, or ), (, , premalignant bone marrow cells or multiple myeloma in a , , Iprozid, Ipronid, Rivivol, Pro Subject, includes administering an autophagy pathway inhibi pilniazida, , , , Niala tor and at least one of a tyrosine kinase inhibitor, a serotonin mide, , , , Phenoxypro modulator, an antidepressant, an anti-anxiety compound, an pazine, Pivalylbenzhydrazine, , Natulan, or antiepileptic, a monoamine oxidase inhibitor, an antibody, a Indicarb, ), or Non-Hydrazines (, Echi non-chemotherapeutic agent and a bisphosphonate. nopsidine, , (ZyVOX, Zyvoxam, 0051. In a further teaching, and autophagy pathway ZyVoxid), or ). inhibitor is a 4-amino quinoline. 0063. In another aspect, monoamine oxidase inhibitors 0052. In a further teaching, a bisphosphonate may include include Valproic Acid, , licorice, Siberian ginseng, at least one of alendronate, pamidronate or Zoledronic acid. Yerba Mate, or Yohimbe. 0053. In an aspect of an embodiment, the monoamine 0064. In another aspect, serotonin include oxidase inhibitor may include a selective monoamine oxidase 5-HT agonists (, , and ), inhibitor, a monoamine oxidase A inhibitor, a monoamine 5-HT1B receptor agonists (Sumatriptan, rizatriptan, and oxidase B inhibitor or a nonselective monoamine oxidase naratriptan), 5-HT, receptoragonists (Sumatriptan, rizatrip inhibitor. tan, and naratriptan), 5-HT, receptor (Lasmiditan), 0054. In an aspect of an embodiment, the method may 5-HT, receptor agonists (LSD, mescaline, psilocin, DMT, include treating with an autophagy inhibitor, and angiogen and 2C-B), 5-HT, receptor agonists (Lorcaserin), 5-HT esis inhibitor, a bone breakdown inhibitor, an osteoclast or receptor agonist, and 5-HT7 receptor agonists. osteoblast activity inhibitor, and in immune signal modulator. 0065. In another aspect, serotonin antagonists include 0055. In an aspect of an embodiment, a kit may include a 5-HT1A antagonists, 5-HT1B receptor antagonists, 5-HT1D vessel or vessels containing purified 4-amino quinoline (for receptor antagonists, 5-HT1F receptor antagonists, 5-HT2A example chloroquine) and at least one of at least one of a receptor antagonists, 5-HT2C receptor antagonists, 5-HT4 purified tyrosine kinase inhibitor, a purified selective seroto receptor antagonists, and 5-HT7 receptor antagonists. nin reuptake inhibitor (SSRI), a purified heterocyclic antide 0066. According to embodiments, at least one process pressant, a purified monoamine oxidase inhibitor, purified an may include a step of selecting or identifying a patient in need US 2012/0076770 A1 Mar. 29, 2012

of treatment. According to embodiments a patient in need of pathways associated with the action of serotonin, an agent treatment may be selected or identified as a patient presently that is synergistic or an agent that is additive with serotonin diagnosed as having monoclonal gammopathy of unknown modulation. significance, premalignant bone, multiple myeloma, bone 0076. In embodiments cells, could be regulated by preneo dysplasia, myeloma, amyloidosis, or myelodysplasia. plastic differentiation, neoplastic angiogenesis or stem cell According to embodiments, patients presently diagnosed function. For example bone marrow stem cell function may be diagnosed or identified via methods well known the includes differentiation of pre-osteoblasts into osteoblasts. skilled artisans. Such methods well known to skilled artisans 0077. In another embodiment, regulating elements resid may include physical examination, immunological detection ing in the bone marrow may include at least one of altering the methods, polymerase chain reaction (PCR)-based methods, proliferation, genetic stability, Survival, and/or function of reverse transcriptase-PCR(RT-PCR)-based methods, South cellular elements residing in bone or bone marrow. ern, Northern, or Western analysis, flow cytometry, reverse (0078 We have developed methods of ex vivo treatment of phrase protein microarray (RPMA), proteomics, genomics, bone marrow aspirate samples (PCT/US2009/004608). We radiological testing processes or combinations thereof. describe methods to screen a large series of kinase and cell 0067 Embodiments relate to methods of treating mono signaling inhibitors in fresh, living patient's myeloma cells clonal gammopathy of unknown significance (MGUS). Addi within the tumor microenvironment within 4 hours of collec tional embodiments include methods of treating multiple tion. The technology provides a method to magnetically sort, myeloma. Still other embodiments include methods of treat in a multiplexed high throughput manner, cellular samples ing myelodysplasia. with concomitant analysis of plasma cells and non-plasma 0068. In another embodiment potential therapeutics cells. Employing these methods we have compiled drug include molecular inhibitors (e.g. Sunitinib, Dasatinib, Erlo inhibitory data for 35 human MM bone marrow aspirate tinib), chemotherapeutics (e.g. Dexamethasone, Rapamycin, samples using ex vivo functional Screening. This information Bcl-2 inhibitor), or exogenous ligands (e.g. SCF, IGF-1 and/ has provided insights into new therapies or combinations of or cytokines (e.g. IL-6). Ideally, the potential therapeutics therapies for treatment of MM. target a wide range of growth, proSurvival, autophagy and 0079 Bone marrow aspirates were treated with unique angiogenesis-related pathways. Exemplary candidate thera drug combinations of Chloroquine and HA14-1 (Bcl-2 peutics include, but are not limited to, Avastin (bevacizumab), inhibitor), Chloroquine and Rapamycin, or Chloroquine and Gleevec (imatinib), Lapatinib, Iressa, Tarceva, Sutent (Suni tyrosine kinase inhibitors such as Sunitinib/Dasatinib/Lapa tinib), Dasatinib (Sprycel), Nexavar (Sorafenib), Revlimid, tinib, etc., permitting the simultaneous evaluation of treat Cucurbitacin I, A77 1726, AG 490, AG 1296, AGL 2043, ment effects on both myeloma (diseased) and non-diseased Bcr-abl inhibitor, HNMPA-(AM)3, IGF-IR inhibitor, Lck cells (FIG. 1). We were able to measure compensatory up inhibitor, LFM-A13, TGFB inhibitor, CD20 antibody, Bort regulation of cell signaling pathways by reverse phase protein eZomib, , Chloroquine, Dasatinib, Dexametha microarray as a prognostic indicator of drug resistance. In sone, Erlotinib, Gefitinib, BCL-inhibitor, Honokiol, IGF-IR addition this method allowed the differential effect of treat inhibitor II, Imatinib, Lapatinib, Mekl & 2 inhibitor, Melato ment on the CD138+ and non-CD138+ cell populations to be nin, Midostaurin, Nilotinib, NVP-TKI258-CU-2, Nilotinib, quantitated. This method may be used for determining toxic , RAD, Rapamycin, Resveratrol, Sorafenib, ity on normal cells in individual patients for therapeutic deci Sunitinib, IL-6 ligand, IGF-1 ligand and SCF/C-kit ligand. S1O.S. 0069. In another embodiment a method of treating bone 0080 We propose a means of treating any stage of mul disease in a Subject may include administering a serotonin tiple myeloma (where the myeloma cells are growth inhib modulator to a subject alone or in combination with other ited, Suppressed, or killed, to a greater extent compared to the therapies. non-myeloma cells) with the combination of an autophagy 0070. In another embodiment, regulating elements resid inhibitor with a non-chemotherapeutic agent (such as a ing in the bone marrow may include altering the proliferation, tyrosine kinase inhibitor, small molecule inhibitor or a thera genetic stability, survival, and/or function of cellular ele peutic antibody with examples listed in Table 1 below). We ments residing in bone or bone marrow. also propose the combination of an autophagy inhibitor with 0071. In another embodiment, treating may include regu non-chemotherapeutic agents for the treatment of patients lating bone, for example bone cells, bone marrow cells, bone with myeloma pre-cursors diseases, Monoclonal Gammopa stroma, gut cells, platelet cells, brain cells, or ventromedial thy, Multiple Gammopathy of Unknown origin Syndrome cells. (MGUS), amyloidosis, plasmacytoma, or any other plasma 0072. In another embodiment, modulating of serotonin cell related disease. may include at least one of administering a serotonin modu I0081. This work can quantitatively measure the phospho lator in an effective amount to modulate serotonin levels or rylation, cleavage or total forms of kinases, phosphatases and the effects mediated by serotonin. other cell signaling proteins in bone marrow aspirate and 0073. In another embodiment, wherein, bone breakdown bone marrow core samples for treatment regimen stratifica inhibitors, or inhibitors that modulate osteoclast and osteo tion. Specific inhibitors, such as gefitinib, erlotininb, and blast activity. surafinib, or combinations of inhibitors with steroids (dexam 0074. In another embodiment, serotonin modulator may ethasone) and/or autophagy inhibitors can be tested ex vivo include at least one of a tyrosine kinase inhibitor, a selective using a patient's bone marrow aspirate to predict which serotonin reuptake inhibitor (SSRI), a heterocyclic antide patient will respond to a particular therapy or combination. pressant, a monoamine oxidase inhibitor, an antidepressant, The multiplexed nature of the reverse phase protein microar an anti-anxiety compound, an anti-epileptic and an antibody. ray technology permits quantitative measurement of multiple 0075. In another embodiment, treating may include treat cell signaling proteins. This work can be used to generate a ing with at least one of an agent that modulates the signaling functional multiple myeloma or leukemia classifier based on US 2012/0076770 A1 Mar. 29, 2012

drug target activation and test the hypothesis that cell signal 0092. In this specification, “a” and “an and similar ing activation portraits can predict a priori which targeted phrases are to be interpreted as “at least one' and “one or therapies will best cause cell death. more. 0082. This work can provide simultaneous assessments of 0093. The disclosure of this patent document incorporates treatment effects on diseased and non-diseased cell popula material which is subject to copyright protection. The copy tions. For example, Non-plasma cells and plasma (myeloma) cells can be concomitantly studied for therapeutic efficacy for right owner has no objection to the facsimile reproduction by an individual patient. Analysis of both diseased and non anyone of the patent document or the patent disclosure, as it diseased cell populations, under the same conditions, with the appears in the Patent and Trademark Office patent file or same treatments, can be used to predict potential toxicity as records, for the limited purposes required by law, but other well as efficacy. wise reserves all copyright rights whatsoever. 0083. While not intending to be bound to any particular 0094. While various embodiments have been described mechanism, Applicants propose a mechanism in FIG.1. The above, it should be understood that they have been presented biologic mechanisms involved in the pathogenesis of mul by way of example, and not limitation. It will be apparent to tiple myeloma (MM)-induced osteolytic bone disease are less persons skilled in the relevant art(s) that various changes in well understood. Physiological interactions between the sero form and detail can be made therein without departing from toninergic and skeletal systems are implicated by clinical the spirit and scope. In fact, after reading the above descrip observations 1. The RPMA used in this invention has tion, it will be apparent to one skilled in the relevant art(s)how revealed a new role for serotonin signaling in myeloma/ to implement alternative embodiments. Thus, the present MGUS osteolytic bone disease. embodiments should not be limited by any of the above 0084. We propose a means of treating or preventing brittle described exemplary embodiments. In particular, it should be bone disease or osteolytic bone disease which comprises a noted that, for example purposes, the above explanation has serotonin modulator alone, or in combination with, an focused on the example(s) serotonin modulators. However, autophagy inhibitor and/or a non-chemotherapeutic agent one skilled in the art will recognize that embodiments of the (examples listed in Table 1 below). invention could be serotonin agonists, serotonin antagonists, 0085. The monoamine serotonin 5-hydroxytryptamine or both. (5-HT) has previously been investigated as a neurotransmit ter, synthesized by a two-step pathway in which 0095. In addition, it should be understood that any figures hydroxylase is the rate-limiting . Circulating 5-HT is which highlight the functionality and advantages, are pre principally stored in platelet-dense granules. Aggregated sented for example purposes only. The disclosed architecture immunoglobulins derived from all the IgG subclasses, iso is sufficiently flexible and configurable, such that it may be lated from healthy controls or myeloma patients, induce utilized in ways other than that shown. For example, the steps platelet granules release in the absence of antigen or particu listed in any flowchart may be re-ordered or only optionally late matter, in a dose dependent manner 2. used in some embodiments. I0086. The brainstem-derived serotonin (BDS) positively (0096. Further, the purpose of the Abstract of the Disclo regulates bone mass following binding to 5-HT2C receptors sure is to enable the U.S. Patent and Trademark Office and the on Ventromedial hypothalamic neurons. This is opposed by public generally, and especially the Scientists, engineers and platelet-derived serotonin (PDS) which induces bone lysis practitioners in the art who are not familiar with patent or and osteoclast activation. legal terms or phraseology, to determine quickly from a cur 0087 Immunoglobulins have been shown to induce plate sory inspection the nature and essence of the technical dis let release a) when participating in immune reactions as anti closure of the application. The Abstract of the Disclosure is gen-antibody complexes or b) by nonimmune mechanisms not intended to be limiting as to the scope in any way. Such as coating of glass or polymethylmethacrylate beads. 0097 Finally, it is the applicant's intent that only claims 0088 MM patients with evidence of osteolytic lesions that include the express language “means for or “step for be exhibited an increase in the concentration of serum tryp interpreted under 35 U.S.C. 112, paragraph 6. Claims that do tophan and serotonin 3, while that of tyrosine, , not expressly include the phrase “means for or “step for are and noradrenaline was decreased 3. not to be interpreted under 35 U.S.C. 112, paragraph 6. 0089. We found that bone marrow cells from patients with osteolytic multiple myeloma has higher levels of Serotonin, Example 1 RANK, Beta Arrestin and DEPTRO compared to non-os teolytic myeloma patients (FIG. 2). Increased circulating 0.098 Bone marrow aspirates were treated with unique serotonin levels released from platelets by immunoglobulin drug combinations of Chloroquine and HA14-1 (Bcl-2 complexes may alter the RANK/RANKL ratio in the BM inhibitor), Chloroquine and Rapamycin, or Chloroquine and environment and promote MM osteolytic lesion. tyrosine kinase inhibitors such as Sunitinib/Dasatinib/Lapa 0090 These data indicate that the 5-HT system plays an tinib, etc., permitting the simultaneous evaluation of treat important role in bone homeostasis through effects on osteo ment effects on both myeloma (diseased) and non-diseased clast function and that the serotonin system is involved in the cells (FIG. 1). Compensatory up-regulation of cell signaling pathogenesis of MM-induced bone disease. Therefore sero pathways was measured by reverse phase protein microarray tonin regulation is a new therapeutic target for preventing or as a prognostic indicator of drug resistance. In addition this treating osteolytic bone disease associated with multiple method allowed the differential effect of treatment on the myeloma or other conditions. CD138+ and non-CD138+ cell populations to be quantitated. 0091. The following references are included provide This method may be used for determining toxicity on normal background information as an aid to explain the present cells in individual patients for therapeutic decisions. The embodiments: results are shown in FIGS. 2-6. US 2012/0076770 A1 Mar. 29, 2012

TABLE 1. TABLE 1-continued Autophagy combination therapy example agents. Autophagy combination therapy example agents. Inhibitors Inhibitors 17-DMAG Asparagine (ASn) 8-hydroxy Guanosine Leupeptin AKT Inhibitor IV Serotonin or serotonin related inhibitor AKT inhibitor X Serotonin modulator agents such as serotonin reuptake AKT inhibitor XI inhibitors, or serotonin receptor antagonists AMPK Inhibitor, Compound C Bisphosphonates and other nitrogenous or non-nitrogenous BAY 1 1-7082 inhibitors such as Clodronate or Zoledronic Acid Bcr-abl Inhibitor Collagenase inhibitors such as Matrix Metalloproteinase Inhibitors Carfilzomib Caspase-3 Inhibitor VII Caspase-8 inhibitor I Caspase-9 inhibitor II REFERENCES CGPO41251 (Midostaurin) Chloroquine (0099. 1. Rosen, Nature Medicine (2009), 15:2, 145-6 Cox II Inhibitor Dasatinib 0100 2. Zimmermann, The Journal of Clinical Investiga Dexamethasone tion (1975), 56,828-834 EGFR inhibitor II, BIBX1382 0101 3. Kurup, International Journal of Neuroscience, EGFRErb-2Erb-4 Inhibitor (2003), 113:9, 1221-1240. ERK inhibitor II, Negative control What is claimed is: ERK inhibitor III erotinib 1. A method of treating bone disease comprising adminis FGF/VEGF Receptor Tyrosine Kinase Inhibitor, PD173074 tering a serotonin modulator to a Subject. Gefitinib 2. The method of claim 1, wherein said treating comprises Glycogen Phosphorylase Inhibitor Granzyme B inhibitor I at least one of the following: HA14-1 a. increasing bone density; HNMPA-(AM). (Insulin Receptor TKI inhibitor) b. decreasing bone density; Honokoil c. maintaining bone density; and HSP90 Inhibitor GF-1RInhibitor II d. regulating elements associated with bone marrow. GF-1RPPP 3. A method of claim 2 where said regulating comprises matinib altering at least one of the following: preneoplastic differen matinib tiation of bone marrow cells, neoplastic angiogenesis of bone ak2. Inhibitor II ak3 Inhibitor I marrow cells, or bone marrow stem cell function. JNK Inhibitor I, (L)-Form 4. The method of claim 1, wherein said serotonin modula K2S29 tor alters at least one of the following: Lapatinib a. ratio of serotonin in platelets to plasma; LY294OO2 MAPK Inhibitor PD169316 b. concentration of Serotonin in the bone marrow; Mek 1 & 2 inhibitor SL327 c. serotonin receptor activity of cells within the bone mar row; d. production of serotonin by cells associated with the NVP-BEZ235 NVP-Raf-265 bone; NVP-LBHS89 e. intracellular signaling pathways associated with seroto NVP-AMN107 (Nilotinib) nin; NVP-TKI2S8-CU-2 5. The method of claim 4 wherein said serotonin modulator PARP Inhibitor XI, DR2313 PD153035 (EGFR Inhibitor) comprises at least one of the following: PD98059 (MEK inhibitor) a. a tyrosine kinase inhibitor, PDGF Receptor Tyrosine Kinase Inhibitor I b. a selective serotonin reuptake inhibitor (SSRI); PI3-KC. Inhibitor IV c. a heterocyclic antidepressant; PI3-Ky Inhibitor II DX, AM114 d. a monoamine oxidase inhibitor, RADOO1 e. an antidepressant; Rapamycin f, an anti-anxiety compound; Resveratrol Sorafinib g. an anti-epileptic; and Src Kinase Inhibitor II h. an antibody. Sunitinib 6. The method of claim 5 wherein the monoamine oxidase Terphenyl (FWF416) inhibitor is a selective monoamine oxidase inhibitor, a VEGF Receptor Tyrosine Kinase Inhibitor III, KRN633 Wortmannin monoamine oxidase A inhibitor, a monoamine oxidase B ZM 336372 (c-Raf inhibitor) inhibitor or a nonselective monoamine oxidase inhibitor. hydroxychloroquine 7. The method of claim 1, further comprising administer 3-methyladenie clomipramine ing at least one of ethyl pyruvate a. an autophagy inhibitor, glycyrrhizin b. a non-chemotherapeutic agent; c. an angiogenesis inhibitor; US 2012/0076770 A1 Mar. 29, 2012

d. a bone breakdown inhibitor; e. cells; and e. a osteoclast or osteoblast activity inhibitor; and f. blood. fan immune signal modulator. 16. The method of claim 14, wherein said determining 8. The method of claim 1, further comprising reducing comprises: serotonin from platelet cells, gastrointestinal cells, neural a. determining the amount of serotonin in a known normal cells, immune cells, bone marrow microenvironment cells or sample: cancer cells. b. determining the amount of serotonin in said biological 9. The method of claim 7, wherein said “bone breakdown sample: inhibitor” is administered in an effective amount to modulate c. comparing said amount of serotonin in said known nor at least one of bone cell activity, stem cell activity, gas mal sample to the amount of serotonin in said biological trointestinal cell activity, cancer cell activity, platelet cell sample. activity, and neural cell activity. 17. The method of claim 14, further comprising treating 10. The method of claim Error! Reference source not said Subject based on said determination. found... wherein said bone disease comprises at least one of: 18. The method of claim 14, further comprising regulating a.brittle bone disease; bone remodeling in the Subject including modulating seroto b. multiple myeloma; nin in the Subject based on said diagnosing. c. osteogenesis imperfecta (OI) 19. The method of claim 14, wherein said assaying step d. osteolytic bone disease; comprises assays evaluating post translational modification e. amyloidosis: of signaling proteins, caspase cleavage, poly(ADP-ribose) f. monoclonal gammopathy; polymerase (PARP) cleavage or dye exclusion/uptake. g. alterations in bone marrow hematopoetic precursor 20. The method of claim 19, wherein said evaluating is cells; and selected from the group consisting of reverse phase microar h. myelodysplasia. ray (RPMA), ELISA, flow cytometry, Immunohistochemis 11. The method of claim 10 wherein said subject may try, Immunoassay, high resolution mass spectroscopy, and include at least one of a chordate, mammal, primate, and Suspension bead array. human. 21. A method of treating monoclonal gammopathy of 12. The method of claim 10 wherein myeloma cells are unknown significance (MGUS), premalignant bone marrow inhibited, Suppressed, or killed to a greater extent as com cells or multiple myeloma in a subject, comprising adminis pared to the non-myeloma cells. tering: 13. The method of claim 1, further comprising selecting a a. an autophagy pathway inhibitor, and Subject in need of treatment. b. at least one of a tyrosine kinase inhibitor, a serotonin 14. A method of diagnosing a subject for a bone disease modulator, an antidepressant, an anti-anxiety com comprising: pound, an antiepileptic, a monoamine oxidase inhibitor, a. assaying a biological sample of the Subject; an antibody, a non-chemotherapeutic agent and a bis b. determining the amount of serotonin in said biological phosphonate. sample; and 22. The method of claim 21, wherein said autophagy path c. determining a disease state based on said amount of way inhibitor is a 4-amino quinoline. serotonin. 23. The method of claim 21 wherein said bisphosphonate 15. The method of claim 14, wherein said biological may include at least one of alendronate, pamidronate or sample comprises at least one of the following: Zoledronic acid. a. bone marrow aspirate; 24. The method of claim 21, wherein said treatment retards b. tissue; the progression from a pre-disease state to multiple myeloma. c. blood serum; d. whole blood; c c c c c