Patent Application: US-201515120850-A

Abstract:
this disclosure relates to compositions and methods for recruiting brown adipocytes in vitro and in vivo from brown adipocyte progenitor cells found in human skeletal muscle . methods for treating metabolic disease are also provided . additionally , methods for treating hypothermia are provided . in some embodiments , the brown adipocyte recruiter is a human protein or peptide . in other embodiments the brown adipocyte recruiter may be a non - human protein or peptide . in still other embodiments , the brown adipocyte recruiter is a small molecule or natural product .

Description:
as used herein , “ agent - activated ” means that the bat progenitor cell or cells have been treated with one or more agents as described herein and are at least partially committed to differentiate into brown adipocytes . the cells can be autologous , allogeneic , or xenogeneic . “ brown adipogenesis ” means generation of brown adipocytes from bat progenitor cells in vivo , in vitro , or partially in vivo and partially in vitro . it should be noted that brown adipogenesis may be induced , i . e ., the so - called “ bat progenitor cells ” before induction by , e . g ., one or more agents disclosed herein , was not necessarily committed to differentiate into brown adipocytes and may be reprogrammed or transdifferentiate into brown adipocytes from a stem cell or a somatic cell . “ recruiting brown adipocytes ” means promoting or enhancing differentiation of bat progenitor cells into brown adipocytes , and / or increasing the amount or concentration of brown adipocytes , in vivo and / or in vitro . provided herein are agents ( e . g ., compounds , proteins , biologicals , and the like ) that can promote the differentiation of bat progenitor cells into brown adipocytes and / or induce the expression of the ucp1 gene in vitro , in vivo , or both . such agents can be identified by screening compounds , proteins , biologicals , and the like . for example , in some embodiments bat progenitor cells ( e . g ., those isolated from human skeletal muscle ) can be used to screen agents for the ability to induce expression of the ucp1 gene and / or differentiation of the bat progenitor cells into brown adipocytes . agents identified in this manner can be used for a variety of research , diagnostic and therapeutic purposes , including , for example , treatment of metabolic diseases such as obesity , type 2 diabetes , insulin - resistance , dyslipidemia , and the like . in some embodiments , an agent identified by an assay according to the present disclosure is optimized for improvement of its physico - chemical and / or pharmacokinetic properties . expression of ucp1 , fabp4 ( ap2 ), pparγ2 , mttfa , pgc - 1α , and / or cox iv in bat progenitor cells in vitro and in vivo can be enhanced according to methods provided in the present disclosure . in some embodiments , exposure to adipogenic media can be used to stimulate increased expression of ucp1 , fabp4 ( ap2 ), pparγ2 , mttfa , pgc - 1α , and / or cox iv in bat progenitor cells . accordingly , in some embodiments the following agents , or combinations thereof , can be used to promote the differentiation of bat progenitor cells into brown adipocytes and / or induce the expression of ucp1 , fabp4 ( ap2 ), pparγ2 , mttfa , pgc - 1α , and / or cox iv in bat progenitor cells in vitro , in vivo , or both : a pde3 inhibitor ( e . g ., siguazodan ), a pde4 inhibitor ( e . g ., rolipram ), a derivative of prostaglandin f2 ( pgf2 ) such as 9β , 11α - prostaglandin f2 or 9α , 11β - prostaglandin f2 , a pepdide derived ( e . g ., a portion ) from the pituitary adenylate cyclase - activating polypeptide ( pacap , adcyap1 , uniprot p18509 ) gene such as the pacap propeptide of 55 aa ( aa 25 - 79 ), bdnf ( brain - derived neurotrophic factor ), a tgr5 agonist such as oleanolic acid , bmp - 7 , a flavonoid such as kaempferol ( kmp , cas number 520 - 18 - 3 ), a stimulator of soluble guanylate cyclase ( sgc ) such as riociguat ( bay 63 - 2521 , cas 625115 - 55 - 1 ), fibroblast growth factors ( such as fgf7 ( fibroblast growth factor - 7 , kgf , keratinocyte growth factor ), fgf10 ( fibroblast growth factor - 10 , kgf - 2 , keratinocyte growth factor - 2 ), or fgf13 ( fibroblast growth factor - 13 )), bnp ( b - type natriuretic peptide ), a trpm8 ( cmri ) ligand such as menthol or icilin , a bombesin peptide such as from the toad ( uniprot p84214 ), cntf ( ciliary neurotrophic factor , uniprot p05231 ), interleukin - 6 ( il - 6 ), orexin b , sdf - 1γ ( cxcl12 ), or guanfacine hydrochloride . further , in other embodiments additional agents or combinations thereof that can be used to promote the differentiation of bat progenitor cells into brown adipocytes and / or induce the expression of ucp1 include prostaglandin j2 ( pgj2 ), 24 ( s )- hydroxycholesterol , forms of vitamin d such as 1 , 25 - dihydroxyvitamin d3 or 24 , 25 - dihydroxyvitamin d3 , and a cyclooxygenase inhibitor such as diflunisal . in still other embodiments , additional agents or combinations thereof that can be used to promote the differentiation of bat progenitor cells into brown adipocytes and / or induce the expression of ucp1 include bone morphogenetic proteins such as bmp5 ( bone morphogenetic protein 5 , uniprot p22003 ) and bmp6 ( bone morphogenetic protein 6 , uniprot p22004 ), platelet - derived growth factor receptor - like protein ( pdgfrl , uniprot q15198 ), vascular endothelial growth factor d ( vegf - d , figf , uniprot o43915 ), cytl1 ( cytokine - like protein 1 , uniprot q9nrr1 ), scg2 ( secretogranin - 2 , uniprot p13521 ), nptx2 ( neuronal pentraxin - 2 , uniprot p47972 ), olfml2b ( olfactomedin - like protein 2b , uniprot q68bl8 ), tfpi2 ( tissue factor pathway inhibitor 2 , uniprot p48307 ), ifne ( interferon epsilon , uniprot q86wn2 ), a prostaglandin f2 - α receptor ( ptgfr , prostanoid fp receptor , uniprot p43088 ) ligand such as prostaglandin f2 , cntf ( ciliary neurotrophic factor ), interleukin - 6 ( il - 6 , uniprot p05231 ), interleukin - 15 ( il - 15 , uniprot p40933 ), cxcl12 (( chemokine ( c - x - c motif ) ligand 12 ), stromal cell - derived factor 1 , sdf1 , uniprot p48061 isoform sdf - 1g / uniprot p48061 - 3 ), and / or a ligand of atypical chemokine receptor 3 ( ackr3 , cmkor1 , cxcr7 , gpr159 , rdc1 , uniprot p25106 ) such as sdf1 ( cxcl12 ). in still further embodiments , other agents or combinations thereof that can be used to promote the differentiation of bat progenitor cells into brown adipocytes and / or induce the expression of ucp1 include a biguanide such as metformin , an antihistamine such as famotidine , an antidopaminergic such as tiapride hydrochloride , spiperone , thiethylperazine , a microtubule modulator such as colchicine , a rauwolfia alkaloid or derivative of such as reserpine or syrosingopine , a potassium channel ligand such as minoxidil , probenecid , or a calcium channel antagonist such as felodipine . in some embodiments , treatment of a subject , including a human subject , with one or a combination of agents shown here , results in an increase in the production of ucp1 mrna or protein in the subject &# 39 ; s skeletal muscle . for example , treatment of subjects with rosiglitazone can , in some embodiments , induce the appearance or differentiation of brown adipocytes in skeletal muscle , enhance expression of the ucp1 gene in existing brown adipocytes in or near skeletal muscle ( between myofibers , at the surface of and / or adjacent to skeletal muscle tissue ), or both . in some embodiments the appearance or differentiation of brown adipocytes in skeletal muscle can be induced in a subject suffering from a metabolic disease . the brown adipocytes can provide a glucose sink with high mitochondrial and cellular respiration and fatty acid oxidation rates , dissipating energy as heat ( uncoupled oxidative phosphorylation ). the subject metabolic rate can be enhanced , and a decrease in body weight can be induced . induction of the appearance or differentiation of brown adipocytes can also yield improvements in insulin sensitivity , blood glucose homeostasis and cardiovascular disease risk factors . brown adipocytes may further secrete factors that contribute to reaching a healthy energy balance and low body fat levels , increased insulin sensitivity and improved blood glucose homeostasis or cardiovascular health . accordingly , in some embodiments the agents disclosed herein , or combinations thereof , can be used for treatment of a subject , including a human subject . in some aspects , these agents may promote the differentiation of bat progenitor cells into brown adipocytes . in other aspects these agents may induce the expression of ucp1 , fabp4 ( ap2 ), pparγ2 , mttfa , pgc - 1α , and / or cox iv in bat progenitor cells in vitro , in vivo , or both . in some aspects the treated metabolic disease may be obesity , type ii diabetes , insulin resistance , hyperinsulinemia , hypertension , hyperlipidemia , hepatosteatosis , fatty liver , non - alcoholic fatty liver disease , hyperuricemia , polycystic ovarian syndrome , acanthosis nigricans , hyperphagia , endocrine abnormalities , triglyceride storage disease , bardet - biedl syndrome , laurence - moon syndrome , prader - willi syndrome , neurodegenerative diseases , and alzheimer &# 39 ; s disease . in other embodiments , agents may be used to activate isolated , autologous bat progenitor cells that are then used for treatment of a subject , including a human subject . gene chip studies were performed to identify molecular pathways that play a role in the differentiation of cd31 - progenitor cells into brown adipocytes and / or the induction of the expression of ucp1 . cd31 - cells were isolated from human skeletal muscle biopsies as described previously in wo2013071063 which is incorporated herein by reference , and were used in two studies : ( 1 ) camp study : cd31 - cells were differentiated as described in wo2013071063 and incorporated herein by reference ( control ) plus addition of vehicle ( control 1 sample ) or camp ( camp sample ); and ( 2 ) rosiglitazone study : cd31 - cells were differentiated as described previously in wo2013071063 except that rosiglitazone was omitted from the adipogenic medium ( control 2 sample ). rosiglitazone was added only to the second sample ( rosiglitazone sample ) in this study . as discussed above , these agents have been shown to promote the differentiation of cd31 - cells into brown adipocytes and the expression of ucp1 . total rna was purified from these samples , and transcriptional profiles were assessed with illumina human wg - 6 beadchip ( expression analysis , inc ., durham , n . c .). results were analyzed with ingenuity pathway analysis 7 . 0 ( trial version ). these results were used to determine what molecular pathways are involved in the differentiation of cd31 - cells into brown adipocytes , and , more importantly , what molecular targets can be used for the development of agents that promote the appearance of brown adipocytes and the expression of ucp1 . based on this work , the following mechanisms and agents were found to promote brown adipocyte development from bat progenitor cells : a pparγ ligand ( e . g ., rosiglitazone ), a pde3 inhibitor ( e . g ., siguazodan ), a pde4 inhibitor ( e . g ., rolipram ), bmp7 ( bone morphogenetic protein 7 , uniprot p18075 ), bmp5 ( bone morphogenetic protein 5 , uniprot p22003 ), bmp6 ( bone morphogenetic protein 6 , uniprot p22004 ), fgf7 ( fibroblast growth factor - 7 , kgf , keratinocyte growth factor ), fgf10 ( fibroblast growth factor - 10 , kgf - 2 , keratinocyte growth factor - 2 ), bnp ( b - type natriuretic peptide ), fgf13 ( fibroblast growth factor - 13 ), bdnf ( brain - derived neurotrophic factor ), a stimulator of soluble guanylate cyclase ( sgc ) ( e . g ., riociguat ( bay 63 - 2521 , cas 625115 - 55 - 1 ), a trpm8 ( cmri ) ligand ( e . g ., menthol , icilin ), platelet - derived growth factor receptor - like protein ( pdgfrl , uniprot q15198 ), vascular endothelial growth factor d ( vegf - d , figf , uniprot o43915 ), cytl1 ( cytokine - like protein 1 , uniprot q9nrr1 ), scg2 ( secretogranin - 2 , uniprot p13521 ), nptx2 ( neuronal pentraxin - 2 , uniprot p47972 ), olfml2b ( olfactomedin - like protein 2b , uniprot q68bl8 ), tfpi2 ( tissue factor pathway inhibitor 2 , uniprot p48307 ), ifne ( interferon epsilon , uniprot q86wn2 ), and a prostaglandin f2 - α receptor ( ptgfr , prostanoid fp receptor , uniprot p43088 ) ligand such as prostaglandin f2 . still other mechanisms / agents that were found to promote brown adipocyte development from bat progenitor cells based on gene chip data include : a peptide derived from the pituitary adenylate cyclase - activating polypeptide ( pacap , adcyap1 , uniprot p18509 ) gene such as pacap propeptide of 55 aa ( aa 25 - 79 ) ( 200 nm - 2 μm ), cntf ( ciliary neurotrophic factor ), interleukin - 6 ( il - 6 , uniprot p05231 ), interleukin - 15 ( il - 15 , uniprot p40933 ), cxcl12 ( chemokine ( c - x - c motif ) ligand 12 ), stromal cell - derived factor 1 ( sdf1 , uniprot p48061 ) isoform sdf - 1g / uniprot p48061 - 3 ), and / or a ligand of atypical chemokine receptor 3 ( ackr3 , cmkor1 , cxcr7 , gpr159 , rdc1 , uniprot p25106 ) such as sdf1 ( cxcl12 ). cd31 - cells can be used as a tool to identify agents ( e . g ., compounds , proteins , biologicals , and the like ) that induce the differentiation of these cells into brown adipocytes or modulate the expression of ucp1 . for example , an rt - pcr based approach can be used to measure ucp1 mrna levels which may be affected by certain agents . this allows the identification of agents that can enhance the differentiation of cd31 - cells into brown adipocytes and / or the expression of ucp1 by enhancing the transcription of the ucp1 gene and / or by stabilizing the ucp1 transcript . for example , a pparγ ligand like rosiglitazone can be used to promote the differentiation of cd31 - progenitor cells into brown adipocytes ( fig1 - 19 , 20a , 21 , 22 ). another example is the use of the recombinant protein , human bmp - 7 ( fig1 - 19 , 21 ). a robust method , previously disclosed in wo2013071063 and incorporated herein by reference , was used for detection of cd31 - cell differentiation into brown adipocytes by simultaneously quantifying mrna species corresponding to the brown adipocyte marker ucp1 , the adipocyte marker pparγ2 , and the “ housekeeping ” gene cyclophilin a which was used as the internal control . this method permits analysis of a large number of samples to identify agents that enhance the differentiation of cd31 - cells into brown adipocytes . when differentiated into brown adipocytes cd31 - cells express much higher levels of ucp1 and pparγ2 mrna for a given level of cyclophilin a . ucp1 and pparγ2 mrna levels normalized to cyclophilin a mrna levels give an indication of the level of differentiation of the cd31 - cells into brown adipocytes , independent of the total number of cells in the sample . quantification of ucp1 , pparγ2 and cyclophilin a mrna by multiplexed taqman real - time pcr was thus used to quantify differentiation of the cd31 - cells into brown adipocytes . using this method the following agents were identified or confirmed to promote the differentiation of bat progenitor cells into brown adipocytes and / or induce the expression of ucp1 , fabp4 ( ap2 ), pparγ2 , mttfa , pgc - 1α , and / or cox iv in bat progenitor cells in vitro , in vivo , or both : a pparγ ligand like rosiglitazone , a pde3 inhibitor like siguazodan , a pde4 inhibitor like rolipram , a derivative of prostaglandin f2 ( pgf2 ) like 9β , 11α - prostaglandin f2 , a pepdide derived from the pituitary adenylate cyclase - activating polypeptide ( pacap , adcyap1 , uniprot p18509 ) gene like the pacap propeptide of 55 aa ( aa 25 - 79 ), bdnf ( brain - derived neurotrophic factor ), a tgr5 agonist like oleanolic acid , bmp - 7 , kaempferol ( kmp , cas number 520 - 18 - 3 ), a stimulator of soluble guanylate cyclase ( sgc ) like riociguat ( bay 63 - 2521 , cas 625115 - 55 - 1 ), fgf7 ( fibroblast growth factor - 7 , kgf , keratinocyte growth factor ), fgf10 ( fibroblast growth factor - 10 , kgf - 2 , keratinocyte growth factor - 2 ), bnp ( b - type natriuretic peptide ), a trpm8 ( cmri ) ligand like menthol or icilin , bombesin , cntf ( ciliary neurotrophic factor ), interleukin - 6 ( il - 6 ), orexin b , sdf - 1γ ( cxcl12 ), and fgf13 ( fibroblast growth factor - 13 ). except otherwise indicated , all organic and inorganic chemicals of analytical or molecular biology grade were purchased from sigma chemical co . ( st louis , mich . ), life technologies ( grand island , n . y . ), genscript , prospec , lifetein , anaspec . rosiglitazone was purchased from cayman chemical (# 71742 ) and recombinant human bmp7 ( rhbmp7 ) was from r & amp ; d systems ( 100 μg / ml , 6 . 3 μm , # 354 - bp - 010 ). cells were seeded at 10 , 000 per cm 2 in 0 . 2 % gelatin coated plates ( 48 - well tissue culture , chemglass # cls - 3500 - 048 ), cultured until confluency ( 2 - 4 days ) at 37 ° c . in endothelial cell growth medium - 2 ( egm2 ) ( bulletkit growth medium , lonza # cc - 3162 ) and until differentiation ( 10 - 16 more days ). after 2 or 3 days in egm2 medium the cells were induced to differentiate by replacing the medium with an adipogenic medium , which is a modification of the adipogenic medium described by rodriguez et al . [ 21 ] and may or may not contain a differentiation inducing agent ( e . g ., pparγ agonist ). the mdm described above contains : dmem / ham &# 39 ; s f - 12 50 / 50 mix ( 3 . 151 g / l , 17 . 5 mm d - glucose , 3 . 651 g / l l - glutamine ) ( cellgro # 10 - 090 - cv ), 5 μg / ml ( 0 . 86 μm ) insulin , 1 μm dexamethasone , 100 μm 3 - isobutyl - 1 - methylxanthine , 0 . 2 nm 3 , 3 ′, 5 - triiodo - l - thyronine , 10 μg / ml ( 127 nm ) transferrin , and 1 % penicillin - streptomycin . if rosiglitazone is used as a differentiation - inducing agent , it can be supplied at 1 μm or any other concentration sufficient to induce differentiation of bat progenitor cells into adipocytes . for cell expansion studies , confluent cells grown in egm2 medium only were detached by treatment with trypsin - edta for 3 - 5 min at 37 ° c ., and then split 1 : 3 or 1 : 4 and cultured as described above . quantification of ucp1 and pparγ2 mrna by quantitative reverse transcription , real - time pcr total rna was prepared from cells using purelink rna isolation kit ( invitrogen # 12183 - 016 ) first strand cdna were synthesized using the high capacity cdna reverse transcription kit ( applied biosystems , foster city , calif .) and random primers . quantitative real - time pcr was performed using an applied biosystems steponeplus ™ instrument , taqman gene expression master mix ( applied biosystems # 4369016 ), and custom taqman gene expression probes and primers for human uncoupling protein - 1 “ ucp1 ” ( genbank nm_021833 ) and for human peptidylprolyl isomerase a “ cyclophilin a ” ( genbank nm_021130 ) . custom taqman gene expression reagents were also developed for simultaneous measurement of peroxisome proliferator - activated receptor gamma , transcript variant 2 ( pparγ2 ) ( genbank nm_015869 ) in a multiplexed fashion ( with ucp1 and cyclophilin a ): ucp1 fam - mgb probe : tca agg ggt tgg tac ctt cc ( seq id no . : 1 ), sense primer : cac taa cga agg acc aac gg ( seq id no . : 2 ), and antisense primer : ttc cag gat cca agt cgc aa ( seq id no . : 3 ). cyclophilin a ned - mgb probe : act gcc aag act gag tgg tt ( seq id no . : 4 ), sense primer : caa atg ctg gac cca aca ca ( seq id no . : 5 ), and antisense primer : tca ctt tgc caa aca cca ca ( seq id no . : 6 ). pparg2 vic - mgb probe : tca caa gaa atg acc atg gtt g ( seq id no . : 7 ), sense primer : agc gat tcc ttc act gat aca c ( seq id no . : 8 ), and antisense primer : cca gaa tgg cat ctc tgt gt ( seq id no . : 9 ). cyclophilin a was used as a control to account for any variations due to the efficiency of reverse transcription . arbitrary units were determined by normalizing target mrna levels to cyclophilin a mrna levels ( based on cts ). data are expressed as means ± sem . significances were evaluated using the unpaired student &# 39 ; s t - test . significances were set at p & lt ; 0 . 05 . pictures of cells were taken using a hand - held digital camera ( nikon coolpix 950 ) and inverted microscope ( nikon tms ) used for cell culture observations ; images were optimized using adobe photoshop elements 8 functions for auto contrast and auto levels . the section headings and subheadings used in this specification are for organizational purposes only and are not to be construed as limiting the subject matter described in any way . further , while the present teachings are described in conjunction with various embodiments , it is not intended that the present teachings be limited to such embodiments . on the contrary , the present teachings encompass various alternatives , modifications , and equivalents as will be appreciated by those of skill in the art . differentiation of brown adipocyte progenitors into brown adipocytes can be detected through quantification of ucp1 protein by immunohistochemistry ( ihc ). culturing and differentiation of cd31 - cells into brown adipocytes were performed using adipogenic differentiation medium lacking ( minimal differentiation medium , mdm ) or containing 1 μm rosiglitazone ( reference differentiation medium , rdm ). after 15 days of differentiation cells were fixed with 4 % paraformaldehyde pbs ph 7 . 4 , and incubated with a ucp1 antibody ( abcam ab23841 ) and alexafluor 488 goat anti - rabbit antibody to quantify relative ucp1 levels ( green ) according to standard protocols . prior to fixation of cells , nuclei were labeled with 5 μm dapi ( blue ) for 10 minutes . each treatment condition was evaluated in triplicate in a 96 - well plate corresponding to approximately 360 - 480 cells for each data point in total . the incell 1000 developer toolbox software was used to develop an automated cell detection script to measure ucp1 signal intensity , using the nuclei and cytoplasm detection algorithms . as a readout , total intensity of ucp1 signal within the cell was used , normalized to cell number . in some embodiments , agents or combinations thereof that were identified using this technique include famotidine , tiapride hydrochloride , guanfacine hydrochloride , reserpine , minoxidil , spiperone , diflunisal , syrosingopine , probenecid , metformin , thiethylperazine , colchicine , and felodipine . bodipy fluorescent dye - labeled neutral lipids become incorporated in cytoplasmic lipid droplets allowing analysis of cellular fatty acid uptake and adipocyte differentiation by fluorescent cellular imaging . cells are incubated with c1 - bodipy ® 500 / 510 c12 ( molecular probes # d - 3823 ) for 3 to 6 hours before imaging on a microplate - based high - throughput , high - content , brightfield and fluorescence cellular imager and analyzer ( cyntellect celigo ® or ge healthcare in cell analyzer ). various aspects of the present disclosure may be used alone , in combination , or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is therefore not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings . for example , aspects described in one embodiment may be combined in any manner with aspects described in other embodiments . use of ordinal terms such as “ first ,” “ second ,” “ third ,” etc ., in the claims to modify a claim element does not by itself connote any priority , precedence , or order of one claim element over another or the temporal order in which acts of a method are performed , but are used merely as labels to distinguish one claim element having a certain name from another element having a same name ( but for use of the ordinal term ) to distinguish the claim elements . also , the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting . the use of “ including ,” “ comprising ,” or “ having ,” “ containing ,” “ involving ,” and variations thereof herein , is meant to encompass the items listed thereafter and equivalents thereof as well as additional items . the present disclosure provides among other things novel compositions capable of recruiting brown adipocytes in vitro and in vivo . while specific embodiments of the subject disclosure have been discussed , the above specification is illustrative and not restrictive . many variations of the disclosure will become apparent to those skilled in the art upon review of this specification . the full scope of the disclosure should be determined by reference to the claims , along with their full scope of equivalents , and the specification , along with such variations . all publications , patents and patent applications referenced in this specification are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication , patent or patent application were specifically indicated to be so incorporated by reference . m . klingenspor , cold - 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