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MODULATING ADIPOSE TISSUE AND ADIPOGENESIS

20180009903 · 2018-01-11

    Inventors

    Cpc classification

    International classification

    Abstract

    The invention relates to the field of obesity and related metabolic diseases. More specifically, the invention relates to methods of reducing aggrecanase activity or antigen in mammals in order to enhance brown adipose tissue (BAT) development, to promote conversion of white adipose tissue (WAT) into BAT in vivo, and to limit triglyceride accumulation and steatosis in the liver. The invention also relates to a strategy of neutralization or depletion of ADAMTS5 as a strategy to inhibit adipogenesis and more specifically, the invention relates to a method of reducing aggracanase-2 (ADAMTS5, A Disintegrin and Metalloproteinase with Thrombospondin motif 1; member 5) antigen and/or activity in mammals in order to impair differentiation of precursor cells into mature adipocytes (i.e., adipogenesis).

    Claims

    1. A method for treating of an ADAMTS5 associated disorder in a subject by administering an ADAMTS5 inhibitor, wherein the ADAMTS5 associated disorder is an overweight disorder or a metabolic disorder which is obesity.

    2. The method according to claim 1, wherein the ADAMTS5 associated disorder is an overweight disorder or a metabolic disorder selected from obesity, type II diabetes, insulin resistance, disorder associated with insufficient insulin activity, disrupted thermogenesis in overweight, liver triglyceride accumulation, hepatosteatosis, fatty liver and non-alcoholic fatty liver disease.

    3. The method according to claim 1, whereby the ADAMTS5 inhibitor is an antigen binding protein comprising at least one immunoglobulin variable domain capable of binding to human ADAMTS5.

    4. The method according to claim 1, whereby the inhibitor is selected from a specific polyclonal antibody, a monoclonal antibody, a full-length antibody, a binding fragment of an antibody and a surrogate of an antibody capable of binding to human ADAMTS5.

    5. The method according to claim 1, whereby the inhibitor is a binding fragment of an antibody or a surrogate of an antibody selected from the group consisting of aFab′, F(ab′)2, Fab, Fv, vIgG, scFv fragments and surrobodies, rIgG, disulfide-stabilized Fv antibodies (dsFv), diabodies, triabodies, and single domain antibodies, such as a camelized antibody or nanobody or humanized camel or shark antibody or nanobody and capable of binding to human ADAMTS5.

    6. The method according to claim 1, whereby the ADAMTS5 inhibitor is an ADAMTS5 antigen-binding fragment of a monoclonal antibody selected from a scFV, Fab, Fab2, F(ab′)2, Fv or dAB and capable of binding to human ADAMTS5.

    7. The method according to claim 1, whereby the ADAMTS5 inhibitor is a chimeric or humanized antibody or antigen-binding fragment thereof and capable of binding to human ADAMTS5.

    8. The method according to claim 1, whereby the ADAMTS5 inhibitor specifically binds to ADAMTS5.

    9. The method according to claim 1, whereby the ADAMTS5 inhibitor specifically binds to human ADAMTS5.

    10. The method according to claim 1, whereby inhibitor is a monoclonal antibody comprising a heavy chain comprising CDRH1, CDRH2 and CDRH3 and a light chain comprising CDRL1, CDRL2 and CDRL3, wherein the complementarity determining regions (CDRs) of the heavy chain are selected from the group of: a) CDRH1 having at least about 80% sequence identity to amino acid sequence DAWMD; b) CDRH2 having at least about 70% sequence identity to amino acid sequence EIRHKANDHAIFYXESVKG; and c) CDRH3 having at least about 70% sequence identity to amino acid sequence TYYYGSSYGYCDV or PFAY; and the complementarity determining regions of the light chain are selected from the group of: d) CDRL1 having at least about 70% sequence identity to amino acid sequence KASQSVGTTIV or RTSENIYSYLA; e) CDRL2 having at least about 70% sequence identity to amino acid sequence NAKTLAE or SASNRXT; and f) CDRL3 having at least about 70% sequence identity to amino acid sequence QQYSSYPFT or QHHYGTPWT.

    11. The method according to claim 1, whereby the inhibitor is a monoclonal antibody comprising a heavy chain comprising CDRH1, CDRH2 and CDRH3 and a light chain comprising CDR1, CDRL2 and CDRL3, wherein the complementarity determining regions (CDRs) of the heavy chain are selected from: (a) CDRH1 is amino acid sequence DAWMD; (b) CDRH2 is select from amino acid sequence EIRHKANDHAIFYAESVKG, EIRNKANNHARHYAESVKG, EIRHKANDYAIFYDESVKG, EIRHKANDHAIFYDESVKG, or DIRNTANNHATFYAESVKG, EIRHKANDHAIFYDESVKG; and (c) CDRH3 is TYYYGSSYGYCDV or PFAY; and the complementarity determining regions of the light chain are selected from: (d) CDRL1 is select from amino acid sequence KASQSVGTTIV, RTSENIYSYLA, or KASQNVGTAVV; (e) CDRL2 is select from amino acid sequence NAKTLAE, SASNRHT, SASTRYT, or SASNRYT; and (f) CDRL3 is select from amino acid sequence QQYSSYPFT, QHHYGTPWT, QQYVNYPFT, or QQYTSYPFT.

    12. The method according to claim 1, whereby the inhibitor is a monoclonal antibody comprising six CDRs wherein: TABLE-US-00005 (SEQ ID NO: 2) a) CDRH1 is DAWMD, (SEQ ID NO: 13) CDRH2 is EIRNKANNHARHYAESVKG, and (SEQ ID NO: 18) CDRH3 is TYYYGSSYGYCDV and (SEQ ID NO: 20) CDRL1 is RTSENIYSYLA, (SEQ ID NO: 22) CDRL2 is NAKTLAE and (SEQ ID NO: 27) CDRL3 is QHHYGTPWT; or (SEQ ID NO: 2) b) CDRH1 is DAWMD, (SEQ ID NO: 15) CDRH2 is EIRHKANDHAIFYDESVKG, and (SEQ ID NO: 5) CDRH3 is PFAY and (SEQ ID NO: 19) CDRL1 is KASQSVGTTIV, (SEQ ID NO: 23) CDRL2 is SASNRHT and (SEQ ID NO: 29) CDRL3 is QQYTSYPFT.

    13. (canceled).

    14. The method according to claim 1, whereby the inhibitor is a nucleic acid molecule in a vector to express a therapeutic agent.

    15. The method according to claim 1, whereby the ADAMTS5 inhibitor comprises an antibody, or antigen-binding fragment thereof, that: a) binds to ADAMTS5 with a dissociation constant (K D) of 150 pM or less, as determined by real-time biospecific interaction analysis (BIA) using surface plasmon resonance (SPR) technology, or with an IC50 of 100 pM or less; or b) binds to a neutralizing epitope of human ADAMTS5 with an affinity of at least about 5×10.sup.4 liter/mole as measured by an association constant (Ka).

    16. (canceled).

    17. The method according to claim 1, whereby the treatment of the subject induces one or more primary efficacy endpoints or direct efficacy endpoints of a) increase in thermogenesis in brown adipose tissue, b) increase in insulin sensitivity of skeletal muscle, white adipose tissue, or liver; c) increase in glucose tolerance, d) increase in basal respiration, maximal respiration rate, or uncoupled respiration, e) increase in metabolic rate, f) decrease in hepatosteatosis or g) decrease in baseline in body weight.

    18. The method according to claim 1, which can modulate one or more of the following biological activities: thermogenesis in adipose cells, differentiation of adipose cells, and insulin sensitivity of adipose cells.

    19. The method according to claim 1, wherein the treatment prevents or reduces the de novo adipogenesis or prevents or reduces de novo fat development.

    20. The method according to claim 1, wherein the treatment impairs differentiation of adipocyte precursor cells into mature adipocytes (hyperplasia) in a mammalian subject.

    21. The method according to claim 1, whereby the ADAMTS5 inhibitor is selected from the group consisting of a peptide, a peptidomimetic, an antibody, a double-stranded RNA, an aptamer, a small interfering RNA (siRNA), a peptide fragment of ADAMTS5 and a ribozyme

    22. The method according to claim 21, whereby the peptide, peptidomimetic, antibody, double-stranded RNA, aptamer, small interfering RNA (siRNA), peptide fragment of ADAMTS5 or ribozyme therapy against ADAMTS5 is delivered by gene therapy and in vivo expressed in a subject.

    23. (canceled).

    24. An ADAMTS5 inhibitor selected from: a. an antigen binding protein comprising at least one first immunoglobulin variable domain capable of binding to human ADAMTS5; b. a nucleic acid molecule in a vector to express a therapeutic agent; and c. a peptide, a peptidomimetic, an antibody, a double-stranded RNA, an aptamer, a small interfering RNA (siRNA), a peptide fragment of ADAMTS5 or a ribozyme.

    Description

    DRAWING DESCRIPTION

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0188] FIG. 1: UCP-1 expression in SC and GN WAT of WT (white bars) and ADAMTS5−/− (black bars) mice kept on SFD (A) or HFD (B) for 15 weeks.

    [0189] Data are means ±SEM of 8 to 12 determinations. Values are corrected for the housekeeping gene β-actin and subsequently normalized to SC WAT of WT mice on either SFD or HFD. *p<0.05 and **p<0.01 for ADAMTS5−/− versus WT.

    [0190] FIG. 2: Western blotting for UCP-1 with protein extracts of SC or GN WAT of WT (+/+) and ADAMTS5 deficient (−/−) mice.

    [0191] (A)Diet study: WT and ADAMTS−/− mice were kept on SFD or HFD for 15 weeks. Sample of BAT is applied at 200-fold lower concentration that those of SC and GN WAT.

    [0192] (B)Cold exposure: WT and ADAMTS−/− mice on SFD were kept for 2 weeks at 24° C. or at 4° C.

    [0193] β-actin was used as internal standard. Two representative samples are shown for each group.

    [0194] FIG. 3: Effect of ADAMTS5 on liver steatosis. Liver sections of WT or ADAMTS5 deficient (KO) mice kept on SFD or HFD for 15 weeks are stained with haemotoxylin/eosin (A) and total steatose area (B) as well as liver triglyceride (TG) content (C) were quantified. *P<0.05

    [0195] FIG. 4: Effect of ADAMTS5 gene silencing on in vitro differentiation of 3T3-F442A preadipocytes. (A-D) Time course of the expression of ADAMTS5 (A), ADAMTS4 (B), biglycan (C) and versican (D) during differentiation of 3T3-F442A cells with shRNA against ADAMTS5 (open squares) or scrambled shRNA (black squares). (E) Oil Red O staining at day 12. The scale bar corresponds to 200 μm. (F) Quantification of Oil Red O staining. (G-I) Time course of the expression of the adipogenic markers aP2 (G), PPARγ (H) and Pref←1 (I). Data are means ±SEM of 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001 versus control.

    [0196] FIG. 5: Differentiation of murine embryonic fibroblasts into mature adipocytes. (A) PCR genotyping of MEF derived from ADAMTS5+/+ (WT, 642 bp) or ADAMTS5−/− (KO, 374 bp) mice. Oil Red O staining (B) and quantification (C) of WT and KO MEF at day 12 of differentiation. The scale bar in panel (B) corresponds to 200 μm. (D-G) Time course of the expression of ADAMTS5 (D) or ADAMTS4 (E) and the adipogenic markers aP2 (F) and PPARγy2 (G) during differentiation ADAMTS5+/+ (open circles) and ADAMTS5−/− (closed circles) MEF. (H-K) Gene expression profile of aggrecan (H), biglycan (I), brevican (J) and versican (K) at days 0 and 12. Data are means ±SEM of 2 independent experiments performed in triplicate. *p<0.05, **p<0.01, *** p<0.001 versus control.

    TABLE-US-00001 TABLE 1 Effect of ADAMTS-5 deficiency on organ weight and fat mass of mice kept on standard fat diet (SFD) or high fat diet (HFD) for 15 weeks. SFD HFD ADAMTS-5.sup.+/+ ADAMTS-5.sup.−/− ADAMTS-5.sup.+/+ ADAMTS-5.sup.−/− (n = 13) (n = 12) (n = 9) (n = 11) SC fat weight (mg) 297 ± 21.0 306 ± 38.5 1428 ± 80.5  1198 ± 88.3  GN fat weight (mg) 655 ± 47.5 582 ± 89.9 2274 ± 93.5  1998 ± 72.9* BAT (mg) 87 ± 4.5   140 ± 13.1*** 266 ± 28.3  428 ± 32.6** Liver (mg) 1191 ± 27.7   1399 ± 42.3** 3852 ± 292     2203 ± 201*** Pancreas (mg) 341 ± 18.4 391 ± 14.5 280 ± 14.6   378 ± 13.7*** Spleen (mg) 75 ± 4.9 83 ± 5.2 117 ± 13.1 103 ± 4.9  Lung (mg) 168 ± 6.7  176 ± 8.2  213 ± 24.7 189 ± 10.9 Heart (mg) 172 ± 4.6   212 ± 8.9** 179 ± 7.4   219 ± 9.0** Kidneys (mg) 398 ± 11.1  499 ± 26.4** 447 ± 20.8 486 ± 12.8 Brain (mg) 452 ± 6.1  463 ± 8.8  456 ± 9.8  460 ± 6.0  Data are means ± SEM of 9-13 experiments in each group. *p < 0.05, **p < 0.01, ***p < 0.001 versus ADAMTS-5.sup.+/+ mice kept on the corresponding diet SC, subcutaneous; GN, gonadal; BAT, brown adipose tissue

    TABLE-US-00002 TABLE 2 Effect of ADAMTS5 deficiency on expression of “browning” markers in adipose tissues of mice kept on standard fat diet (SFD) or high fat diet (HFD) for 15 weeks ADAMTS5.sup.+/+ ADAMTS5.sup.−/− SC GN BAT SC GN BAT SFD UCP-1 1.0 ± 0.6 0.002 ± 0.0005 1185 ± 80.2.sup.  42.0 ± 13*    0.02 ± 0.005*   1141 ± 94.4 UCP-2  1.0 ± 0.09 1.1 ± 0.07 1.5 ± 0.06 2.5 ± 0.1*** 3.2 ± 0.1***    0.5 ± 0.02*** Cidea  1.2 ± 0.25 0.04 ± 0.005 268 ± 9.6.sup.  15.5 ± 3.7***   0.2 ± 0.03***   212 ± 9.2** Pgc1a 1.05 ± 0.1  0.4 ± 0.06 12.3 ± 1.7  2.2 ± 0.29*  0.93 ± 0.16*  12.2 ± 2.2 Prdm16 1.04 ± 0.1  0.5 ± 0.03 3.8 ± 0.4  1.50 ± 0.09*  0.65 ± 0.05**   2.8 ± 0.4 HFD UCP-1 1.0 ± 0.7 0.03 ± 0.008 2887 ± 120.6  28.8 ± 8.4**  0.2 ± 0.06** 3184 ± 450 UCP-2  1.0 ± 0.07 2.5 ± 0.1  0.5 ± 0.02 3.3 ± 0.3*** 4.2 ± 0.2***   2.5 ± 0.3*** Cidea 1.1 ± 0.2 0.03 ± 0.002 114 ± 5.5.sup.  4.9 ± 0.6***  0.2 ± 0.02*** 126 ± 18 Pgc1a 1.06 ± 0.15 0.21 ± 0.03  8.2 ± 0.62 2.1 ± 0.25** 0.6 ± 0.1**  11.6 ± 1.6 Prdm16 1.02 ± 0.07 0.28 ± 0.05  1.61 ± 0.077 1.3 ± 0.091*  0.6 ± 0.05***  2.4 ± 0.3* Data are means ± SEM of 6-12 determinations. Values are corrected for the housekeeping gene β-actin and subsequently normalized to SC AT of ADAMTS-5.sup.+/+ mice on SFD or HFD. *p < 0.05, **p < 0.01, ***p < 0.001 versus ADAMTS-5.sup.+/+ mice kept on the corresponding diet. SC, subcutaneous; GN, gonadal; BAT, brown adipose tissue

    TABLE-US-00003 TABLE 3 Effect of cold exposure on adipose tissues in wild-type (ADAMTS-5.sup.+/+) and ADAMTS-5 deficient (ADAMTS-5.sup.−/−) mice. 24° C. 4° C. (n = 6) ADAMTS-5.sup.+/+ ADAMTS-5.sup.−/− ADAMTS-5.sup.+/+ ADAMTS-5.sup.−/− (n = 3) (n = 3) (n = 6) (n = 6) Body weight (g) Start 20.8 ± 0.3   21.2 ± 0.4 21.0 ± 0.4   21.2 ± 0.5  Week 1 22.1 ± 0.8   22.4 ± 0.5 22.2 ± 0.6   20.5 ± 0.6  Week 2 22.7 ± 0.7   23.1 ± 0.6 22.6 ± 0.6   20.1 ± 0.8* Food intake (g/day) Week 1 3.9 ± 0.1   4.5 ± 0.01 8.4 ± 0.2  8.8 ± 0.4 Week 2 3.7 ± 0.2   3.9 ± 0.1 9.0 ± 0.2  8.9 ± 0.4 SC fat (mg) 229 ± 13.2   226 ± 27.2 175 ± 13.6   235 ± 10.1* GN fat (mg) 396 ± 64.5   403 ± 95.5 220 ± 14.5  185 ± 19.5 BAT (mg) 64 ± 0.7  110 ± 6.7 82 ± 2.2   125 ± 13.2** Data are means ± SEM; *p < 0.05, **p < 0.01 versus ADAMTS-5.sup.+/+ mice kept on the corresponding temperature. SC, subcutaneous; GN, gonadal; BAT, brown adipose tissue

    TABLE-US-00004 TABLE 4 Effect of ADAMTS5 knock-down (KD) in 3T3-F442A preadipocytes on de novo adipogenesis following injection in NUDE mice kept on high fat diet for 4 weeks. Control ADAMTS5 KD (n = 4) (n = 4) Body weight start (g) 18.1± 0.8  20.9 ± 0.8   Body weight end (g) 24.3 ± 0.6   25.1 ± 0.7   SC fat weight (mg) 206 ± 13.4 198 ± 18.0 GN fat weight (mg) 315 ± 17.7 348 ± 36.4 de novo fat pad weight (mg) 24.5 ± 1.3   14.1 ± 1.1*  Data are means ± SEM; *p < 0.01