Patent Description:
Dupuytren's disease, also known as palmar fibromatosis or in its established disease stage Dupuytren's contracture, is a disease associated with the buildup of extracellular matrix materials such as collagen on the connective tissue of the hand (the palmar fascia) causing it to thicken and shorten with the result that the fingers curl into the palm.

Dupuytren's disease is a common fibrotic disorder (Hindocha, <NUM>). The mean age of treatment for the disease is <NUM> years (Chen, <NUM>), with onset approximately <NUM> years earlier. It exhibits a strong hereditary basis (Hurst, <NUM>). Dupuytren's disease causes the fingers to curl irreversibly into the palm, leading to significant impairment of hand function.

There is no approved treatment for early disease. Once patients have established deformities, the mainstay of treatment is surgical excision (fasciectomy) of the diseased tissue or cords (Davis, <NUM>). Patients with advanced disease are treated by surgical excision of diseased tissues. Surgery is recommended when patients develop flexion deformities of the digits of <NUM> degrees or more of the finger joints and suffer impaired hand function (Rayan, <NUM>). Between <NUM>-<NUM>% of patients develop recurrence over <NUM> years following surgery (Ullah, <NUM>; van Rijssen, <NUM>) and are treated with more extensive surgery that involves excision of the diseased tissue and the overlying skin (dermofasciectomy). Post-operatively, patients require <NUM>-<NUM> months of hand therapy and splintage (Hughes, <NUM>; Larson, <NUM>). Complications occur in about <NUM>% of surgical patients (Bulstrode, <NUM>; Crean, <NUM>).

Alternative, less invasive techniques have been developed to disrupt the cords of diseased tissue with either a needle (Beaudreuil, <NUM>) or collagenase digestion (Hurst, <NUM>). However, recurrence rates are high, affecting <NUM>% of patients treated with percutaneous needle fasciotomy (van Rijssen, <NUM>) and <NUM>% of those treated with collagenase (Peimer, <NUM>) at <NUM> years. The complication rate is <NUM>% following needle fasciotomy (Crean, <NUM>) and over <NUM>% after collagenase injection (Hurst, <NUM>).

In the United Kingdom, the vast majority of patients with established disease and finger contractures are treated surgically (Davis, <NUM>). Over <NUM>% of the <NUM>,<NUM> patients who have surgery for Dupuytren's disease per annum in the United Kingdom undergo fasciectomy. Recurrence rates are of the order of <NUM>% within <NUM> years of fasciectomy and the costs for dermofasciectomy for recurrent disease are much higher (Ullah, <NUM>). Neither surgical fasciectomyor or collagenase injection was found to be an effective use of health care resources (Chen, <NUM>).

Intralesional steroid injection and radiotherapy are two additional possible treatments for Dupuytren's disease. Intralesional steroid injection has been proposed based on a retrospective study of <NUM> patients with early Dupuytren's disease treated with steroid injection into the nodules at <NUM> week intervals (Ketchum, <NUM>). However, this treatment has found limited acceptance. Radiotherapy has also been used although <NUM>-<NUM>% of patients developed long term adverse effects, including dry skin, increased desquamation, skin atrophy, telangiectasia, erythema, altered heat and pain sensation (Seegenschmiedt, <NUM>; Pohl, <NUM>; Betz, <NUM>). Based on the published data The National Institute for Health and Care Excellence (NICE) does not recommend radiotherapy for Dupuytren's disease (NICE, <NUM>).

Therefore, there is a need to develop an effective therapy to prevent progression of early Dupuytren's disease while avoiding the necessity for invasive procedures. Also, there is a need to prevent the development of recurrent disease following surgery, needle fasciotomy, or collagenase injection in patients with established finger contractures.

Although one approach was suggest by Verjee, <NUM>, which studied the cellular mechanisms leading to the development of Dupuytren's disease and identified TNF as potential therapeutic target, there is still the need to develop further effective therapies which avoid invasive procedures and which prevent the development of recurrent disease in patients with established finger contractures.

The present invention relates to the embodiments as characterized in the claims. Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human body by therapy.

The subject invention provides a method of treating a patient suffering from a localized fibrotic condition, namely Dupuytren's disease, which comprises administering to the patient an amount of a TNF receptor <NUM> (TNFR2) antagonist effective to treat the patient, wherein the TNFR2 antagonist is.

<FIG>: Immune cells are present in Dupuytren's myofibroblast-rich tissue and release pro-inflammatory cytokines. (A) Flow cytometric analysis of cells isolated from freshly disaggregated Dupuytren's tissue. Intracellular α-SMA-positive (myofibroblasts; mean ± SD: <NUM> ± <NUM>%), cell surface CD68-positive/CD163-negative (classically activated M1 macrophages; mean ± SD: <NUM> ± <NUM>%), CD68-positive /CD163-positive (alternatively activated M2 macrophages; mean ± SD: <NUM> ± <NUM>%) and CD117-positive (mast cells; mean ± SD: <NUM> ± <NUM>% cells were quantified. ) (B) Serial histological sections of Dupuytren's tissue stained for α-SMA+ (myofibroblasts), CD68+ (monocytes) and chymase+ (mast cells) (Scale bar, <NUM>. )
<FIG>: TNF selectively induces IL-<NUM> mRNA expression in palmar dermal fibroblasts. 1ng/ml rhTNF stimulation for <NUM> selectively induced IL-<NUM> mRNA expression in dermal palmar fibroblasts (PF-D) at <NUM> hours. rhTNF did not have any effect on non-palmar dermal fibroblasts from Dupuytren's patients (NPF-D), or palmar dermal fibroblasts from normal individuals without Dupuytren's disease (PF-N). n=<NUM> patients for all cell types. **P<<NUM>
<FIG>: Myofibroblasts from patients with Dupuytren's disease (MF-D) respond to neutralizing anti-IL-<NUM> in a dose-dependent manner. (A) Anti-IL-<NUM> downregulates relative COL1A1 and α-SMA mRNA expression; (B) Anti-IL-<NUM> downregulates the relative expression of TNF receptor <NUM> (TNFR1) and TNF receptor <NUM> (TNFR2) (C) Anti-IL-<NUM> downregulates relative expression of mRNA of IL-<NUM> and its cell surface receptor ST2L. All values were normalized to fold change compared to untreated MF-D. n=<NUM> for <NUM>. 04ug/ml and 4ug/ml anti-IL-<NUM> and n=<NUM> for <NUM>. 4ug/ml anti-IL-<NUM>. IgG isotype control for anti-IL-<NUM> showed no effect in the relative expression of the genes at the corresponding doses tested. Data expressed as mean ± SEM. *P < <NUM>, **P < <NUM>, ***P < <NUM>, ****P<<NUM>. Methods: <NUM>×<NUM><NUM> cells were cultured in monolayer and treated with rhTNF (<NUM>-01A, Peprotech), neutralizing anti-TNF (MAB2101, R&D), neutralizing anti-TNF receptor <NUM> (MAB625, R&D), neutralizing anti-TNF receptor <NUM> (MAB726, R&D), anti-TNF/TNF receptors isotype control (MAB002, R&D), neutralizing anti-IL-<NUM> (<NUM>-P261, Peprotech) or isotype control (<NUM>-P00, Peprotech). The total RNA was extracted from each sample using a QIAshredder, followed by QIAamp RNeasy Mini Kit (<NUM>, Qiagen) with on-column RNase-Free DNase set (<NUM>, Qiagen) according to the manufacturer's instructions. RNA was eluted in 30µl RNase-free water provided and quantified using a NanoDrop ND-<NUM> spectrophotometer (NanoDrop Technologies), ensuring a <NUM>/<NUM> and <NUM>/<NUM> ratios ><NUM>. For real-time reverse transcription PCR, Inventoried TaqMan® Gene expression Assays were used for α-SMA (Hs00426835-g1), COL1A1 (Hs00164004-m1), TNFR1 (Hs01042313-m1) and TNFR2 (Hs00961749-m1), IL-<NUM>(Hs00369211_m1) and ST2 (Hs00545033_m1) (Applied Biosystems) with Reverse Transcriptase qPCR™ Mastermix No ROX (RT-QPRT-032XNR, Eurogentec). A total of 10µl of reaction mixture containing 2µl of RNA at 50ng/ml, 5µl of 2x buffer, <NUM>. 5µl Taqman probe, <NUM>. 05µl of Reverse Transcriptase enzyme with RNase inhibitors and <NUM>. 45µl RNase free water were added to each well of a <NUM> well plate. Samples were run on the ABI VAii 7TM Real-Time PCR System (Applied Biosystems). Expression was normalized to GAPDH (Hs02758991-g1, Applied Biosystems) and compared to the level of gene expression in baseline respective cell types, which were assigned the value of <NUM> using delta delta CT analysis performed with SDS software (Applied Biosystems). <FIG>: Immune cells are present in Dupuytren's nodules and secrete cytokines. <FIG>: Characterization of cells in Dupuytren's nodules by FACS. The majority of the cells present are myofibroblasts, there are significant numbers of CD45+ immune cells, with macrophages, including classically activated (M1) and alternatively activated (M2) phenotypes, T cells and mast cells. <FIG>: Immunostaining of Dupuytren's nodules. The majority of the cells are α-SMA positive myofibroblasts with interspersed CD68+ macrophages and tryptase positive mast cells. <FIG>: Chemokines secreted by freshly disaggregated cells from Dupuytren's nodules. Chemokine levels were detected by electrochemiluminescence assays in the supernatants of freshly disaggregated Dupuytren's nodular cells after <NUM> hours. n=<NUM> patient samples. CCL2 and CXCL10 are known chemoattractants for macrophages and CXCL8 (IL-<NUM>), CCL26 and CXCL10 for mast cells.

<FIG>: Dupuytren's disease is a localized inflammatory disorder characterized by the secretion of cytokines, including TNF, which leads to increased expression of TNFR2 in palmar fibroblasts and myofibroblasts from patients with Dupuytren's disease. <FIG>: A range of cytokines are secreted, including TNF and IL-<NUM>. Cytokines released by freshly isolated nodular cells in monolayer culture for <NUM> hours were measured using electrochemiluminescence. N=<NUM> samples for TGFβ1 and <NUM> for all other cytokines.

<FIG>: Cytokine levels do not depend on cell concentration. TNF secreted by varying numbers of freshly disaggregated cells from Dupuytren's nodules incubated for <NUM> hours in <NUM> of culture medium (DMEM) and <NUM>% fetal bovine serum. The levels of TNF were determined by ELISA. <FIG>: Cytokines in the plasma of patients with Dupuytren's disease compared with those secreted by freshly disaggregated nodular cells. Plasma levels of TNF, IL-1β, IL-<NUM> and IL-<NUM> were much lower in the systemic circulation. <FIG>: Characterization of cells in Dupuytren's nodules secreting TNF. The cells expressing TNF by FACS included macrophages, both classically and alternatively activated mast cells and T cells. <FIG>: Palmar dermal fibroblasts but not non-palmar dermal fibroblasts from the same individuals with Dupuytren's disease show increased expression of TNFR2 but not TNFR1 on treatment with TNF. Dupuytren's disease only occurs in the palm of genetically susceptible individuals. Exposure to physiologically relevant levels (<NUM>. 1ng/ml) of TNF of the palmar dermal fibroblasts from these patients resulted in increased expression of the inducible TNFR2 whilst expression of TNFR1 is reduced in these cells at both mRNA and protein level when exposed. <FIG>: Immunostaining of TNFR1 and TNFR2 in Dupuytren's nodules. The majority of the cells in Dupuytren's nodules express both TNFR1 and TNFR2. <FIG>: Palmar dermal fibroblasts and myofibroblasts show increased expression of TNFR2 but not TNFR1 on treatment with TNF. Non-palmar dermal fibroblasts from the same individuals with Dupuytren's disease show decreased expression of TNFR2. Quantification of immunofluorescent staining of matched cells from <NUM> donors. <NUM> cells were assessed from each patient. <FIG>: IL-<NUM> produced by myofibroblasts acts on mast cells and alternatively activated (M2) macrophages leading to increased TNF expression. <FIG>: Myofibroblasts from Dupuytren's nodules express IL-<NUM>. The majority of the cells expressing IL-<NUM> by FACS are myofibroblasts. <FIG>: Immunofluorescence staining of ST2 and IL-<NUM> freshly isolated myofibroblasts from Dupuytren's nodules. ST2 labels the cell surface whilst the IL-<NUM> is seen both within the nucleus and cytoplasm. <FIG>: Freshly isolated mast cells and macrophages from Dupuytren's nodules express ST2, the receptor for IL-<NUM>. Immunofluorescence co-staining. <FIG>: Mast cell lines show increased TNF secretion on exposure to IL-<NUM> in a dose-dependent manner. <FIG>: Only alternatively activated macrophages (M2) derived from human monocytes and pre-treated with TNF show increased secretion of TNF on exposure to IL-<NUM> in a dose-dependent manner. <FIG>: Palmar fibroblasts but not non-palmar fibroblasts from patients with Dupuytren's disease differentiate into myofibroblasts and show increased expression of IL-<NUM> and ST2 on exposure to TNF. <FIG>: Only palmar fibroblast differentiate into myofibroblasts as evidenced by increased α-SMA at mRNA and protein levels and increased COL1A1 mRNA expression on treatment with <NUM>. 1ng/ml TNF. <FIG>: Only palmar fibroblast show increased expression of IL-<NUM> and ST2 at both mRNA and protein levels whilst non-palmar fibroblasts show reduced expression of ST2 on exposure to TNF. <FIG>: Palmar fibroblasts show increased expression of nuclear and cytoplasmic IL-<NUM> and ST2 on treatment with TNF. Quantification of immunofluorescent staining of matched cells from <NUM> donors. <NUM> cells of each type were assessed from every patient. <FIG>: Inhibition of TNF, TNFR2 or IL-<NUM> down regulates the myofibroblast phenotype, with a combination of TNFR2 and IL-<NUM> being most effective. <FIG>: Anti-IL-<NUM> down regulates the expression of α-SMA and ST2 at both the mRNA and protein level and COL1A1 at the mRNA level in myofibroblasts from patients with Dupuytren's disease in a dose-dependent manner. Data from non-responders not shown. <FIG>: Only inhibition of TNF or TNFR2 but not TNFR1 downregulates the expression of α-SMA, COL1-A1, IL-<NUM> and ST2 at mRNA level and Il-<NUM> and ST2 also at protein level in myofibroblasts from responsive myofibroblasts from patients with Dupuytren's disease. Data from non-responders not shown. <FIG>: Venn diagram showing the relative efficacy of TNF or IL-<NUM> or TNFR2 inhibition. α-SMA was down regulated in myofibroblasts <NUM> of <NUM> patient samples (<NUM>%) by anti-TNF, <NUM> of <NUM> patient samples (<NUM>%) by anti-IL-<NUM> and in <NUM> of <NUM> samples by anti-TNFR2. Therefore, combined anti-TNF and anti-IL-<NUM> would be effective in <NUM> out of <NUM> patient samples (<NUM>%) and anti-TNFR2 and anti-IL-<NUM> in <NUM> of <NUM> samples (<NUM>%). <FIG>: Inhibition of expression of TNFR2, ST2 and most effectively TNFR2+ST2 down regulates myofibroblast phenotype
<FIG>: Proposed mechanism of action of IL-<NUM>. TNF secreted by resident immune cells, including macrophages and mast cells, converts precursor cells into myofibroblasts. As the cells differentiate into myofibroblasts, they secrete IL-<NUM>. This in turn acts on the immune cells, leading to further TNF production through a positive feedback loop, resulting in chronic localized inflammation and a fibrotic response. <FIG>: Proposed mechanism of action of IL-<NUM>. TNF secreted by resident immune cells, including macrophages and mast cells, converts precursor cells into myofibroblasts. As the cells become myofibroblasts, they secrete IL-<NUM>, which acts on the immune cells, leading to further TNF production, driving a positive feedback loop and a chronic fibrotic response. The IL-<NUM> also acts on the myofibroblasts via ST2 and further enhances IL-<NUM> expression.

As mentioned above, the localized fibrotic condition is Dupuytren's disease.

In another embodiment, the localized fibrotic condition is early disease state Dupuytren's disease. In another embodiment, the localized fibrotic condition is established disease state Dupuytren's disease.

As mentioned above, the TNFR2 antagonist is.

In one embodiment, the TNFR2 antagonist is an antibody selected from the group consisting of chimeric antibodies, humanized antibodies, human antibodies, and antigen binding fragments of chimeric humanized and human antibodies.

In one embodiment the TNFR2 antagonist is a bispecific antibody selected from the group consisting of.

In one embodiment, the TNFR2 antagonist is administered orally, intralesionally, by intravenous therapy or by subcutaneous, intramuscular, intraarterial, intracavitary, intracranial, or intraperitoneal injection. In another embodiment, the TNFR2 antagonist is administered by intravenous injection. In an additional embodiment, the TNFR2 antagonist is administered orally.

In one embodiment, the TNFR2 antagonist is injected directly into the affected tissue. In another embodiment, the TNFR2 antagonist is injected to a site of maximal cellularity or maximal inflammation.

In one embodiment, the TNFR2 antagonist is administered daily. In another embodiment, the TNFR2 antagonist is administered weekly. In a further embodiment, the TNFR2 antagonist is administered monthly. In an additionally embodiment, the TNFR2 antagonist is administered once every three months, once every <NUM> months, or once every <NUM> months.

In one embodiment, the TNFR2 antagonist is administered in an amount between about <NUM> and about <NUM>.

In another embodiment, the method further comprises administering a therapeutically, prophylactically or progression-inhibiting amount of a DAMP antagonist and/or an AGE inhibitor to the patient. In one embodiment, a DAMP antagonist is administered and the DAMP antagonist is an Alarmin antagonist.

In an additional embodiment, the Alarmin antagonist is one or more of an antagonist of HMGBl, an antagonist of S100A8, an antagonist of S100A9, an antagonist of SI00A8/<NUM>, and a heat shock protein.

For the foregoing embodiments, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. In addition, the elements recited in the packaging and pharmaceutical composition embodiments can be used in the method and use embodiments described herein.

The active compounds for use according to the invention may be provided in any form suitable for the intended administration. Suitable forms of the pre- or prodrug or functionally active protein produced as an active pharmaceutical ingredient, through recombinant DNA technology, include pharmaceutically (i.e. physiologically) acceptable salts, formulations, and excipients, known to those skilled in the art, for the compound(s) for use according to the invention.

As used herein, and unless stated otherwise, each of the following terms shall have the definition set forth below.

As used herein, "effective" as in an amount effective to achieve an end means the quantity of a component that is sufficient to yield an indicated therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this disclosure. For example, an amount effective to treat a localized fibrotic condition. The specific effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.

As used herein, an "amount" of a compound as measured in milligrams refers to the milligrams of compound present in a preparation, regardless of the form of the preparation. An "amount of compound which is <NUM>" means the amount of the compound in a preparation is <NUM>, regardless of the form of the preparation. Thus, when in the form with a carrier, the weight of the carrier necessary to provide a dose of <NUM> compound would be greater than <NUM> due to the presence of the carrier.

As used herein, "about" in the context of a numerical value or range means ±<NUM>% of the numerical value or range recited or claimed.

As used herein, to "treat" or "treating" encompasses, e.g., inducing inhibition, regression, or stasis of the disorder and/or disease. As used herein, "inhibition" of disease progression or disease complication in a subject means preventing or reducing or reversing the disease progression and/or disease complication in the subject.

By early disease, early disease stage, or early disease state it is meant that indications of disease are present, e.g. histological markers or more particularly clinical nodules in tissue, but in the absence of, for example, palpable cord or significant contracture. By early Dupuytren's disease, early disease stage Dupuytren's disease or early disease state Dupuytren's disease, it is meant that indications of Dupuytren's disease are present, for example histological markers or more particularly clinical nodules in palmar and/or digital tissue, and a flexion deformity of less than or equal to <NUM> degrees at any joint in the digit.

By established disease stage or established disease state, it is meant that clinical nodules are present, palpable cord is present and contracture is evident. By established disease stage Dupuytren's disease, it is meant that clinical nodules are present on the palm and digits of the hand and a flexion deformity of greater than <NUM> degrees at any joint in the digit.

Varying histological stages of Dupuytren's disease have been categorised in the literature, most succinctly by Rombouts, <NUM> and later authors, into three distinct stages: <NUM>) a proliferative stage with high cellularity and the presence of mitotic <FIG>) a fibrocellular stage characterised by high cellularity but no mitotic figures and the presence of reticulin network; and <NUM>) a fibrous stage with few cells separated by broad bundles of collagen fibres. Stage <NUM>) disease is believed to correlate with early disease stage as discussed above (i.e. presence of nodules but no contracture) and Dupuytren's stages <NUM>) and <NUM>) is believed to correlate with our Established Disease Stage (characterized by digital contracture). During early and early established disease stages, active myofibroblasts are collected in the established nodules and cords, especially in relation to the MCP and PIP joints and these drive the progression of flexion contractures of the digit.

The antagonists for use according to the present invention may be injected directly into the affected tissue. The antagonists for use according to the present invention may be injected to a site of maximal cellularity or maximal inflammation.

This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

By systematically unraveling the signaling pathways, TNF was identified as a novel therapeutic target to down regulate myofibroblasts, the cells responsible for matrix deposition and contraction in Dupuytren's disease (DD) (Verjee, <NUM>). Anti-TNF drugs have been used for more than <NUM> years to treat inflammatory conditions including rheumatoid arthritis, psoriatic arthritis, juvenile arthritis, Crohn's colitis, ankylosing spondylitis and psoriasis. Although these drugs can reduce the disease-associated inflammation, they do not reverse the underlying mechanisms that drive inflammation. As a result, they have to be administered at regular intervals. Whilst TNF inhibition could be used clinically to treat early Dupuytren's disease or to prevent recurrence, it will also likely need to be injected repeatedly on a regular basis, as for rheumatoid arthritis (Taylor, <NUM>). A survey showed a high acceptance rate for one injection per year but this fell sharply when frequency of injection was increased to <NUM> per year (Table <NUM>).

Targeting the pathway that drives chronicity will likely reduce the frequency of anti-TNF injections necessary to control progression of the disease. IL-<NUM> is likely one of the important factors responsible for the chronic inflammation seen in Dupuytren's disease and related disorders such as frozen shoulder and Peyronie's disease.

IL-<NUM> is the most recently described member of the IL-<NUM> family of cytokines and plays an important role in fibrotic disorders in a variety of tissues (Palmer, <NUM>). Its expression is limited to fibroblasts, myofibroblasts, smooth muscle, epithelial and dendritic cells (Schmitz, <NUM>) and is markedly increased by pro-inflammatory cytokines (Xu, <NUM>). It has been shown to play a key role in fibrotic disorders in a variety of tissues, including the skin (Rankin, <NUM>) and gut (Sponheim, <NUM>). In active lesions of ulcerative colitis, myofibroblasts are the major source of IL-<NUM> (Kobori, <NUM>). IL-<NUM> can activate inflammatory cells, including mast cells and macrophages via the ST2L/IL1RAP receptor to secrete pro-inflammatory cytokines, in particular TNF, and systemic anti-TNF therapy can reduce circulating IL-<NUM> levels (Pastorelli, <NUM>). Fibroblasts also secrete IL-<NUM> in response to mechanical strain in vitro (Kunisch, <NUM>). This is particularly pertinent as strain is crucial to the development and persistence of myofibroblasts; on loss of tension, they disassemble their α-SMA within hours (Hinz, <NUM>). However, the precise role of IL-<NUM> in driving musculoskeletal and other localized fibrotic diseases such as endometriosis, abdominal adhesions, adhesive capsulitis, hypertrophic scars or keloid scars, Ledderhose disease and Peyronie's disease is not clear.

Whilst best known as effector cells in allergic responses, mast cells are now recognised as important physiological regulators of the innate and adaptive immune response, smooth muscle contraction and wound healing (Bischoff, <NUM>). Mast cells constitutively express the IL-<NUM> receptor ST2/IL1-RAP, and on exposure to IL-<NUM>, secrete pro-inflammatory cytokines including TNF without degranulation (Moulin, <NUM>). Whilst the differentiation and function of myofibroblasts can be regulated by mast cells (Gailit, <NUM>), the precise contribution of mast cell derived pro-inflammatory cytokines in driving myofibroblast formation in Dupuytren's disease disease and other localized fibrotic disorders has not been established.

Dupuytren's tissue has been shown to be composed mainly of myofibroblasts and about <NUM>% of all cells comprise macrophages, predominantly of the M1 phenotype. Significant numbers of mast cells have been found in Dupuytren's disease tissue (<FIG>).

Analysis of supernatants from freshly disaggregated Dupuytren's tissue for a panel of cytokines and chemokines using Mesoscale Discovery (MSD) and detected IL-<NUM> (<FIG>), CCL2, CXCL8 (IL-<NUM>), CXCL10, and CCL26 (<FIG>) are presented in <FIG> and <FIG>. The latter three are known chemokines for mast cells, which also require IL-<NUM> as a growth factor. Elevated CXCL10 and CCL2 levels are consistent with the preponderance of classically activated M1 macrophages found in the Dupuytren's tissue (<FIG>). These data suggest that macrophages and mast cells may be attracted to the Dupuytren's tissue by locally produced chemokines.

IL-<NUM> was detected in the supernatant of freshly disaggregated Dupuytren's tissue (<NUM> ± <NUM>. 32pg/ml) as shown in <FIG>.

The best characterized human mast cell lines are LAD2, HMC1. <NUM> and HMC1. Exposure of all three cell lines to recombinant human IL-<NUM> (rhIL-<NUM>) resulted in a dose dependent secretion of TNF (<FIG>). Concentrations of IL-<NUM> of the order released by freshly disaggregated tissue (10pg/ml) led to TNF production at concentrations similar to those secreted ex vivo by freshly disaggregated cells from Dupuytren's nodules (Verjee, <NUM>).

Only palmar dermal fibroblasts (PF-D) from patients with Dupuytren's disease expressed IL-<NUM> on exposure to TNF (<FIG>) while TGF-β1 indiscriminately induced expression of IL-<NUM> in both palmar and non-palmar dermal fibroblasts (NPF-D) from these patients and in dermal fibroblasts from normal non-Dupuytren's controls (PF-N).

Treatment with anti-IL-<NUM> resulted in a downregulation of the myofibroblasts phenotype in a dose-dependent manner (<FIG>). Inhibition of IL-<NUM> also resulted in reduction of IL-<NUM> and ST2 (a receptor for IL-<NUM>) expression by myofibroblasts, again in a dose-dependent manner (<FIG>). The interaction between the IL-<NUM> and TNF pathways was confirmed as anti-IL-<NUM> resulted in reduced expression of the receptors for TNF, TNFR1 and TNFR2 (<FIG>).

<FIG> notably and unexpected demonstrates that <NUM>% of patients responded to anti-TNF and anti-IL-<NUM>. Additionally, it was also unexpectedly shown that <NUM>% of patients respond to anti-TNFR2 and anti-IL-<NUM>. Therefore, applicants have shown that targeting TNFR2 and IL-<NUM> is superior and advantageous compared to using anti-TNF and anti-IL-<NUM>. This could not have been predicted and was unexpected. IL-<NUM> stimulates TNF production by classically activated macrophages and mast cells recruited during fibrosis. Elevated local levels of TNF lead to the synthesis of more IL-<NUM> by palmar fibroblasts as they differentiate into myofibroblasts. This in turn promotes further TNF production, creating a positive feedback loop and a chronic fibrotic response. Furthermore, IL-<NUM> enhances the expression of its ST2 receptor on myofibroblasts, thereby inducing a positive autocrine feedback loop (<FIG>).

Periodic administration of a therapeutically effective amount of an IL-<NUM> antagonist to a patient suffering from Dupuytren's disease successfully treats the patient.

Periodic administration of a therapeutically effective amount of an IL-<NUM> antagonist to a patient suffering from frozen shoulder successfully treats the patient.

Periodic administration of a therapeutically effective amount of an IL-<NUM> antagonist to a patient suffering from periarticular fibrosis successfully treats the patient.

Periodic administration of a therapeutically effective amount of an IL-<NUM> antagonist to a patient suffering from keloid or hypertrophic scars successfully treats the patient.

Periodic administration of a therapeutically effective amount of an IL-<NUM> antagonist to a patient suffering from endometriosis successfully treats the patient.

Periodic administration of a therapeutically effective amount of an IL-<NUM> antagonist to a patient suffering from abdominal adhesions successfully treats the patient.

Periodic administration of a therapeutically effective amount of an IL-<NUM> antagonist to a patient suffering from Ledderhose disease successfully treats the patient.

Periodic administration of a therapeutically effective amount of an IL-<NUM> antagonist to a patient suffering from Peyronie's disease successfully treats the patient.

Periodic administration of a therapeutically effective amount of an IL-<NUM> antagonist to a patient suffering from peritendinous adhesions successfully treats the patient.

Co-administration of a therapeutically effective amount of an IL-<NUM> antagonist and a therapeutically effective amount of a TNF-α antagonist to a patient suffering from Dupuytren's disease successfully treats the patient.

Periodic administration of an IL-<NUM> antagonist as an add-on therapy for a human patient afflicted with Dupuytren's disease who is already receiving a TNF-α antagonist provides a clinically meaningful advantage and is more effective (provides at least an additive effect or more than an additive effect) in treating the patient than when the TNF-α antagonist is administered alone (at the same dose).

Periodic administration of a TNF-α antagonist as an add-on therapy for a human patient afflicted with Dupuytren's disease who is already receiving an IL-<NUM> antagonist provides a clinically meaningful advantage and is more effective (provides at least an additive effect or more than an additive effect) in treating the patient than when the IL-<NUM> antagonist is administered alone (at the same dose).

The add-on therapies also provides efficacy (provides at least an additive effect or more than an additive effect) in treating the patient without undue adverse side effects or affecting the safety of the treatment:.

Disclosed herein is the use of an IL-<NUM> antagonist in addition to or in combination with a TNF-α antagonist for the treatment of Dupuytren's disease.

Periodic administration of a IL-<NUM> antagonist in combination with a TNF-α antagonist to a human patient afflicted with Dupuytren's disease provides increased efficacy (provides at least an additive effect or more than an additive effect) in treating the patient than when a TNF-α antagonist is administered alone or when an IL-<NUM> antagonist is administered alone (at the same dose). The combination therapy also provides efficacy (provides at least an additive effect or more than an additive effect) in treating the patient without undue adverse side effects or affecting the safety of the treatment.

The combination therapy provides a clinically meaningful advantage and is more effective (provides at least an additive effect or more than an additive effect) in treating the patient than when the IL-<NUM> antagonist or a TNF-α antagonist is administered alone (at the same dose) in the following manner:.

Cultured myofibroblasts from patients with Dupuytren's disease were used up to passage <NUM>. <NUM>,<NUM> cells were mixed with 100µl of Nucleofector Kit for Human Dermal Fibroblast transfection reagent (VPD-<NUM>, Lonza) and <NUM> silencer select siRNA (Applied Biosystem), then electroporated using the AMAXA nucleofection 2b Device (Lonza) to transfect the siRNA probes. Inventoried silencer-select reagents and respective non-targeting negative controls were used for TNFR1 (<NUM>, siRNA ID s14265), TNFR2 (<NUM>, siRNA ID s14270), IL1RL1 (<NUM>, s17532, Applied Biosystems).

Negative control siRNAs <NUM>(<NUM>) and <NUM> (<NUM>) (Applied Biosystems) were used with sequences that do not target any gene product and provide a baseline to compare siRNA-treated samples. Cells were immediately transferred to a <NUM>-well plate with <NUM> OptiMEM (<NUM>, Life Technologies) without serum, pre-warmed to <NUM> in an incubator with <NUM>% CO2. After <NUM> the transfection medium was washed three times with Phosphate Buffered Saline, before being replaced by DMEM with <NUM>% FBS and <NUM>% penicillin/streptomycin and incubated for another <NUM> in a <NUM> incubator with <NUM>% CO2. RT-PCR analysis was used to quantify knockdown of gene as previously described.

TNFR1 expression is effectively down regulated by siRNA knockdown of TNFR1, TNFR1+TNFR2 or TNFR1+ST2 knockdown. TNFR2 expression is reduced by siRNA knockdown of TNFR2, TNFR1+TNFR2 or TNFR2+ST2 knockdown. ST2 expression is reduced by siRNA knockdown of ST2, TNFR1+ST2 or TNFR2+ST2 knockdown. Myofibroblast phenotype is down regulated as evidenced by α-SMA expression by siRNA knock down of TNFR2 (but not TNFR1) or ST2 and most effectively by siRNA knockdown of TNFR2+ST2 at mRNA and protein levels. Expression of COL1A1 mRNA, another marker of the myofibroblast phenotype, is reduced by siRNA knockdown of TNFR2 (but not TNFR1), ST2 or TNFR2+ST2 (<FIG>).

Example <NUM> (Reference example)Co-administration of a therapeutically effective amount of an IL-<NUM> antagonist and a therapeutically effective amount of a TNFR2 antagonist to a patient suffering from Dupuytren's disease successfully treats the patient.

Periodic administration of an IL-<NUM> antagonist as an add-on therapy for a human patient afflicted with Dupuytren's disease who is already receiving a TNFR2 antagonist provides a clinically meaningful advantage and is more effective (provides at least an additive effect or more than an additive effect) in treating the patient than when the TNFR2 antagonist is administered alone (at the same dose).

Periodic administration of a TNFR2 antagonist as an add-on therapy for a human patient afflicted with Dupuytren's disease who is already receiving an IL-<NUM> antagonist provides a clinically meaningful advantage and is more effective (provides at least an additive effect or more than an additive effect) in treating the patient than when the IL-<NUM> antagonist is administered alone (at the same dose).

Disclosed herein is the use of an IL-<NUM> antagonist in addition to or in combination with a TNFR2 antagonist for the treatment of Dupuytren's disease.

Periodic administration of a IL-<NUM> antagonist in combination with a TNFR2 antagonist to a human patient afflicted with Dupuytren's disease provides increased efficacy (provides at least an additive effect or more than an additive effect) in treating the patient than when a TNFR2 antagonist is administered alone or when an IL-<NUM> antagonist is administered alone (at the same dose). The combination therapy also provides efficacy (provides at least an additive effect or more than an additive effect) in treating the patient without undue adverse side effects or affecting the safety of the treatment.

Claim 1:
TNF receptor <NUM> (TNFR2) antagonist for use in treating a patient suffering from an early or established disease state Dupuytren's disease, wherein the TNFR2 antagonist is
a) an antibody, or antigen binding fragment of an antibody, that specifically binds to, and inhibits activation of, a TNFR2;
b) a soluble form of TNF that specifically binds to TNFR2 and inhibits TNF from binding to the TNFR2; or
c) a bispecific antibody comprising at least one antigen binding domain which binds to and inhibits activation of, a TNFR2.