ES2149776T3

Morphogen-induced modulation of inflammatory response

Abstract

THE PRESENT INVENTION REFERS TO METHOD AND COMPOSITIONS TO MITIGATE THE DESTRUCTIVE EFFECTS ON TISSUES ASSOCIATED WITH THE INFLAMMATORY RESPONSE TO DAMAGE TO A TISSUE FROM A MAMMAL. THE METHODS AND COMPOSITIONS CONSIST OF THE ADMINISTRATION OF A THERAPEUTICALLY EFFECTIVE CONCENTRATION OF A MORPHOGEN OR OF A MORPHOGEN STIMULATING AGENT THAT MITIGATES THE DESTRUCTION OF TISSUES MEDIATED BY AN IMMUNE CELL.

ES2149776T3, drawing sheet 1 — Sheet 1 of 6

Term

Term ended

Projected expiry passed 28 August 2012, 14.1 years ago.

25 claims: 11 independent, 14 dependent

1
ES 2 149 776 T3 REIVINDICACIONES 1. El uso de un morfogeno que comprende una proteína dimera, que induce morfogénesis especifica de tejido en un mamáfero (por ejemplo, el ser humano) y comprende un par de polipáeptidos plegados, cuyas secuencias de aminoáacidos comprenden en cada caso una secuencia que comparte por lo menos el 70 % de homologáa en la secuencia de aminoáacidos con el dominio de siete cisteánas de la secuencia C-terminal de la OP-1 humana (restos 38-139 de identificacioán de secuencia No. 5), para la fabricaciáon de un medicamento para:(a) modular una respuesta inflamatoria;o (b) aliviar los efectos de destrucciáon de tejido asociados con la respuesta inflamatoria a la lesioán hástica;o (c) incrementar la viabilidad del tejido mamífero lesionado o danado (por ejemplo, tejido retirado in vivo en un mamáfero).
2
Uso de acuerdo con la reivindicaciáon 1, para modular una respuesta inflamatoria en tejidos de mamáferos, en el que la modulaciáon de dicha respuesta inflamatoria comprende:(a) alivio de fibrosis o de formacioán de tejido de cicatrizaciáon;y/o (b) inhibicioán de adherencia de las cáelulas efectoras inmunitarias al endotelio vascular;y/o (c) alivio de inflamaciáon intersticial;y/o (d) alivio de edema o eritema;y/o (e) alivio de necrosis del tejido inflamatorio.
3
Uso de acuerdo con la reivindicaciáon 1 o la reivindicacioán 2, en el que la respuesta inflamatoria estaá asociada con lesiáon hástica (pudiendo tener la lesiáon una causa, por ejemplo, quámica, mecáanica, bioláogica o inmunitaria).
4
Uso de acuerdo con una cualquiera de las reivindicaciones precedentes, en el que dicho medicamento se administra:(a) para la profilaxis o el tratamiento de la lesiáon de dicho tejido;y/o (b) en una cantidad eficaz para inhibir la fibrogáenesis o estimular la regeneracioán especáfica de tejido de dicho tejido lesionado o danado;y/o (c) para inhibir la perdida o la reducción de la funcionalidad de dicho tejido;y/o (d) de modo oral, parenteral o toápico;y/o (e) disperso en adhesivo láquido para tejidos;y/o (f) en aerosol.
5
Uso de acuerdo con la reivindicaciáon 3 oá 4, en el que la lesiáon de dicho tejido incluye:(a) lesiáon por isquemia y reperfusioán;o (b) lesioán hiperáoxica.
6
Uso de acuerdo con la reivindicaciáon 5(a), en el que dicha lesiáon por isquemia y reperfusioán estáa:(a) asociada con paro cardáaco, oclusiáon pulmonar, oclusiáon arterial (por ejemplo, de la arteria renal), oclusioán coronaria o apoplejáa oclusiva;o (b) asociada con cirugáa o la reducciáon o interrupciáon del flujo sanguáneo en un áorgano durante un procedimiento clánico (por ejemplo, enterectomáa de caráotida, derivaciáon de arteria coronaria, un procedimiento de injerto de tejido, un trasplante de oárgano o una terapia fibronolática);o (c) asociada con infarto cerebral, infarto de miocardio, asfixia o parada cardiorrespiratoria.
7
Uso de acuerdo con la reivindicaciáon 5(b), en el que la lesiáon hiperáoxica estaá asociada con el tratamiento de:(a) un nacimiento prematuro;o (b) enfisema;o (c) asfixia. ES 2 149 776 T3
8
Uso de acuerdo con una cualquiera de las reivindicaciones precedentes, en el que el tejido es pulmonar, cardíaco, hepatico, neural, pancreático, sinovial, cutáneo o renal, por ejemplo, en el que:(a) el tejido es tejido renal y el mamífero padece nefritis glomerular, aterosclerosis o diabetes, o (b) el tejido es tejido cardíaco y el mamífero padece aterosclerosis o enfermedad vascular, o (c) el tejido es tejido pancreíatico y el mamífero padece diabetes, o (d) el tejido es tejido sinovial y el mamífero padece artritis, (e) el tejido es tejido cutíaneo y el mamífero padece dermatitis o psoriasis, (f ) el tejido es tejido pulmonar y el mamífero padece bronquitis, enfisema, fibrosis pulmonar idiopaítica, asma, asfixia o síndrome de disnea en el adulto.
9
Uso de acuerdo con la reivindicaciíon 3 oí 4, en el que la lesioín del tejido incluye:(a) una respuesta inflamatoria mediada por las cíelulas del sistema inmunitario;o (b) una enfermedad inflamatoria (por ejemplo, una enfermedad inflamatoria croínica);o (c) una respuesta inmunitaria anoímala;o edema o eritema.
10
Uso de acuerdo con la reivindicaciíon 9(a), 9(c) o 9(d), en el que la lesiíon de tejido incluye:(a) una respuesta inflamatoria aguda generalizada;o (b) inflamacioín de las vías respiratorias.
11
Uso de acuerdo con la reivindicaciíon 9(b), en el que la enfermedad inflamatoria incluye:(a) una enfermedad autoinmunitaria;o (b) artritis, psoriasis, dermatitis, enfermedad inflamatoria del intestino o diabetes.
12
Uso de acuerdo con la reivindicaciíon 10, en el que:(a) en la reivindicaciíon 10(a) la respuesta inflamatoria aguda estía asociada con asfixia o síndrome de disnea en el adulto;o (b) en la reivindicaciíon 10(b) la inflamaciíon de las vías respiratorias estaí asociada con bronquitis críonica, enfisema, fibrosis pulmonar idiopaítica o asma.
13
Uso de acuerdo con la reivindicaciíon 11, en el que:(a) en la reivindicaciíon 11(a) la enfermedad autoinmunitaria es una enfermedad neurodegenerativa (por ejemplo, esclerosis muíltiple o esclerosis lateral amiotríofica);o (b) en la reivindicaciíon 11 (b) la artritis es reumatoide, degenerativa o psoriaísica.
14
Uso de acuerdo con cualquiera de las reivindicaciones 3 a 13, en el que la modulaciíon de la respuesta inflamatoria se efectuía por administraciíon del medicamento antes o despuíes de la lesiíon, por ejemplo antes o despuíes de la reducciíon o interrupciíon del flujo sanguíneo o despuíes de la reanudacioín del flujo sanguíneo, cuando la lesioín es una lesiíon por isquemia y reperfusioín.
15
Uso de acuerdo con la reivindicaciíon 6 (b), en el que dicha intervenciíon clínica es un procedimiento de injerto de tejido (por ejemplo, piel, míedula oísea o tejido de la mucosa gastrointestinal) o de trasplante de un árgano (por ejemplo;pulmon, corazán, rinon, hígado o pancreas).
16
Uso de acuerdo con la reivindicaciíon 15, en el que la modulaciíon de la respuesta inflamatoria es afectada por la administracioán del medicamento:(a) en el momento o despuáes de la extirpaciáon del áorgano o tejido de un donante hospedante;o (b) durante el almacenaje o transporte del oárgano o tejido previo al implante en el receptor hospedante;o ES 2 149 776 T3 (c) en el momento o despuáes del implante en el receptor hospedante.
17
Uso de acuerdo con cualquiera de las reivindicaciones precedentes, en el que dicho medicamento comprende el morfágeno disperso en un vehículo acuoso fisiolágicamente aceptable, apropiado para la administraciáon parenteral a un mamáfero.
18
Uso de acuerdo con cualquiera de las reivindicaciones precedentes, en el que la secuencia de aminoáacidos de dichos polipáeptidos comprende una secuencia que:(a) comparte por lo menos el 80 % de homologáa con la secuencia de dicho dominio con siete cisteánas de la OP-1 humana;o (b) comparte por lo menos el 60 % de identidad aminoáacida, por ejemplo, por lo menos el 65 % de identidad con la secuencia de dicho dominio con siete cisteánas de la OP-1 humana;o (c) comprende la secuencia del dominio con siete cisteánas de la OP-1 humana, OP-1 de ratáon, OP-2 de humana, OP-2 de ratáon o 60A, ocurre naturalmente o es una variante biosintáetica de cualquiera de las mencionadas, siempre y cuando cualquiera de dichas variantes induzca la morfogeánesis especáfica de tejido en dichos mamáferos;o (d) comprende una secuencia de aminoaácidos seleccionada de la identificaciáon de secuencia Nos. 9, 10, 11, 12, 13, 14, 26, 27, 28 áo 29 (CBMP2A(fx), CBMP2B(fx), DPP(fx), Vg1(fx), Vgr-1(fx), GDF-1(fx), BMP3, BMP5, BMP6 u OPX), ocurre naturalmente o es una variante biosinteática de cualquiera de las mencionadas, siempre y cuando cualquiera de dichas variantes induzca la morfogáenesis especáfica de tejido en dichos mamáferos;o (e) comprende una secuencia de aminoácidos de los restos 38 a 139 de la identificacion de secuencia N° 5, 6, 7 u 8, o los restos 354 a 455 de la identificacián de secuencia N° 24, ocurre naturalmente o es una variante biosintáetica de cualquiera de las mencionadas, siempre y cuando cualquiera de dichas variantes induzca la morfogáenesis especáfica de tejido en dichos mamáferos.
19
Una composiciáon farmacáeutica para uso en el alivio de la lesiáon asociada con la exposicioán del tejido de mamáferos a concentraciones táoxicas de oxágeno, que comprende un morfáogeno disperso en un veháculo acuoso fisioloágicamente aceptable a una concentracioán eficaz para aliviar extravasaciáon de cáelulas efectoras inmunitarias, inflamacioán intersticial, edema, necrosis o fibrogáenesis en tejidos de mamáferos en riesgo de padecer o que padecen dicha lesiáon, comprendiendo dicho morfáogeno una proteána dámera que induce la morfogáenesis especáfica de tejido en mamáferos, comprendiendo dicha proteána dámera un par de polipáeptidos plegados, cuyas secuencias de aminoaácidos comprenden una secuencia que comparte por lo menos el 70% de homologáa en la secuencia de aminoáacidos con el dominio de siete cisteánas de la regiáon C-terminal de la OP-1 humana (restos 38-139 de identificaciáon de secuencia No. 5), comprendiendo dicha composiciáon ademaás un anticoagulante o un agente inhibidor con un radical libre de oxágeno.
20
Una composicioán farmacáeutica para administraciáon táopica a tejido epitelial de mamáferos, que comprende un morfoágeno disperso en un veháculo dermatoloágicamente aceptable o en un adhesivo para la superficie del tejido, biocompatible y no irritante, (por ejemplo, hidroxipropilcelulosa) a una concentraciáon eficaz para aliviar la extravasaciáon de cáelulas efectoras inmunitarias, inflamacioán intersticial, edema, necrosis o fibrogáenesis en tejido de mamáferos en riesgo de padecer o que padecen dicha lesioán, comprendiendo dicho morfáogeno una proteána dámera que induce morfogáenesis especáfica de tejido en un mamáfero, comprendiendo dicha proteána dámera un par de polipáeptidos plegados, cuyas secuencias de aminoaácidos comprenden una secuencia que comparte por lo menos el 70 % de homologáa en la secuencia de aminoaácidos con el dominio de siete cisteánas de la regiáon C-terminal de la OP-1 humana (restos 38-139 de identificacioán de secuencia No. 5).
21
Una soluciáon conservante para mantener la viabilidad ex vivo de cáelulas o tejido de mamáferos o un oárgano de mamáferos, que comprende una formulacioán fluida que tiene una presioán osmáotica sustancialmente equivalente a la presioán osmáotica de las cáelulas vivas de mamáferos y un morfoágeno disperso en dicho fluido a una concentracioán eficaz para aliviar la extravasaciáon de cáelulas efectoras inmunitarias, inflamacioán intersticial, edema, necrosis o fibrogáenesis en dichas cáelulas, tejido u áorgano, comprendiendo dicho morfoágeno una proteána dámera que induce morfogáenesis especáfica de tejido en un mamáfero, comprendiendo dicha proteána dámera un par de polipáeptidos plegados, cuyas secuencias de aminoáacidos comprenden una secuencia que comparte por lo menos el 70 % de homologáa en la secuencia de aminoáacidos con el dominio de siete cisteánas de la regiáon C-terminal de la OP-1 humana (restos 38-139 de identificaciáon de secuencia No. 5). ES 2 149 776 T3
22
Una solución, de acuerdo con la reivindicación 21, que comprende además un azúcar, un anticoagulante o un agente inhibidor de radicales libres de oxígeno;o formulada ademas para mantener sustancialmente normales los niveles de ATP en dichas cóelulas, tejido u óorgano.
23
La composicioón de la reivindicacioón 19 óo 20 o la solucioón de la reivindicacióon 21 oó 22, en la que el morfóogeno es como se define en la reivindicacioón 18.
24
Un metodo ex vivo para proteger un tejido vivo de mamíferos u organo trasplantado de mamíferos contra una lesioón asociada con la respuesta inflamatoria de cóelulas activas efectoras inmunitarias, que comprende la etapa de proporcionar a dicho tejido u óorgano un morfóogeno que comprende una proteóna dómera que induce morfogóenesis especófica de tejido en un mamófero, comprendiendo dicha proteóna dómera un par de polipóeptidos plegados, cuyas secuencias de aminoaócidos comprenden una secuencia que comparte por lo menos el 70 % de homologóa en la secuencia de aminoóacidos con el dominio de siete cisteónas de la regioón C-terminal de la OP-1 humana (restos 38-139 de identificacioón de secuencia No. 5).
25
El móetodo de reivindicacioón 24, en el que:(a) el tejido u oórgano es como se define en la reivindicacióon 15;y/o (b) el morfoógeno es como se define en la reivindicacioón 18;(c) el morfóogeno es proporcionado en la forma de una solucióon como se define en la reivindicacioón 21 óo 22. NOTA INFORMATIVA: Conforme a la reserva del art. 167.2 del Convenio de Patentes Europeas (CPE) y a la Disposición Transitoria del RD 2424/1986, de 10 de octubre, relativo a la aplicación del Convenio de Patente Europea, las patentes europeas que designen a España y solicitadas antes del 7-10-1992, no producirán ningún efecto en España en la medida en que confieran proteccion a productos químicos y farmacéuticos como tales. Esta informacioín no prejuzga que la patente estíeonoincluída en la mencionada reserva.

Independent claims25

1,712 paragraphs in 105 sections, as filed

ES 2 149 776 T3

DESCRIPTION

Morphogen-induced modulation of the inflammatory response.

Field of the invention

The present invention relates generally to a method for modulating the inflammatory response induced in a mammal after injury to tissue. For example, this invention relates to a method of alleviating immune-mediated tissue destruction associated with the inflammatory response.

Foundations of the Invention

The body's inflammatory response to tissue injury can cause serious tissue destruction, resulting in loss of tissue function. Damage to cells as a result of the effects of the inflammatory response, for example, by immune-mediated tissue destruction, has been implicated as the cause of a reduction or loss of tissue function in joint diseases (e.g. rheumatoid arthritis and osteoarthritis) and of various organs, including the kidneys, pancreas, skin, lungs, and heart. For example, glomerular nephritis, diabetes, inflammatory bowel diseases, vascular diseases such as atherosclerosis and vasculitis, and skin diseases such as psoriasis and dermatitis are believed to be largely the result. of an unwanted acute inflammatory reaction and fibrosis. A number of these diseases, including arthritis, psoriasis, and inflammatory bowel diseases, are considered chronic inflammatory diseases. Damaged tissue is also often replaced by fibrotic tissue, for example scar tissue, which further reduces the function of the tissue. Rejection of a transplanted graft or organ is also believed to be caused primarily by the action of the body's immune / inflammatory response system.

Following an initial injury or injury to tissue, immune-mediated tissue destruction usually occurs. Secondary damage, caused by the inflammatory response, is usually the cause of significant tissue damage. Among the factors suspected of mediating these harmful effects are those associated with the modulation of the body's inflammatory response that occurs after tissue injury, for example cytokines such as interleukin 1 (IL 1) and humoral necrosis factor. (TNF), and free radicals derived from oxygen such as superoxide anions. These humoral agents are produced by adherent neutrophilic leukocytes or endothelial cells and have been identified in ischemic areas after reperfusion. In addition, TNF levels increase in humans after myocardial infarction.

There is a range of lung diseases that are characterized by inflammation of the respiratory tract, including chronic bronchitis, emphysema, idiopaotic pulmonary fibrosis, and asthma. Another type of inflammatory disorders related to the lungs is that of inflammatory diseases characterized by a widespread and generalized acute inflammatory response, such as adult dyspnoea syndrome. Another dysfunction associated with the inflammatory response is that generated in response to injury caused by hyperoxia, for example, by prolonged exposure to lethally high concentrations of oxygen (95-100% O2). Similarly, a reduced blood supply in a tissue (and consequently a reduced level or a complete lack of oxygen in the tissue), as described below, can also induce a primary lesion in the tissue that stimulates the response. inflammatory.

It is well known that mammalian cells deprived of oxygen are damaged. In fact, interruption of blood supply, either partial (hypoxia) or complete (ischemia) and the consequent inflammatory responses may be the most important cause of coagulative necrosis or cell death in human disease. Complications of atherosclerosis, for example, are generally the result of ischemic cell injury to the brain, heart, small intestine, kidneys, and lower extremities. Highly differentiated cells, such as cells of the proximal convolute tubule of the kidney, cardiac myocytes, and neurons of the central nervous system, all depend on aerobic respiration to produce ATP, the energy necessary to carry out their respective specialized functions. When ischemia limits oxygen supply and ATP is depleted, affected cells can suffer irreversible damage. The consequent inflammatory responses to this initial injury produce additional damage to the affected tissue. Examples of this hypoxia or ischemia are the partial or total loss of blood supply in the entire body, in an organ within the body, or in a region within an organ, such as occurs in cardiac arrest, pulmonary embolism, occlusion of a renal artery, coronary occlusion or an occlusive cerebral embolism.

Tissue damage associated with ischemia and reperfusion injury is believed to be comprised of the

ES 2 149 776 T3 initial cellular damage induced by the deprivation of oxygen in the cells and the subsequent restoration of the irrigation in them, as well as the damage caused by the response of the body to this initial damage. It is believed that a reperfusion injury may be the cause of dysfunction in the endothelium of the vasculature as well as injury to the surrounding tissue. In idiopathic pulmonary fibrosis, for example, scar tissue accumulates in the wall of the lung tissue, inhibiting the elasticity of the tissue. Tissue damage associated with a hyperexic injury is believed to follow a similar mechanism, where the initial damage is mediated primarily by the presence of theogenic oxygen metabolites followed by an inflammatory response to the initial injury.

Likewise, tissues and organs for transplantation are also exposed to the tissue-destroying effects associated with the inflammatory response of the recipient's body after transplantation. It is currently believed that the initial destructive response is due in large part to reperfusion injury to the transplanted organ after it has been transplanted to the organ recipient.

Therefore, the success of an organ or tissue transplant depends in large part on the preservation of tissue activity (for example, viability of the tissue or organ) at the time of excision of the organ, during storage of the excised organ. , and at the time of transplantation. To date, the preservation of organs such as the lungs, the pancreas, the heart and the liver continues to be a major obstacle to the successful transplantation of these organs. US Patent No. 4,952,409 describes a liposome containing a superoxide dismutase to inhibit injury caused by reperfusion. US Patent No.° 5,002,965 describes the use of gyneolides, known antagonists of platelet activating factor, to inhibit the injury caused by reperfusion. As described, these two types of factors act mainly by inhibiting the release and / or inhibiting the damaging effect of oxygen free radicals. A number of patents have also been issued on the use of immunosuppressants to inhibit graft rejection. A representative list includes US Patent Nos.° 5,104,858, 5,008,246, and 5,068,323. A serious problem with many immunosuppressants is their low therapeutic index, which requires the administration of high doses that can be accompanied by significant toxic side effects.

Rheumatoid arthritis and osteoarthritis are prevalent diseases characterized by crogenic inflammation of the synovial membrane covering the affected joint. A serious consequence of crogenic inflammatory joint disease (eg rheumatoid arthritis) and degenerative arthritis (eg osteoarthritis) is loss of function in affected joints. This loss of function is mainly due to the destruction of the main structural components of the joint, cartilage and bone, and the subsequent loss of the correct anatomy of the joint. As a consequence of chronic disease, joint destruction occurs which can lead to permanent and irreversible joint damage and loss of function. Current treatment methods for severe cases of rheumatoid arthritis typically include removal of the synovium, for example a synovectomy. Surgical synovectomy has many limitations, including the risk of surgical intervention in se, and the fact that the surgeon is often unable to completely remove the diseased membrane. Usually the diseased tissue that remains regenerates, causing the same symptoms that the surgery tried to alleviate.

Psoriasis is a chronic, recurrent disease characterized by scales and of unknown etiology, which manifests as a chronic inflammation of the skin. Erythematous eruptions, often in papules or plaques, and usually manifesting as silvery-white scales, can appear anywhere on the skin, but usually appear on the scalp, elbows, knees, and lower back. The disease usually occurs in adults, but children can also be affected. Patients suffering from psoriasis have a much higher incidence of arthritis (psoriatic arthritis), and widespread exfoliation and even death can threaten affected individuals.

Current treatment methods include the topical or intralesional application of corticosteroids, the topical administration of keratolytic agents, and the use of tar and UV light in the affected areas. There is no ideal therapy, and it is rare that the patient is not treated with various alternatives during the course of relapse and remission of the disease. Although a systematic treatment can induce a rapid resolution of psoriasis lesions, suppression often requires a progressive increase in dose, sometimes with toxic side effects, and a reduction therapy can cause rebound phenomena with the extension of the lesions. possibly even exfoliated.

Inflammatory bowel disease (IBD) includes a class of clinical mucosal gas disorders3

ES 2 149 776 T3 trointestinal characterized by chronic inflammation and severe ulceration of the mucosa. The two main disorders in this classification are ulcerative colitis and regional enteritis (Crohn's disease). Like oral mucositis, disorders classified under IBD are associated with severe mucosal ulceration (which frequently penetrates the wall of the intestine and forms narrowing and fistulas), severe mucosal and submucosal inflammation and edema, and fibrosis (for example , a formation of cicatrizing tissue that interferes with the protective function of the acid of the gastrointestinal wall). Other types of IBD include regional ileotis and proctitis. Clonically, patients with fulminant IBD can become seriously ill with massive diarrhea, blood loss, dehydration, weight loss, and fever. The prognosis of the disease is not favorable and resection of the diseased tissue is frequently required.

This invention finds its application in a method of protecting mammalian tissue, particularly human tissue, from damage associated with the inflammatory response that follows injury to the tissue. The inflammatory reaction can occur in response to an initial injury or injury to the tissue. The original lesion may have a chemical, mechaonic, biological, or immunological cause. Another application is in methods and compositions to protect tissue against the destructive effects associated with chronic inflammatory diseases, including arthritis (eg, rheumatoid arthritis or osteoarthritis), psoriaotic arthritis, psoriasis and dermatitis, inflammatory bowel disease, and other autoimmune diseases. . Another application is found in methods and compositions to enhance the viability of mammalian tissues and organs for transplantation, such as the protection of transplanted organs against immune-mediated tissue destruction, such as tissue damage associated with ischemic and reperfusion injuries. This tissue damage can occur during the removal and transport of the donated tissue or organ, as well as after the restoration of blood supply in the recipient after transplantation of the organ or tissue.

The invention also finds application in a method of alleviating tissue damage associated with ischemic and reperfusion injury in a mammal following oxygen deprivation in tissue in the mammal. Other applications include providing a method of alleviating tissue damage associated with ischemic and reperfusion injury in a human who has suffered hypoxia or ischemia following cardiac arrest, pulmonary embolism, renal artery occlusion, coronary occlusion, or cerebral embolism. stop. Yet another application is to provide a method of alleviating tissue damage associated with hyperoxia-related injuries, more specifically, for example, lethally elevated concentrations of oxygen.

Yet another application of this invention is to provide a method for modulating inflammatory responses in general, particularly those induced in a human after tissue damage.

These and other objects and features of the invention will become apparent through the description, drawings, and claims that follow.

Kurvilla et al. (1991) 88 Proc. Natl. Acad. Sci. USA 2918-2921, Lefer et al. (1990), 249 Science 61-64; Shepard et al., EP 0,269,408; Nathan et al., WO90 / 00900; and Bentz et al., US Patent 4,971,952 describe studies examining whether TGF-β can mitigate specific types of ischemia and reperfusion or inflammatory damage in mammalian cells or tissues. TGF-β is not a member of the class of proteins defined herein as morphogens.

Oppermann et al., WO91 / 05802; Kuberasampath et al., WO89 / 09787; Oppermann et al., WO89 / 09788 and Oppermann et al., WO92 / 07073 show that OP-1 and related proteones can be used in stimulating tissue-specific regeneration in cartilage and bone tissue of mammals. In particular, these references show that OP-1, when adsorbed onto a suitable support matrix, induces the developmental cascade of molecular and cellular events that culminate in endochondrial bone morphogenesis. Biologically active OP-1 preparations show that it is, at best, moderately soluble in physiologically compatible solutions. Matrix devices loaded with OP-1 are thus disclosed very useful in inducing local morphogenesis of bone and / or cartilage.

Cohen et al., WO92 / 15323 shows that OP-1 induces tissue-specific morphogenesis in various tissues of the mammalian body, and this morphogen can be used in the replacement or repair of damaged tissues.

ES 2 149 776 T3

Compendium of the invention

The present invention is defined in the claims. It finds utility in a method of alleviating the tissue destruction effects associated with the activation of inflammatory responses after tissue injury. The method comprises the step of providing the affected tissue with a therapeutically effective concentration of a morphogenic proteon ("morphogen", as defined in this document) after a hostic injury or in advance of the hostic injury, and sufficient to inhibit or significantly reduce the effects of homic destruction of the inflammatory response.

They are also described herein as therapeutic treatment compositions and methods comprising the step of administering to a mammalian a therapeutically effective amount of a morphogenic proteone ("morphogen"), as defined herein, after or prior to homic injury. to said host injury, for a time and at a concentration sufficient to inhibit the effects of host destruction associated with the body's inflammatory response, including repair of damaged tissue and / or inhibition of further damage.

Also described in this document are compositions and therapeutic treatment methods to protect tissues and organs from the effects of host destruction of the inflammatory response, which include administering to the mammal, after a home injury or before said injury, a compound that stimulates in vivo a therapeutically effective concentration of an endogenous morphogen in the mammalian body that is sufficient to protect tissue from the effects of host destruction associated with the inflammatory response, including repair of damaged tissue and / or inhibition of additional damage. These compounds are defined herein as morphogen-stimulating agents, and include substances that, when administered to a mammal, act on the cells of one or more tissues or organs that are normally responsible for or capable of producing a morphogen and / or secreting a morphogen, and cause the endogenous level of the morphogen to be altered. The agent can act, for example, by stimulating the expression and / or secretion of an endogenous morphogen.

As indicated herein, the term "ischemic and reperfusion injury" refers to the initial damage associated with oxygen deprivation in a cell and the subsequent damage associated with the inflammatory response when oxygen supply to the cell is restored. As indicated herein, the term "hyperoxia-induced injury" refers to tissue damage associated with prolonged exposure to lethally high doses of oxygen, for example, greater than 95% O2, including hostic damage associated with the inflammatory response to the toxic high dose of oxygen. Therefore, as used herein, the term "toxic concentrations of oxygen" refers to the hostic damage associated with injury induced both by lethally low concentrations of oxygen (including complete lack of oxygen), and by lethally high concentrations. of oxygen. The term "mitigate" means to protect against, reduce, and / or eliminate unwanted host destruction, particularly cell-mediated host destruction of the immune system. Tissue destruction may be the response to an initial home injury, which may have a mechaonic, chemical, or immunological origin. The expression "enhance the viability of" living tissues or organs, as used herein, means to protect against, reduce and / or eliminate the loss or diminution of function of the organ or tissue as a result of homic death, particularly homic death. mediated by cells of the immune system. "Transplanted" living tissue encompasses both tissue transplants (for example, in the case of bone marrow transplants) and tissue grafts. Finally, an "oxygen free radical inhibiting agent" means a molecule capable of inhibiting the release of oxygen free radicals and / or inhibiting their damaging effects on tissues.

Described herein are methods and compositions for mitigating ischemic and reperfusion injury to mammalian tissue as a result of oxygen deprivation and subsequent reperfusion of tissue oxygen. A method to mitigate the tissue-destroying effects associated with hyperoxia is also described. Also described in this document are methods and compositions to maintain the viability of tissues and organs, particularly living tissues and organs to be transplanted, including protecting them against injury by ischemia and reperfusion, along with methods to protect tissues and organs against the effects. of homic destruction caused by chronic inflammatory diseases, such as arthritis, psoriasis, dermatitis, including contact dermatitis, IBD and other chronic inflammatory diseases of the gastrointestinal tract, as well as the tissue-destroying effects associated with other known autoimmune diseases, such as diabetes, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and other neurodegenerative autoimmune diseases.

Morphogen can be administered to damaged tissue after initial injury to the tissue. The morphogen can be administered directly to the tissue, e.g. by injection into the site of damaged tissue or

ES 2 149 776 T3 by topical administration, or it can be administered indirectly, eg, systemically orally or parenterally. Alternatively, as described above, an agent capable of stimulating the expression and / or secretion of an endogenous morphogen can be administered to the mammal. Preferably, the agent can stimulate an endogenous morphogen in cells associated with damaged tissue. Alternatively, the expression and / or secretion of morphogen can be stimulated in distant tissue and transported to the damaged tissue by the circulatory system.

Morphogen can also be administered to tissue at risk of damage due to cell-mediated tissue destruction of the immune system. Examples of such tissues include tissue grafts and organ and tissue transplants, as well as any tissue or organ that is to undergo a surgical or other clinical procedure that is likely to inhibit blood flow to the tissue or in other cases it will induce an inflammatory response. Here the morphogen or morphogen-stimulating agent is administered to the patient preferably prior to injury induction, eg, as a prophylactic, to provide a cytoprotective effect for tissue at risk.

When the tissue at risk consists of a tissue or organ to be transplanted, the tissue or organ to be transplanted is preferably exposed to the morphogen prior to transplantation. Most preferably, the tissue or organ is exposed to the morphogen prior to its removal from the donor, providing the donor with a composition consisting of a morphogen or morphogen-stimulating agent. Alternatively, or in addition, once removed from the donor, the organ or tissue is placed in a preservative solution containing a morphogen or morphogen-stimulating agent. Furthermore, it is also preferable to provide the transplant recipient with a morphogen or morphogen-stimulating agent just prior to or simultaneously with the transplant. In all cases, the morphogen or morphogen-stimulating agent can be administered directly to the tissue at risk, either by injection or topical administration to the tissue, or it can be provided systemically, either orally or parenterally.

The morphogens described in this document are expected to be of great use to enhance the viability of any organ or living tissue to be transplanted. Morphogens can be used for particular benefit in lung, heart, liver, kidney, or pancreas transplants, as well as in bone marrow, skin, gastrointestinal mucosa, and other living tissue transplantation and / or grafting.

When the patient suffers from a chronic inflammatory disease, such as diabetes, arthritis, psoriasis, IBD, and the like, the morphogen or morphogen-stimulating agent is preferably administered at regular intervals as a prophylactic, to prevent and / or inhibit the historical damage normally. associated with disease during periods of worsening. As before, the morphogen or morphogen stimulating agent can be administered directly to the tissue at risk, for example by administration of an injection or topically, or indirectly, systemically, eg, oral or parenteral administration.

Among the morphogens useful in this invention are the proteones originally identified as osteogenic proteones, such as OP-1, OP-2 and CBMP2 proteones, as well as proteones with an afon amino acid sequence such as DDP (from Drosophila), Vgl ( of Xenopus), Vgr-1 (of mouse, see document US 5,011,691 in Oppermann et al.), GDF-1 (mouse, see Lee (1991) PNAS 88: 4250-4254), all of them presented in Table II and Sequences with identification numbers 5-14), and the recently identified 60 A protein (from Drosophila, Sec. With NID No. 24, see Wharton et al. (1991) PNAS 88: 9214-9218.) Members of this family, which include members of the TGF- β, share a high homology of amino acid sequences in their C-terminal regions. The proteins are translated as precursors, having a peptide signal sequence in the N-terminal region, usually of less than about 30 residues, followed by a "pro" domain that is cut to give the mature protein sequence. The signal peptide is cleaved rapidly in the translation process, at the cleavage site that can be predicted in a given sequence using the Von Heijne method [(1986) Nucleic Acids Research 14: 4683-4691.) Table 1, below, describes the various morphogens identified to date, including their nomenclature as used in this document, their NID references (No. identification) of Sec., and publication sources for the amino acid sequences of the whole proteon not included in the List of Sec.

ES 2 149 776 T3 “OP-1” “OP-2” “CBMP2” “DPP (fx)” “Vgl (fx)” “Vgr-1 (fx)”

TABLE I

Generically refers to the group of morphogenically active proteins expressed from part or all of the DNA sequence encoding the OP1 protein, including species and allelic variants, eg, human OP-1 ("hOP-1 ", Seq with NID No. 5, amino acid sequence of mature protein), or mouse OP-1 (" mOP-1 ", Seq with NID No. 6, amino acid sequence of mature protein.). The conserved skeleton of seven cystenes is defined by residues 38 to 139 of Sec. with NID. Nos. 5 and 6. The cDNA sequences and amino acids encoding all protein are provided in Seq. With Nid. Nos. 16 and 17 (hOP1) and in Sec. With Nid. Nos. 18 and 19 (mPO1). Mature proteins are defined by residues 293-431 (hOP1) and 292-430 (mOP1). The "pro" regions of the proteins, which are cleaved to give the mature, morphogenic active proteins, are essentially defined by residues 30-292 (hOP1) and residues 30-291 (mOP1).

Generically refers to the group of active proteins expressed from part or all of the DNA sequence encoding the OP-2 protein, including species and allelic variants, eg, human OP-2 ("hOP- 2 ”, Sec with NID N° 7, amino acid sequence of mature protein), or ratatoen OP-2 ("mOP-2", Seq. With NID N° 8, amino acid sequence of mature protein.). The conserved seven cysteine skeleton is defined by residues 38 to 139 of Sec. With NID. Nos. 7 and 8. The cDNA sequences and amino acids encoding all protein are provided in Seq. With Nid. Nos. 20 and 21 (hOP2) and in Sec. With Nid. Nos. 22 and 23 (mPO2). Mature proteins are essentially defined by residues 264-402 (hOP2) and 261-399 (mOP2). The "pro" regions of the proteins, which are cut to give the mature, morphogenic active proteins, are essentially defined by residues 18-263 (hOP2) and residues 18-260 (mOP2). (There is also another 21 residue cleavage site upstream for both OP-2 proteins.)

Generically refers to the group of morphogenic active proteins expressed from a DNA sequence encoding CBMP2 proteins, including species and allelic variants, eg, human CBMP2A ("CBMP2A (fx)", Sec with NID N° 9), or human CBMPB2 DNA ("CBMP2B (fx)" Sec. With NID N° 10). The sequence of amino acids that encode all the protein, referred to in the literature as BMP2A and BMP2B or BMP2 and BMP4, appears in Wozney, et al. (1988) Science 242: 1528-1534. The pro domain for BMP2 (BMP2A) probably includes residues 25-248 or 25-282; mature protein, residues 249-396 or 283-396. The pro domain for BMP4 (BMP2B) probably includes residues 25-256 or 25-292; mature protein, residues 257-408 or 293-408.

It refers to the protein sequences encoded by the Drosophila DPP gene and which define the conserved backbone of seven cystenes (Seq. With NID. No.11). The amino acid sequence of the entire protein appears in Padgett, et al (1987) Nature 325: 81-84. The pro domain extends from the signal peptide cleavage site to residue 456; mature protein is probably defined by residues 457-588.

It refers to the protein sequences encoded by the Xenopus Vgl gene and which define the conserved backbone of seven cysteines (Seq. With NID. No.12). The amino acid sequence of the entire protein appears in Weeks (1987) Cell 51: 861-867. The pro domain likely extends from the signal peptide cleavage site to residue 246; mature protein is defined by residues 247-360.

Refers to the protein sequences encoded by the murine Vgr-1 gene and defines the conserved backbone of seven cysteines (Seq. With NID. No.13). The amino acid sequence of the entire protein appears in Lyons, et al, (1989) PNAS 86: 4554-4558. The pro domain extends from the signal peptide cleavage site to residue 299; mature protein is probably defined by residues 300-438.

ES 2 149 776 T3

TABLE I (Cont.) “GDF-1 (fx)” “60A” “60- (fx)” “BMP3 (fx)” “BMP5 (fx)” “BMP6 (fx)”

Refers to the protein sequences encoded by the human GDF-1 gene and defining the conserved skeleton of seven cisternae (Seq. With NID. No.14). The cDNA and amino acid sequence of the entire protein is provided in Seq. With NID. No. 32. The pro domain likely extends from the signal peptide cleavage site to residue 214; the mature protein is probably defined by residues 215-372.

Generically refers to the group of morphogenically active proteins expressed from part or all of the DNA sequence (from Drosophila gene 60A) that encodes protein 60A (see Seq. With NID. No. 24 where provides the cDNA and amino acid sequence that encodes the entire protein).

refers to the protein sequences that define the backbone of seven conserved cysteines (residues 354 to 455 of Seq. with NID N° 24.) The pro domain likely extends from the signal peptide cleavage site to residue 324; the mature protein was probably defined by residues 325-455.

Refers to the protein sequences encoded by the human BMP3 gene and defining the conserved seven cysteine backbone (Seq. With NID. No.26). The amino acid sequence of the entire protein appears in Wozney et al. (1988) Science 242: 15281534. The pro domain extends from the signal peptide cleavage site to residue 290; the mature protein is defined by residues 291-472.

Refers to the protein sequences encoded by the human BMP5 gene and defining the conserved seven cysteine skeleton (Seq. With NID n° 27). The amino acid sequence of the complete protein appears in Celeste, et al. (1991) PNAS 87: 9843-9847. The pro domain can span from the signal peptide cleavage site to residue 316; the mature protein was probably defined by residues 317-454.

Refers to the protein sequences encoded by the human BMP6 gene and defining the conserved seven cysteine skeleton (Seq. With NID n° 28). The amino acid sequence of the complete protein appears in Celeste, et al. (1990) PNAS 87: 9843-5847. The pro domain can range from the signal's peptide segmentation point to residue 374; the mature protein can include residues 375-513.

OP-2 proteins possess an additional cysteine residue in this region (e.g., see remainder 41 of the secs. With NID n° 7 and 8) apart from the conserved cysteine skeleton that they have in common with the other proteins of this family. The GDF-1 protein possesses an insertion of four amino acids within the conserved skeleton (residues 44-47 of sec. With NID n° 14) but this insertion probably does not interfere with the relationship of the cysteines in the folded structure. Furthermore, the CBMP2 proteins lack an amino acid residue within the cysteine backbone.

Morphiogens are inactive in their reduced form, but are active when they are oxidized homodimers and when they are oxidized together with other morphogens of this invention (eg, when they are heterodimers). Thus, as defined here, a morphiogen is a dimeric protein made up of a pair of polypeptide chains, where each polypeptide chain comprises at least by the backbone of six C-terminal cysteines defined by residues 43-139 of the sec. with NID n° 5, including functionally equivalent arrangements of these cysteines (eg, insertions or deletions of amino acids that alter the linear arrangement of the cysteines in the sequence but not their relationship in the folded structure), such that when the polypeptide chains are folded , the dimeric protein species comprising the pair of polypeptide chains has the appropriate three-dimensional structure, including the appropriate intrachain or interchain disulfide bonds, such that the protein is capable of acting as a morphiogen as defined herein. Specifically, morphiogens are generally capable of performing all of the following biological functions in a morphogenically permissive environment: stimulating the proliferation of progenitor cells; stimulate the differentiation of progenitor cells; stimulate the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells, including the "redifferentiation" of transformed cells. In addition, it is also expected that these morphiogens are capable of inducing the redifferentiation of the cells involved under the appropriate environmental conditions.

ES 2 149 776 T3

In a preferred aspect, the morphogens of this invention consist of one of two species of amino acid genetic sequences: Generic Sequence 1 (Seq. With NID N° 1) or Generic Sequence 2 (Sec. With

NID No. 2); where each Xaa indicates one of the 20 L isomers of naturally occurring α-amino acids, or a derivative thereof. Generic Sequence 1 consists of the conserved skeleton of six cysteines and Generic Sequence 2 comprises the conserved skeleton of six cysteines plus the additional cysteine identified in OP-2 (see remainder 36 Sec. With NID N° two). In another preferred aspect, these sequences also consist of the following additional sequence at their N-terminus:

Cys Xaa Xaa Xaa Xaa (Seq. ID No, 15)

The preferred amino acid sequences within the preceding genetic sequences are: Generic Sequence 3 (Seq. With NID N° 3), Generic Sequence 4 (Sec. With NID N° 4) Generic Sequence 5 (Sec. With NID N° 30) and Generic Sequence 6 (Sec. With NID N° 31), listed below. These Generic Sequences include the homologies shared by the different preferred members of this family of morpheogens identified in Table II, as well as the variation of amino acid sequences between them. Specifically, Generic Sequences 3 and 4 are amino acid sequences composed of the following proteins presented in Table II and identified in Seq. With NID N° 5-14: Human OP-1 (hOP-1, Sec. With NID N° 5 and 16-17), Ratoen OP-1 (mOP-1, Sec. With NID N° 6 and 18-19), human and rataen OP-2 (Sec. With NID N° 7, 8 and 20-22), CBMP2A (Sec. With NID N° 9), CBMP2B (Sec. With NID N° 10), DPP (from Drosophila, Sec. With NID N° 11), Vgl (from Xenopus, Sec. With NID N° 12), Vgr-1 (from rataen, Sec. With NID N° 13), and GDF-1 (from rataen, Sec. With NID N° 14). Generic sequences include both amino acid identity shared by the sequences in Table II and alternative residues at variable positions within the sequence. Note that these genetic sequences allow an additional cysteine at position 41 or 46 in Generic Sequences 3 and 4 respectively, providing a suitable cysteine backbone where inter- or intra-molecular disulfide bonds can form, and that they contain certain fundamental amino acids that influence in the tertiary structure of proteins.

Generic Sequence 3

Leu Tyr Val Xaa Phe 1 5

Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 10

Xaa Ala Pro Xaa Gly Xaa Xaa Ala 15 20

Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 25 30

Xaa Pro Xaa Xaa Xaa xaa Xaa

Xaa Xaa Xaa Asn Kís Ala Xaa Xaa 40 45

ES 2 149 776 T3

Gene Sequence 3 (Cont.)

<td>Xaa</td><td>Xaa</td><td colspan="2">Leu Xaa</td><td>Xaa fifty</td><td colspan="2">Xaa Xaa</td><td>Xaa</td>

Xaa Xaa Xaa

Xaa Cys Gly

Leu Xaa Xaa Xaa

Xaa Val Xaa Leu Xaa 80 xaa Met Xaa Val xaa

Cys Xaa where each Xaa is independently selected from a group of one or more specific amino acids defined as follows: "Res." means "remainder" and Xaa in res. 4 = (Ser, Asp or Glu); Xaa in res. 6 = (Arg, Gln, Ser or Lys); Xaa in res. 7 = (Asp, or Glu); Xaa in res. 8 = (Leu or Val); Xaa in res. 11 = (Gln, Leu, Asp, His or Asn); Xaa in res. 12 = (asp., Arg or Asn); Xaa in res. 14 = (Ile or Val); Xaa in res. 15 = (Ile or Val); Xaa in res. 18 = (Glu, Gln, Leu, Lys, Pro or Arg); Xaa in res. 20 = Tyr or Phe); Xaa in res. 21 = (Ala, Ser, Asp, Met, His, Leu or Gln); Xaa in res. 23 = (Tyr, Asn or Phe); Xaa at res 26 = (Glu, His, Tyr, Asp or Gln); Xaa in res. 28 = (Glu, Lys, Asp or Gln); Xaa in res. 30 = (Ala, Ser, Pro or Gln); Xaa in res. 31 = (Phe, Leu or Tyr); Xaa in res. 33 = (Leu or Val); Xaa in res. 34 = (Asn, Asp, Ala or Thr); Xaa in res. 35 = (Ser, Asp, Glu, Leu or Ala); Xaa in res. 36 = (Tyr, Cys, His, Ser or Ile); Xaa in res. 37 = (Met, Phe, Gly or Leu); Xaa at res 38 = (Asn or Ser); Xaa at res 39 = (Ala, Ser or Gly); Xaa in res. 40 = Thr, Leu or Ser); Xaa in res. 44 = (Ile or Val); Xaa in res. 45 = Val or Leu); Xaa in res. 46 = (Gln or Arg); Xaa at res 47 = (Thr, Ala or Ser); Xaa in res. 49 = (Val or Met); Xaa in res. 50 = (His or Asn); Xaa in res. 51 = (Phe, Leu, Asn, Ser, Ala or Val); Xaa in res. 52 = (Ile, Met, Asn, Ala or Val); Xaa in res. 53 = (Asn, Lys, Ala or Glu); Xaa in res. 54 = (Pro or Ser); Xaa in res. 55 = (Glu, Asp, Asn, or Gly); Xaa in res. 56 = (Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa in res. 57 = (Val, Ala or Ile); Xaa in res. 58 = (Pro or Asp); Xaa in res. 59 = (Lys or Leu); Xaa in res. 60 = (Pro or Ala); Xaa in res. 63 = (Ala or Val); Xaa in res. 65 = (Thr or Ala); Xaa in res. 66 = (Gln, Lys, Arg or Glu); Xaa in res. 67 = (Leu, Met or Val); Xaa in res. 68 = (Asn, Ser or Asp); Xaa in res. 69 = (Ala, Pro or Ser); Xaa in res. 75 = (Phe, Tyr, or Leu); Xaa in res. 76 = (Asp or Asn); Xaa in res. 77 = (Asp, Glu, Asn or Ser); Xaa in res. 78 = (Ser, Gln, Asn or Tyr); Xaa in res. 79 = (Ser, Asn, Asp or Glu); Xaa in res. 80 = (Asn, Thr or Lys); Xaa in res. 82 = (Ile or Val); Xaa in res. 84 = (Lys or Arg); Xaa in res. 85 = (Lys, Asn, Gln or His); Xaa in res. 86 (Tyr or His); Xaa in res. 87 = (Arg, Gln or Glu); Xaa in res. 88 = (Asn, Glu or Asp); Xaa in res. 90 = (Cal, Thr or Ala); Xaa in res. 92 = (Arg, Lys, Val, Asp or Glu); Xaa in res. 93 = Ala, Gly or Glu); and Xaa in res. 97 = (His or Arg).

ES 2 149 776 T3

Generic Sequence 4

<td rowspan="2">Xaa Xaa twenty Xaa</td><td rowspan="2">Xaa To Tyr</td>

where each Xaa is independently selected from a group of one or more specific amino acids defined as follows: "Res." means "remainder" and Xaa in res. 2 = (Lys or Arg); Xaa in res. 3 = (Lys or Arg); Xaa in res. 4 = (His or Arg); Xaa in res. 5 = (Glu, Ser, His, Gly, Arg or Pro); Xaa in res. 9 = (Ser, Asp or Glu); Xaa in res. 11 = (Arg, Gln, Ser or Lys); Xaa in res. 12 = (Asp, or Glu); Xaa in res. 13 = (Leu or Val); Xaa in res. 16 = (Gln, Leu, Asp, His or Asn); Xaa in res. 17 = (Asp, Arg or Asn); Xaa in res. 19 = (Ile or Val); Xaa in res. 20 = (Ile or Val); Xaa in res. 23 = (Glu, Gln, Leu, Lys, Pro or Arg); Xaa in res. 25 = (Tyr or Phe); Xaa in res. 26 = (Ala, Ser, Asp, Met, His, Leu or Gln); Xaa in res. 28 = (Tyr, Asn or Phe); Xaa at res 31 = (Glu, His, Tyr, Asp or Gln); Xaa in res. 33 = (Glu, Lys, Asp or Gln); Xaa in res. 35 = (Ala, Ser, Pro or Gln); Xaa in res. 36 = (Phe, Leu or Tyr); Xaa in res. 38 = (Leu or Val); Xaa in res. 39 = (Asn, Asp, Ala or Thr); Xaa in res. 40 = (Ser, Asp, Glu, Leu or Ala); Xaa in res. 41 = (Tyr, Cys, His, Ser or Ile); Xaa in res. 42 = (Met, Phe, Gly or Leu); Xaa in res. 43 = (Asn or Ser); Xaa at res 44 = (Ala, Ser or Gly); Xaa in res. 45 = Thr, Leu or Ser); Xaa in res. 49 = (Ile or Val); Xaa in res. 50 = Val or Leu); Xaa in res. 51 = (Gln or Arg); Xaa at res 52 = (Thr, Ala or Ser); Xaa in res. 54 = (Val or Met); Xaa in res. 55 = (His or Asn); Xaa in res. 56 = (Phe, Leu, Asn, Ser, Ala or Val); Xaa in res. 57 = (Ile, Met, Asn, Ala or Val); Xaa in res. 58 = (Asn, Lys, Ala or Glu); Xaa in res.

ES 2 149 776 T3 = (Pro or Ser); Xaa in res. 60 = (Glu, Asp, Asn, or Gly); Xaa in res. 61 = (Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa in res. 62 = (Val, Ala or Ile); Xaa in res. 63 = (Pro or Asp); Xaa in res. 64 = (Lys or Leu); Xaa in res. 65 = (Pro or Ala); Xaa in res. 68 = (Ala or Val); Xaa in res. 70 = (Thr or Ala); Xaa in res. 71 = (Gln, Lys, Arg or Glu); Xaa in res. 72 = (Leu, Met or Val); Xaa in res. 73 = (Asn, Ser or Asp); Xaa in res. 74 = (Ala, Pro or Ser); Xaa in res. 80 = (Phe, Tyr, or Leu); Xaa in res. 81 = (Asp or Asn); Xaa in res. 82 = (Asp, Glu, Asn or Ser); Xaa in res. 83 = (Ser, Gln, Asn or Tyr); Xaa in res. 84 = (Ser, Asn, Asp or Glu); Xaa in res. 85 = (Asn, Thr or Lys); Xaa in res. 87 = (Ile or Val); Xaa in res. 89 = (Lys or Arg); Xaa in res. 90 = (Lys, Asn, Gln or His); Xaa in res. 91 (Tyr or His); Xaa in res. 92 = (Arg, Gln or Glu); Xaa in res. 93 = (Asn, Glu or Asp); Xaa in res. 95 = (Cal, Thr or Ala); Xaa in res. 97 = (Arg, Lys, Val, Asp or Glu); Xaa in res. 98 = Ala, Gly or Glu); and Xaa in res. 102 = (His or Arg).

Similarly, Gene Sequence 5 (Seq. With NID No. 30) and Gene Sequence 6 (Seq. With NID No. 31) include the homologues shared by all members of the family of morphogenic proteins identified in Table II. . Specifically, Genomic Sequences 5 and 6 are amino acid sequences composed of human OP-1 (hOP-1, Seq. With NID N° 5 and 16-17), mouse OP-1 (mOP-1, Sec. With NID N° 6 and 18-19), human and mouse OP-2 (Sec. With NID N° 7, 8 and 20-22, CBMP2A (Sec. With NID N° 9), CBMP2B (Sec. With NID N° 10), DPP (from Drosophila, Sec. With NID N° 11), Vgl (from Xenopus, Sec. With NID N° 12), Vgr-1 (from mouse, Sec. With NID N° 13), and GDF-1 (from mouse, Sec. With NID N° 14), human BMP3 (Sec. With NID N° 26), human BMP5 (Sec. With NID N° 27), human BMP6 (Seq. With NID N° 28) and 60A (from Drosophila, Sec. With NID N° 24-25). The genomic sequences include both the amino acid identity shared by these sequences in the domain of the C-terminal end, defined by the skeletons of six and seven cisternae (Generic Sequences 5 and 6 respectively) as the alternative residues of the variable positions within the sequence. As for Gene Sequences 3 and 4, Gene Sequences 5 and 6 allow for an additional cysteone at position 41 (Gene Sequence 5) or at position 46 (Gene Sequence 6), providing a suitable cysteone backbone where disulfide bonds can form. inter or intramolecular, and that it contains certain fundamental amino acids that influence the tertiary structure of proteones.

Gene Sequence 5

Leu Xaa Xaa Xaa Phe

Xaa Xaa Xaa Gly Trp Xaa Xaa Trp xaa 10

ES 2 149 776 T3

Generic Sequence 5 (Cont.)

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 55 60

Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 65

Xaa Xaa Xaa Leu Xaa Xaa Xaa

Xaa Xaa Xaa Xaa Val Xaa Leu Xaa

Xaa xaa Xaa Xaa Met Xaa Val Xaa

Xaa Cys Xaa Cys Xaa where each Xaa is independently selected from a group of one or more specific amino acids defined as follows: "Res." means "remainder" and Xaa in res. 2 = (Tyr or Lys); Xaa in res. 3 = (Val or Ile); Xaa in res. 4 = (Ser, Asp or Glu); Xaa in res. 6 = (Arg, Gln, Ser, Lys or Ala); Xaa in res. 7 = (Asp, Glu or Lys); Xaa in res. 8 = (Leu, Val or Ile); Xaa in res. 11 = (Gln, Leu, Asp, His, Asn or Ser); Xaa in res. 12 = (Asp, Arg, Asn or Glu); Xaa in res. 14 = (Ile or Val); Xaa in res. 15 = (Ile or Val); Xaa in res. 16 = (Ala or Ser); Xaa in res. 18 = (Glu, Gln, Leu, Lys, Pro or Arg); Xaa in res. 19 = (Gly or Ser); Xaa in res. 20 = (Tyr or Phe); Xaa in res. 21 = (Ala, Ser, Asp, Met, His, Gln, Leu or Gly); Xaa in res. 23 = (Tyr, Asn or Phe); Xaa at res 26 = (Glu, His, Tyr, Asp, Gln or Ser); Xaa in res. 28 = (Glu, Lys, Asp, Gln or Ala); Xaa in res. 30 = (Ala, Ser, Pro, Gln or Asn); Xaa in res. 31 = (Phe, Leu or Tyr); Xaa in res. 33 = (Leu, Val or Met); Xaa in res. 34 = (Asn, Asp, Ala, Thr or Pro); Xaa in res. 35 = (Ser, Asp, Glu, Leu, Ala or Lys); Xaa in res. 36 = (Tyr, Cys, His, Ser or Ile); Xaa in res. 37 = (Met, Phe, Gly or Leu); Xaa at res 38 = (Asn, Ser or Lys); Xaa at res 39 = (Ala, Ser, Gly, or Pro); Xaa in res. 40 = Thr, Leu or Ser); Xaa in res. 44 = (Ile, Val or Thr); Xaa in res. 45 = Val, Leu or Ile); Xaa in res. 46 = (Gln or Arg); Xaa in res. 47 = (Thr, Ala or Ser); Xaa in res. 48 = (Leu or Ile); Xaa in res. 49 = (Val or Met); Xaa in res. 50 = (His, Asn or Arg); Xaa in res. 51 = (Phe, Leu, Asn, Ser, Ala or Val); Xaa in res. 52 = (Ile, Met, Asn, Ala, Val or Leu); Xaa in res. 53 = (Asn, Lys, Ala, Glu, Gly or Phe); Xaa in res. 54 = (Pro, Ser or Val); Xaa in res. 55 = (Glu, Asp, Asn Gly, Val or Lys); Xaa in res. 56 = (Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa in res. 57 = (Val, Ala or Ile); Xaa in res. 58 = (Pro or Asp); Xaa in res. 59 = (Lys, Leu or Glu); Xaa in res. 60 = (Pro or Ala); Xaa in res. 63 = (Ala or Val); Xaa in res. 65 = (Thr, Ala or Glu); Xaa in res. 66 = (Gln, Lys, Arg or Glu); Xaa in res. 67 = (Leu, Met or Val); Xaa in res. 68 = (Asn, Ser, Asp or Gly); Xaa in res. 69 = (Ala, Pro or Ser); Xaa in res. 70 = (Ile, Thr, Val or Leu); Xaa in rest. 71 = (Ser, Ala or Pro); Xaa in rest. 72 = (Val, Met or Ile); Xaa in rest. 74 = (Tyr or Phe); Xaa in res. 75 = (Phe, Tyr, Leu or His); Xaa in res. 76 = (Asp, Asn or Leu); Xaa in res. 77 = (Asp, Glu, Asn or Ser); Xaa in res. 78 = (Ser, Gln, Asn, Tyr or Asp); Xaa in res. 79 = (Ser, Asn, Asp, Glu or Lys); Xaa in res. 80 = (Asn, Thr or Lys); Xaa in res. 82 = (Ile, Val or Asn); Xaa in res. 84 = (Lys or Arg); Xaa in res. 85 = (Lys, Asn, Gln, His or Val); Xaa in res. 86 (Tyr or His); Xaa in res. 87 = (Arg, Gln, Glu or Pro); Xaa in res. 88 = (Asn, Glu or Asp); Xaa in res. 90 = (Cal, Thr, Ala or Ile); Xaa in res. 92 = (Arg, Lys, Val, Asp or Glu); Xaa in res. 93 = (Ala, Gly, Glu or Ser); Xaa in res. 95 = (Gly or Ala) and Xaa at res. 97 = (His or Arg).

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Gene Sequence 6

where each Xaa is independently selected from a group of one or more specific amino acids defined as follows: "Res." means "residue" and Xaa at res 2 = (Lys, Arg, Ala or Gln); Xaa in res. 3 = (Lys, Arg or Met); Xaa in res. 4 = (His, Arg or Gln); Xaa in res. 5 = (Glu, Ser, His, Gly, Arg, Pro, Thr or Tyr); beef. 7 = (Tyr or Lys); Xaa in res. 8 = (Val or Ile); Xaa in res. 9 = (Ser, Asp or Glu); Xaa in res. 11 = (Arg, Gln, Ser, Lys or Ala); Xaa in res. 12 = (Asp, Glu or Lys); Xaa in res. 13 = (Leu, Val or Ile); Xaa in res. 16 = (Gln, Leu, Asp, His, Asn or Ser); Xaa in res. 17 = (Asp, Arg, Asn or Glu); Xaa in res. 19 = (Ile or Val); Xaa in res. 20 = (Ile or Val); Xaa in res. 21 = (Ala or Ser); Xaa in res. 23 = (Glu, Gln, Leu, Lys, Pro or Arg); Xaa in res. 24 = (GlyoSer); Xaaenres. 25 = (TyroPhe); Xaa in res. 26 = (Ala, Ser, Asp, Met, His, Gln, Leu or Gly); Xaa in res. 28 = (Tyr, Asn or Phe); Xaa at res 31 = (Glu, His, Tyr, Asp, Gln or Ser); Xaa in res. 33 = (Glu, Lys, Asp, Gln or Ala); Xaa in res. 35 = (Ala, Ser, Pro, Gln or Asn); Xaa in res. 36 = (Phe, Leu or Tyr); Xaa in res. 38 = (Leu, Val or Met); Xaa in res. 39 = (Asn, Asp, Ala, Thr or Pro); Xaa in res. 40 = (Ser, Asp, Glu, Leu, Ala or Lys); Xaa in res. 41 = (Tyr, Cys, His, Ser or Ile); Xaa in res. 42 = (Met, Phe, Gly or Leu); Xaa at res 43 = (Asn, Ser or Lys); Xaa at res 44 = (Ala, Ser, Gly, or Pro); Xaa in res. 45 = Thr, Leu or Ser); Xaa in res. 49 = (Ile, Val or Thr); Xaa in res. fifty = Val, Leu or Ile); Xaa in res. 51 = (Gln or Arg); Xaa in res. 52 = (Thr, Ala

ES 2 149 776 T3 or Ser); Xaa in res. 53 = (Leu or Ile); Xaa in res. 54 = (Val or Met); Xaa in res. 55 = (His, Asn or Arg); Xaa in res. 56 = (Phe, Leu, Asn, Ser, Ala or Val); Xaa in res. 57 = (Ile, Met, Asn, Ala, Val or Leu); Xaa in res. 58 = (Asn, Lys, Ala, Glu, Gly or Phe); Xaa in res. 59 = (Pro, Ser or Val); Xaa in res. 60 = (Glu, Asp, Asn Gly, Val or Lys); Xaa in res. 61 = (Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa in res. 62 = (Val, Ala or Ile); Xaa in res. 63 = (Pro or Asp); Xaa in res. 64 = (Lys, Leu or Glu); Xaa in res. 65 = (Pro or Ala); Xaa in res. 66 = (Ala or Val); Xaa in res. 70 = (Thr, Ala or Glu); Xaa in res. 71 = (Gln, Lys, Arg or Glu); Xaa in res. 72 = (Leu, Met or Val); Xaa in res. 73 = (Asn, Ser, Asp or Gly); Xaa in res. 74 = (Ala, Pro or Ser); Xaa in res. 75 = (Ile, Thr, Val or Leu); Xaa in rest. 76 = (Ser, Ala or Pro); Xaa in rest. 77 = (Val, Met or Ile); Xaa in rest. 79 = (Tyr or Phe); Xaa in res. 80 = (Phe, Tyr, Leu or His); Xaa in res. 81 = (Asp, Asn or Leu); Xaa in res. 82 = (Asp, Glu, Asn or Ser); Xaa in res. 83 = (Ser, Gln, Asn, Tyr or Asp); Xaa in res. 84 = (Ser, Asn, Asp, Glu or Lys); Xaa in res. 85 = (Asn, Thr or Lys); Xaa in res. 87 = (Ile, Val or Asn); Xaa in res. 89 = (Lys or Arg); Xaa in res. 90 = (Lys, Asn, Gln, His or Val); Xaa in res. 91 (Tyr or His); Xaa in res. 92 = (Arg, Gln, Glu or Pro); Xaa in res. 93 = (Asn, Glu or Asp); Xaa in res. 95 = (Cal, Thr, Ala or Ile); Xaa in res. 97 = (Arg, Lys, Val, Asp or Glu); Xaa in res. 98 = (Ala, Gly, Glu or Ser); Xaa in res. 100 = (Gly or Ala) and Xaa at res. 102 = (His or Arg).

Sequences of particular utility as morphogens in this invention include the C-terminus domains, eg, C-terminus amino acid residues 96-102 of Vgl, Vgr-1, DPP, OP-1, OP -2, CBMP-2A, CBMP-2B, GDF-1 (see Table II below and Seq. With NID No. 5-14), as well as the proteones that span the domains of the C-terminal end of 60A, BMP3, BMP5 and BMP6 (see Sections with NID N° 24-28), all of which include at least the conserved skeleton of six or seven cysteones. Furthermore, biosynthetic constructs designed from genomic sequences, such as COP-1, 3-5, 7, 16, described in US Patent No.° 5,011,691 are also useful. Other sequences are the inhibin / activin proteones (see, for example, US Patent No.° 4,968,590 and 5,011,691). Accordingly, other useful sequences are those that share at least 70% homology or "similarity" with the amino acid sequence, and preferably if they have 80% homology or similarity with any of the sequences mentioned above. These are expected to include species and alloelic variants and mutants, as well as biosynthetic muteons and new members of this morphogenic family of proteones. Within the family of osseous-related proteones, proteones that display morphogenic activity and in which amino acid changes of preferred sequences include conservative changes, eg, those defined in Dayoff et al., Are especially desirable. Atlas of Protein Sequence and Structure; vol. 5, Suppl. 3, pp. 345-362 (MO Dayoff, ed., Nat'l BioMed. Research Fdn., Washington DC 1979). As used herein, potentially useful sequences are aligned with a known sequence of morphogens by the method of Needleman et al., (1970) J. Mol. Biol., 48: 443-453) and identities are calculated with the Align program (DNAstar, Inc). "Homologous" or "similarity", as used herein, includes the allowed conservative changes defined by Dayoff et al.

Currently most preferred proteon sequences that are useful as morphogens in this invention include those that have greater than 60% identity, preferably when greater than 65% identity, with the amino acid sequence that defines the conserved skeleton of six cysteones of hOP1 (eg, residues 43-139 of seq. with NID n° 5). These preferred moe sequences include both alloelic and species variants of the OP-1 and OP-2 proteones, including the Drosophila 60A proteon. In the same way, in another preferable aspect of the invention, useful morphogens include active proteones constituted by species of polypeptide chains that have the genomic sequence of amino acids here called "OPX", which includes the homologies between the different identified species of OP1 and OP2. (Sec. With NID N° 29).

Useful morphogens in the methods, composition, and devices of this invention include proteones made up of any of the polypeptide chains described above, whether they have been isolated from natural sources or produced by recombinant DNA or other synthetic theoechnics, and include alloelic variants. and of species of these proteones, natural or biosynthetic mutants thereof, and various truncated and fusion constructs. It is expected that mutants with deletions or additions are also active, including those that can alter the conserved C-terminal cysteone skeleton, provided that the alteration does not functionally disrupt the relationship of these cysteones in the folded structure. Accordingly, these active forms are considered the equivalent of the specifically described constructions explained here.

Proteones can include forms that have different glycosylation patterns, different N-terminal ends, a family of proteones related to the above that have regions of homology.

ES 2 149 776 T3 in the amino acid sequence, and active truncated or mutated forms of native or biosynthetic proteins, produced by expression of recombinant DNA in host cells.

Morphogenic proteins can be expressed from intact or truncated cDNA or from synthetic DNA in prokaryotic or eukaryotic host cells, and be purified, cleaved, refolded and dimerized to form morphogenic active compositions. Currently preferred host cells are E. coli or mammalian cells, such as CHO, COS, or BSC cells. A detailed description of the useful morphogens in the methods, compositions, and devices of this invention is published in copending US patent application No.° No. 752,764, filed on August 30, 1991, and No.° No. 667,274, filed on March 11, 1991.

Thus, in view of this description, specialized genetic engineers can isolate genes from cDNA or genoemic libraries of several different species that encode the appropriate amino acid sequences, or construct DNA chains from oligonucleotides, for later express them in various types of host cells, including prokaryotes and eukaryotes, in order to produce large amounts of active proteins capable of protecting tissues and organs from cell-mediated tissue destruction, including the substantial inhibition of such damage and / or the regeneration of damaged tissue in a wide variety of mammals, including the Humans.

The foregoing and other objects, functions and advantages of the present invention will be better illustrated by the detailed description that follows.

Brief description of the illustrations

Fig 1 shows the cardioprotective effects of morpheogen (hOP1) in a rat model with myocardial ischemia and reperfusion, as evidenced by the lower loss of myocardial creatine kinase in rats treated with hOP1;

Fig 2 shows the effects of 20 µg of morphogen (hOP1) administered 24 hours before rat heart isolation on endothelium-dependent vasorelaxation to acetylcholine after ischemia and reperfusion-induced injury;

Fig 3 shows the effect of morphogen (hOP1) on the adherence of neutroephils to the endothelium of the LTB4-stimulated mesenteric artery in rats with activated neutrophils;

Fig 4 (A and B) are schematic representations of inhibition by morphogens of early mononuclear phagocetic multinuclearization in vivo;

Fig 5 illustrates the effect of a morphogen (eg, OP-1) and a placebo control on the formation of a mucosal lesion; and

Fig 6 (AD) illustrates the effects of a morphegen (eg, OP-1, Fig. 6A and 6C) and TGF-β (Fig. 6B and 6D) on the production of collagen (6A and 6B) and acid hyalureonic (6C and 6D) in primary fibroblast cultures.

Detailed description of the invention

Surprisingly, the morphogens described herein have now been found to be effective agents for alleviating the tissue-killing effects associated with the body's inflammatory response to tissue injury. In particular, as disclosed herein, morphogens are capable of alleviating the effects of tissue necrosis associated with subsequent inflammatory responses that occur after initial tissue injury.

When a tissue injury occurs, caused by bacteria, trauma, chemicals, heat, or any other phenomenon, the body's inflammatory response is stimulated. In response to signals emitted by damaged cells (eg, cytokines), extravascularization of immune effector cells is induced. Under normal circumstances, these invasive immune effector cells kill the infectious agent and / or infected or damaged cells (by releasing killer substances such as superoxides, perforins, and other antimicrobial agents stored in granules), eliminating tissues and organisms dead (via phagocytosis), release different modifiers of the biological response that promote rapid healing and wound coverage (which often leads to the formation of fibrous scar tissue), and later, when the area is

ES 2 149 776 T3 well cured, they leave the point of the initial aggression. Once the point is considered normal, the local release of inflammatory cytokines ceases, and the manifestation of adhesion molecules in the endothelium of the vessel returns to basal levels. In some cases, however, the heat of these interactive signals and cellular systems, which are designed to capture and retain infectious agents that multiply rapidly, act to the detriment of the body, killing additional surrounding healthy tissue. This unnecessary additional tissue death further compromises the function of the organ and sometimes leads to the death of the individual. Furthermore, the resulting scar tissue that forms can often interfere with normal tissue function, as occurs for example in the case of idiopathic pulmonary fibrosis, IBD, and organ cirrhosis.

The vascular endothelium constitutes the first barrier between circulating immune effector cells and extravascular tissues. Extravasation of these circulating cells requires that they bind to vascular endothelial cells, cross the base membrane, and enter damaged tissues, eg, by phagocytosis or protease-mediated degradation of the extracellular matrix. Without being limited to a particular theory, it is believed that the morpheogens of this invention may in part modulate the inflammatory response by modulating the connection of immune effector cells to the luminal side of the endothelium of blood vessels at or near sites of tissue damage. themselves and / or inflammatory lesions. Since the method reduces or prevents the attachment of immune effector cells at these sites, it also prevents subsequent release of tissue-destroying agents by these same immune effector cells at sites of bowel damage or inflammatory lesions. Since the connection of immune effector cells to the endothelium must precede their extravascularization, the method also prevents the initial or continued entry of these cells into extravascular sites of tissue destruction or in ongoing inflammatory lesions. Therefore, the invention is not only related to a method of reducing or preventing immune cell-mediated cell destruction at extravacular sites of recent hestic destruction, but is also related to a method of preventing or reducing the continuous entry of cells. Immune effectors at extravascular sites of ongoing inflammatory cascades. As those skilled in the art will appreciate, the morpheogens of this invention may also be viewed in mechanisms that disrupt the functional interaction of immune effector cells with endothelium where adhesion molecules are induced by means other than in response to a hestic injury.

One source of bowel injury is induced by exposure of the cell to toxic oxygen concentrations, such as ischemia and reperfusion injury (oxygen deprivation), and following hyperoxic injury (lethally elevated oxygen concentrations). Accordingly, the process of the present invention provides a method of alleviating injury-induced bone damage after ischemia and reperfusion or hyperoxia-induced injury, comprising the step of administering to the injured individual a therapeutic amount of a morphegen before, during, or after treatment. damage to the affected tissue. When toxic concentrations of oxygen can be deliberately induced, as in a surgical or clinical procedure, it is preferable to administer the morphogen prior to induction.

Furthermore, the morphogens described herein, in contrast to fibrogenic growth factors such as TGF-β, stimulate heroic morphogenesis and do not stimulate fibrosis or scar tissue formation (see Example 9 below). Consequently, in addition to inhibiting the tissue destruction effects associated with the inflammatory response, morpheogens further improve the viability of damaged tissues and / or organs by stimulating the regeneration of damaged tissue and preventing fibrogenesis.

The morpheogens described herein may further inhibit epithelial cell proliferation (see Example 10 below). This activity of morphogens may also be particularly useful in the treatment of psoriasis and other inflammatory diseases involving epithelial cell populations.

Detailed descriptions of suitable morphogens that are useful in the methods and compositions of this invention, as well as methods for their administration and application, and numerous non-limiting examples that 1) illustrate the suitability of morphogens and morphogen-stimulating agents are provided below. described as therapeutic agents that protect tissues from the effects of tissue destruction associated with the body's inflammatory response; and 2) provide assays to test candidate morpheogens and morpheogen-stimulating agents for their efficacy.

ES 2 149 776 T3

I. Useful Morphogens

As defined here, a protein is morphogenic if it is capable of inducing the evolutionary cascade of cellular and molecular events that culminate in the formation of new tissue specific for the organism and consists at least of the conserved skeleton of six cysteines of the C-terminal end or its functional equivalent (see mine above). Specifically, morphogens are generally capable of performing all of the following biological functions in a morphogenically permissive environment: stimulating the proliferation of progenitor cells; stimulate the differentiation of progenitor cells; stimulate the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells, including the "redifferentiation" of transformed cells.

Details of how useful morphogens were first identified in the method of this invention, as well as a description of how to make, use, and test them for morphogenic activity are published in USSN 667,274. filed March 11, 1991, and USSN 752,764, filed August 30, 1991. As described therein, morphogens can be purified from material from natural sources or from material produced recombinantly from prokaryotic or eukaryotic host cells, using the genetic sequences published therein. On the other hand, new morphogenic sequences can be identified by following the procedures published there.

Proteins of particular utility are those consisting of the naturally derived sequences listed in Table II. Other useful sequences are biosynthetic constructs, such as those published in US Patent 5,011,691 (eg, COP-1, COP-3, COP-4, COP-5, and COP- 16).

Accordingly, the morphiogens useful in the methods and compositions of this invention can also be described with morphogenically active proteins that have amino acid sequences that share 70% homology (similarity), or preferably 80%, with any of the sequences described mine. above, where “homology” conforms to the definition provided above.

Morphogens useful in the method of this invention can also be described by any of the 6 genetic sequences described herein (Generic Sequences 1, 2, 3, 4, 5 and 6). Generic Sequences 1 and 2 can also include at their N-terminal end the sequence

Cys Xaa Xaa Xaa Xaa {Seq. ID No. 15)

Table II, presented below, compares the amino acid sequences of the active regions of native proteins that have been identified as morphiogens, including human OP-1 (hOP-1, Sec. With NID N° 5 and 16-17), Matoin OP-1 (mOP-1, Sec. With NID N° 6 and 18-19), human and mouse OP-2 (Sec. With NID N° 7, 8 and 20-23), CBMP2A (Sec. With NID N° 9), CBMP2B (Sec. With NID N° 10), BMP3 (Sec. With NID N° 26), DPP (from Drosophila, Sec. With NID N° 11), Vgl (from Xenopus, Sec. With NID N° 12), Vgr-1 (from Ratoin, Sec. With NID N° 13), GDF-1 (from Ratoin, Sec. With NID N° 14, 32 and 33), Protein 60A (from Drosophila, Sec. With NID N° 24 and 25), BMP5 (Sec. With NID N° 27) and BMP6 (Sec. With NID N° 28). Essentially, the stain sequences aligned according to the method of Needleman et al., (1970) J. Mol. Biol., 48: 443-453) and calculated with the Align program (DNAstar, Inc). In the table, three dots indicate that the amino acid at that position is the same as the amino acid in hOP-1. Three dashes indicate the absence of amino acids at that position, and are included for the purpose of illustrating homologies. For example, amino acid residue 60 of CBMP-2A AND CBMP-2B is missing. Of course, the two amino acid sequences in this region include Asn-Ser (residues 58, 59), and CBMP-2A includes Lys and Ile, while CBMP-2B contains Ser and Ile.

ES 2 149 776 T3

TABLE II

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100 ** Between residues 56 and 57 of BMP3 is a valine residue; Between residues 43 and 44 of GDF-1 was the amino acid sequence Gli-Gli-Pro-Pro.

As is apparent from comparing the above amino acid sequences, significant amino acid sequence changes can be made within genomic sequences while retaining morphogenic activity. For example, while the GDF-1 proteon sequence presented in Table II shares only about 50% amino acid identity with the hOP-1 sequence described in the text, the GDF-1 sequence shares more than 70% of amino acid sequence homology (or "similarity") to the hOP-1 sequence, where "homology" or "similarity" includes allowed changes in amino acids conserved within the sequence, as defined by Dayoff, et al., Atlas of Protein Sequence and Structure vol 5, supp. 3, pp 345-352, (MO Dayoff, ed. Natl. BioMed. Res. Fd'n, Washington, DC 1979).

Currently, the most preferred proteon sequences that are useful as morphogens in this invention include those that have more than 60% identity, preferably more than 65% identity, with the amino acid sequence that defines the conserved skeleton of 6 cysteones of hOP-1 (eg, residues 43-139 of Seq. With NID N° 5). These preferred mine sequences include both alloelic and species variant variants of the OP-1 and OP-2 proteones, including Drosophila proteon 60A. Consequently, in another preferred aspect, the invention includes morphogens containing polypeptide chain species with the genomic amino acid sequence referred to herein as "OPX",

ES 2 149 776 T3 and which defines the seven cysteine backbone, and takes into account the identities between the various OP1 and OP2 proteins identified in mice and humans. OPX is presented in Seq. With NID N° 29. As described, each Xaa at a given position is independently selected for residues occurring at the corresponding position in the sequence of the C-terminal end of the mouse or human OP1 and OP2 protein (see Seq. NID N° 5-8 and / or Seq. NID N° 16-23)

II. Formulations and Methods for Administering Therapeutic Agents

Morphogens can be delivered to an individual by any suitable method, with direct administration (eg, locally via injection or topical administration to a site in the tissue) or systemic (eg, by the perenteral or oral route) being preferred. In cases where the morphogen is supplied by the parenteral route, such as by the intravenous, subcutaoneal, intramuscular, intraorbital, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, buccal, rectal, vaginal, intranasal routes, or by means of a aerosol, it is preferred that the morphogen is part of an aqueous solution. The solution is physiologically acceptable so that in addition to being a vehicle for the delivery of the desired morphogen to the patient, the solution does not adversely affect the patient's electrolyte or volume balance. The aqueous medium for morphogen can therefore be constituted of normal physiological saline solution (9.85% NaCl, 0.15M), pH7-7.4. The aqueous solution containing the morphogen can be made, for example, by dissolving the protein in acetonitrile containing 50% ethanol and 0.1% trifluoroacetic acid (ATF) or 0.1% HCl, or equivalent solvents. Then, for example, one volume of the solution is added to ten volumes of phosphate buffered saline (PBS) which may include 0.1-0.2% human serum albumin (SAH). The resulting solution is extensively vortexed. If desired, the solubility of a given morphogen can be increased by linking it to a suitable molecule. For example, the association of a mature dimer with the pro domain of a morphogen keeps the morphogen soluble in a physiological buffer. In fact, endogenous protein is believed to be transported in this way. Another molecule capable of increasing solubility and which is particularly useful for oral administration is caseoin. For example, the addition of 0.2% caseoin increases the solubility of the active mature form of OP-1 by 80%. Other components found in milk and other whey proteins can also be helpful.

Useful solutions for parenteral administration can be prepared by any of the methods well known in this pharmaceutical field, described for example in Remington's Pharmaceutical Sciences (Gennaro, A., ed), Mack Pub. 1990. Formulations can include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. Formulations for direct administration, in particular, can include glycerol and other high viscosity compositions to help maintain the morphogen at the desired locus (site). Biologically compatible, preferably resorbable, polymers, including, for example, hyaluroonic acid, collagen, tricalcium phosphate, polybutyrate, polymers of lactide and glycolide, and copolymers of lactide / glycolide, may be useful excipients to control release of the in vivo morphogen. . Other parenteral delivery systems that are potentially useful for these morphogens include ethylene vinyl acetate copolymer particles, osmootic pumps, infusion systems that are implanted, and liposomes. Formulations for administration by inhalation contain as excipients, for example, lactose, or they can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl-ether, glycocholate or deoxycholate, or solutions in oil to be administered in the form of nasal drops. or as a gel to be applied intranasally. Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysilicylate for rectal administration, or chitric acid for vaginal administration.

Suppositories for rectal administration can also be prepared by mixing the morphogen or morphogen-stimulating agent with a non-irritating excipient such as cocoa butter or other compositions that are solid at room temperature and liquid at body temperature.

Formulations for topical administration to the skin surface can be prepared by dispersing the morphogen or morphogen-stimulating agent with an acceptable dermatological vehicle such as a lotion, cream, ointment, or soap. Of particular utility are vehicles capable of forming a film or thin layer on the skin to localize the application and inhibit its removal. For topical administration to internal tissue surfaces, the morphogen can be dispersed in a liquid tissue adhesive medium or other substances known to increase adsorption to a tissue surface. For example, hydroxypropyl cellulose or fibrinogen / thrombin solutions can be used for this to advantage. Alternatively, solutions that form a layer on fabrics, such as formulations containing pectin, can be used.

ES 2 149 776 T3

Alternatively, the morphogens described herein can be administered orally. Oral administration of proteones as drugs is not generally practiced, since most proteones are easily broken down by digestive enzymes and acids in mammalian digestive systems before they can be absorbed into the blood. However, the here described morphogens are topically stable in acids and resistant to degradation by proteases (see for example, US Pat.° 4,968,590). Furthermore, at least one morphogen, OP-1, has been identified in extracts of mammary glandules, colostrum, and 57-dollar milk. Furthermore, OP-1 purified from an extract of the mammary glandular is morphoenically active. Specifically, this proteon induces endochondrial bone formation in mammals when implanted subcutaoneally in association with a suitable matrix material and can be measured using a normal in vivo bone assay, such as that disclosed in US Patent No.° 4,968,590. In addition, the morphogen is also detected in the blood. Finally, the soluble form of the morphogen, eg the mature morphogen associated with the pro domain is morphoenically active. These results indicate that oral and parenteral administration are viable methods for administering morphogen to an individual. Furthermore, while the mature forms of some of the morphogens described here are poorly soluble, the form of the morphogen found in milk (and in extracts of the mammary gland and colostrum) is rapidly soluble, possibly due to the association of the mature form. , morphoenically active with part or all of the pro domain of the intact sequence, and / or by association with one or more components of milk. Consequently, the compounds provided herein can be associated with molecules capable of increasing their solubility in vitro and in vivo.

In cases where the morphogen or morphogen stimulating agent constitutes part of a solution for preserving a tissue or organ, any commercially available preservation solution may be used to advantage. For example, useful solutions that are known in the art include Collins's solution, Wisconsin solution, Belzer's solution, Eurocollins solution, and lactated Ringer's solution. Generally, an organ conservation solution usually possesses one or more of the following properties: (a) an osmotic pressure substantially equal to that of the interior of a mammalian cell (solutions are topically hyperosmolar, and have K + and / or Mg ++ ions present in sufficient quantities to produce an osmotic pressure slightly higher than that within a mammalian cell); (b) the solution is topically capable of maintaining substantially normal levels of ATP in cells; and (c) the solution usually allows optimal maintenance of glucose metabolism in cells. Organ preservation solutions may also contain anticoagulants, energy sources such as glucose, fructose and other sugars, metabolites, heavy metal chelating agents, glycerol and other high viscosity materials to increase survival at low temperatures, radical inhibiting agents. free of oxygen, and a pH indicator. A detailed description of preservation solutions and useful components can be found, for example, in US Patent No.° 5.002.965.

The compounds provided herein can also be associated with molecules capable of targeting the morphogen or morphogen-stimulating agent to a desired tissue. For example, an antibody, antibody fragment, or other binding proteon that specifically interacts with a molecule on the surface of the desired tissue co-cell can be used. Useful guide molecules can be designed, for example, using single chain binding site technology, disclosed for example in US Pat.° 5.091.513.

As described above, the morphogens provided herein share significant homology in the sequence of the active domains at the C-terminus. In contrast, the sequences topically diverge significantly in the sequences that define the pro domain. Therefore, the pro domain is believed to be specific to each morphogen. As described above, it is also known that the various morphogens identified to date are differentially expressed in different tissues. Therefore, without being limited to a specific theory, it is likely that under the natural conditions of the body, selected morphogens act topically in a given tissue. Therefore, part or all of the pro domains that have been identified and associated with the active form of the morphogen in solution may act as guide molecules for the morphogens described herein. For example, pro domains can interact specifically with one or more molecules in the specific tissue to direct the morphogen associated with that pro domain to that tissue. Therefore, another guide molecule useful for guiding the morphogen to a tissue of interest is part or all of the pro-domain of a morphogen. For example, part or all of the GDF-1 pro domain can be used to guide a morphogen into nervous tissue. Alternatively, part or all of the OP-1 or CBMP2 pro domain can be used to guide a morphogen to bone tissue, both of which are proteones that are naturally associated with bone tissue.

The morphogens described here are useful in providing neuroprotective effects that alleviate damage to neural pathways associated with the body's immune / inflammatory response to injury.

ES 2 149 776 T3 initial nerve tissues. As used here, "a neural pathway" refers to a circuit of nerves for the passage of electrical signals from a source to a specific site in a cell, and includes both the central nervous system (CNS) and the peripheral nervous system ( PNS). The pathway includes the neurons through which the electrical impulse is transported, encompassing groups of interconnected neurons, the nerve fibers formed by branches of neural axons, and the glial cells that surround and stain associated with neurons. An inflammatory response to nerve tissue injury may occur following trauma to nerve tissue, caused for example by autoimmune dysfunction (including autoantibodies), neoplastic injury, infection, chemical or mechaine trauma, or other disease. An example of an inflammatory disease related to the nerves is multiple sclerosis. Damage to the neural pathway may also occur as a result of a reduction or interruption, eg, occlusion, of the blood supply to the nerves, as occurs in an emboil effusion (eg, hypoxia-induced ischemia or injury) , or by other trauma to the neighboring nerve or material. Furthermore, some of the damage associated with a number of primary brain tumors also appears to be immune in nature. The application of the morphiogen directly to the cells to be treated, or the delivery of the morphogen systemically to the mammal, for example, intravenously or indirectly by oral administration, can be used to alleviate and / or inhibit the immune-type response to a neural injury. Alternatively, the administration of an agent capable of stimulating the expression and / or secretion of the morphoigen in vivo can also be used, preferably at the site of injury. In cases where a lesion is to be induced, such as during surgery or other aggressive clinical treatments, the morphiogen or agent may be delivered prior to inducing the lesion to provide a neuroprotective effect to the nervous tissue at risk.

In cases where it is proposed to use morphoigen as a therapeutic agent to alleviate tissue damage associated with an immune / inflammatory condition of the CNS, an additional problem should be considered: overcoming the so-called “blood-brain barrier”, the capillary wall structure of the brain that acts as an effective screen for all but some selected categories of molecules present in the blood preventing them from passing to the brain. The blood-brain barrier can be effectively bypassed by direct infusion of the morphogen or morphiogen-stimulating agent into the brain. Alternatively, the morphiogen or morphiogen-stimulating agent can be modified to enhance its transport across the blood-brain barrier. For example, certain truncated forms of the morphogen or of a morphiogen-stimulating agent may be more successful. Alternatively, the morphiogen or morphiogen-stimulating agent can be modified to make it lipophilic, or it can be conjugated to another molecule that is naturally transported across the barrier, using normal methods known to those skilled in the art, such as those described in Pardridge, Endocrine Reviews 7: 314-330 (1985) and US Patent No.° 4.801.575.

Finally, the morphogens or stimulating agents of the morphiogens provided herein can be administered alone or in combination with other molecules known to be beneficial in the compositions and treatment methods described herein, including but not limited to anticoagulants, agents that inhibit free radicals of oxygen, salicylic acid, vitamin D, and other anti-inflammatory agents. Treatments for psoriasis may also include treatment with ultraviolet light, zinc ioxide, and retinoids.

The compounds provided herein can be formulated into pharmaceutical preparations by mixing them with pharmaceutically acceptable and non-thioxic excipients and carriers. As indicated above, such compositions can be prepared for parenteral administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; or intranasal, particularly in the form of powders, nasal drops or aerosols.

The compositions can be formulated for parenteral or oral administration to humans and other mammals in therapeutically effective amounts, e.g., amounts that provide adequate concentrations for a time sufficient to alleviate the tissue-destroying effects associated with anti-inflammatory responses, including fabric protection in anticipation of fabric damage.

As would be appreciated by those familiar with this field, the concentration of the compounds described in a therapeutic composition will vary depending on a number of factors, including the dose of the drug to be administered, the chemical characteristics (e.g. , hydrophobicity) of the compounds used, and the route of administration. The preferred dose of the drug to be administered is likely to depend on variables such as the type and degree of progression of tissue damage, the general state of health of the particular patient, the relative biological efficacy of the selected compound, the formulation of the excipients for the compound, and their route of administration. In general terms, the compounds of this invention can be supplied in an aqueous solution in buffer.

ES 2 149 776 T3 containing about 0.001% to 10% weight / volume of the compound for parenteral administration. Topical dose ranges are from about 10 ng / kg to about 1 g / kg of body weight per day; a preferred dose range is from about 0.1 µg / kg to 100 mg / kg of body weight per day. Optimally, the dose of morphogen delivered is between 0.1 and 100 µg of protein per kilogram of patient weight. No obvious morphogen-induced pathological lesions have been observed when mature morphogen (eg, OP-1, 20 μg) is administered daily for 21 days to rats with normal growth. Furthermore, 10 μg of the morphogen in systemic injections (eg, OP-1) injected daily for 10 days into normal newborn mice does not produce any gross abnormalities.

In administering morphogens systemically using the methods of the present invention, it is preferred to use a large volume loading dose at the beginning of treatment. Treatment is then continued with a maintenance dose. Subsequent administration can be determined by monitoring blood levels of morphogen at intervals.

When a tissue injury is deliberately induced, as occurs for example in a surgical procedure, it is preferred to deliver the morphogen just before or at the same time as the induction of trauma. Preferably, the morphogen is administered prophylactically in surgical settings.

Alternatively, an effective amount of an agent capable of stimulating endogenous levels of a morphogen can be administered by any of the routes described above. For example, an agent capable of stimulating the production of the morphogen and / or its secretion from cells in affected tissues and / or transplanted tissues can be delivered to a mammal, eg, by direct administration of the agent to the tissue to be treated. . A method for identifying and analyzing agents capable of modulating endogenous morphogen levels in a given tissue is described generally in Example 15 here, and in detail in pending document USSN 752,859, filed August 30, 1991. In short, compounds that are candidates can be identified and analyzed by incubating the compound in vitro with a tissue or cells under test, for a time sufficient to allow the compound to effect production, that is, the expression and / or secretion of the compound. morphogen produced by the cells of that tissue.

For the purposes of the present invention, the morphogens described above, effective in alleviating tissue damage associated with ischemic and reperfusion injury (or the agents that stimulate them, collectively referred to herein as "therapeutic agents") are administered before or during restoration of oxygen (eg, restoration of blood supply, reperfusion). In cases where treatment is for an existing injury, the therapeutic agent is preferably administered as an intravenous infusion given acutely after a hypoxic or ischemic condition occurs. For example, the therapeutic agent can be administered by intravenous infusion immediately after a cerebral infarction, a myocardial infarction, asphyxia, or a cardiorespiratory arrest. In cases where the ischemic or hypoxic injury is deliberately and / or unavoidably induced as part of, for example, a surgical procedure, where the blood supply to an organ or system of organs is deliberately and / or temporarily interrupted, e.g. , carootid enterectomy, aortocoronary bypass, grafts, organ transplants, fibrinolytic therapy, etc., The therapeutic agent is preferably delivered just before or at the same time as the reduction of oxygen in the tissue occurs. Preferably, the morphogen is administered prophylactically in a surgical setting.

Similarly, in cases where hyperoxia-induced lesions have previously occurred, the morphogen is administered once the diagnosis is made. In cases where hyperoxic injury can be induced, for example, during treatment of premature newborns, or patients suffering from pulmonary diseases such as emphysema, the therapeutic agent is preferably administered prior to the administration of oxygen, e.g., prophylactically.

III. Examples

Example 1

Identification of tissues expressing morphogens

Determination of the distribution of morphogens in tissues can be used to identify different morphogens expressed in a particular tissue, as well as to identify new related morphogens. Tissue distribution can also be used to identify useful morphogen-producing tissues that can be used in the selection and identification of candidate agents that

ES 2 149 776 T3 stimulate morpheogens. Morpheogens (or their mRNA transcripts) are easily identified in different tissues using normal methodologies and minimal modifications in tissues where expression may be low. For example, the distribution of a protein can be determined using normal Western blot analysis or immunofluorescence techniques, and antibodies specific to the morphogen (s) of interest. Similarly, the transcript distribution of morphogen can be determined using normal Northern hybridization protocols and transcript-specific probes.

Any probe capable of specifically hybridizing to a transcript and of distinguishing the transcript of interest from other related transcripts can be used. Because the morphogens described here share high sequence homology in their active domains at the C-terminus, the best way to determine the tissue distribution of a morphogen-specific transcript may be to use a region-specific probe. pro of the immature protein and / or the N-terminal region of the mature protein. Another useful sequence is the 3 'end of the noncoding region that lies on both sides and immediately follows the terminating elbow. These parts of the sequence vary substantially among the morphogens of this invention, and are therefore specific for each protein. For example, a particularly useful Vgr-1 specific probe sequence is the PvuII-SacI fragment, a 265 base pair fragment that encodes both a part of the untranslated pro region as well as the N-terminal region. of the mature sequence (see Lyons et al. (1989) PNAS 86: 4554-4558 for a description of the cDNA sequence). Similarly, particularly useful mOP-1-specific probe sequences are the BstXI-BglI fragment, a 0.68 Kb sequence that covers approximately two-thirds of the pro region of mOP-1; a StuI-StuI fragment, a 0.2 Kb sequence immediately preceding the cysteine-7 domain in the opposite direction of expression; and the Ear1-Pst1 fragment, a 0.3 Kb fragment that contains a part of the 3 'untranslated sequence (See Sequence With NID N° 18, where the region pro is essentially defined by residues 30-291). Similar approaches can be used, for example, with hOP-1 (sequence With NID N° 16) or human or ratane OP-2 (Sequence NID N° 20 and 22).

Using these specific sequences for morphogens, which can be synthetically constructed or derived from cloned sequences, transcripts of morphogens can be identified in mammalian tissues using standard methodologies well known to those skilled in the art. In short, total RNA is prepared from various adult murine tissues (eg, liver, kidney, testes, heart, brain, thymus, and stomach) using standard methodology such as the method of Chomczyaski et al. (1987) Anal Biochem 162: 156-159) and described below. Poly A + RNA is prepared using cellulose-oligo (dT) chromatography (eg, type 7 from Pharmacia LKB Biotechnology, Inc.). Poly A + RNA (generally 15 μg) from each tissue is fractionated on a 1% agarose / formaldehyde gel and transferred to a Nytran membrane (Schleicher & Schuell). After transferring, the membrane is baked at 80°C and RNA is tightly bound to the membrane using ultraviolet light (generally 30 seconds at 1 mW / cm2). Before hybridization, the appropriate probe is heat denatured. Hybridization is carried out in a lucite cylinder that rotates in an apparatus with a cylindrical bottle at a speed of approximately 1 rev / min for approximately 15 hours at 37°C using a hybridization mixture of 40% formamide, 5 x Denhardts, 5 x SSPE and 0.1% SDS. Following hybridization, the non-specific beads are removed from the membranes by washing them in 0.1% SSPE, 0.1% SDS at 50°C.

Examples demonstrating the tissue distribution of various morpheogens, including Vgr-1, OP-1, BMP2, BMP3, BMP4, BMP5, GDF-1, and OP-2, in developing and adult tissues are disclosed in USSN. 752,764 pending and in Ozkaynak et al. (1991) Biochem. Biophys. Res. Commn. 179: 116-123, and in Oskaynak et al. (1992) (JBC, in press). Using the general probe methodology described here, Northern blot hybridizations using probes specific to these morphogens to probe brain, spleen, lung, heart, liver, and kidney tissues have indicated that kidney-related tissues appear to be the most common. main source of expression for OP-1, being the tissues of the brain, heart, and lung secondary sources. Using this specific probe methodology, mRNA of OP-1 was also identified in salivary glands, specifically in rat parapetid glands. Lung tissue appears to be the main source of expression for Vgr-1, BMP5, BMP4, and BMP3. Lower levels of Vgr-1 have also been observed in kidney and heart tissues, while the liver appears to be a source of secondary expression for BMP5 and the spleen appears to be a source of secondary expression for BMP4. GDF-1 appears to be expressed primarily in brain tissues. So far, OP-2 appears to be expressed mainly in young embryonic tissues. Specifically, Northern blots of murine embryos and 6-day-old animals from birth demonstrate abundant OP-2 expression in 8-day embryos. Expression is significantly reduced in 17-day embryos and is not detected in animals after birth.

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Example 2

Active morphogens in body fluids

The expression of OP-1 has been identified in saliva (specifically the parootid gland of the rat, see Example 1), human blood serum, and in various forms of milk, including extracts of the mammary gland, colostrum, and bovine milk of 57 days. Furthermore, and as described in the text of USSN 923,780, the proteon extracted from body fluids is morphoenically active. The discovery that morphogen is naturally present in milk and saliva, together with the known observation that the active, mature form of OP-1 is stable in acidic medium and resistant to proteases, indicate that oral administration is an alternative route. Useful for the therapeutic administration of morphogen to a mammal. Oral administration is the topically preferred mode of delivery in prolonged or prophylactic therapies. Furthermore, the identification of morphogens in all forms of milk, including colostrum, suggests that proteone may play a significant role in tissue development, including skeletal development, in the young.

2.1 Detection of morphogens in milk

OP-1 was partially purified from an extract of rat mammary glands and bovine colostrum and 57-day milk by passing these fluids through a series of chromatographic columns, (eg, cation exchange, affinity and reverse phase). At each step the eluent was collected into fractions that were assayed for the presence of OP-1 with normal immunoblotting, the immunoreactive fractions were then pooled and further purified. The final partially purified product was screened for the presence of OP-1 with the Western blot technique, using OP-1 specific antisera, and tested for in vivo and in vitro activity.

The purified OP-1s from the different milk sources were characterized by Western blotting techniques using antibodies raised against OP-1 and BMP2. Antibodies were prepared following standard immunological protocols widely known in the art, and as described in general terms in Example 15, below, using OP-1 and BMP2 produced by full-length E. coli as immunogens. In all cases, purified OP-1 reacted only with the anti-OP-1 antibody and not with the anti-BMP2 antibody.

The morphogenic activity of OP-1 purified from mammary gland extract was evaluated in vivo essentially following the rat model assay described in US Patent No. 4,968,590. Briefly, a sample was prepared from each OP-1 immunoreactive fraction of the final OP-1 product derived from mammary glandular extract by lyophilizing a portion (33%) of the fraction and resuspending the protein at 220 μ! 50% acetonitrile / 0.1% TFA. After vortexing, 25 mg of collagen matrix was added. Samples were lyophilized overnight, and implanted in Long Evans rats (Charles River Laboratories, Wilmington, MA, 29 to 35 days old). Each fraction was implanted in duplicate. For more details on the implantation procedure, see, for example, US Patent No. 4,968,590. After 12 days, the implants were removed and evaluated for new bone formation by histological observation, as described in US Patent No. 4,968,590. In all cases, the immunoreactive fractions were osteogeonically active.

2.2 Detection of morphogen in serum

It is possible to detect a morphogen in serum using antibodies specific for the morphogen. The assay can be performed using any standard immunological assay, such as Western blotting (immunoblotting) and the like. Preferably, the determination is carried out using an affinity column to which an antibody specific for the morphogen has been bound and through which the sample serum is then poured, to selectively extract the morphogen of interest. The morphogen is then eluted. An appropriate elution buffer can be emporically determined by determining the proper binding and elution conditions first with a control (eg. purified morphogen, produced recombinantly). The fractions are then assayed for the presence of morphogen with normal immunoblot technique, and the results are confirmed by N-terminal sequencing. Preferably, the affinity column is prepared using monoclonal antibodies. Morphogen concentrations in serum or other fluid samples can then be determined using standard proteon quantification techniques, including spectrophotometric absorbance, or by conjugated antibody quantification.

ES 2 149 776 T3

The following is a sample protocol to identify OP-1 in serum. Following this general methodology, other morphogens can be detected in body fluids, including serum. Identification of the morphogen in serum further indicates that systemic administration is an appropriate way to deliver therapeutic concentrations of a morphogen to a person, and that morphogens may well behave systemically as endocrine-like factors. Finally, using this protocol, fluctuations in endogenous morphogen levels can be detected, and these altered levels can be used as an indicator of hostic dysfunction. Alternatively, fluctuations in morphogen levels can be determined by monitoring morphogen transcription levels, either by the normal method of northern blot analysis, as described in Example 1, or by in situ hybridization, using a labeled probe capable of to specifically hybridize to the morphogen mRNA, and the normal protocols of hybridization to RNA clearly described in the article and described in general terms in Example 1.

OP-1 was detected in human serum using the following assay. A monoclonal antibody produced against recombinant mammalian OP-1, using standard immunological techniques, well described clearly in the literature and described in general terms in Example 15, was immobilized by passing the antibody over an agarose-activated gel (eg. Affi-Gel7, from Bio-Rad Laboratories, Richmond, CA, prepared according to manufacturer's instructions) and used to purify OP-1 from serum. Human serum was then passed over the column and eluted with 3M K-thiocyanate. Then, the K-thiocyanate fractions were dialyzed in 6M urea, 20mM PO4, pH 7.0, applied to a C8 column HPLC, and were eluted with a gradient of 25-50% acetonitrile / 0.1% TFA over 20 minutes. Mature and recombinantly produced OP-1 homopolymers elute between 20 and 22 minutes. The fractions were then collected, which were analyzed for the presence of OP-1 following normal immunoblot technique using an antibody specific for OP-1 as in Example 2.A.

The levels of administered or endogenous morphogen can be monitored in the therapies described herein by comparing the amount of morphogen present in a sample of body fluids with a predetermined reference value, for example, to evaluate the efficiency of a therapeutic protocol, and the like. Furthermore, fluctuations in the levels of antibodies against endogenous morphogen can be detected with this method, most likely in serum, using an antibody or other binding proteon capable of specifically interacting with the antibody against endogenous morphogen. For example, detected fluctuations in levels of antibody to endogenous morphogen can be used as indicators of a change in tissue status. For example, when damaged tissue regenerates and the function of the tissue or organ returns to "normal" and, if no additional hostic damage is detected, lower doses of morphogen may be necessary and a higher level of antibody can be measured. to circulating morphogen.

Example 3

Effect of morphogen after the beginning of the ischemic process

The cardioprotective effect of morphogens after ischemic and reperfusion injury in a mammal can easily be evaluated with a rat model. In this example, the morphogen (eg OP-1) is administered immediately before the onset of the ischemic process in rats with experimentally induced myocardial infarctions, essentially following the method of Lefer, et al. (1990) Science 249: 61-64 and (1992) J. Mol. Cell. Cardiol. 24: 385-393. Briefly, the loss of myocardial tissue function after ischemia and reperfusion was evaluated by measuring the loss of myocardial creatine kinase (CK) activity and the loss of endothelium-dependent vasorelaxation function (see Example 4, móas under).

In a first group of ether anesthetized rats, the left coronary artery proximal to the first main branch was occluded with a silk ligation in order to induce a myocardial infarction (MI). The ligation was removed 10 minutes after occlusion to allow coronary reperfusion. Hereafter, this first group will be referred to as the "myocardial infarction" (MI) group. A second group of rats received the same procedure with the exception that the coronary artery was not occluded, and consequently, a myocardial infarction did not occur. Hereafter, this second group will be referred to as the "false myocardial infarction" (FALSE MI) group.

The first group of rats, the IM group of rats, was again subdivided into three sub-groups. 2 µg of morphogen (OP-1) were injected intravenously into the first subgroup of rats IM 10 minutes after ligation, immediately before reperfusion; in the second group of IM rats,

ES 2 149 776 T3 injected 20 µg of OP-1 intravenously 10 minutes after ligation, immediately before reperfusion; and in the third subgroup of IM rats (control) only one vehicle was injected, eg. 0.9% NaCl, same as for rats treated with OP-1.

After twenty-four hours, the hearts of all rats were removed and the creatine kinase (CK) levels of the left ventricular (the infarcted region) and the interventricular septum (the non-ischeemic control region) were determined following normal methods. When comparing the differences in CK activity in both regions, the loss of the amount of CK activity in the infarcted region was used as an index of cardiac cell injury in the infarcted region.

As shown in Figure 1, the data indicate that morpheogens (eg, OP-1) may offer a significant cardioprotective effect when administered to ischemic tissue. In the figure, CK loss is graphed as the difference in specific CK activity between the interventricular septum and the left ventricular.

The loss of CK activity by the subgroup of IM rats that received 2 μg of OP-1 immediately before reperfusion demonstrated some level of protection when compared to control IM rats that received vehicle injections alone, when levels of both sub-groups are measured against and compared with the levels obtained for the control of IM FALSE. Significant cardioprotection was observed in the subgroup that received 20 μg of OP-1 immediately before reperfusion when compared to the control IM rats that received vehicle injections alone, when the levels of both subgroups were measured against and compared with the levels obtained for the control of FALSE IM.

These data indicate that OP-1 offers important cardiac protection when administered after ischemia and before reperfusion.

A variation on this example can also be performed by administering morpheogen to the animal prior to induction of ischemia. Experiments can be performed in normal or immune-compromised rats to assess the cardioprotective effects of morphogen administered prior to ischemia.

Example 4

Vasodilation of cardiac tissue with myocardial infarction treated with morphogen

Certain vasodilators such as acetylcholine (Ach) and adenosine diphosphate (ADP, an immune mediator) exert vasodilatation activity only in the presence of intact endothelium, which is stimulated to release a substance called endothelium-derived relaxing factor (EDRF). If the endothelium is injured so that EDRF is not released, vasodilation does not occur in response to these endothelium-dependent agents. In contrast, several other vasodilators, including nitroglycerin (NTG) and nitroprusside, are independent dilators of the endothelium, since they directly dilate blood vessels.

The present example demonstrates the ability of OP-1 to prevent cardioendothelium-dependent loss of relaxation activity (EDR) in the coronary microvasculature after reperfusion of the ischeemic myocardium, and its ability to reduce myocardial injury 24 hours after treatment of the morphogenic. In synthesis, 2 to 24 hours after morpheogen treatment, injury is induced by ischemia and reperfusion in isolated rat hearts, the reperfused hearts are vasodilated with ACh or NTG. If morphogenic treatment has not been performed, the injured tissue should inhibit ACh-induced vasodilation, but not NTG-induced vasodilation. Morpheogen treatment is anticipated to improve ACh-induced vasodilation in reperfused hearts.

Consequently, 48 adult Sprague-Dawley rats (250-330 g) were divided into eight groups of 6 rats each. Twelve rats were subjected to false myocardial infarctions (FALSE MI), as described in Example 3. The hearts of the remaining 36 rats were isolated as follows: a set of twelve rats was injected intravenously with 20 µg of OP-1 24 hours before isolation of the heart; Another group of rats was injected intravenously with 20 µg of OP-1 2 hours before isolation of the heart; the final group was injected with the vehicle only (eg NaCl 0, 9%). The rats were then anesthetized with sodium pentobarbital (35 mg / kg, intraperitoneal); their hearts were isolated and perfused by the Langerdoff method at a constant flow (15 ml / min) with an oxygenated Krebs-Henseleit solution (Aoki et al. (1988) J. Pharmacol. 95-35).

ES 2 149 776 T3

Each group of rats was then divided into two subgroups of six rats each. Twenty minutes before reperfusion, the coronary vasodilator response was measured by inducing constriction with 0.05 μmol U-44619 (9,11-methanoepoxy-postaglandin H2) followed by a vasodilating agent 3 minutes later: subgroup one - 15 nmol ACh; subgroup two - 15 nmol NTG and the increase in the level of coronary perfusion pressure (CPP) was measured as an indication of vasodilation. When CPP levels returned to normal values, the hearts were subjected to ischemia reducing coronary infusion to 15% of control flow for 30 minutes, then reestablishing normal flow, that is, reperfusion, for an additional 20 minutes.

The vasodilator response was then measured again by constriction and administration of a vasodilator agent as previously described.

The results of these experiments are shown in Fig. 2. Before the ischemic event, both ACh and NTG produced normal vasorelaxant results in all events. Hearts that received OP-1 24 hours before ischemia demonstrated an approximately 70% response to ACh, whereas hearts that received OP-1 two hours before ischemia demonstrated a 55% response to ACh. The group that received the vehicle alone demonstrated a 40% response to ACh. Finally, the control group that did not undergo ischemia demonstrated an ACh response of approximately 95%. This demonstrates that endothelium-dependent vasodilators exert a reduced vasodilator response after ischemia and reperfusion in the rat heart. Even more, OP-1 conserved considerably the dependent dilatation of the endothelium when administered 24 hours before the induction of myocardial ischemia. No defect in vasodilation occurred in response to direct vasodilator (NTG); NTG-induced vasodilator activities were 95% of baseline in ischemic-exposed hearts and 100% of baseline in nonischemic hearts. Example 5

Effect of morphogen on neutrophil adherence

The role of neutroophil adherence in endothelial dysfunction and the cardioprotective effects of morphogens in modulating this activity can be evaluated using a normal polymorphonuclear (PMN) adherence assay as described by Lefer et al. (1992) J. Mol. Cell. Cardiol. 24: 385-393. Briefly, segments of the superior mesenteric artery were isolated from rats that had been treated with morphogen (OP-1, 20 µg) or 0.9% NaCl, 24 hours prior to artery isolation. The segments were cleaned, cut into transverse rings 1 to 2 mm long, and these were subsequently opened and incubated in a KH solution at 37° C, pH 7.4. Neutrophils were prepared and fluorescently labeled using standard procedures (eg, leukocytes were isolated from rats essentially following the procedure of Pertroft et al. (1968) Exp. Cell. Res. 50: 355-368, washed in phosphate buffered saline (PBS), were purified by gradient centrifugation, and were labeled by the method of Yuan et al. (1990) Microvasc Res 40: 218-229.

Labeled neutrophils were then added to the open ring baths and activated with 100 nM leukotriene B4 (LTB4). The rings were incubated for 20 minutes and the number of neutrophils adhered to the endothelial surface was determined visually with a fluorescent microscope.

As shown in Figure 3, unstimulated PMNs (ie, PMNs alone) added to the baths did not adhere significantly to the vascular endothelium. In the rings obtained from rats injected with 0.9% NaCl, the activation of neutrophils with LTB4 (100 nM) significantly increased the number of PMN that adhered to the endothelium (P <0.001). OP-1 (20 μg administered 24 hours before) considerably inhibited the adherence of PMN activated with LTB4 (P <0.01 of the control). Example 6

In-vivo models for protection against ischemia and reperfusion in lung, nerve and kidney tissues

Other tissues severely affected by injury due to ischemia and reperfusion include neural tissue, kidney tissue, and lung tissue. The effect of morphogens on the palliation of ischemia and reperfusion injury in these tissues can be evaluated using methodologies and models known to those with experience in this field and which are described below. Likewise, it is provided

ES 2 149 776 T3 also a methodology to evaluate the protective effects of a morphoigen tissue on injured tissues of the lung after a lesion produced by hyperoxia.

For example, the rabbit emboil attack model offers a useful method to evaluate the effect of morphiogens on tissue lesions after cerebral ischemia and reperfusion. The protocol presented below is essentially that of Phillips et al. (1989) Annals of Neurology 25: 281-285. Simply put, New England white rabbits (2 to 3 kg) are anesthetized and placed on a respirator. The intracranial circulation is then selectively catheterized using the Seldinger technique. A baseline cerebral angiography is then performed, using a digital subtraction unit. The distal internal cariotid artery or its branches are then selectively embolized with 0.085 ml of 18-hour-old autologous thrombi. Arterial occlusion is documented with repeated angiograms immediately after embolization. After a period of time sufficient to induce cerebral infarcts (15 minutes or 90 minutes), reperfusion is induced by administering a bolus of a reperfusion agent such as the TPA analog known as Fb-FB-CF (e.g., 0, 8 mg / kg for 2 minutes).

The effect of morphogen on cerebral infarcts can be evaluated by administering various concentrations of morphogens, eg. OP-1, at different times before or after embolization and / or reperfusion. The rabbits are sacrificed 3 to 14 days after embolization and their brains are prepared for neuropathological examination, fixing them by immersion in 10% neutral buffered formalin for at least 2 weeks. The brains are then cut in a coronal plane at 2-3 mm intervals, numbered, and shipped for normal histological processing in paraffin, and the degree of neural tissue necrosis is visually determined.

The renal protective effects of morphiogens on ischemic and reperfusion injury can be determined using the mouse model presented by Oueliette et al. (1990) J. Clin. Invest. 85: 766-771. In summary, renal ischemia is surgically induced in exogenously reared male Swiss mice aged 35 to 45 days, performing a normal right nephroctomy and occluding the artery reaching the left kidney for 10 to 30 minutes with a microaneurysm forceps. The morphogen can then be administered parenterally, at various times before or after occlusion and / or reperfusion. The effects of morphiogen can then be evaluated by biological estimation and histological estimation using standard techniques well known in this field.

The tissue protective effects manifested by morphoigen on tissues exposed to high lethal oxygen concentrations can be determined by the following procedure. Adult rats (275 to 300 g) first receive the morphiogen (eg HOP1) or only the vehicle, and are then exposed to oxygen at 96 to 98% as described in Rinaldo et al. (1983) Am. Rev. Respir. Dis. 130: 1065 to induce hyperoxia. The animals are kept in plastic cages (38 cm x 48 cm x 21 cm). A cage containing 4 to 5 animals is placed in a 75 liter watertight Plexiglas chamber. An atmosphere of 96 to 98% oxygen is maintained by the delivery of O2 gas (liquid O2). Adjust the gas flow in the chamber to maintain at least 10 air exchanges / hour, a temperature of 22 ± 1° C, minimum levels of condensation in the cage, and a carbon dioxide concentration of <0.5% measured with a mass spectrophotometric medical gas analyzer.

After 72 hours, all survivors are observed in room air for 1.5 hours and for longer periods of time to assess the degree of respiratory distress and cyanosis induced by the initial insult and subsequent cell-mediated immune damage. At the end of the test, the number of survivors is noted and the treated group is compared with the untreated control using the chi-squared ratio test. Several of the surviving animals from each group are randomly selected for histological processing of lung tissue.

Lung tissue for histology processing is fixed with an infusion of buffered 10% formalin through a tracheal tube at a constant pressure of 20 cm H2O. After fixation for 24 to 48 hours, sections of each lobe are cut and subsequently stained with hematoxylin and eosin. Coded slides are then examined, preferably double-blind, for signs of pathological changes, such as edema, interstitial cellularity, and inflammatory response.

Example 7

Inhibition of the cellular and humoral inflammatory response by morphoUgens

The morphiogens described herein inhibit multinucleation of mononuclear phagocytic cells under conditions in which these cells would normally be activated, eg, in response to tissue injury.

ES 2 149 776 T3 or the presence of a foreign substance. For example, if there is no morphogen, an implanted substrate material (e.g. implanted subcutaneously) composed of, for example, mineralized bone, a ceramic such as titanium oxide or any other substrate that causes multinucleated giant cell formation, it is quickly surrounded by giant multinucleated cells, eg, activated phagocytes stimulated to respond and destroy the foreign object. However, in the presence of morphogene, the recruited cells remain in their mononuclear precursor form and the maximal material is not altered. Figure 4 illustrates this effect of morphogens, in a graphical representation of the histological results of a subcutaneously implanted titanium oxide substrate. In the figure, "mg" means gigantic mononucleated cells and "ob" means osteoblasts. The substrate represented in Figure 4B was implanted together with the morphogen (OP-1) and newly formed osteoblasts around the substrate are evident. In contrast, the substrate represented in Fig. 4A was implanted without morphogene and the formation of large numbers of multinucleated giant cells around the substrate is evident. Therefore, the effect of morphogen in inhibiting excessive loss of bone mass in a mammal may also include inhibiting the activation of these cells.

Furthermore, the morphogens described herein also suppress stimulated antibody production in response to a foreign antigen in a mammal. Specifically, when bovine bone choleagene matrix was implanted alone into a bone site in a rat, a normal antibody response to choleagene was stimulated in the rat and could be determined by normal anti-bovine choleagene ELISA experiments performed on samples. blood collected at four week intervals after implantation (eg, between 12 to 20 weeks). Serum anti-choleagene antibody titers, measured with ELISA essentially following the procedure described by Nagler-Anderson et al. (1986) PNAS 83: 7443-7446, increased consistently throughout the experiment. However, when the matrix was implanted together with a morpheogen (eg, OP-1, dispersed in the matrix and adsorbed thereto, essentially as described in US Patent No. 4,968,590) the production of anti-bovine choleagene antibodies was considerably suppressed. This ability of morpheogen to suppress the humoral response is further evidence of the usefulness of morphegen in alleviating the bone damage associated with autoimmune diseases, including diseases caused by the antibodies themselves, such as rheumatoid arthritis.

Example 8

Protection of the mucosa of the intestinal tract, through morphogens, against ulcerations and inflammations

Oral mucositis is an inflammatory disease of the gastrointestinal tract in which ulcerations of the mucosa of the mouth occur as a result of, for example, radiation therapy or chemotherapy. Although not typically a typically chronic disease, the tissue-destroying effects of oral mucositis are similar to those of inflammatory bowel disease such as IBD (inflammatory bowel disease). The example described below demonstrates the efficacy with which morpheogens protect the oral mucosa from oral mucositis in hamsters, including both inhibiting inflammatory ulcerations and increasing the regeneration of ulcerated tissues. For detailed information on the protocol used, see Sonis, et al., (1990) Oral Surg. Oral Med. Oral Pathol. 69: 437-443. Based on these data, the morphogens described in this document should prove effective in the treatment of chronic inflammatory diseases, including: IBD, arthritis, psoriasis and psoriatic arthritis, multiple sclerosis, and the like.

A set of Syrian Golden Hamsters (6 to 8 weeks old, from Charles River Laboratories, Wilmington, Massachusetts) was divided into 3 test groups: Group 1 was a control group treated with saline only (i.e. placebo); the other two groups were treated with a low dose of morphogens (100 ng), and with a high dose of morphogens (1 µg) (Groups 2 and 3, respectively). The morphogen doses were given in a 30% ethanol solution. Each group contained 12 animals.

Beginning on day 0 and continuing through day 5, Groups 2 and 3 received two daily applications of morphogen. On day 3, induction of mucositis began in all groups. On days 3 and 5, 5-fluorouracil (60 mg / kg) was injected intraperitoneally. On day 7, and using an 18 gauge needle, the mucosa of the right buccal pouch of the animals was superficially irritated. In untreated animals, by day 10 severe ulcerative mucositis had been induced in at least 80% of the animals.

In each administration of the control vehicle (placebo) or of the morphogen, the administration was carried out by first lightly drying the mucosa of the cheek pouch, and then uniformly applying the vehicle or the morphogen on the surface of the mucosa. To keep morpheogen in contact with the

ES 2 149 776 T3 mucosa a coating based on hydroxypropyl cellulose was used. This coating provided at least 4 hours of contact time.

On day 12, two animals from each group were sacrificed for histological studies. The mucosa of the right buccal bursa and the underlying connective tissue were dissected, then fixed in 10% formalin using normal dissection and histological procedures. The specimens were mounted on paraffin and prepared for histological examination. These sections were then stained with hematoxylin and eosin and were examined blindly by three oral pathologists with experience in the study of histological sections from heamsters, being scored, also blind, according to a normal mucositis panel. The degree of atrophy, cellular infiltration, disintegration of connective tissue, degree of ulceration and epithelialization were evaluated.

During 21 days, the mean mucositis score shown by each experimental group was determined daily by means of photographs and visual examination of the pouch of the right cheek of the mouth. Differences between groups were determined using a normal "t" test, eg. Student's "t" test. In addition, the data from the three groups were contrasted by comparing the number of animals showing severe mucositis, using statistical Chi-squares analysis. Likewise, the statistical significance of the differences between the average daily weights was determined.

The experimental results are shown in Fig. 5, which graphically represents the effect of morpheogen (at high doses: squares; at low doses: diamonds) and placebo: circles) on the mean mucositis scores. Both low and high doses of morphogen significantly inhibit lesion formation, proportionally to the dose. In addition, the results of the histological examinations showed significantly lower levels of liver atrophy, cellular debris, and immune effector cells, including lower amounts of activated macrophages and neutroephiles, in the morpheogen-treated animals compared to the untreated control animals.

Example 9

Effect of morphogens on fibrogoenesis and scar tissue formation

The morphogens described herein induce tissue morphogenesis in damaged or lost tissues. The ability of these proteins to regenerate new tissue increases their anti-inflammatory effect. A series of in vitro experiments are described below that demonstrate the ability of morpheogens to induce migration and accumulation of mesenchymal cells. Furthermore, experiments show that morphogens, unlike TGF-β, do not stimulate fibrogenesis or the formation of scar tissue. Specifically, morphogens do not stimulate the production of collagen, hyalureonic acid (HA), or metalloproteinases in primary fibroblasts, all of which are necessary for fibrogenesis or the formation of scar tissue. In contrast, TGF-β, a known inducer of fibrosis, but not of tissue morphogenesis, does stimulate the production of these fibrosis markers.

The chemotaxis and migration of the mesenchymal progenitor cells was measured in modified Boyden chambers, essentially as described by Fava. RA et al, (1991), in J. Exp. Med. 173: 1121-1132, using polycarbonate filters with holes of 2, 3 and 8 microns in order to quantify the migration of progenitor cells of neutrophils, monocytes and fibroblasts. Chemotaxis was measured at a variety of morpheogen concentrations, namely: OP-1 10-20Ma10-12M. For neutrophilic and monocyte progenitor cells, concentrations of 10-18 a10-17M OP-1 induced, in a systematic way, a maximum migration, while, in the case of progenitor cells of fibroblasts, maximum migration was observed at concentrations of 10-14Ma10-13M from OP-1. In all cases, chemotactic activity could be inhibited with anti-OP-1 antibodies. With TGF-β, similar migratory activities were measured and observed.

The effect of morphogene on fibrogenesis was determined by evaluating the production of hyalureonic acid (HA), choleagene, collagenase, and heroic inhibitor of metalloproteinases (ITMP), by fibroblasts.

From child foreskin explants, human fibroblasts were obtained and maintained in monolayer cultures using normal culture procedures. (See, for example, (1976) J. Exp. Med. 144: 1188-1203). To describe it succinctly, fibroblasts were cultured in a maintenance medium composed of Eagle's MEM, and supplemented with non-essential amino acids, asceorbic acid.

ES 2 149 776 T3 (50 μg / m!), NaHCO buffers3 and HEPES (pH 7.2), penicillin (100 U / ml), streptomycin (100 μg / m!), amphotericin B (1 μg / m!), and CFS (fetal calf serum) at 9% inactivated by the hot. Fibroblasts used as target cells with which to measure chemotaxis were kept in glass Petri dishes of 150 mm diameter. The fibroblasts used in the experiments to measure the synthesis of collagen, hyaluronic acid, collagenase and hostic metalloproteinase inhibitor (TIMP) were cultured in tissue culture plastic Petri dishes of 100 mm diameter.

The effects of morphogen on the production of hyaluronic acid, collagen, collagenase and ITMP by fibroblasts were determined by normal tests. (See, for example, Posttethwaite et al. (1989) J. Clin. Invest. 83: 629-636, Posttethwaithe (1988) J. Cell Biol. 106: 311-318, and Clark et al. (1985) Arch. Bio -chem. Biophys. 241: 36-44). In these assays, fibroblasts were transferred to 24-well tissue culture plates with a density of 8 x 104 cells per well. These fibroblasts were cultured confluently, for 72 hours, in a maintenance medium containing 9% CFS, and subsequently cultured in serum-free culture medium for 24 hours. The culture medium was then removed from all wells, added with various concentrations of OP-1 (in soluble or mature form, produced by recombination) or TGF © 1 (R&D Systems, Minneapolis) in 50 μl of PBS, to in order to triple the wells containing the confluent fibroblast monolayers. In the experiments in which the production of collagenase and TIMP was measured, a maintenance medium (450 μl) containing 5% FCS was added to each well, extracting the culture supernatant from each well 48 hours later and stored west at -70°C until the time of the test. In the experiments in which the production of HA was evaluated, a maintenance medium (450 μ ^ containing 2.5% FCS was added to each well, culturing for 48 hours. In the experiments in which the production was measured of collagen by the fibroblasts, a maintenance medium (450 μ ^ free of serum and non-essential amino acids was added to each well, culturing for 72 hours. HA production by fibroblasts was measured by labeling newly synthesized glycosaminoglycans (GAGs) with [3H] -acetate during the last 24 hours of culture and quantifying the radioactivity released after its incubation with hyaluronidase from Streptomyces hyaluronicus (ICN Biochemical, Cleveland, Ohio), which degrades hyaluronic acid in a specific way. Total collagen production by fibroblasts was measured by performing an analysis of collagenase-sensitive proteones, which reflects the incorporation of [3H] -proline during the last 24 hours of culture, in the newly synthesized collagen. The levels of collagenase and TIMP protein in fibroblast culture supernatants were measured by specific ELISAs.

As can be seen in Fig. 6, OP-1 does not stimulate a significant production of collagen or HA, compared to TGF-β. In the figure, panel A shows the effect of OP-1 on collagen production, panel B shows the effect of TGF-β on collagen production, and panels C and D show the effects of OP- 1 (panel C) and TGF-β (panel D) on HA production. The morphogen results were identical whether the soluble or mature form of OP-1 was used. In contrast, the latent form of TGF-β (that is, the form of TGF-β that tends to associate forming pro domains of TGF-β) was not shown to be active.

Example 10

Inhibition, by morphogens, of epithelial cell proliferation

This example demonstrates the ability of morphogens to inhibit the proliferation of epithelial cells in vitro, as determined by the uptake of 3H-thymidine using culture cells from a mink lung epithelium cell line (ATCC N° CCL 64), and normal mammalian co-cell culture procedures. To describe it succinctly, the cells were cultured in confluence, in Eagle's mononymous essential medium (EMEM) supplemented with 10% FBS (fetal calf serum), 200 units / ml of penicillin, and 200 µg / ml of streptomycin; this culture medium, together with the cells, was used to seed a 48-well cell culture plate, at a cell density of 200,000 cells per well. Once confluence was reached in this culture, the medium was replaced with 0.5 ml of EMEM supplemented with 1% FBS and penicillin / streptomycin, incubating it at 37°C for 24 hours. Morphogen assay samples in EMEM supplemented with 5% FBS were then added to the wells, and the cells were incubated for another 18 hours. After this incubation, 1.0 μθί of3H-thymidine in 10 µl to each well, and the cells were incubated at 37 ° C for 4 hours. The medium was then removed and the cells were washed once with ice cold phosphate buffered saline, and the DNA was precipitated by adding 0.5 ml of 10% TCA to each well, and incubating at room temperature for 15 minutes. . Afterwards, the cells were washed three times with ice-cold distilled water, and they were lysed with 0.5 ml of 0.4 M NaOH; the lysate from each well was transferred to a scintillation vial, and the radioactivity was measured by a scintillation counter (Smith-Kline Beckman).

ES 2 149 776 T3

The results are presented in Table III, mine below. The antiproliferative effect of the various morphogens tested was expressed as counts of3H-thymidine (x 1000) integrated in DNA, and these were compared with untreated cells (negative control) and with TGF-β (1 ng), a locally acting factor known to inhibit proliferation of epithelial cells. COP-5 and COP-7 are biosynthetic products that have previously been shown to have osteogenic activity, capable of inducing the complete cascade that leads to endochondrial bone formation in a normal rat bone experiment (see US Pat. USA N° 5,011,691). Morphogens significantly inhibit epithelial cell proliferation. Similar experiments, carried out with the morphiogens COP-16, bOP (purified bone osteogenic protein, a dimeric protein composed of CBMP2 and OP1), and recombinant OP-1, also inhibit cell proliferation. bOP and COP-16 also induce endochondrial bone formation (see US Patents No.° 4,968,590 and N° 5.011.691).

TABLE III

<td></td><td>Thymidine uptake (x 1000)</td>

<td>control</td><td> 50,048, 53,692</td>

Example 11

Treatment with morphogens of a systemic inflammatory disease

The following example provides an adjuvant-induced model of arthritis in rats demonstrating the efficacy of morphiogens in the treatment of arthritis and other systemic inflammatory diseases. Adjuvant-induced arthritis in rats induces a systemic inflammatory disease in which bone and cartilage undergo changes similar to those seen in rheumatoid arthritis, only in a much shorter period of time (see, for example, Pearson (1964) Arth. Rheum. 7:80). For a detailed description of the protocol used, see Walz, et al., (1971) J. Pharmac. Exp. Ther. 178: 223-231.

To describe it succinctly, female Sprague-Dawley rats (in this case, from Charles River Laboratories, Wilmington, Massachusetts) are randomly distributed, forming three groups: control, low-dose morphigen (1-10 μg / kg of body weight body weight and day), and morphigenic at high doses (10-20 μg / kg of body weight and day), called Groups 1, 2 and 3, respectively.

Arthritis is induced by an adjuvant in all three groups, injecting 0.05 ml of a 1.5% Mycobacterium butyricum suspension (dead bacteria) in mineral oil into the subplantar surface of the right fore claw. On day 18 after the injection of this adjuvant, the limb volumes of both hind limbs are determined. In the absence of morphogenic treatment, a systemic arthritic condition has already been induced in adjuvant-injected rats at this time, as indicated by significant swelling of the non-injected hind limbs (<2.3 ml volume , measured by mercury displacement). Subsequently, claw edema determinations and radiograph scores of the noninjected hind limb are made. Beginning on day 1, Group 2 and 3 rats are given a daily dose of morphiogen orally. Limb volumes are determined on days 29 and 50 after adjuvant injection; Likewise, the size of the edema is determined by measuring the difference in volume compared to day 18. On day 50, radiographs of the uninjected hind limb of each rat are taken, and joint injuries are evaluated using an arbitrary scale of 1 to 10 (1 = no injuries; 10 = maximal injuries). Using a normal "t" test, differences between the control group and the morphiogen-treated groups (edema on day 29, edema on day 50, and radiograph scores on day 50) are analyzed. Morphogen-treated rats show uniformly less joint lesions (ie, less edema and radiograph scores), compared to untreated control rats.

ES 2 149 776 T3

As another alternative example, Group 2 and 3 rats are given a daily dose of morphogens, starting at day 18 and running through day 50, in order to demonstrate the efficacy of morphogens in the treatment of arthrotic animals. .

Example 12

Morphogen inhibition of localized edema

The following example demonstrates the efficacy of morphogens in inhibiting a localized inflammatory response in a normal model of edema in rats. Experimental rats (here, Long-Evans rats from Charles River Laboratories, Wilmington, Massachusetts) are distributed, forming three groups: Group 1, a negative control, to which only one vehicle is administered; Group 2, a positive control, administered a well-known and characterized anti-inflammatory agent (specifically, indomethacin); and Group 3, to which a morphogen is administered.

Groups 2 and 3 can be subdivided into subgroups to test low, medium, and high doses (for example, Group 2: 1.0 mg / kg, 3.0 mg / kg, and 9.0 mg / kg of indomethacin; Group 3: 0.1-5 μg, 5-20 μg, and 20-50 μg of morphogen). 60 Minutes after indomethacin or morphogen was administered to Group 2 or 3 rats (for example, by injection into the tail vein, or through an oral catheter), inflammation was induced in all rats by a subplantar injection of a 1% carrageenan solution (50 μ!) in the right posterior claw. Three hours after carrageenan administration, claw thickness is measured as an indication of edema (ie, swelling) and an inflammatory response induced by the injected carrageenan solution.

Three hours after the carrageenan injection, significant swelling is evidenced in rats not treated with morphogen. After euthanasia, inflammation is also measured using normal histological procedures: specifically, the right posterior claw of each animal is sectioned at the level of the ankle joint, the bone is weighed, and then the footpad tissue is fixed in a 10% formalin neutral buffered solution, preparing samples, stained with hematoxylin and eosin, for visual examination.

The rats treated with morphogen show an induction of edema, subsequent to the injection of carrageenan, notably lower than that shown by the untreated rats.

Example 13

Morphogen treatment of allergic encephalomyelitis

The following example demonstrates the efficacy of morphogens in the treatment of Experimental Allergic Encephalomyelitis (EAE) in a rat. EAE is a well-characterized animal model that can serve as a reference for multiple sclerosis, an autoimmune disease. For a detailed description of the protocol used, see Kuruvilla, et al., (1991) PNAS 88: 2918-2921.

To describe it succinctly, EAE is induced in rats (eg, Long-Evans rats, Charles River Laboratories, Wilmington, Massachusetts) by injecting a homogenate of CNS tissue (eg, spinal modula) in complete Freund's adjuvant ( CFA) at days -44, -30 and 0 (last day of immunization), by subcutaoneal via at three points on the back of the animal. Morphogen is administered daily by intraperitoneal injection, beginning at day -31. Preferably, a series of morphogen dosage ranges (low, medium and high, as in Example 12 described above) were evaluated. Control rats are administered only the vehicle that accompanies the morphogen (eg 0.9% NaCl, or buffered saline). Rats are examined daily for signs of disease, scoring them on an ascending severity scale from 0 to 4.

In the absence of treatment with the morphogen, at days +7 to +10 significant neurological dysfunctions are evident (specifically, weakness in the fore and hind limbs that progresses to total paraolysis of the hind limbs). In order to evaluate the degree of tissue necrosis, hematological analyzes, serum chemistry profiles and histological analyzes are performed, and histology is carried out, using normal procedures. Morphogen treatment significantly inhibits the neurological dysfunction normally evident in animals affected by EAE. Furthermore, the histopathological markers normally associated with EAE are absent in morphogen-treated animals.

ES 2 149 776 T3

Example 14

Morphogen treatment of collagen-induced arthritis

The following example demonstrates the efficacy of morphogens in inhibiting the inflammatory response in collagen-induced arthritis (CIA) in a rat. CIA is a well-characterized animal model that can serve as a reference for rheumatoid arthritis, an autoimmune disease. The published protocol is essentially the one published in Kuruvilla et al., (1991) PNAS 88: 2918-2921. To describe it succinctly, CIA is induced in rats (e.g., Long-Evans rats, Charles River Laboratories, Wilmington, Massachusetts) by multiple intradermal injection of Type II bovine coloagene (in doses of 100 μg) in CFA ( 0.2 ml), on day 1. The animals are divided into two groups: Group 1, control animals, to which only the vehicle is administered, and Group 2, animals treated with morphogen, which, preferably, is subdivided into low, medium and high dosage ranges. as in Example 13, described above. The morphogen is administered daily (by injection into the tail vein), starting at different days after the collagen injection, specifically, starting at days 7, 14, 28, 35 and 42. Throughout the entire experiment , the animals were visually assessed and the claw thickness and body weight were monitored. The animals are sacrificed at day 60 and the proximal and distal joints of the extremities, as well as the ear, the school and the spinal medulla, are prepared for histological evaluation as described in Examples 12 and 13. As a variation of the experiment, the morphogen can be administered for prescribed periods, for example, in periods of five days, starting at different days after the injection of the cologen (for example, at days 0-4, 7-11, 14-18 , 28-32).

In the absence of morphogen treatment, arthrotic disease is usually induced 30 days after collagen injection. In animals treated with morphogen, the CIA is suppressed and the histopathological changes normally evidenced by control animals with induced CIA (specifically, accumulations of activated mononuclear inflammatory cells and fibrous connective tissue) are absent. Furthermore, and consistent with the results obtained in Example 7 described above, serum titers of anti-collagen antibodies are also significantly suppressed in animals treated with morphogen.

Example 15

Assay for the detection of candidate compounds that alter the levels of endogenous morphogens

Candidate compounds that can be administered to alter the level of a given morphogen can be found by the following screening assay; In this test, the level of morphogen production by a type of cell that produces measurable levels of said morphogen is determined both by incubating and without incubating the cell in a culture in which the compound is also present, in order to evaluate the effects that the compound has on the cell. This can be achieved by detecting morphogen at the proteon or RNA level. A more detailed description can also be found in USSN 752,861.

15.1 Cell culture growth

Cell cultures of the kidney, adrenal capsules, urinary bladder, brain, or other organs can be prepared as widely described in the literature. For example, kidneys from neonates, newborns, young or adult rodents (mouse or rat) can be explanted and used in organ cultures as complete or partial tissues (1 to 4 mm). Primary tissue cultures and established cell Aeneas also derived from kidney, adrenal capsules, urinary bladder, brain, breast, or other tissues can be established in multi-well plates (6 or 24 wells) according to conventional cell culture techniques and they are cultured in the absence or presence of serum for a period of time (from 1 to 7 days). Coellules can be cultured for example in Eagle's modified Dulbecco's medium (Gibco, Long Island, NY) containing serum (eg Gibco's 1% to 10% fetal calf serum) or in serum-free medium, As desired or in a defined medium (containing eg insulin, transferrin, glucose, albumin or other growth factors).

Samples to analyze the level of morphogen production include culture supernatants or cell lysates collected periodically and evaluated for OP-1 production by immunoblot analysis (Sambrook et al, eds, 1989, Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor, NY) or a portion of the cell culture itself, periodically harvested and used to prepare polyA + RNA for RNA analysis. To monitor the de novo OP-1 prosthesis, (again pro40

ES 2 149 776 T3 yield) some cultures are labeled according to conventional procedures with a mixture of 35S-methionine /35S-cysteine for 6 to 24 hours and then evaluated for the OP-1 synthesis by conventional immunoprecipitation methods.

15.2 Determination of Morphogenic Protein Level

To quantify the production of a morphogenic proteon by a type of cell, an immunoassay to detect the morphogen can be performed using a monoclonal or polyclonal antibody specific for that proteon. For example, OP-1 can be detected using a polyclonal antibody specific for OP-1 in an ELISA assay as described below:

1 μg / 100 μl of OP-1 specific affinity purified polyclonal rabbit IgG is added to each well of a 96-well plate and incubated at 37°C for one hour. The wells are washed four times with 0.167 sodium borate buffer with 0.15 M NaCl (BSB), pH 8.2, containing 0.1% Tween 20®. To minimize nonspecific binding, the wells are blocked by filling them completely with 1% bovine serum albumin (BSA) in BSB and incubating it for 1 hour at 37°C. The wells are then washed four times with BSB containing 0.1% Tween 20®. An alloquot of 100 μ! An appropriate dilution of each of these cell culture supernatant test samples is added to each cell in triplicate and incubated at 37 ° C for 30 minutes. After incubation, 100 μl of biotinylated rabbit anti-OP-1 serum (a stock solution of approximately 1 mg / ml diluted 1: 400 in BSB containing 1% BSA before use) is added to each well and incubated at 37°C for 30 minutes. The wells are then washed four times with BSB containing 0.1% Tween 20®. 100 μl of alkaline strepavidin (Southern Biotechnology Associates, Inc., Birmingham, Alabama; diluted 1: 2000 in BSB containing 0.1% Tween 20® before use) is added to each well and incubated at 37 ° C for 30 minutes. Plates are washed four times with 0.5 M Tris buffered saline (TBS), pH 7.2, 50 μl of substrate (ELISA Amplification System, Life Technologies, Inc., Bethesda, MD) is added to each well. and incubated at room temperature for 15 minutes. Then 50 µl of amplifier (from the same amplifier system kit) is added and incubated for another 15 minutes at room temperature. The reaction is stopped with the addition of 50 μl of 0.3 M sulfuric acid. Record the OD at 490 nm of the solution in each well. To quantify OP-1 in culture medium, the normal OP-1 curve is run in parallel with the test samples.

The polyclonal antibody can be prepared as follows: Each rabbit is given primary immunization of 100 μg / 500 μl of an OP-1 monomer produced by E.coli (amino acids 328-431 in sequence identification No. 5) in SDS at 0 , 1% mixed with 500 μl of complete Freund's adjuvant. The antigen is injected subcutaneously at different sites on the back and sides of the animal. The rabbit is boosted in the same manner after one month using the incomplete Freund adjuvant. Seven days later blood samples are taken from the ear vein. Two additional boosts are given and blood samples are taken at one month intervals until antibody to OP-1 is detected in serum using an ELISA assay. The rabbit is then boosted every month with 100 μg of antigen and blood is obtained (15 ml each time) on days seven and ten after the boost.

Specific monoclonal antibodies can be prepared for a given morphogen as follows. A mouse is given two injections of an OP-1 monoomer produced by E.coli. The first injection is given subcutaneously and contains 100 µ ”'of OP-1 in complete Freund's adjuvant. The second injection is given intraperitoneally and contains 50 μg of OP-1 in incomplete Freund's adjuvant. The mouse then receives a total of 230 μg of OP-1 (amino acids 307-431 in sequence identification No. 5) in four intraperitoneal injections given repeatedly over an eight-month period. One week before fusion, both mice are boosted intraperitoneally with 100 μg of OP-1 (307-431) and 30 μg of N-terminal peptide (Ser293-Asn309-Cys) conjugated through the addition of cysteone to bovine serum albumin with the crosslinking agent SMCC. This reinforcement was repeated five days (IP), four days (IP), three days (IP) and one day (IV), before the merger. The spleen cells of the mouse are then fused to the myeloma cells (e.g. 653) at a ratio of 1: 1 using PEG 1500 (Boehringer Mannheim) and the fusion of the cells is plated and selected for OP- specific antibodies. 1 using OP-1 (307-431) as antigen. Cell fusion and monoclonal selection would then be according to normal procedures well described in widely used textbooks.

The invention may be carried out in other specific forms and the present embodiments should consequently be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated in the appended claims.

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Sequence list (1) GENERAL INFORMATION:

(i) APPLICANTS: KUBERASAMPATH, THANGAVEL

PANG, ROY HL

OPPERMANN, HERMANN RUEGER, DAVID C.

COHEN, CHARLES M.

OZKAYNAK, ENGIN SMART, JOHN (ii) TITLE OF INVENTION: MORPHOGEN-INDUCED MODULATION OF THE INFLAMMATORY RESPONSE (iii) NUMBER OF SEQUENCES: 33 (iv) ADDRESS:

(A) NAME: CREATIVE BIOMOLECULES (B) ADDRESS: 35 SOUTH STREET (C) CITY: HOPKINTON (D) STATE: MASSACHUSETTS (E) COUNTRY: UNITED STATES (F) ZIP CODE:

(v) COMPUTER READABLE FORMAT:

(A) MEDIUM TYPE: floppy disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) APPLICATION PROGRAM: Patent in Release 1.0,

Version 1.25 (vii) DATA FROM A PREVIOUS APPLICATION:

(A) APPLICATION NUMBER: US 667,274 (B) FILING DATE: MARCH 11, 1991 (vii) DATA FROM A PREVIOUS APPLICATION:

(A) APPLICATION NUMBER: US 753.059 (B) FILING DATE: AUGUST 30, 1991 (vii) DATA FROM A PREVIOUS APPLICATION:

(A) APPLICATION NUMBER: US 752,764 (B) SUBMISSION DATE: AUGUST 30, 1991 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE NO.1 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 97 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) TYPE OF MOLECULES: protein (ix) CHARACTERISTIC:

(A) NAME: Generic sequence 1 (D) OTHER INFORMATION: Each Xaa indicates one of the 20 natural L isomers of α-amino acids or a derivative of them.

(xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 1:

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Xaa Xaa Xaa Xaa Xaa Xaa

<td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa 10</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa fifteen</td><td colspan="2">Xaa Xaa</td>

(2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. two:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 97 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) TYPE OF MOLECULES: protein (ix) CHARACTERISTIC:

(A) NAME: Generic sequence 2 (D) OTHER INFORMATION: Each Xaa indicates one of the 20 natural L isoomers of α-amino acids or a derivative of them.

(xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. two:

Xaa Xaa Xas xaa Xaa Xaa 1 5

<td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa 10</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa fifteen</td><td>Xaa</td><td>Xaa</td>

<td>Xaa</td><td>Xaa</td><td>xaa twenty</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Cys 25</td><td>Xaa</td><td>Xaa</td><td>Xaa</td>

<td>xaa 40</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>xaa Four. Five</td><td>xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa</td><td>Xaa fifty</td>

ES 2 149 776 T3 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE NO. 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 97 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) TYPE OF MOLECULES: protein (ix) CHARACTERISTIC:

(A) NAME: Generic Sequence 3 (D) OTHER INFORMATION: wherein each Xaa is independently selected from a group of one or more specific amino acids as defined in the specification.

(xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 3:

Leu Tyr Val Xaa Phe

<td>fifteen Xaa</td><td>Tyr</td><td>Cys</td><td>xaa</td><td>Gly</td><td>twenty Xaa</td><td>Cys</td><td>Xaa</td>

<td>Xaa</td><td>Xaa</td><td>Xaa</td><td>xaa</td><td>fifty Xaa</td><td>Xaa</td><td>Xaa</td><td>Cys</td>

(2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 102 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) TYPE OF MOLECULES: protein (ix) CHARACTERISTICS:

(A) NAME: Generic Sequence 4 (D) OTHER INFORMATION: wherein each Xaa is independently selected from a group of one or more specific amino acids as defined in the specification.

EN 2 149 776 T3 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF THE SEQUENCE NO. 4:

100 (2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 5:

(i) SEQUENCE CHARACTERISTICS:

(D) LENGTH: 139 amino acids (E) TYPE: amino acids (F) TOPOLOGY: linear (iii) TYPE OF MOLECULES: protein (ix) CHARACTERISTICS:

(A) NAME: hOP-1 (mature form) (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 5:

ES 2 149 776 T3

<td>Aan 10</td><td>Arg</td><td>To be</td><td>Lys</td><td>Thr</td><td>Pro fifteen</td><td>Lys</td><td>Asn</td><td>Gln</td>

<td>Glu</td><td>To twenty</td><td>Leu</td><td>Arg</td><td>Met</td><td>To</td><td>Asn 25</td><td>Val</td><td>To</td>

<td>To be</td><td>Phe</td><td>Arg</td><td>Asp</td><td>Leu fifty</td><td>Gly</td><td>Trp</td><td>Gln</td><td>Asp</td>

(2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 139 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) TYPE OF MOLECULES: protein (ix) CHARACTERISTICS:

(A) NAME: mOP-1 (mature form) (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 6:

<td>Asn 10</td><td>Arg</td><td>To be</td><td>Lys</td><td>Thr</td><td>Pro fifteen</td><td>Lys</td><td>Asn</td><td>Gln</td>

<td>Glu</td><td>To twenty</td><td>Leu</td><td>Arg</td><td>Met</td><td>To</td><td>To be 25</td><td>Val</td><td>To</td>

ES 2 149 776 T3

<td>To be</td><td>Phe</td><td>Arg</td><td>Asp</td><td rowspan="2">Leu fifty Pro</td><td>Gly</td><td>Trp</td><td>Gln</td><td>Asp</td>

(2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 7:

(i) SEQUENCE CHARACTERISTICS:

(D) LENGTH: 139 amino acids (E) TYPE: amino acids (F) TOPOLOGY: linear (ii) TYPE OF MOLECULE: proteone (ix) CHARACTERISTIC:

(A) NAME: hOP-2 (mature form) (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 7:

<td>Pro 10</td><td>Lys</td><td>Lys</td><td>To be</td><td>Asn</td><td>Glu fifteen</td><td>Leu</td><td>Pro</td><td>Gln</td>

<td>To</td><td>Asn twenty</td><td>Arg</td><td>Leu</td><td>Pro</td><td>Gly</td><td>lie 25</td><td>Phe</td><td>Asp</td>

<td>To be</td><td>Phe</td><td>Gln</td><td>Asp</td><td rowspan="2">Leu fifty Pro</td><td>Gly</td><td>Trp</td><td>Leu</td><td>Asp</td>

ES 2 149 776 T3

(2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 8:

(i) SEQUENCE CHARACTERISTICS:

(G) LENGTH: 139 amino acids (H) TYPE: amino acids (I) TOPOLOGY: linear (ii) TYPE OF MOLECULE: proteone (ix) CHARACTERISTIC:

(A) NAME: mOP-2 (mature form) (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 8:

<td>Pro 10</td><td>Lys</td><td>Lys</td><td>Thr</td><td>Asn</td><td>Glu fifteen</td><td>Leu</td><td>Pro</td><td>His</td>

<td>Pro</td><td>Asn twenty</td><td>Lys</td><td>Leu</td><td>Pro</td><td>Gly</td><td>lie 25</td><td>Phe</td><td>ASp</td>

<td>To be</td><td>Phe</td><td>Arg</td><td>Asp</td><td>Leu fifty</td><td>Gly</td><td>Trp</td><td>Leu</td><td>Asp</td>

ES 2 149 776 T3

Lys Hia Arg Asn Met Val Val Lys Ala

130 135

Cye Gly Cys His (2) SEQUENCE IDENTIFICATION INFORMATION NO. 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 96 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) TYPE OF MOLECULES: protein (ix) CHARACTERISTIC:

(A) NAME: CBMP-2A (fx) (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 9:

<td>Asp</td><td>Val</td><td>Gly</td><td>Trp fifteen</td><td>Asn</td><td>Asp</td><td>Trp</td><td>lie</td><td>Val twenty</td><td>To</td><td>Pro</td>

<td>Thr Four. Five</td><td>Asn</td><td>ais</td><td>To</td><td>lie</td><td>Val fifty</td><td>Gln</td><td>Thr</td><td>Leu</td><td>Val</td><td>Asn 55</td>

Cys Arg 100 (2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 101 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) TYPE OF MOLECULES: protein (ix) CHARACTERISTIC:

(A) NAME: CBMP-2B (fx) (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 10:

ES 2 149 776 T3

(2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. eleven:

(i) SEQUENCE CHARACTERISTICS:

(J) LENGTH: 102 amino acids (K) TYPE: amino acids (L) TOPOLOGY: linear (ii) TYPE OF MOLECULE: proteone (ix) CHARACTERISTIC:

(A) NAME: DPP (fx) (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO. eleven:

(2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 12: (i) SEQUENCE CHARACTERISTICS:

(M) LENGTH: 102 amino acids (N) TYPE: amino acids

ES 2 149 776 T3 (O) TOPOLOGY: linear (ii) TYPE OF MOLECULA: protein (ix) CHARACTERISTIC:

(A) NAME: Vgl (fx) (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 12:

<td>Asp</td><td>val</td><td>Gly</td><td>Trp fifteen</td><td>Gln</td><td>Asn</td><td>Trp Val</td><td>lie twenty</td><td>To</td><td>Pro</td>

<td>To be Four. Five</td><td>Asn</td><td>His</td><td>To</td><td>lie</td><td>Leu fifty</td><td>Gln Thr</td><td>Leu</td><td>Val</td><td>His 55</td>

(2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 102 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) TYPE OF MOLECULES: protein (ix) CHARACTERISTIC:

(A) NAME: Vgr-1 (fx) (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 13:

<td>Asp</td><td>Val</td><td>Gly</td><td>Trp fifteen</td><td>Gln</td><td>Asp</td><td>Trp</td><td>I have</td><td>lie twenty</td><td>To</td><td>Pro</td>

<td>Thr Four. Five</td><td>Asn</td><td>His</td><td>To</td><td>lie</td><td>Val fifty</td><td>Gln</td><td>Thr</td><td>Leu</td><td>Val</td><td>His 55</td>

ES 2 149 776 T3

Leu Tyr Phe Asp Asp Asn Ser Asn Val lie Leu

Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys

Gly Cys His 100 (2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 106 amino acids (B) TYPE: proteon (C) CHAINS: uonic (D) TOPOLOGY: linear (ii) MOLECULE TYPE: proteone (vi) ORIGINAL SOURCE (A) ORGANISM: human (F) TISSUE TYPE : cerebral (ix) FEATURE:

(G) OTHER INFORMATION:

/ product: “GDF-1 (fx)” (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 14:

Cys Arg 105 (2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. fifteen:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

ES 2 149 776 T3 (B) TYPE: amino acids (C) CHAINS: uinic (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. fifteen:

Cys Xaa Xaa Xaa Xaa

5 (2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1,822 base pairs (B) TYPE: nucleic acid (C) STRINGS: uine (D) TOPOLOGY: linear (ii) MOLECLE TYPE: cDNA (vi) ORIGINAL SOURCE (A) ORGANISM: homo sapiens (F ) TYPE OF TISSUE: hippocampus (ix) FEATURE:

(A) NAME / PASSWORD: CDS (B) LOCATION: 49..1341 (D) OTHER INFORMATION: / normal name is “hOP1” (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 16:

CGTGCGGGCC CGGAGCCCGG AGCCCGGGTA CCGCGTACáC CCGGCCCG ATC CAC CTG Het His Val

ES 2 149 776 T3

His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys lie 150 155 160

CCA GAA GGG GAA GCT GTC ACG GCA GCC GAA TTC CGG ATC TAC AAG GAC Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg lie Tyr Lys Asd

165 170 175

ES 2 149 776 T3

<td>TAC</td><td>AGA</td><td>AAC</td><td>ATG</td><td>GTG</td><td>GTC</td><td>CGG</td><td>GCC</td><td>TGT</td><td>GGC</td><td>TGC</td><td>CAC</td><td colspan="3">TAGCTCCTCC</td><td></td>

873

921

969

1017

1065

1113

1161

1209

1257

1305

1351

GAGAATTCAG ACCCTTTGGG GCCAAGTTTT TCTGGATCCT CCATTGCTCG CCTTGGCCAG 1411 GAACCAGCAG ACCAACTGCC TTTTGTGAGA CCTTCCCCTC CCTATCCCCA ACTTTAAAGG 1471 TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CAGTGGCAGC 1531 ATCCAATGAA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AAAAAACAAC 1591

ES 2 149 776 T3

GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TGCACGGACT 1651

CGTTTCCAGA GGTAATTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG GGAGGAAGGG 1711

GGCCTGGCAA GGGÜIGGGCA CATTGG7GTC TCTGCGAAAG GAAAATTGAC CCGGAAGTTC 1771

CTGTAATAAA TGTCACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAAA A 1622 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE N° 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 431 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: proteone (ix) CHARACTERISTIC:

(A) OTHER INFORMATION: / product = “OPI-PP” (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 17:

<td>Asp</td><td>To</td><td>ASP</td><td>Het</td><td>Val</td><td>Het</td><td>To be</td><td>Phe</td><td>Val</td><td>HANDLE</td><td>Leu</td><td>Val</td><td>Glu</td><td>His</td><td rowspan="2">Asp</td><td rowspan="2">Lys</td>

ES 2 149 776 T3

lie lie

Leu

Arg

240

To be

370 375 380

Phe lie Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln

385 390 395 400

Leu Asn Ala lie Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val lie

405 410 415

Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420 425 430

ES 2 149 776 T3 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE NO. 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1,873 base pairs (B) TYPE: nucleic acid (C) STRINGS: uonic (D) TOPOLOGY: linear (ii) MOLECLE TYPE: cDNA (vi) ORIGINAL SOURCE (A) ORGANISM: Muridae (F) TYPE OF TISSUE: embryonic (ix) CHARACTERISTIC:

(A) NAME / PASSWORD: CDS (B) LOCATION: 104..1393 (D) OTHER INFORMATION: / note: “MOP1 (cDNA)” (ix) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 18:

CTGCAGCAAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG CCACCTGGGC

<td>CGG</td><td colspan="2">CGCGGGC</td><td colspan="2">CCGCTGCC</td><td colspan="2">CC GGATC</td><td>GCGC</td><td>G TA</td><td>GAGC</td><td>CGGC</td><td>GCG</td><td>ATG</td><td>CAC</td><td>GTG</td><td>CGC</td>

115

163

211

259

307

355

403

ES 2 149 776 T3

155 160

GGC GAA CGG GTG ACC GCA GCC GAA TTC AGG ATC TAT AAG GAC TAC ATC

Gly Glu Arg Val Thr Ala Ala Glu Phe Arg lie Tyr Lys Asp Tyr lie 165 170 175 180

CGG GAG CGA TTT GAC AAC GAG ACC TTC CAG ATC ACA GTC TAT CAG GTG

Arg Glu Arg Phe Asp Asn Glu Thr Phe Gln lie Thr Val Tyr mn Val

185 190 195

CTC CAG GAG CAC TCA GGC AGG GAG TCG GAC CTC TTC TTC CTG GAC AGC

Leu Gln Glu His Ser Gly Arg Glu Ser Asp Leu Phe Leu Leu Asp Ser

200 205 210

451

499

547

595

643

691

739

787

835

883

931

979

1027

ES 2 149 776 T3

<td>AAC ATG GTG GTC CGG</td><td>GCC TGT</td><td colspan="2">GGC TGC CAC TAGCTCTTCC TGAGACCCTG</td><td> 1413</td>

425 430

ACCTTTGCGG GGCCACACCT TTCCAAATCT TCGATGTCTC ACCATCTAAG TCTCTCACTG 1473 CCCACCTTGG CGAGGAGAAC AGACCAACCT CTCCTGAGCC TTCCCTCACC TCCCAACCGG 1533 AAGCATGTAA GGGTTCCAGA AACCTGAGCG TGCAGCAGCT GATGAGCGCC CTTTCCTTCT 1593 GGCACGTGAC GGACAAGATC CTACCAGCTA CCACAGCAAA CGCCTAAGAG CAGGAAAAAT 1653 GTCTGCCAGG AAAGTGTCCA GTGTCCACAT GGCCCCTGGC GCTCTGAGTC TTTGAGGAGT 1713 AATCGCAAGC CTCGTTCAGC TGCAGCAGAA GGAAGGGCTT AGCCAGGGTG GGCGCTGGCG TCTGTGTTGA 1773 AGGGAAACCA AGCAGAAGCC ACTGTAATGA TATGTCACAA TAAAACCCAT 1833 GAATGAAAAA AAAAAAAAAA AAAAAAAAAA AAAAGAATTC 1873

ES 2 149 776 T3 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE NO. 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 430 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) TYPE OF MOLECULES: proteone (ix) CHARACTERISTIC:

(D) OTHER INFORMATION: / product = “mOP1-PP” (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 19:

130 135 140

Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser

145 150 155 160

Lys lie Pro Glu Gly Glu Arg Val Tht Ala Ala Glu Phe Arg lie Tyr

165 170 175

Lys Asp Tyr lie Arg Glu Arg Phe Asp Asn Glu Thr Phe Gln lie Tht 180 165 190

Val Tyr Gln Val Leu Gln Glu His Ser Gly Arg Glu Ser Asp Leu Phe 195 200 205

ES 2 149 776 T3

420 425 430 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE NO. twenty:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1,723 amino acids (B) TYPE: nucleic acid (C) STRINGS: uonic (D) TOPOLOGY: linear (ii) MOLECLE TYPE: cDNA (vi) ORIGINAL SOURCE:

(A) ORGANISM: homo sapiens (F) TISSUE TYPE: hippocampus (ix) CHARACTERISTIC:

ES 2 149 776 T3 (A) NAME / PASSWORD: CDS (B) LOCATION: 490..1696 (D) OTHER INFORMATION: / note “hOP2 (cDNA)” (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. twenty:

ggcgccggca gagcaggagt ggctggagga gctgtggttg GAGCAGGAGG TGGCACGGCA 60

GGCCTGGAGG GCTCCCTATG AGTGGCGGAG ACGGCCCAGG AGCCGCTGGA GCAACAGCTC 120

CCACACCGCA CCAAGCGGTG GCTGCAGGAG CTCGCCCATC GCCCCTCCGC TGCTCGGACC 180

GCGGCCACAG CCGCACTGGC GGCTACGGCG GCGACAGAGG CATTGGCCGA GAGTCCCACT 240

CCGCAGAGTA GCCCCGGCCT CGAGGCGGTG GCGTCCCGGT CCTCTCCGTC CAGCAGCCAG 300

GACAGCTGTC GCGCGGCGGG GCTCCAGGGA CCGCGCCTGA GGCCGGCTGC CCGGCCGTCC 360

CGCCCCGCCC CGCCGCCCGC CGCCCGCCGA CCCCAGCCTC CTTGCCGTCG GGGCGTCCCC 420

AGGCCCTGGG TCGGCCGCGG AGCCGATGCG CGCCCGCTGA GCGCCCCAGC TGAGCGCCCC 460

CGGCCTGCC ATG ACC GCG CTC CCC GGC CCG CTC TGG CTC CTG GGC CTG 528

Met Thr Ala Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu l 5 10

<td>To</td><td>Leu fifteen</td><td>Cys Wing</td><td>Leu</td><td>Gly</td><td>Gly twenty</td><td>Gly</td><td>Gly Pro</td><td>Gly Leu 25</td><td>Arg</td><td>Pro</td><td>Pro Pro</td>

<td>Arg</td><td>Glu</td><td>lie Leu</td><td>To fifty</td><td>Val</td><td>Leu</td><td>Gly</td><td>Leu Pro 55</td><td>Gly Arg</td><td>Pro</td><td>Arg</td><td>Pro Arg 60</td><td></td>

ES 2 149 776 T3

290 295 300

ES 2 149 776 T3

<td>Cys</td><td>Being Phe 335</td><td>Pro</td><td>Leu</td><td>Asp</td><td>To be 340</td><td>Cys</td><td>Het</td><td>Asn</td><td>To</td><td>Thr 3. 4. 5</td><td>Asn</td><td>His</td><td>To</td><td>lie</td>

GCC TGC GGC TGC CAC T GAGTCAGCCC GCCCAGCCCT ACTGCAG Ala Cys Gly Cys His

400

1440

14S8

1536

1584

1632

1680

1723 (2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. twenty-one:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 402 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: proteone (ix) CHARACTERISTIC:

(A) OTHER INFORMATION: / product = “hOP2-PP” (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. twenty-one:

ES 2 149 776 T3

<td>Leu</td><td>To fifty</td><td>Val</td><td>Leu</td><td>Gly</td><td>Leu</td><td>Pro 55</td><td colspan="2">Gly Arg</td><td>Pro</td><td>Arg</td><td>Pro 60</td><td>Arg</td><td>To</td><td>Pro</td><td>Pro</td>

ES 2 149 776 T3

Cys His (2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1,926 base pairs (B) TYPE: nucleic acid (C) STRINGS: uonic (D) TOPOLOGY: linear (iii) MOLECLE TYPE: cDNA (iv) ORIGINAL SOURCE:

(A) ORGANISM: Muridae (F) TISSUE TYPE: embryonic (x) CHARACTERISTIC:

(A) NAME / PASSWORD: CDS (B) LOCATION: 93..1.289 (D) OTHER INFORMATION: / note “mOP2 cDNA” (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 22:

GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC CCGACCAGCT ACCAGTGGAT GCGCGCCGGC TGAAAGTCCG AG ATG GCT ATG CGT

Met Ala Met Arg

104

152

200

ES 2 149 776 T3

230 235 240

ES 2 149 776 T3

305

GAC CTT GGC TGG CTG GAC TGG GTC ATC GCC CCC CAG GGC TAC TCT GCC

Asp Leu Gly Trp Leu Asp Trp Val lie Wing Pro Gln Gly Tyr Ser Wing

310 315 320

TAT TAC TCT GAG GGG GAG TGT GCT TTC CCA CTG GAC TCC TGT ATG AAC

Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys Met Asn 325 330 335 340

GCC ACC AAC CAT GCC ATC TTG CAG TCT CTG GTG CAC CTG ATG AAG CCA

Wing Thr Asn His Wing lie Leu Gln Ser Leu Val His Leu Met Lys Pro

345 350 355

GAT GTT GTC CCC AAG GCA TGC TGT GCA CCC ACC AAA CTG AGT GCC ACC

Asp Val Val Pro Lys Ala Cys Cys AlaPro Thr Lys Leu Ser Ala Thr

360 365 370

TCT GTG CTG TAC TAT GAC AGC AGC AAC AAT GTC ATC CTG CGT AAA CAC

Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val lie Leu Arg Lys His

375 380 385

CGT AAC ATG GTG GTC AAG GCC TGT GGC TGC CAC TGAGGCCCCG CCCAGCATCC Arg Asn Met Val Val Lys Ala Cys Gly Cys His

390 395

TGCTTCTACT ACCTTACCAT CTGGCCGGGC CCCTCTCCAG AGGCAGAAAC CCTTCTATGT TATCATAGCT CAGACAGGGG CAATGGGAGG CCCTTCACTT CCCCTGGCCA CTTCCTGCTA AAATTCTGGT CTTTCCCAGT TCCTCTGTCC TTCATGGGGT TTCGGGGCTA TCACCCCGCC CTCTCCATCC TCCTACCCCA AGCATAGACT GAATGCACAC AGCATCCCAG AGCTATGCTA ACTGAGAGGT CTGGGGTCAG CACTGAAGGC CCACATGAGG AAGACTGATC CTTGGCCATC CTCAGCCCAC AATGGCAAAT TCTGGATGGT CTAAGAAGGC CGTGGAATTC TAAACTAGAT

872

920

968

1016

1064

1112

1160

1208

1256

1309

1369

1429

1489

1549

1609

1669

ES 2 149 776 T3

GATCTGGGCT CTCTGCACCA TTCATTGTGG CAGITGGGAC AITTTTAGGT ATAACAGACA i 729

CATACACTTA GATCAATGCA TCGCTGTACT CCTTGAAATC AGAGCTACCT TGTTAGAAAA 1789

AGAATCAGAG CCAGGTATAG CGGTGCATGT CATTAATCCC AGCGCTAAAG AGACAGAGAC 1849

AGGAfiAATCT CTGTGAGTTC AAGGCCACAT AGAAAGAGCC TGTCTCGGGA GCAGGAAAAA 1909

AAAAAAAAAC GGAATTC 1926 (2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 2. 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 399 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (D) (v) TYPE OF MOLECULES: protein (xi) CHARACTERISTIC:

(D) OTHER INFORMATION: / product “mOP2-PP” (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 2. 3:

135 140 145

ES 2 149 776 T3

ES 2 149 776 T3 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE NO. 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1,368 base pairs (B) TYPE: nucleic acid (C) STRINGS: uonic (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTIC:

(A) NAME / PASSWORD: CDS (B) LOCATION: 1..1368 (D) OTHER INFORMATION: normal name “60A” (x) PUBLICATION INFORMATION:

(A) AUTHORS: WHARTON, KRISTIA .; THOMSEN, GERALD H.,

GELBERT, WILLIAM M.

(B) TITLE: DROSOPHILA, 60A GENE ...

(C) MAGAZINE: PROC. NAT'L ACAD. SCI. USAVOLUMEN: 88 (E) RELEVANT REMAINS IN THE INFORMATION FOR THE IDENTIFICATION OF SEQUENCE NO. 3, FROM 1 TO 1,368 (D) PAGES 9214-9218 DATE: OCTOBER 1991 (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO. 24:

<td>ATG Met 1</td><td>TCG To be</td><td>GGA Gly</td><td>CTG Leu</td><td>CGA Arg 5</td><td>AAC Asn</td><td>ACC Thr</td><td>TCG To be</td><td>GAG Glu</td><td>GCC To 10</td><td>GTT Val</td><td>GCA To</td><td>GTC Val</td><td>CTC Leu</td><td>GCC To fifteen</td><td>CBT To be</td><td></td>

<td>Leu</td><td>Gly</td><td>Leu</td><td>Gly twenty</td><td>Het</td><td>Val</td><td>Leu</td><td>Leu</td><td>Het 25</td><td>Phe</td><td colspan="2">Val Ala</td><td>Thr</td><td>Thr 30</td><td>Pro</td><td>Pro</td><td></td>

<td>Gln</td><td>Thr fifty</td><td>lie</td><td>Mee</td><td>His</td><td>Arg</td><td>Val 55</td><td>Leu</td><td>To be</td><td>Glu</td><td>Asp</td><td>ASp 60</td><td>Lys</td><td>Leu</td><td>A $ p</td><td>Val</td>

ES 2 149 776 T3

290 295 300

ES 2 149 776 T3

(2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 25:

(i) SEQUENCE CHARACTERISTICS:

(D) LENGTH: 455 amino acids (E) TYPE: amino acids (F) TOPOLOGY: linear (vi) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 25:

ES 2 149 776 T3

Het Ser Gly Leu Arg Asn Thr Ser Glu Ala Val Ala Val Leu Ala Ser 15 10 15

Leu Gly Leu Gly Het Val Leu Leu Het Phe Val Ala Thr Thr Pro Pro 20 25 30

Wing Val Glu Wing Thr Gln Ser Gly lie Tyr lie Asp Asn Gly Lys Asp 35 40 45

<td>Gln</td><td>Thr fifty</td><td>lie</td><td>Het</td><td>His</td><td>Arg</td><td>Val 55</td><td>Leu</td><td>To be</td><td>Glu</td><td>Asp</td><td>Asp 60</td><td>Lys</td><td>Leu</td><td>Asp</td><td>Val</td>

150 155 160

ES 2 149 776 T3

(2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (vii) MOLECULE TYPE: protein (viii) ORIGINAL SOURCE:

(A) ORGANISM: Homo Sapiens (xii) FEATURE:

(A) NAME / KEY: protein (B) LOCATION: 1..102 (D) OTHER INFORMATION: / note “BMP 3” (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 26: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 104 amino acids (B) TYPE: amino acids (C) CHAINS: unique

ES 2 149 776 T3 (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: proteone (ix) CHARACTERISTIC:

(A) NAME / KEY: proteone (B) LOCATION: 1..104 (D) OTHER INFORMATION: / note “BMP 3” (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 26:

Thr Val Glu Ser Cys Ala Cys Arg 100 (2) SEQUENCE IDENTIFICATION INFORMATION NO. 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 102 amino acids (B) TYPE: amino acids (C) CHAINS: uonic (D) TOPOLOGY: linear (ii) MOLECLE TYPE: proteone (ix) ORIGINAL SOURCE:

(A) ORGANISM: Homo Sapiens (ix) FEATURE:

(A) NAME / PASSWORD: proteone (B) LOCATION: 1..102

OTHER INFORMATION: / note = “BMP5” (x) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 27:

ES 2 149 776 T3

Arg Ser Cys Gly Cys His 100 (2) SEQUENCE IDENTIFICATION INFORMATION NO. 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 102 amino acids (B) TYPE: amino acids (C) CHAINS: Single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULES: protein (ix) CHARACTERISTICS:

(A) NAME / KEY: protein (B) LOCATION: 1..102

OTHER INFORMATION: / note = “BMP6” (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 28:

cys Arg Lys His Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp Gln 15 10 15

<td>Asp</td><td>Trp</td><td>Xle</td><td>I have</td><td>To</td><td>Pt</td><td>Lys</td><td>Gly</td><td>Tyr</td><td>To</td><td>To</td><td>Handle</td><td rowspan="2">Tyr</td><td>Cya</td><td rowspan="2">Asp</td><td rowspan="2">Gly</td>

90 95

Arg Ala Cys Cly Cys His 100

ES 2 149 776 T3 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE NO. 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 102 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULES: protein (ix) CHARACTERISTIC:

(A) NAME / KEY: protein (B) LOCATION: 1..102 (E) OTHER INFORMATION: OPX label

Note: "Where Xaa at each position is independently selected from the residues that occur at the corresponding position in the C terminal sequence of mouse or human OP1 or OP2 (see sequence info. Nos. 5, 6, 7 and 8 oré 16, 18, 20 and 22).

(xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 29:

<td>ASp</td><td>Xaa</td><td>To be</td><td>Xaa</td><td>Handle</td><td>Val</td><td>Xaa</td><td>Leu</td><td>Xaa</td><td>Lys</td><td>Xaa</td><td rowspan="2">Arg</td><td>Asn</td><td>Het</td><td>Val</td><td>Val</td>

100 (2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 97 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) TYPE OF MOLECULES: protein (ix) CHARACTERISTIC:

(A) NAME: genetic sequence 5 (D) OTHER INFORMATION: wherein each Xaa is independently selected from a group of one or more specific amino acids as defined in the specification.

(xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 30:

ES 2 149 776 T3

(2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE N ° 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 102 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) TYPE OF MOLECULES: proteone (ix) CHARACTERISTIC:

(A) NAME / KEY: genomic sequence 6 (D) OTHER INFORMATION: wherein each Xaa is independently selected from a group of one or more specific amino acids as defined in the specification.

(xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NO. 31:

ES 2 149 776 T3

INFORMATION FOR THE IDENTIFICATION OF SEQUENCE NO. 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1,238 base pairs, 372 amino acids (B) TYPE: nucleic acids, amino acids (C) STRINGS: uanic (D) TOPOLOGY: linear (ii) MOLECLE TYPE: cDNA (iii) ORIGINAL SOURCE (A) ORGANISM : human (F) TISSUE TYPE: cerebral (xi) CHARACTERISTICS:

(A) NAME / PASSWORD: CDS (B) LOCATION:

(A) AUTHORS: Lee, Se-Jin (B) TITLE: Growth Expression / Differentiation Factor 1 (C) JOURNAL: Proc. Nat'l Acad. Sci.

(D) VOLUME: 88 (E) RELEVANT REMAINS: 1 to 1238 (F) PAGES 4,250-4,254 (G) DATE: MAY 1991

EN 2 149 776 T3 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF THE SEQUENCE NO. 32:

GGGGACACCG GCCCCGCCCT CAGCCCACTG GTCCCGGGCC GCCGCGGACC CTGCGCACTC 60

TCTGGTCATC GCCTGGGAGG AAG ATG CCA CCG CCG'CAG CAA GGT CCC TGC GGC 113

Het Pro Pro Pro Gln Gln Gly Pro Cys Gly 1 5 10

<td>Leu</td><td>Leu</td><td>Leu fifteen</td><td colspan="2">Wing Leu</td><td>Leu twenty</td><td>Leu</td><td>Pro</td><td colspan="2">Ser Leu</td><td>Pro 25</td>

ES 2 149 776 T3

ES 2 149 776 T3

<td>CBT</td><td>CGC</td><td colspan="8">TAACCCGGCC CGGGCAGGGA CCCGGGCCCA ACAATAAATG CCGCCTGC</td><td> 1238</td>

Cys Arg 372 (2) INFORMATION FOR IDENTIFICATION OF SEQUENCE NO. 33:

(i) SEQUENCE CHARACTERISTICS:

(E) LENGTH: 372 amino acids (F) TYPE: amino acids (G) CHAINS: uanic (H) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) SOURCE ORIGINAL:

(A) ORGANISM: human (F) TYPE OF TISSUE: cerebral (ix) CHARACTERISTIC:

(A) NAME / PASSWORD: CDS (B) LOCATION:

Met Pro Pro Pro Gln Gln Gly Pro Cys Gly 1 5 10

ES 2 149 776 T3

<td>His</td><td>His</td><td>Leu</td><td>Leu</td><td>Leu fifteen</td><td>Leu</td><td>Leu</td><td>To</td><td>Leu</td><td>Leu 2Q</td><td>Leu</td><td>Pro</td><td>To be</td><td>Leu</td><td>Pro 25</td>

<td>Gln</td><td>To</td><td>Leu</td><td>Gly</td><td>Leu Four. Five</td><td>Arg</td><td>Asp</td><td>Glu</td><td>Pro</td><td>Gln fifty</td><td>Gly</td><td>To</td><td>Pro</td><td>Arg</td><td>Leu 55</td>

ES 2 149 776 T3

Cys Arg 372

Contents105

418 members in 16 offices

This recordMember, by publication datePriority claim6 more offices are in the list below

Priority claims15

Priority claims
Document	Office	Kind	Date
19910752764	United States of America	–	1991-08-30
19910752861	United States of America	–	1991-08-30
19910753059	United States of America	–	1991-08-30
75276491	United States of America	A	1991-08-30
75276491	United States of America	A	1991-08-30
75286191	United States of America	A	1991-08-30
75286191	United States of America	A	1991-08-30
75305991	United States of America	A	1991-08-30
75305991	United States of America	A	1991-08-30
752861	–	–	–
753059	–	–	–
92919544	–	–	–
US19910752764	–	–	–
US19910752861	–	–	–
US19910753059	–	–	–

Members418

Family members, oldest first
Document	Office	Kind
WO8909787A2	World Intellectual Property Organization (WIPO)	A2	1989-10-19
WO8909788A2	World Intellectual Property Organization (WIPO)	A2	1989-10-19
AU3444989A	Australia	A	1989-11-03
AU3530589A	Australia	A	1989-11-03
WO8909787A3	World Intellectual Property Organization (WIPO)	A3	1990-02-08
WO8909788A3	World Intellectual Property Organization (WIPO)	A3	1990-02-08
EP0362367A1	European Patent Office (EPO)	A1	1990-04-11
EP0372031A1	European Patent Office (EPO)	A1	1990-06-13
US4943233A	United States of America	A	1990-07-24
WO9010018A1	World Intellectual Property Organization (WIPO)	A1	1990-09-07
AU5174790A	Australia	A	1990-09-26
US4968590A	United States of America	A	1990-11-06
US4975526A	United States of America	A	1990-12-04
EP0411105A1	European Patent Office (EPO)	A1	1991-02-06
JPH03500655A	Japan	A	1991-02-14
CA2027259A1	Canada	A1	1991-04-18
CA2042577A1	Canada	A1	1991-04-18
US5011691A	United States of America	A	1991-04-30
WO9105802A1	World Intellectual Property Organization (WIPO)	A1	1991-05-02
AU6648190A	Australia	A	1991-05-16
JPH03502579A	Japan	A	1991-06-13
EP0448704A1	European Patent Office (EPO)	A1	1991-10-02
JPH03504736A	Japan	A	1991-10-17
AU618357B2	Australia	B2	1991-12-19
CA2094027A1	Canada	A1	1992-04-19
JPH04502336A	Japan	A	1992-04-23
US5108753A	United States of America	A	1992-04-28
WO9207004A1	World Intellectual Property Organization (WIPO)	A1	1992-04-30
WO9207073A1	World Intellectual Property Organization (WIPO)	A1	1992-04-30
AU8900091A	Australia	A	1992-05-20
AU8941791A	Australia	A	1992-05-20
EP0411105A4	European Patent Office (EPO)	A4	1992-05-20
EP0448704A4	European Patent Office (EPO)	A4	1992-07-08
AU627850B2	Australia	B2	1992-09-03
AU628050B2	Australia	B2	1992-09-10
CA2104678A1	Canada	A1	1992-09-12
CA2363965A1	Canada	A1	1992-09-17
WO9215323A1	World Intellectual Property Organization (WIPO)	A1	1992-09-17
AU1754392A	Australia	A	1992-10-06
US5162114A	United States of America	A	1992-11-10
US5171574A	United States of America	A	1992-12-15
US5182365A	United States of America	A	1993-01-26
CA2116560A1	Canada	A1	1993-03-18
CA2116562A1	Canada	A1	1993-03-18
WO9304692A1	World Intellectual Property Organization (WIPO)	A1	1993-03-18
WO9305172A1	World Intellectual Property Organization (WIPO)	A1	1993-03-18
CA2116559A1	Canada	A1	1993-04-01
WO9305751A2	World Intellectual Property Organization (WIPO)	A2	1993-04-01
AU2564592A	Australia	A	1993-04-05
AU2862492A	Australia	A	1993-04-05
AU3176293A	Australia	A	1993-04-27
WO9305751A3	World Intellectual Property Organization (WIPO)	A3	1993-05-13
US5250302A	United States of America	A	1993-10-05
US5258494A	United States of America	A	1993-11-02
US5266683A	United States of America	A	1993-11-30
CA2138270A1	Canada	A1	1993-12-23
WO9325246A1	World Intellectual Property Organization (WIPO)	A1	1993-12-23
EP0575555A1	European Patent Office (EPO)	A1	1993-12-29
AU4599793A	Australia	A	1994-01-04
CA2141554A1	Canada	A1	1994-02-17
CA2141555A1	Canada	A1	1994-02-17
CA2141556A1	Canada	A1	1994-02-17
WO9403075A2	World Intellectual Property Organization (WIPO)	A2	1994-02-17
WO9403200A1	World Intellectual Property Organization (WIPO)	A1	1994-02-17
WO9403600A1	World Intellectual Property Organization (WIPO)	A1	1994-02-17
AU4795193A	Australia	A	1994-03-03
AU4797193A	Australia	A	1994-03-03
AU4995593A	Australia	A	1994-03-03
CA2144513A1	Canada	A1	1994-03-31
CA2144514A1	Canada	A1	1994-03-31
CA2144515A1	Canada	A1	1994-03-31
WO9406399A1	World Intellectual Property Organization (WIPO)	A1	1994-03-31
WO9406420A2	World Intellectual Property Organization (WIPO)	A2	1994-03-31
WO9406447A2	World Intellectual Property Organization (WIPO)	A2	1994-03-31
WO9406449A2	World Intellectual Property Organization (WIPO)	A2	1994-03-31
AU5129293A	Australia	A	1994-04-12
AU5129393A	Australia	A	1994-04-12
AU5162393A	Australia	A	1994-04-12
AU5290893A	Australia	A	1994-04-12
CA2147598A1	Canada	A1	1994-05-11
WO9410203A2	World Intellectual Property Organization (WIPO)	A2	1994-05-11
AU648997B2	Australia	B2	1994-05-12
AU5590094A	Australia	A	1994-05-24
EP0601106A1	European Patent Office (EPO)	A1	1994-06-15
EP0601129A1	European Patent Office (EPO)	A1	1994-06-15
EP0601135A1	European Patent Office (EPO)	A1	1994-06-15
US5324819A	United States of America	A	1994-06-28
JPH06506360A	Japan	A	1994-07-21
WO9410203A3	World Intellectual Property Organization (WIPO)	A3	1994-08-18
WO9406420A3	World Intellectual Property Organization (WIPO)	A3	1994-09-01
WO9406447A3	World Intellectual Property Organization (WIPO)	A3	1994-09-01
WO9406449A3	World Intellectual Property Organization (WIPO)	A3	1994-09-01
US5344654A	United States of America	A	1994-09-06
CA2157387A1	Canada	A1	1994-09-15
WO9420539A1	World Intellectual Property Organization (WIPO)	A1	1994-09-15
AU6358894A	Australia	A	1994-09-26
US5354557A	United States of America	A	1994-10-11
EP0575555A4	European Patent Office (EPO)	A4	1994-11-02
WO9403075A3	World Intellectual Property Organization (WIPO)	A3	1994-11-10
JPH06510432A	Japan	A	1994-11-24

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