Title:  Use of relaxin treat diseases related to vasoconstriction

United States Patent:  6,723,702

Issued:  April 20, 2004

Inventors:  Conrad; Kirk P. (Cranberry Township, PA); Lewis; Martyn (Menlo Park, CA); Unemori; Elaine N. (Oakland, CA); Huang; Xinfan (Menlo Park, CA); Tozzi; Carol A. (Jackson, NJ)

Assignee:  RAS Medical, Inc. (San Mateo, CA); The University of Medicine and Dentistry of New Jersey - Robert Wood (New Brunswick, NJ); The University of Pittsburgh - of the Commonwealth System of Higher (Pittsburgh, PA)

Appl. No.:  780752

Filed:  February 9, 2001

Abstract

The invention related to methods of treating disease related to vasoconstriction that is a major factor in hypertensive vascular diseases and vasodilation, generally comprising administering to an individual an effective amount of a pharmaceutically active relaxin. Relaxin functions to increase both vasodilation and angiogenesis in males as well as females, and is useful in treating a wide variety of diseases relating to vasoconstriction.

SUMMARY OF THE INVENTION

The present invention provides methods for treating diseases or disorders related to vasoconstriction, generally comprising administering a formulation comprising a therapeutically effective amount of relaxin. The formulation may be administered by injection, or by sustained-release modes of administration, over a period of time and in amounts which are effective to treat the vasoconstriction-related disease or disorder. Relaxin can act to increase vasodilation, to increase neovascularization (i.e., stimulate, increase, or promote angiogenesis), or both, thereby alleviating the disorder, or symptoms of the disorder. Thus, the invention further provides methods for increasing vasodilation, and methods of stimulating angiogenesis in an individual, generally comprising administering a formulation comprising a pharmaceutically effective amount of pharmaceutically acceptable carrier having therein therapeutically active relaxin.

The invention further provides methods for treating angiotensin-II (AngII)-mediated vasoconstriction. These methods generally comprise administering a formulation comprising an amount of relaxin effective to reverse, inhibit, or reduce the vasoconstricting effects of AngII.

The invention further provides methods for treating endothelin-1 (ET-1)-mediated vasoconstriction. These methods generally comprise administering a formulation comprising an amount of relaxin effective to reverse, inhibit, or reduce the vasoconstricting effects of ET-1. In some embodiments, the methods comprise increasing endothelin type B receptor activation in a cell in a blood vessel by administering relaxin to the individual.

The invention further provides methods for treating an ischemic condition, generally comprising administering a formulation comprising an amount of relaxin effective to stimulate or promote angiogenesis and/or vasodilation, thereby treating the ischemic condition. The methods are useful in treating a variety of ischemic conditions. In some embodiments, methods are provided for treating an ischemic condition which arises as a result of myocardial infarct. In other embodiments, methods are provided for treating an ischemic condition associated with a wound. Thus, the invention further provides methods for promoting wound healing.

The invention further provides methods for stimulating angiogenic and/or vasodilatory cytokine expression generally comprising administering a formulation comprising an amount of relaxin effective to vasodilate blood vessels and/or stimulate or promote angiogenic cytokine production. In some embodiments, the methods provide for stimulating expression of basic fibroblast growth factor (bFGF) and/or vascular endothelial cell growth factor (VEGF). Such methods are useful in treating a wide variety of diseases which can be treated by increasing blood flow at or near the site of disease.

The invention further provides a method of increasing renal vasodilation and hyperfiltration, generally comprising administering a formulation comprising an amount of relaxin. These methods are useful in treating a variety of renal pathologies. Accordingly, the invention further provides methods of treating a renal pathology related to vasoconstriction.

The invention further provides a method of reducing pulmonary hypertension, generally comprising administering a formulation comprising an amount of relaxin.

An advantage of the present invention lies in the fact that the safety profile of relaxin in humans is superior to other agents, such as VEGF and FGF.

A further advantage of the use of relaxin to treat hypertensive vascular diseases is that it is effective in both males and females.

Another object of the invention is a method whereby therapeutically effective amounts of relaxin are repeatedly administered to a patient over a period of time to obtain a beneficial therapeutic result.

Another aspect of the invention is to repeatedly or substantially continuously administer relaxin over a period of time in a manner so as to maintain therapeutic blood levels of relaxin over periods sufficient to obtain therapeutic results.

A feature of the invention is injectable and sustained-release formulations of relaxin which are useful in the method of the invention wherein the formulation comprises a pharmaceutically acceptable carrier and a therapeutically effective amount of relaxin.

These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a disease" includes a plurality of such diseases and reference to "the method" includes reference to one or more methods and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Definitions

As used herein, the terms "disease related to vasoconstriction," "disorder related to vasoconstriction," "disease associated with vasoconstriction," and "disorder associated with vasoconstriction," used interchangeably herein, refer to a disease or condition or disorder that involves vasoconstriction in some manner. The disease may be a disease which is a direct result of vasoconstriction; a disease or condition that is exacerbated by vasoconstriction; and/or a disease or condition that is a sequelae of vasoconstriction. Diseases and disorder related to vasoconstriction include, but are not limited to, pulmonary vasoconstriction and associated diseases and disorders; cerebral vasoconstriction and associated diseases and disorders; peripheral vasoconstriction and associated diseases and disorders; cardiovascular vasoconstriction and associated diseases and disorders; renal vasoconstriction and associated diseases and disorders; and ischemic conditions. Such diseases and disorders include, but are not limited to, chronic stable angina; unstable angina; vasospastic angina; microvascular angina; blood vessel damage due to invasive manipulation, e.g., surgery; blood vessel damage due to ischemia, e.g., ischemia associated with infection, trauma, and graft rejection; ischemia associated with stroke; cerebrovascular ischemia; renal ischemia; pulmonary ischemia; limb ischemia; ischemic cardiomyopathy; myocardial ischemia; reduction in renal function as a result of treatment with a nephrotoxic agent, e.g., cyclosporine A; acute myocardial infarction; ischemic myocardium associated with hypertensive heart disease and impaired coronary vasodilator reserve; subarachnoid hemorrhage with secondary cerebral vasospasm; reversible cerebral vasoconstriction; migraine; disorders relating to uterine vascoconstriction, e.g., preeclampsia of pregnancy, eclampsia, intrauterine growth restriction, inadequate maternal vasodilation during pregnancy; post transplant cardiomyopathy; renovascular ischemia; cerebrovascular ischemia (Transient Ischemic Attack (TIA) and stroke); pulmonary hypertension; renal hypertension; essential hypertension; atheroembolic diseases; renal vein thrombosis; renal artery stenosis; renal vasoconstriction secondary to shock, trauma, or sepsis; liver ischemia, peripheral vascular disease; diabetes mellitus; thromboangiitis obliterans; and burn/thermal injury.

As used herein, the term "relaxin" refers to biologically active (also referred to herein as "pharmaceutically active") relaxin from recombinant or native sources as well as relaxin variants, such as amino acid sequence variants. Naturally occurring biologically active relaxin may be derived from human, murine (i.e., rat or mouse), porcine, or other mammalian sources. The term "relaxin" encompasses human H1 preprorelaxin, prorelaxin, and relaxin; and H2 preprorelaxin, prorelaxin, and relaxin; and recombinant human relaxin. Also encompassed is relaxin modified to increase in vivo half life, e.g., PEGylated relaxin (i.e., relaxin conjugated to a polyethylene glycol), and the like. The term also encompasses relaxin comprising A and B chains having N- and/or C-terminal truncations. In general, in H2 relaxin, the A chain can be varied from A(1-24) to A(10-24) and B chain from B(^- 1-33) to B(10-22); and in H1 relaxin, the A chain can be varied from A(1-24) to A(10-24) and B chain from B(1-32) to B(10-22). Also included within the scope of the term "relaxin" are other insertions, substitutions, or deletions of one or more amino acid residues, glycosylation variants, unglycosylated relaxin, organic and inorganic salts, covalently modified derivatives of relaxin, preprorelaxin, and prorelaxin. Also encompassed in the term is a relaxin analog having an amino acid sequence which differs from a wild-type (e.g., naturally-occurring) sequence, including, but not limited to, relaxin analogs disclosed in U.S. Pat. No. 5,811,395. Possible modifications to relaxin amino acid residues include the acetylation, formylation or similar protection of free amino groups, including the N-terminal, amidation of C-terminal groups, or the formation of esters of hydroxyl or carboxylic groups, e.g., modification of the tryptophan (Trp) residue at B2 by addition of a formyl group. The formyl group is a typical example of a readily-removable protecting group. Other possible modifications include replacement of one or more of the natural amino-acids in the B and/or A chains with a different amino acid (including the D-form of a natural amino-acid), including, but not limited to, replacement of the Met moiety at B24 with norleucine (Nle), valine (Val), alanine (Ala), glycine (Gly), serine (Ser), or homoserine (HomoSer). Other possible modifications include the deletion of a natural amino acid from the chain or the addition of one or more extra amino acids to the chain. Additional modifications include amino acid substitutions at the B/C and C/A junctions of prorelaxin, which modifications facilitate cleavage of the C chain from prorelaxin; and variant relaxin comprising a non-naturally occurring C peptide, e.g., as described in U.S. Pat. No. 5,759,807. Also encompassed by the term "relaxin" are fusion polypeptides comprising relaxin and a heterologous polypeptide. A heterologous polypeptide (e.g., a non-relaxin polypeptide) fusion partner may be C-terminal or N-terminal to the relaxin portion of the fusion protein. Heterologous polypeptides include immunologically detectable polypeptides (e.g., "epitope tags"); polypeptides capable of generating a detectable signal (e.g., green fluorescent protein, enzymes such as alkaline phosphatase, and others known in the art); therapeutic polypeptides, including, but not limited to, cytokines, chemokines, and growth factors. All such variations or alterations in the structure of the relaxin molecule resulting in variants are included within the scope of this invention so long as the functional (biological) activity of the relaxin is maintained. Preferably, any modification of relaxin amino acid sequence or structure is one that does not increase its immunogenicity in the individual being treated with the relaxin variant. Those variants of relaxin having the described functional activity can be readily identified using the in vitro and in vivo assays mentioned above.

As used herein the terms "isolated" and "substantially purified," used interchangeably herein, when used in the context of "isolated relaxin," refer to a relaxin polypeptide that is in an environment different from that in which the relaxin polypeptide naturally occurs. As used herein, the term "substantially purified" refers to a relaxin polypeptide that is removed from its natural environment and is at least 60% free, preferably 75% free, and most preferably 90% free from other components with which it is naturally associated.

The terms "effective amount" and "therapeutic amount" and the like are used interchangeably here to describe a relaxin formulation that is sufficient to treat a disease related to vasoconstriction. The term "effective amount" means a dosage sufficient to provide treatment for the disease state being treated, i.e., a disease related to vasodilation. In general, an effective amount of relaxin is one that is effective to increase vasodilation and/or to increase neovascularization. The term "increase" is used interchangeably herein with "stimulate" and "promote." The Examples provide general guidance for effective amounts used in rats. Those skilled in the art will readily be able to determine effective amounts for use in human subjects, given the guidance in the Examples. In general, a dose is from about 0.1 to 500 .mu.g/kg of body weight per day, about 6.0 to 200 .mu.g/kg, or about 12.0 to 100 .mu.g/kg. For administration to a 70 kg person, the dosage range would be about 7.0 .mu.g to 3.5 mg per day, about about 42.0 .mu.g to 2.1 mg per day, or about 84.0 to 700 .mu.g per day. In some embodiments, for administration to a human, an effective dose is from about 5 .mu.g/kg body weight/day to about 50 .mu.g/kg body weight/day, or from about 10 .mu.g/kg body weight/day to about 25 .mu.g/kg body weight/day. The amount of relaxin administered will, of course, be dependent on the size, sex and weight of the subject and the severity of the disease or condition, the manner and schedule of administration, the likelihood of recurrence of the disease, and the judgment of the prescribing physician.

The terms "subject" or "individual" or "patient," used interchangeably herein, refer to any subject, particularly a mammalian subject, for whom diagnosis or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on. A preferred subject is a human in need of treatment for a disease related to vasoconstriction, particularly a renal disease, and an ischemic condition.

The terms "treatment," "treating," "therapy," and the like are used herein to generally refer to obtaining a desired therapeutic, pharmacologic or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a marnmal, e.g. a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

OVERVIEW OF THE INVENTION

The present invention provides methods of treating diseases related to vasoconstriction, comprising administering to an individual, who has a disease or condition relating to vasoconstriction, an effective amount of relaxin in a pharmaceutical formulation. Relaxin formulations of the invention can act both to increase vasodilation, and to increase formation of new blood vessels. Thus, relaxin can be used to treat a wide variety of conditions related to vasoconstriction. Further, it has been surprisingly found that relaxin, a pregnancy hormone, can function to promote vasodilation and to promote neovascularization in males as well as females. Relaxin can inhibit or reduce angiotensin-II (AngII)-mediated and endothelin-1 (ET-1)-mediated vasoconstriction. It can also promote renal vasodilation and hyperfiltration, which is particularly useful in the context of treating renal disease which result, directly or indirectly, from renal vasoconstriction. It has further been found that relaxin stimulates angiogenic cytokine expression, including bFGF and VEGF, and hence promotes blood vessel formation. It has also been found that relaxin stimulates ischemic wound healing. It has also been found that relaxin is effective in inhibiting progression of pulmonary hypertension. Thus, relaxin's vasodilation- and neovascularization-promoting properties can be used to treat a wide variety of disease conditions arising from vasoconstriction, or inadequate blood supply.

METHODS OF TREATING DISEASES RELATED TO VASOCONSTRICTION

The present invention provides methods for treating diseases related to vasoconstriction. The methods generally comprise administering to an individual in need thereof a pharmaceutical formulation comprising pharmaceutically active relaxin in an amount effective to treat the disease. An effective amount of relaxin is one that is effective to increase, stimulate, or promote, vasodilation; and/or to increase, stimulate, or promote, neovascularization; and/or to promote wound healing; and/or to treat an ischemic condition; and/or to reduce hypertension. The effect of relaxin on vasodilation or neovascularization may be direct or indirect. Modes of administration, amounts of relaxin administered, and relaxin formulations, for use in the methods of the present invention, are discussed below.

An effective amount of relaxin is one that is effective in treating a disease related to vasoconstriction. Whether the disease has been treated is determined by measuring one or more diagnostic parameters indicative of the course of the disease, compared to a suitable control. In the case of an animal experiment, a "suitable control" is an animal not treated with relaxin, or treated with the pharmaceutical formulation without relaxin. In the case of a human subject, a "suitable control" may be the individual before treatment, or may be a human (e.g., an age-matched or similar control) treated with a placebo. Methods for assessing whether a disease related to vasoconstriction has been treated are known in the art, and are described in numerous publications, including, e.g., Young et al., eds. (1996) "Peripheral Vascular Diseases", Mosby-Year Book, Inc. St. Louis, Mo. Additional methods are described hereinbelow.

In some embodiments, the invention provides a method of increasing nitric oxide production in a cell of a blood vessel, comprising administering to an individual a pharmaceutical formulation comprising pharmaceutically active relaxin in an amount effective to increase nitric oxide production in a cell of a blood vessel. Administration of an effective amount of a pharmaceutically active relaxin to an individual in need thereof increases nitric oxide production by at least about 10%, at least about 20%, at least about 30%, at least about 50%, or at least about 75% or more, compared to a suitable control. By administering relaxin to an individual, a cell of a blood vessel in the individual is contacted with relaxin, and nitric oxide production by the cell is increased. A suitable control may be a cell of a blood vessel not contacted with relaxin. Cells of a blood vessel that produce nitric oxide in response to admininstration of relaxin include, but are not limited to, endothelial cells and smooth muscle cells. Methods of measuring nitric oxide production are known in the art; any such method can be used to determine whether nitric oxide production is increased. See, e.g., Gupta et al. (1998) Hepatol. 28:926-931; Hill-Kapturczak et al. (1999) J. Am. Soc. Nephrol. 10:481-491; Lee et al. (2000) Microvasc. Res. 60:269-280; and Berkels et al. (2001) J. Appl. Physiol. 90:317-320. Production of nitric oxide by a cell of a blood vessel effects vasodilation, and is thus useful in treating a disease related to vasoconstriction.

In some embodiments, the invention provides methods of treating hypertension, comprising administering to a patient in need thereof a pharmaceutical formulation comprising pharmaceutically active relaxin in an amount effective to reduce hypertension. In some of these embodiments, methods are provided for treating renal hypertension, particularly by increasing renal vasodilation. In other embodiments, methods are provided for treating pulmonary hypertension.

In some embodiments, the invention provides methods for increasing renal vasodilation. Administration of an effective amount of a pharmaceutically active relaxin to an individual in need thereof increases renal vasodilation by at least about 10%, at least about 20%, at least about 30%, at least about 50%, or at least about 75% or more, compared to a suitable control. Parameters of and methods for assessing whether renal vasodilation is increased following relaxin administration are known in the art, including, but not limited to, measuring one or more of the following parameters: effective renal vascular resistance (ERVR); glomerular filtration rate (GFR); mean arterial pressure (MAP); effective renal plasma flow (ERPF); hematocrit; plasma osmolality; and plasma sodium concentration. Administration of relaxin results in one or more of the following: (1) an increase in values for GFR and ERPF; (2) a decrease in ERVR; (3) a decrease in hematocrit; (4) a decrease in plasma osmolality; (5) a decrease in plasma sodium concentration; and (6) a decrease in serum creatinine. A decrease in hematocrit, plasma osmolality, and plasma sodium concentration are indicative of general vasodilation, resulting in an increase in blood volume and a resulting dilution of red cell number and sodium concentration. Methods for measuring these parameters are well known in the art, and are described in Examples 1, 2, and 9.

In some embodiments, methods are provided for treating pulmonary hypertension. Example 5 provides data showing that administration of relaxin, e.g. by infusion over an extended time period, inhibits progression of pulmonary hypertension, as evidenced by an inhibition of collagen deposition in the vessel wall, and by ameliorative effects on compensatory right ventricular hypertrophy. Administration of an effective amount of a pharmaceutically active relaxin to an individual in need thereof reduces pulmonary hypertension by at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75% or more, when compared to a suitable control. Whether administration of relaxin reduces pulmonary hypertension can be determined using any method known in the art, including, but not limited to, measuring right ventricular pressure (RVP). Thus, the methods are effective to reduce right ventricular pressure by at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, or at least about 50% or more when compared to a suitable control.

In some embodiments, the invention provides methods for increasing or stimulating the expression of angiogenic cytokine production. Administration of an effective amount of a pharmaceutically active relaxin to an individual in need thereof increases angiogenic cytokine production by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 100% (or two-fold), at least about 2.5-fold, at least about 3-fold, at least about 5-fold, or at least about 10-fold or more, compared to a suitable control. Angiogenic factors include, but are not limited to, FGF, including acidic FGF, basic FGF; VEGF, including VEGF-A, VEGF-B, VEGF-C, and synthetic and recombinant forms which possess VEGF activity, specifically angiogenic activity; hepatocyte growth factor (HGF); platelet-derived growth factor (PDGF); placental growth factor; angiopoietin-1; proliferin; insulin-like growth factor-1; granulocyte colony stimulating factor (G-CSF); transforming growth factor-.alpha.; and interleukin-8. Whether angiogenic cytokine production is increased following relaxin administration can be assessed using any method known in the art, including, but not limited to, measuring angiogenic cytokine levels using PCR, as described in Example 3; using an enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), using antibody specific for individual angiogenic factors; and bioassays for specific individual angiogenic factors. See, e.g., Nicosia et al. (1994) Am. J. Pathol. 145:1023-1029; Morishita et al. (1999) Hypertension 33: 1379-1384; Koblizek et al. (1998) Curr. Biol. 8:529-532; Schraufnagel et al. (1992) J. Thorac. Cardiovasc. Surg. 104:1582-1588; and Yoshida et al. (1997) Mol. Cell. Biol. 17:4015-4023.

In some embodiments, the invention provides methods for increasing or stimulating the expression of angiogenic cytokine production. Administration of an effective amount of a pharmaceutically active relaxin to an individual in need thereof increases angiogenic cytokine production by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 100% (or two-fold), at least about 2.5-fold, at least about 3-fold, at least about 5-fold, or at least about 10-fold or more, compared to a suitable control. Angiogenic factors include, but are not limited to, fibroblast growth factor (FGF), including acidic FGF, basic FGF; VEGF, including VEGF-A, VEGF-B, VEGF-C, and synthetic and recombinant forms which possess VEGF activity, specifically angiogenic activity; hepatocyte growth factor (HGF); platelet-derived growth factor (PDGF); placental growth factor; angiopoietin-1; proliferin; insulin-like growth factor-1; granulocyte colony stimulating factor (G-CSF); transforming growth factor-.alpha.; and interleukin-8. Whether angiogenic cytokine production is increased following relaxin administration can be assessed using any method known in the art, including, but not limited to, measuring angiogenic cytokine levels using polymerase chain reaction (PCR), as described in Example 3; using an enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), using antibody specific for individual angiogenic factors; and bioassays for specific individual angiogenic factors. See, e.g., Nicosia et al. (1994) Am. J. Pathol. 145:1023-1029; Morishita et al. (1999)Hypertension 33: 1379-1384; Koblizek et al. (1998) Curr. Biol. 8:529-532; Schraufnagel et al. (1992) J. Thorac. Cardiovasc. Surg. 104:1582-1588; and Yoshida et al. (1997) Mol. Cell. Biol. 17:4015-4023.

Thus, the invention provides methods of treating an ischemic condition. Administration of an effective amount of pharmaceutically active relaxin results in an increase in blood supply to an ischemic tissue. Following administration of relaxin, blood supply (blood flow) to the ischemic tissue is increased by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, or at least about 100%, or more when compared to a suitable control. Whether the blood supply to an ischemic tissue is increased can be measured by any method known in the art, including, but not limited to, thermography; infrared recorder; transcutaneous PO₂, transcutaneous PCO₂, laser Doppler, Doppler waveform, ankle brachial index, pulse volume recording, toe pressure, duplex waveform, magnetic resonance imaging profile, isotope washout, and NAD/NADH fluorometry. Such methods are well known in the art and have been described in numerous publications, including, e.g., Lazarus et al. ((1994) Arch. Dermatol. 130:491) and references cited therein.

In some embodiments, methods are provided for promoting or enhancing wound healing. Administration of an effective amount of a pharmaceutically active relaxin to an individual in need thereof promotes wound healing by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, or at least about 100%, or more when compared to a suitable control, e.g., the amount of necrotic tissue in the wound is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, or at least about 100%, or more when compared to a suitable control. Whether administration of relaxin promotes wound healing can be determined using any method known in the art, including, but not limited to, the methods described in Example 4. For example, the amount of necrotic tissue can be measured; and/or histochemical evaluation of a tissue biopsy can be conducted to determine the presence of and/or to measure the amount of tissue necrosis.

In some embodiments, methods are provided for reducing angiotensin II (AngII)-mediated vasoconstriction. Administration of an effective amount of a pharmaceutically active relaxin to an individual in need thereof reduces AngII-mediated vasoconstriction by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, or at least about 100%, or more when compared to a suitable control. Whether administration of relaxin reduces AngII-mediated vasoconstriction can be determined using any method known in the art for measuring vasodilation.

In some embodiments, methods are provided for reducing ET-1-mediated vasoconstriction. Administration of an effective amount of a pharmaceutically active relaxin to an individual in need thereof reduces ET-1-mediated vasoconstriction by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, or at least about 100%, or more when compared to a suitable control. Whether administration of relaxin reduces ET-1-mediated vasoconstriction can be determined using any method known in the art for measuring vasodilation. In some embodiments, the methods comprise increasing endothelin type B receptor activation in a cell in a blood vessel by administering relaxin to the individual. Methods of determining whether endothelin type B receptor activation has been achieved are described in Example 2. Cells in a blood vessel that would be expected to be affected include, but are not limited to, endothelial cells, and smooth muscle cells.

Administration of relaxin in the hypoxic rat model of pulmonary hypertension described in Example 5 resulted in decreased extracellular matrix (ECM) synthesis in the vessel wall. Thus, in some embodiments, methods are provided for reducing ECM deposition in the vessel wall by at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, or at least about 50% or more when compared to a suitable control.

The methods of the present invention are suitable for treating an individual who has been diagnosed with a disease related to vasoconstriction, who is suspected of having a disease related to vasoconstriction, who is known to be susceptible and who is considered likely to develop a disease related to vasoconstriction, or who is considered likely to develop a recurrence of a previously treated disease relating to vasoconstriction.

Methods of treating diseases related to vasoconstriction can be enhanced by administering relaxin in combination with a known vasodilator and/or known angiogenic compounds. Methods of treating wounds can be enhanced by administering relaxin in combination with known vasodilator and/or angiogenic compounds.

RELAXIN FORMULATIONS

Relaxin formulations suitable for use in the methods of the invention are pharmaceutical formulations comprising a therapeutically effective amount of pharmaceutically active relaxin, and a pharmaceutically acceptable excipient. The formulation is preferably injectable and most preferably designed for intravenous injection.

Any known relaxin formulation can be used in the methods of the present invention, provided that the relaxin is pharmaceutically active. "Pharmaceutically active" relaxin is a form of relaxin which results in increased vasodilation and/or increased angiogenesis when administered to an individual.

Relaxin may be administered as a polypeptide, or as a polynucleotide comprising a sequence which encodes relaxin. Relaxin suitable for use in the methods of the present invention can be isolated from natural sources, may be chemically or enzymatically synthesized, or produced using standard recombinant techniques known in the art. Examples of methods of making recombinant relaxin are found in various publications, including, e.g., U.S. Pat. Nos. 4,835,251; 5,326,694; 5,320,953; 5,464,756; and 5,759,807.

Relaxin suitable for use includes, but is not limited to, human relaxin, recombinant human relaxin, relaxin derived from non-human mammals, such as porcine relaxin, and any of a variety of variants of relaxin known in the art. Relaxin, pharmaceutically active relaxin variants, and pharmaceutical formulations comprising relaxin are well known in the art. See, e.g., U.S. Pat. Nos. 5,451,572; 5,811,395; 5,945,402; 5,166,191; and 5,759,807, the contents of which are incorporated by reference in their entirety for their teachings relating to relaxin formulations, and for teachings relating to production of relaxin. In general, recombinant human relaxin (rhRLX) is identical in amino acid sequence to the naturally occurring product of the human H2 gene, consisting of an A chain of 24 amino acids and a B chain of 29 amino acids.

Relaxin can be administered to an individual in the form of a polynucleotide comprising a nucleotide sequence which encodes relaxin. Relaxin-encoding nucleotide sequences are known in the art, any of which can be used in the methods described herein. See, e.g. GenBank Accession Nos. AF135824; AF076971; NM_-- 006911; and NM_-- 005059. The relaxin polynucleotides and polypeptides of the present invention can be introduced into a cell by a gene delivery vehicle. Generally, gene delivery vehicles can encode either polypeptides or polynucleotides, such as antisense or ribozymes. The gene delivery vehicle may be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 1:185-193; and Kaplitt (1994) Nature Genetics 6:148-153). Gene therapy vehicles for delivery of constructs including a coding sequence of a polynucleotide of the invention can be administered either locally or systemically. These constructs can utilize viral or non-viral vector approaches. Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.

The present invention can employ recombinant retroviruses which are constructed to carry or express a selected nucleic acid molecule of interest. Retrovirus vectors that can be employed include those described in EP 415 731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5, 219,740; WO 93/11230; WO 93/10218; Vile and Hart (1993) Cancer Res. 53:3860-3864; Vile and Hart (1993) Cancer Res. 53:962-967; Ram et al. (1993) Cancer Res. 53:83-88; Takamiya et al. (1992) J. Neurosci. Res. 33:493-503; Baba et al. (1993) J. Neurosurg. 79:729-735; U.S. Pat. No. 4,777,127; and EP 345,242.

Packaging cell lines suitable for use with the above-described retroviral vector constructs may be readily prepared (see PCT publications WO 95/30763 and WO 92/05266), and used to create producer cell lines (also termed vector cell lines) for the production of recombinant vector particles. Within particularly preferred embodiments of the invention, packaging cell lines are made from human (such as HT1080 cells) or mink parent cell lines, thereby allowing production of recombinant retroviruses that can survive inactivation in human serum.

Gene delivery vehicles of the present invention can also employ parvovirus such as adeno-associated virus (AAV) vectors. Representative examples include the AAV vectors disclosed by Srivastava in WO 93/09239, Samulski et al. (1989) J. Vir. 63:3822-3828; Mendelson et al. (1988) Virol. 166:154-165; and Flotte et al. (1993) Proc. Natl. Acad. Sci. USA 90:10613-10617.

Also of interest are adenoviral vectors, e.g., those described by Berkner, Biotechniques (1988) 6:616-627; Rosenfeld et al.(1991) Science 252:431-434; WO 93/19191; Kolls et al. (1994) Proc. Natl. Acad. Sci. USA 91:215-219; Kass-Eisler et al. (1993) Proc. Natl. Acad. Sci. USA 90:11498-11502; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655.

Other gene delivery vehicles and methods may be employed, including polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example Curiel (1992) Hum. Gene Ther. 3:147-154; ligand linked DNA, for example see Wu (1989) J. Biol. Chem. 264:16985-16987; eukaryotic cell delivery vehicles cells; deposition of photopolymerized hydrogel materials; hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655; ionizing radiation as described in U.S. Pat. No. 5,206,152 and in WO 92/11033; nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip (1994) Mol. Cell Biol. 14:2411-2418, and in Woffendin (1994) Proc. Natl. Acad. Sci. 91:1581-1585.

Naked DNA may also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120, PCT Nos. WO 95/13796, WO 94/23697, and WO 91/14445, and EP No. 524 968.

Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al. (1994) Proc. Natl. Acad. Sci. USA 91:11581-11585. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Pat. No. 5,206,152 and PCT No. WO 92/11033.

In general, a daily dose of relaxin may be from about 0.1 to 500 .mu.g/kg of body weight per day, from about 6.0 to 200 .mu.g/kg, or from about 12 to 100 .mu.g/kg. In some embodiments, it is desirable to obtain a serum concentration of relaxin at or above about 1.0 ng/ml, from about 0.5 to about 50 ng/ml, from about 1 to about 20 ng/ml. For administration to a 70 kg person, a dosage may be in a range of from about 2 .mu.g to about 2 mg per day, from about 10 .mu.g to 500 .mu.g per day, or from about 50 .mu.g to about 100 .mu.g per day. The amount of relaxin administered will, of course, be dependent on the subject and the severity of the affliction, the manner and schedule of administration and the judgment of the prescribing physician.

In employing relaxin for treatment of diseases relating to vasoconstriction, any pharmaceutically acceptable mode of administration can be used. Relaxin can be administered either alone or in combination with other pharmaceutically acceptable excipients, including solid, semi-solid, liquid or aerosol dosage forms, such as, for example, tablets, capsules, powders, liquids, gels, suspensions, suppositories, aerosols or the like. Relaxin can also be administered in sustained or controlled release dosage forms (e.g., employing a slow release bioerodable delivery system), including depot injections, osmotic pumps (such as the Alzet implant made by Alza), pills, transdermal and transcutaneous (including electrotransport) patches, and the like, for prolonged administration at a predetermined rate, preferably in unit dosage forms suitable for single administration of precise dosages. The compositions will typically include a conventional pharmaceutical carrier or excipient and relaxin. In addition, these compositions may include other active agents (e.g., other angiogenic agents, other vasodilation-promoting agents), carriers, adjuvants, etc. Generally, depending on the intended mode of administration, the pharmaceutically acceptable composition will contain about 0.1% to 90%, about 0.5% to 50%, or about 1% to about 25%, by weight of relaxin, the remainder being suitable pharmaceutical excipients, carriers, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1995. The formulations of human relaxin described in U.S. Pat. No. 5,451,572, are non-limiting examples of suitable formulations which can be used in the methods of the present invention.

Parenteral administration is generally characterized by injection, either subcutaneously, intradermally, intramuscularly or intravenously, or subcutaneously. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, solubility enhancers, and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, cyclodextrins, and the like.

The percentage of relaxin contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the needs of the subject. However, percentages of active ingredient of 0.01% to 10% in solution are employable, and will be higher if the composition is a solid which will be subsequently diluted to the above percentages. In general, the composition will comprise 0.2-2% of the relaxin in solution.

Parenteral administration may employ the implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained. Various matrices (e.g., polymers, hydrophilic gels, and the like) for controlling the sustained release, and for progressively diminishing the rate of release of active agents such as relaxin are known in the art. See, U.S. Pat. No. 3,845,770 (describing elementary osmotic pumps); U.S. Pat. Nos. 3,995,651, 4,034,756 and 4,111,202 (describing miniature osmotic pumps); U.S. Pat. Nos. 4,320,759 and 4,449,983 (describing multichamber osmotic systems referred to as push-pull and push-melt osmotic pumps); and U.S. Pat. No. 5,023,088 (describing osmotic pumps patterned for the sequentially timed dispensing of various dosage units).

Drug release devices suitable for use in administering relaxin according to the methods of the invention may be based on any of a variety of modes of operation. For example, the drug release device can be based upon a diffusive system, a convective system, or an erodible system (e.g., an erosion-based system). For example, the drug release device can be an osmotic pump, an electroosmotic pump, a vapor pressure pump, or osmotic bursting matrix, e.g., where the drug is incorporated into a polymer and the polymer provides for release of drug formulation concomitant with degradation of a drug-impregnated polymeric material (e.g., a biodegradable, drug-impregnated polymeric material). In other embodiments, the drug release device is based upon an electrodiffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolytic system, etc.

Drug release devices based upon a mechanical or electromechanical infusion pump, are also suitable for use with the present invention. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and the like. In general, the present treatment methods can be accomplished using any of a variety of refillable, non-exchangeable pump systems. Osmotic pumps have been amply described in the literature. See, e.g., WO 97/27840; and U.S. Pat. Nos. 5,985,305 and 5,728,396.

Relaxin may be administered over a period of hours, days, weeks, or months, depending on several factors, including the severity of the disease being treated, whether a recurrence of the disease is considered likely, etc. The administration may be constant, e.g., constant infusion over a period of hours, days, weeks, months, etc. Alternatively, the administration may be intermittent, e.g., relaxin may be administered once a day over a period of days, once an hour over a period of hours, or any other such schedule as deemed suitable.

Formulations of relaxin may also be administered to the respiratory tract as a nasal or pulmonary inhalation aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose, or with other pharmaceutically acceptable excipients. In such a case, the particles of the formulation may advantageously have diameters of less than 50 micrometers, preferably less than 10 micrometers.

Claim 1 of 17 Claims

What is claimed is:

1. A method of treating hypertension, comprising administering to a patient in need thereof a pharmaceutical formulation comprising pharmaceutically active relaxin in an amount effective to reduce hypertension.

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