WO2003009871A2

WO2003009871A2 - Inhibitors of plasminogen activator inhibitor for decreasing body mass

Info

Publication number: WO2003009871A2
Application number: PCT/IB2002/003387
Authority: WO
Inventors: John Lucas; Deno Dialynas
Original assignee: Genset S.A.
Priority date: 2001-07-20
Filing date: 2002-07-18
Publication date: 2003-02-06
Also published as: WO2003009871A3; AU2002326069A1

Abstract

The present invention relates to the field of treating obesity related disorders. Obesity is a public health problem that is serious and widespread. Compounds, PAI Inhibitors, have been identified that reduces weight gain in animals. These compounds should be effective for reducing body mass and for treating obesity-related diseases and disorders. These obesity-related diseases and disorders include hyperlipidemias, atherosclerosis, diabetes, and hypertension.

Description

INHIBITORS OF PLASMINOGEN ACTIVATOR INHIBITOR FOR DECREASING BODY

MASS

FIELD OF THE INVENTION The present invention relates to the field of metabolic research, in particular the discovery of compounds effective for reducing body mass and useful for treating obesity-related diseases and disorders. The obesity-related diseases or disorders envisioned to be treated by the methods of the invention include, but are not limited to, hyperlipidemia, atherosclerosis, diabetes, and hypertension.

BACKGROUND OF THE INVENTION

Obesity is a public health problem that is serious, widespread, and increasing. In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634). Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643). Even modest weight loss ameliorates these associated conditions.

While still acknowledging that lifestyle factors including environment, diet, age and exercise play a role in obesity, twin studies, analyses of familial aggregation, and adoption studies all indicate that obesity is largely the result of genetic factors (Barsh et al (2000) Nature 404:644-651). hi agreement with these studies is the fact that an increasing number of obesity-related receptors are being identified. Some of the more extensively studied receptors include those encoding leptin (ob) and its receptor (db), pro-opiomelanocortin (Pome), melanocortin-4-receptor (Mc4r), agouti protein ( ), carboxypeptidase E (fat), 5-hyά oxytιyptanιine receptor 2C (Htr2c), nescient basic helix-loop-helix 2 (Nhlhl), prohormone convertase 1 EPCSKl), and tubby protein (tubby) (rev'd in Barsh et al (2000) Nature 404:644-651). Recently it was shown that particular carboxyl-terminal of the full-length apM-1 and Arcp30 compounds have unexpected effects in vitro and in vivo, including utility for weight reduction, prevention of weight gain, and control of blood glucose levels. The effects of Arcp30 administration in mammals also include reduction of elevated free fatty acid levels caused by administration of epinephrine, i.v. injection of "intralipid", or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, and weight reduction in mammals consuming a high fat high sucrose diet.

SUMMARY OF THE INVENTION

The instant invention is based on the use of Plasminogen Activator Inhibitors (PAI)

Inhibitors for weight reduction, prevention of weight gain, and control of blood glucose levels in humans and other mammals. In a preferred embodiment, the PAI Inhibitor is able to lower circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.

Further preferred are PAI Inhibitors demonstrating a greater than transient activity and/or have a sustained activity. Further preferred PAI Inhibitors are those that significantly stimulate muscle lipid or free fatty acid oxidation as compared to untreated cells. Further preferred PAI Inhibitors are those that cause C2C12 cells differentiated in the presence of said inhibitor to undergo at least 10%, 20%,

30%, 35%, or 40%) more oleate oxidation as compared to untreated differentiated cells.

Further preferred PAI Inhibitors are those that increase leptin uptake in liver cells (e.g. BPRCL mouse liver cells (ATCC CRL-2217)).

Further preferred PAI Inhibitors are those that significantly reduce the postprandial increase in plasma free fatty acids, particularly following a high fat meal.

Further preferred PAI Inhibitors are those that significantly reduce or eliminate ketone body production, particularly following a high fat meal. Further preferred PAI Inhibitors are those that increase glucose uptake in skeletal muscle cells.

Further preferred PAI Inhibitors are those that increase glucose uptake in adipose cells.

Further preferred PAI Inhibitors are those that increase glucose uptake in neuronal cells.

Further preferred PAI Inhibitors are those that increase glucose uptake in red blood cells. Further preferred PAI Inhibitors are those that increase glucose uptake in the brain.

Further preferred PAI Inhibitors are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal.

Further preferred PAI Inhibitors are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal. Further preferred PAI Inhibitors are those that improve insulin sensitivity.

In another aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said PAI Inhibitor described herein and, alternatively, a pharmaceutical or physiologically acceptable diluent.

In another aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said PAI Inhibitor described herein, insulin, and, alternatively, a pharmaceutical or physiologically acceptable diluent. h a another aspect, the invention features a method of reducing body mass comprising providing or administering to individuals in need of reducing body mass a pharmaceutical or physiologically acceptable composition described in the herein. In preferred embodiments, the identification of said individuals in need of reducing body mass to be treated with a pharmaceutical or physiologically acceptable composition comprises genotyping apMl single nucleotide polymorphisms (SNPs) or measuring apMl polypeptide or mRNA levels in clinical samples from said individuals. Preferably, said clinical samples are selected from the group consisting of plasma, urine, and saliva. Preferably, a PAI inhibitor of the present invention is administered to an individual with at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in blood, serum or plasma levels of the naturally proteolytically cleaved apMl as compared to healthy, non-obese patients.

In another aspect, the invention features a method of preventing or treating an obesity- related disease or disorder comprising providing or administering to an individual in need of such treatment a pharmaceutical or physiologically acceptable composition described herein. Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NBDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (EDDM or Type I diabetes). Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia. Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, AIDS- related weight loss, anorexia, and bulimia, preferred embodiments, said individual is a mammal, preferably a human. In a further aspect, any PAI inhibitor or other molecule or biological activity or disorder described herein may also be specifically excluded from the present invention. hi related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a PAI Inhibitor, wherein said biological response is selected from the group consisting of:

(a) lowering circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids;

(b) lowering circulating (either blood, serum or plasma) levels (concentration) of glucose;

(c) lowering circulating (either blood, serum or plasma) levels (concentration) of triglycerides;

(d) stimulating muscle lipid or free fatty acid oxidation;

(e) increasing leptin uptake in the liver or liver cells;

(f) reducing the postprandial increase in plasma free fatty acids, particularly following a high fat meal; and, (g) reducing or eliminating ketone body production, particularly following a high fat meal;

(h) increasing tissue sensitivity to insulin, particularly muscle, adipose, liver or brain. In further preferred embodiments, the present invention of a pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Noninsulin Dependent Diabetes Mellitus (NTDDM, Type II diabetes).

In further preferred embodiments, the present invention of a pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (TDDM, Type I diabetes).

In further preferred embodiments, the present invention of a pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Noninsulin Dependent Diabetes Mellitus (NTDDM, Type II diabetes). In further preferred embodiments, the present invention of a pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes).

In a further preferred embodiment, the present invention may be used in complementary therapy of NTDDM patients to improve their weight or glucose control in combination with insulin, an insulin secretagogue or an insulin sensitizing agent. Preferably, the insulin secretagogue is 1,1- dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from toϊbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.

Preferably, the insulin sensitizing agent is selected from metformin, ciglitazone, troglitazone and pioglitazone. The present invention further provides a method of improving the body weight or glucose control of NTDDM patients with a PAI inhibitor alone, without insulin, an insulin secretagogue or an insulin sensitizing agent.

In a further preferred embodiment, the present invention may be used in complementary therapy of TDDM patients to improve their weight or glucose control in combination with insulin, an insulin secretagogue or an insulin sensitizing agent. Preferably, the insulin secretagogue is 1,1- dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.

Preferably, the insulin sensitizing agent is selected from metformin, ciglitazone, troglitazone and pioglitazone. The present invention further provides a method of improving the body weight or glucose control of TDDM patients alone, without insulin, an insulin secretagogue or an insulin sensitizing agent.

In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with insulin, the insulin secretagogue or insulin sensitizing agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially

(either before or after the secretagogue or either before or after the sensitizing agent). Accordingly, the present invention further provides for a composition of a pharmaceutical or physiologically acceptable composition of a PAI inhibitor and insulin, an insulin secretagogue or insulin sensitizing agent as a combined preparation for simultaneous, separate or sequential use for the improvement of body weight or glucose control in NTDDM or TDDM patients.

In further preferred embodiments, the present invention of a pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin sensitizer.

In further preferred embodiments, the present invention of a pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Noninsulin Dependent Diabetes Mellitus (NTDDM, Type Tl diabetes).

In further preferred embodiments, the present invention of a pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Insulin Dependent Diabetes Mellitus (TDDM, Type I diabetes).

In another aspect, the invention features a method of making the PAI Inhibitor described herein.

In another aspect, the invention features a use of the PAI Inhibitor described herein for treatment of obesity-related diseases and disorders and/or reducing or increasing body mass.

Preferably, said obesity-related diseases and disorders are selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NTDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (TDDM or Type I diabetes). Diabetes- related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia. In another aspect, the invention features a use of a PAI inhibitor described herein for the preparation of a medicament for the treatment of obesity-related diseases and disorders and/or for reducing body mass. Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NTDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (TDDM or Type I diabetes). Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia. Further included in the invention is a method of treating cachexia, wasting, ATDS-related weight loss, anorexia, and bulimia with a PAI Inhibitor. In preferred embodiments, an individual treated using said methods is a mammal, preferably a human.

In another aspect, the invention features methods of reducing body weight comprising providing to an individual said pharmaceutical or physiologically acceptable composition described herein. Where the reduction of body weight is practiced for cosmetic purposes, the individual has a BMI of at least 20 and no more than 25. In embodiments for the treatment of obesity, the individual may have a BMI of at least 20. One embodiment for the treatment of obesity provides for the treatment of individuals with BMI values of at least 25. Another embodiment for the treatment of obesity provides for the treatment of individuals with BMI values of at least 30. Yet another embodiment provides for the treatment of individuals with BMI values of at least 40. Alternatively, for increasing the body weight of an individual, the BMI value should be at least 15 and no more than 20.

In a preferred embodiment, the invention features a pharmaceutical or physiologically acceptable composition described herein for reducing body mass and/or for treatment or prevention of obesity-related diseases or disorders. Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NTDDM, or Type Tl diabetes) and Insulin Dependent Diabetes Mellitus (TDDM or Type I diabetes). Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia. Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, anorexia, and bulimia. In preferred embodiments, said individual is a mammal, preferably a human. In preferred embodiments, the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping apMl (Adipocyte most abundant transcript 1, GenBank Accession No. XM_003191) single nucleotide polymorphisms (SNPs) or measuring apMl mRNA levels in clinical samples from said individuals. Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.

In another aspect, the invention features a pharmaceutical or physiologically acceptable composition described herein for reducing body weight for cosmetic reasons.

In another aspect, the PAI Inhibitors of the invention are used in methods treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described herein. The PAI Inhibitor may also be used to enhance physical performance during work or exercise or enhance a feeling of general well-being. Physical performance activities include walking, running, jumping, lifting and/or climbing.

The PAI Inhibitor or antagonists thereof may also be used to treat dyslexia, attention-deficit disorder (ADD), attention-deficit/hyperactivity disorder (ADHD), and psychiatric disorders such as schizophrenia by modulating fatty acid metabolism, more specifically, the production of certain long-chain polyunsaturated fatty acids.

It is expressly considered that the PAI Inhibitor of the invention may be provided alone or in combination with other pharmaceutically or physiologically acceptable compounds. Other compounds useful for the treatment of obesity and other diseases and disorders are currently well- known in the art.

In a preferred embodiment, the PAI Inhibitors are useful for, and used in, the treatment of insulin resistance and diabetes using methods described herein and known in the art. More particularly, a preferred embodiments relates to process for the therapeutic modification and regulation of glucose metabolism in an animal or human subject, which comprises administering to a subject in need of treatment (alternatively on a timed daily basis) a PAI Inhibitor (or polynucleotide encoding said compound) in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject, preferably to normal levels.

Further preferred embodiments relate to methods for the prophylaxis or treatment of diabetes comprising administering to a subject in need of treatment (alternatively on a timed daily basis) a PAI Inhibitor (or polynucleotide encoding said compound) in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject.

In a preferred aspect of the methods above and disclosed herein, the amount of PAI Inhibitor administered to an individual is sufficient or effective to bring circulating (blood, serum, or plasma) levels (concentration) of apMl (naturally cleaved or full length) to their normal levels (levels in non- obese individuals). "Normal levels" may be specified as the total concentration of all circulating apMl or the concentration of all circulating proteolytically cleaved apMl only.

Although not binding oneself to a particular mechanism of action, polypeptides involved in metabolism and obesity related disorders and activities as described herein must be proteolytically cleaved for full activity. A preferred polypeptide that must be cleaved for full activity is apMl. By inhibiting an inhibitor of the proteolytic processing, proteolytic cleavage and activation of apMl and other metabolic related polypeptides become more efficient thereby increasing their activity. Another method of increasing this proteolytic efficiency would be to increase the level or concentration of the protease that cleaves the metabolic related polypeptides such as apMl. Preferred proteases that can be substituted for the PAI inhibitors in the methods of the present invention include: tissue plasminogen activator (t-PA) and variants thereof such as Monteplase is a modified tissue type-plasminogen activator (t-PA) constructed by substituting only one amino acid in the epidermal growth factor domain (Cys84~>Ser), TNK-tissue plasminogen activator (TNK-t-

PA), a bioengineered variant of tissue-type plasminogen activator (t-PA), that has a longer half-life than t-PA because the glycosylation site at amino acid 117 (Nl 17Q, abbreviated N) has been shifted to amino acid 103 (T103N, abbreviated T) and is resistant to inactivation by plasminogen activator inhibitor 1 because of a tetra-alanine substitution in the protease domain

(K296A/H297A/R298A/R299A, abbreviated K); Reteplase, a non-glycosylated deletion mutant of wild-type human t-PA which contains only kringle 2 and the protease domain but lacks its kringle 1 and the finger and growth factor domains (the structural changes in reteplase translate into a decreased fibrin binding, a lower affinity to endothelial and liver cells resulting in an extended half- life), Lanoteplase, a deletion mutant of t-PA with a half-life that is circa 10 times greater than alteplase, YM866 is another mutant of t-PA in which the aminoacids 92 to 173 of kringle 1 were deleted and arginine 275 replaced by glutamic acid which confers a longer half-life to the mutant; uPA (urokmase-type plasminogen activator), or guanidinobenzoatase (GB). Additional proteases include trypsin, plasmin, thrombin, adipsin, Clr and matrix metalloproteases such as MMP-1,-2, and -9. These or other proteases that cleave metabolic proteins may be used alone or in combination with a metabolic polypeptide such as apMl, a PAI Inhibitor of the present invention or other appropriate compound(s) (e.g., insulin). Further included in the invention are variants of the above proteases with improved characteristics such as stability and increased half-life.

DETAILED DISCLOSURE OF THE INVENTION

Before describing the invention in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used to describe the invention herein.

Without being limited by theory, the compounds/compounds of the invention are capable of modulating the partitioning of dietary lipids between the liver and peripheral tissues, and are thus believed to treat "diseases involving the partitioning of dietary lipids between the liver and peripheral tissues. " The term "peripheral tissues" is meant to include muscle and adipose tissue. In preferred embodiments, the compounds/compounds of the invention partition the dietary lipids toward the muscle. In alternative preferred embodiments, the dietary lipids are partitioned toward the adipose tissue. In other preferred embodiments, the dietary lipids are partitioned toward the liver. In yet other preferred embodiments, the compounds/compounds of the invention increase or decrease the oxidation of dietary lipids, preferably free fatty acids (FFA) by the muscle. Dietary lipids include, but are not limited to triglycerides and free fatty acids.

Preferred diseases believed to involve the partitioning of dietary lipids include obesity and obesity-related diseases and disorders such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NTDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (TDDM or Type I diabetes). Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia. Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, ATDS-related weight loss, anorexia, and bulimia.

The term " PAI inhibitor" as used herein refers to inhibition of PAI' s inhibition of protease activity, preferably serine or cysteine protease activity, whereby PAI inhibits the proteolytic cleavage of a polypeptide by said protease. Thus, inhibition of PAI would have the result of increasing protease activity, preferably serine or cysteine protease activity. The inhibitors of PAI may bind and inhibit the PAI polypeptide (e.g., antibody or organic compound), may proteolytically cleave or degrade PAI or target PAI for degradation (activated protein C (APC)), inhibit the transcription of PAI mRNA, inhibit PAI mRNA translation, inhibit PAI protein transport, inhibit PAI protein processing/modification, or act in another manner. Embodiments of the present invention may be limited to one or more of these activities/mechanisms. Embodiments of the present invention may also specifically exclude one or more of these activities/mechanisms. Inhibitors may be specified in terms of the degree of inhibition of PAI activity (as described above). Preferred inhibitors reduce PAI activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Preferably the PAI inhibitor is an inhibitor of Plasminogen activator inhibitor type I. A PAI inhibited by the PAI inhibitor may be specified as any of the Plasminogen activator inhibitors disclosed herein, preferably Plasminogen activator inhibitor type I, or elsewhere known in the art. Moreover, a PAI inhibited by the inhibitor may be specifically excluded from the present invention. Moreover, any PAI inhibitor of the present invention or known elsewhere in the art may be specifically excluded from the present invention.

The term "Plasminogen activator inhibitor" or "PAI" preferably refers to Plasminogen activator inhibitor type I (GenBank accession P05121) and may be specifically limited thereto. Alternatively, a different Plasminogen activator inhibitor or Serpin may be specifically included or excluded from the present invention. These PAIs and Serpins include but are not limited to, those disclosed herein, of GenBank accessions: AAH10860, XP_051249, XP_051248, XP_004828, XP_051250, P05120in: PCT applications WO00052160A1, WO00138534A2, WO09940183A1, WO09833890A1, WOH6324A2, WO09816643A1, and WO09405804A1; and U.S. Patents US06013448, US05929210, US05422090, US05747645, US05444153, or US04923807.

The terms "comprising", "consisting of and "consisting essentially of are defined according to their standard meaning. A defined meaning set forth in the M.P.E.P. controls over a defined meaning in the art and a defined meaning set forth in controlling Federal Circuit case law controls over a meaning set forth in the M.P.E.P. With this in mind, the terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term.

The term "obesity" as used herein is defined in the WHO classifications of weight

(Kopelman (2000) Nature 404:635643). BMI is body mass index and is kg/m². Underweight individuals have a BMI less than 18.5 (thin); a healthy BMI is 18.5-24.9 (normal); grade 1 overweight BMIs range from 25.0 to 29.9 (overweight); the grade 2 overweight BMI is 30.0-39.0

(obesity); grade 3 overweight is greater than or equal to 40.0 BMI (morbid obesity). Waist circumference can also be used to indicate a risk of metabolic complications where in men a circumference of greater than or equal to 94 cm indicates an increased risk, and greater than or equal to 102 cm indicates a substantially increased risk. For women, a waist circumference greater than or equal to 88 cm indicates an increased risk, and greater than or equal to 88 cm indicates a substantially increased risk. The waist circumference is measured in cm at midpoint between lower border of ribs and upper border of the pelvis. Other measures of obesity include, but are not limited to, skinfold thickness which is a measurement in cm of skinfold thickness using calipers, and bioimpedance, which is based on the principle that lean mass conducts current better than fat mass because it is primarily an electrolyte solution; measurement of resistance to a weak current (impedance) applied across extremities provides an estimate of body fat using an empirically derived equation.

The term "diabetes" as used herein is intended to encompass the usual diagnosis of diabetes made from any of the methods included, but not limited to, the following list: symptoms of diabetes (eg. polyuria, polydipsia, polyphagia) plus casual plasma glucose levels of greater than or equal to 200 mg/dl, wherein casual plasma glucose is defined any time of the day regardless of the timing of meal or drink consumption; 8 hour fasting plasma glucose levels of less than or equal to 126 mg/dl; and plasma glucose levels of greater than or equal to 200 mg/dl 2 hours following oral administration of 75 g anhydrous glucose dissolved in water.

The term "impaired glucose tolerance (IGT)" as used herein is intended to indicate that condition associated with insulin-resistance that is intermediate between frank, NTDDM and normal glucose tolerance (NGT). A high percentage of the IGT population is known to progress to NTDDM relative to persons with normal glucose tolerance (Sad et al., New Engl J Med 1988; 319:1500-6 which disclosure is hereby incorporated by reference in its entirety). Thus, by providing therapeutics and methods for reducing or preventing IGT, i.e., for normalizing insulin resistance, the progression to NTDDM can be delayed or prevented. IGT is diagnosed by a procedure wherein an affected person's postprandial glucose response is determined to be abnormal as assessed by 2-hour postprandial plasma glucose levels. In this test, a measured amount of glucose is given to the patient and blood glucose levels measured regular intervals, usually every half hour for the first two hours and every hour thereafter. In a "normal" or non-IGT individual, glucose levels rise during the first two hours to a level less than 140 mg/dl and then drop rapidly. In an IGT individual, the blood glucose levels are higher and the drop-off level is at a slower rate.

The term "Insulin-Resistance Syndrome" as used herein is intended to encompass the cluster of abnormalities resulting from an attempt to compensate for insulin resistance that sets in motion a series of events that play an important role in the development of both hypertension and coronary artery disease (CAD), such as premature atherosclerotic vascular disease. Increased plasma triglyceride and decreased HDL-cholesterol concentrations, conditions that are known to be associated with CAD, have also been reported to be associated with insulin resistance. Thus, by providing therapeutics and methods for reducing or preventing insulin resistance, the invention provides methods for reducing and/or preventing the appearance of insulin-resistance syndrome.

The term "polycystic ovary syndrome (PCOS)" as used herein is intended to designate that etiologically unassigned disorder of premenopausal women, affecting 5-10% of this population, characterized by hyperandrogenism, chronic anovulation, defects in insulin action, insulin secretion, ovarian steroidogenesis and fibrinolysis. Women with PCOS frequently are insulin resistant and at increased risk to develop glucose intolerance or NTDDM in the third and fourth decades of life

(Dunaif et al. (1996) J Clin Endocrinol Metab 81:3299 which disclosure is hereby incorporated by reference in its entirety). Hyperandrogenism also is a feature of a variety of diverse insulin-resistant states, from the type A syndrome, through leprechaunism and lipoatrophic diabetes, to the type B syndrome, when these conditions occur in premenopausal women. It has been suggested that hyperinsulinemia per se causes hyperandrogenism. Insulin-sensitizing agents, e.g., troglitazone, have been shown to be effective in PCOS and that, in particular, the defects in insulin action, insulin secretion, ovarian steroidogenosis and fibrinolysis are improved (Ehrman et al. (1997) J Clin Invest 100: 1230 which disclosure is hereby incorporated by reference in its entirety), such as in insulin- resistant humans. The term "insulin resistance" as used herein is intended to encompass the usual diagnosis of insulin resistance made by any of a number of methods, such as the intravenous glucose tolerance test or measurement of the fasting insulin level. It is well known that there is an excellent correlation between the height of the fasting insulin level and the degree of insulin resistance. Therefore, one could use elevated fasting insulin levels as a surrogate marker for insulin resistance for the purpose of identifying which normal glucose tolerance (NGT) individuals have insulin resistance. Another way to do this is to follow the approach as disclosed in The New England Journal of Medicine, No. 3, pp. 1188 (1995) (which disclosure is hereby incorporated by reference in its entirety), i.e. to select obese subjects as an initial criterion for entry into the treatment group. Some obese subjects have impaired glucose tolerance (IGT) while others have normal glucose tolerance (NGT). Since essentially all obese subjects are insulin resistant, i.e. even the NGT obese subjects are insulin resistant and have fasting hyperinsulinemia. Therefore, the target of the treatment according to the present invention can be defined as NGT individuals who are obese or who have fasting hyperinsulinemia, or who have both.

A diagnosis of insulin resistance can also be made using the euglycemic glucose clamp test.

This test involves the simultaneous administration of a constant insulin infusion and a variable rate glucose infusion. During the test, which lasts 3-4 hours, the plasma glucose concentration is kept constant at euglycemic levels by measuring the glucose level every 5-10 minutes and then adjusting the variable rate glucose infusion to keep the plasma glucose level unchanged. Under these circumstances, the rate of glucose entry into the bloodstream is equal to the overall rate of glucose disposal in the body. The difference between the rate of glucose disposal in the basal state (no insulin infusion) and the insulin infused state, represents insulin mediated glucose uptake. In normal individuals, insulin causes brisk and large increase in overall body glucose disposal, whereas in NTDDM subjects, this effect of insulin is greatly blunted, and is only 20-30%) of normal. In insulin resistant subjects with either IGT or NGT, the rate of insulin stimulated glucose disposal is about halfway between normal and NTDDM. For example, at a steady state plasma insulin concentration of about 100 μU/ml (a physiologic level) the glucose disposal rate in normal subjects is about 7 mg/kg/min. In NTDDM subjects, it is about 2.5mg/kg/min., and in patients with IGT (or insulin resistant subjects with NGT) it is about 4-5 mg/kg/min. This is a highly reproducible and precise test, and can distinguish patients within these categories. It is also known that as subjects become more insulin resistant, the fasting insulin level rises. There is an excellent positive correlation between the height of the fasting insulin level and the magnitude of the insulin resistance as measured by euglycemic glucose clamp tests and, therefore, this provides the rationale for using fasting insulin levels as a surrogate measure of insulin resistance.

The term "agent acting on the partitioning of dietary lipids between the liver and peripheral tissues" refers to a compound that modulates the partitioning of dietary lipids between the liver and the peripheral tissues as previously described. Preferably, the agent increases or decreases the oxidation of dietary lipids, preferably free fatty acids (FFA) by the muscle. Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an obesity-related disease or disorder such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NTDDM, or Type II diabetes) and Insulin Dependent Diabetes

Mellitus (TDDM or Type I diabetes). Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia. Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, anorexia, and bulimia. The terms "response to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues" refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacoMnetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual. The terms "side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues" refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors. "Side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues " can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.

The term "cosmetic treatments" is meant to include treatments with compounds or compounds of the invention that increase or decrease the body mass of an individual where the individual is not clinically obese or clinically thin. Thus, these individuals have a body mass index (BMI) below the cut-off for clinical obesity (e.g. below 25 kg/m²) and above the cut-off for clinical thinness (e.g. above 18.5 kg/m²). In addition, these individuals are preferably healthy (e.g. do not have an obesity-related disease or disorder of the invention). "Cosmetic treatments" are also meant to encompass, in some circumstances, more localized increases in adipose tissue, for example, gains or losses specifically around the waist or hips, or around the hips and thighs, for example. These localized gains or losses of adipose tissue can be identified by increases or decreases in waist or hip size, for example.

The term "preventing" as used herein, refers to administering a compound prior to the onset of clinical symptoms of a disease or condition so as to prevent a physical manifestation of aberrations associated with obesity or other disorder described herein. Alternatively, the term "preventing" can also be used to signify the reduction, or severity, of clinical symptoms associated with a disease or condition.

The term "treating" as used herein refers to administering a compound after the onset of clinical symptoms.

The term "in need of treatment" as used herein refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, etc in the case of humans; veterinarian in the case of animals, including non-human mammals) that an individual or animal requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver ' s expertise, but that include the knowledge that the individual or animal is ill, or will be ill, as the result of a condition that is treatable by the compounds of the invention. The term "perceives a need for treatment" refers to a sub-clinical determination that an individual desires to reduce weight for cosmetic reasons as discussed under "cosmetic treatment" above. The term "perceives a need for treatment" in other embodiments can refer to the decision that an owner of an animal makes for cosmetic treatment of the animal. The term "individual" or "patient" as used herein refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans. The term may specify male or female or both, or exclude male or female. The term "non-human animal" refers to any non-human vertebrate, including birds and more usually mammals, preferably primates, animals such as swine, goats, sheep, donkeys, horses, cats, dogs, rabbits or rodents, more preferably rats or mice. Both the terms "animal" and "mammal" expressly embrace human subjects unless preceded with the term "non-human".

The instant invention encompasses the use of PAI Inhibitors in the partitioning of free fatty acid (FFA) and as an important new tool to control energy homeostasis. Of the tissues that can significantly remove lipids from circulation and cause FFA oxidation, muscle is quantitatively the most important. PAI Inhibitors are a unique and novel pharmacological tool that controls body weight without interfering with food intake.

Optionally, "obesity-related activity" can be selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity, or an activity within one of these categories.

By "lipid partitioning" activity is meant the ability to effect the location of dietary lipids among the major tissue groups including, adipose tissue, liver, and muscle. By "lipid metabolism" activity is meant the ability to influence the metabolism of lipids. By "insulin-like" activity is meant the ability of PAI Inhibitor to modulate the levels of glucose in the plasma.

The term "significantly greater " as used herein refers to a comparison of the activity of a

PAI Inhibitor in an obesity-related assay compared with untreated cells or animals in the same assay. "Significantly" as it is used herein, means statistically significant as it is typically determined by those of ordinary skill in the art. For example, data are typically calculated as a mean + SEM, and a p-value < 0.05 is considered statistically significant. Statistical analysis is typically done using either the unpaired Student's t test or the paired Student's t test, as appropriate in each study.

Examples of a significant change in activity as a result of the presence of a PAI Inhibitor of the invention compared to the absence of a PAI Inhibitor or presence of a negative control include an increase or a decrease in a given parameter of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,

45%), 50%, 55%, 60%), 65%, 70%, or 75%. One or more, but not necessarily all, of the measurable parameters will change significantly in the presence of PAI Inhibitor as compared to in the absence of a PAI Inhibitor. Representative "obesity-related assays" include, but are not limited to, methods of measuring the postprandial response, methods of measuring free fatty acid oxidation, and methods of measuring weight modulation. In preferred embodiments, the post-prandial response is measured in non-human animals, preferably mice. In preferred embodiments changes in dietary lipids are measured, preferably free fatty acids and/or triglycerides. In other embodiments, other physiologic parameters are measured including, but not limited to, levels of glucose, insulin, and leptin. In yet other preferred embodiments weight modulation is measured in human or non-human animals, preferably rodents (rats or mice), primates, canines, felines or porcines on a high fat/sucrose diet.

Optionally, "obesity-related activity" includes other activities not specifically identified herein. In general, "measurable parameters" relating to obesity and the field of metabolic research can be selected from the group consisting of free fatty acid levels, free fatty acid oxidation, triglyceride levels, glucose levels, insulin levels, leptin levels, food intake, weight, leptin and lipoprotein binding, uptake and degradation and LSR expression.

In these obesity-related assays, preferred PAI Inhibitors of the invention would cause a significant change in at least one of the measurable parameters selected from the group consisting of post-prandial lipidemia, free fatty acid levels, triglyceride levels, glucose levels, free fatty acid oxidation, and weight. The invention is drawn, inter alia, to isolated, purified PAI Inhibitor. PAI Inhibitors of the invention are useful for reducing or increasing (using antagonists of PAI Inhibitors) body weight either as a cosmetic treatment or for treatment or prevention of obesity-related diseases and disorders. When used for cosmetic treatments, or for the treatment or prevention of obesity-related diseases, disorders, or conditions, one or more PAI Inhibitor can be provided to a subject. Thus, various PAI Inhibitors can be combined into a "cocktail" for use in the various treatment regimens. The PAI Inhibitors of the present invention can also be used in combination with other compounds to treat a disorder or cause/induce a biological activity described herein, e.g., insulin.

Inhibitors of Plasminogen Activator Inhibitor PAI Inhibitors can be divided into classes based on chemical structure and mode of action.

Small molecules that decrease the expression or secretion of PAI include: select compounds with benzothiophene, triphenyl ethylene, and benzopyran base structures, vitamins C and E, TNF-alpha inhibitors, TGF-beta family inhibitors, ceramide, anthracyclins, Angiotensin Converting Enzyme (ACE) inhibitors, Angiotensin Receptor antagonists, cAMP agonists, Rho inhibitors, statins, EGF receptor antagonists, aldosterone inhibitors, estrogen, serotonin receptor inhibitors, vastatin/ oxysterol-type, and PPAR-alpha and gamma agonists. Peptides or proteins that decrease expression or secretion of PAI include: adrenomedullin, transcriptional inhibitors of PAI, and their agonists. Small molecules that act as competitive inhibitors of PAI include: dextran sulfate and compounds with diketopiperazine, benzil, benzophenone, substituted and unsubstituted biaryl, benzyl ether, and thioether base structures. Peptides or proteins that directly contact PAI to inhibit its activity include the competitive inhibitors elastase and peptides derived from Sp-40,40, Alpha-1 Acid glycoprotein, and vitronectin, as well as the noncompetitive inhibitor Matrix MetalloProteinase (MMP)-3. PAI- specific antibodies or PAI-specific antibody fragments may also be used as competitive PAI inhibitors. PAI message is contacted and targeted for degradation by antisense polynucleotides complementary to PAI message and by PAI RNA-Binding Protein (PATRBP)-l .

Preferred inhibitors of PAI include those that interfere with PAI expression or secretion. Each PAI inhibitor of this class can be additionally excluded from the invention individually or as a group.

Selected benzothiophene-based PAI inhibitors are made as disclosed in U.S. Patents 4133814, 4418068, and 4380635, which disclosures are hereby incoφorated by reference in their entireties. In general, the process starts with benzo(b)-thiophene having a 6-hydroxyl group and a 2- (4-hydroxyphenyl) group. Protection, acylation, and deprotection of this compound are described in the above disclosures. Preferred substituent and functional groups are disclosed herein (Benzothiophene Structure A) and in U.S. Patent 5731328, disclosure of which is hereby incorporated by reference in its entirety. Benzothiophene Structures A and B show exemplary benzothiophene-based inhibitors. Synthesis of selected triphenyl ethylene-based PAI inhibitors is disclosed in U.S. Patent

5047431, disclosure of which is hereby incorporated by reference in its entirety. Preferred substituent and functional groups are disclosed herein (Triphenyl ethylene Structure C) in U.S. Patent 5792798, which is also incorporated by reference in its entirety. Particularly preferred is (E)- l-[4'-(2-dimethylaminoethoxy) phenyl]-l-(3-hydroxyphenyl)-2-phenyl-but-l-ene citrate. Triphenyl ethylene Structure C shows exemplary triphenyl ethylene-based inhibitors.

Benzopyran-based inhibitors are made as disclosed in PCT W09310741 and U.S. Patent 5446061, which disclosures are hereby incorporated by reference. Preferred substituent and functional groups are disclosed herein (Benzopyran Structure D) and in U.S. Patent 5980138, disclosure of which is hereby incoφorated by reference in its entirety. Particularly preferred is 2-[4- [2-( 1 -piperidino) ethoxyjphenyl] -3 -(4-hydroxyphenyl)-4-methyl-7-hydroxy-2H- 1 -benzopyran. Benzopyran Structure D shows an exemplary benzopyran-based PAI inhibitor.

The activity of benzothiophene, triphenyl ethylene, and benzopyran PAI inhibitors may be tested on human umbilical vein endothelial cell (HUVEC) cultures as described in U.S. Patents 5731328, 5792798, and 5980938. Briefly, HUVEC cells are cultured in standard endothelial cell growth medium and treated with InM IL-1 beta and a given dose of any of the above inhibitors overnight. Secreted PAI-1 was detected using the Imubind Plasma PAI-1 ELISA (American Diagnostic Inc. #822/1 S). Each of these compounds has been shown to be very effective at inhibiting PAI secretion from endothelial cells in the 0.5 to 10 nM range.

Vitamins C and E reduce serum levels of PAI. PAI expression increases upon exposure to reactive oxygen species. Therefore, vitamins C and E may exert an effect on PAI via their antioxidant properties. Vitamin C at 500mg-2g/day and Vitamin E at about 5001.UVday are effective to reduce serum PAI levels and activity. These compounds are readily available to the public and may be used alone or in combination with other PAI inhibitors. Vitamins C and E, and any other compound disclosed herein, may be excluded from the present invention.

Tumor Necrosis Factor (TNF) -alpha signaling increases PAI expression. Therefore, preferred PAI inhibitors are antagonists of TNF-alpha signaling. Each antagonist of TNF-alpha signaling can additionally be excluded from the invention individually or as a class. Daunorubicin and other anthracycline derivatives attenuate basal PAI expression, as well as PAI expression in response to TNF-alpha. Anthracyclines may decrease PAI expression by increasing ceramide generation. The structure of daunorubicin is disclosed herein. Methods of making and administering daunorubicin are described in U.S. Patents 3875010 and 6087340, disclosures of which are hereby incoφorated by reference in their entireties. HMG-Co A reductase antagonists also decrease both constitutive and TNF-alpha-induced PAI expression. Examples of HMG-CoA reductase antagonists include statins, particularly atorvastatin and fluvastatin. Atorvastatin and fluvastatin are effective inhibitors of PAI expression in cultured endothelial cells at a concentration of 2uM and typical dosages for humans range from 5-80mg/day. Fluvastatin synthesis is described in U.S. Patent 5356896, disclosure of which is hereby incoφorated by reference in its entirety. Structures of preferred statin molecules are disclosed herein. Further included inhibitors of PAI expression are soluble polypeptide fragments comprising the ligand-binding site of the TNF-alpha receptor, neutralizing antibodies against TNF-alpha or its receptor, antigen-binding fragments thereof, and other compounds that block TNF-alpha binding to the TNF-alpha receptor. Additionally included TNF-alpha antagonists include antisense polynucleotides complementary to the polynucleotide sequence of TNF-alpha or TNF-alpha receptor.

Factors belonging to the Transforming Growth Factor (TGF)-beta superfamily increase PAI expression. Such factors include TGF-beta, Osteogenic Protein (OP)-l, and BMP. Therefore, preferred PAI inhibitors of the invention are antagonists of TGF-beta signaling. Each antagonist of signaling by the TGF-beta superfamily can additionally be excluded from the invention individually or as a class. Preferred antagonists of TGF-beta signaling include serum glycoproteins such as prolactin and fetuin and TGF-beta inhibiting fragments thereof. Fetuin directly binds and inhibits TGF-beta family members. The IC₅₀ of fetuin for TGF-beta is 1-2 uM and a dosage of about 5- 40mg/kg is effective, depending on the individual case. The full-length sequence of fetuin follows: MKSLVLLLCLAQLWGCHSAPHGPGLΓYRQPNCDDPETEEAALVAΓDYΓNQNLPWGYKHTLN QROEVKVWPQQPSGELFETEIDTLETTCHVLDPTPVARCSVRQLKEHAVEGDCDFQLLKLDG

KFSVVYAKCDSSPDSAEDVRKVCQDCPLLAPLNDTRVVHAAKAALAAFNAQNNGSNFQLE EISRAQLVPLPPSTYVEFTVSGTDCVAKEATEAAKCNLLAEKQYGFCKATLSEKLGGAEVA VTCTVFQTQPVTSQPQPEGANEAVPTPWDPDAPPSPPLGAPGLPPAGSPPDSHVLLAAPPG HQLHPvAHYDLRHTFMGVVSLGSPSGEVSHPRKTRTVVQPSVGAAAGPV PCPGRIRHFKV . Pharmaceutical compositions of the polypeptide are described in PCT WO9830583, disclosure of which is hereby incoφorated by reference in its entirety. Intracellular proteins that inhibit TGF-beta signaling include but are not limited to Brain Factor (BF)-l

(MLDMGDRKEV ITPKSSFSINSLWEGLQNDNIIHASHGHHNSHHPQHHHHHHHHHHHPP

PPAPQPPPPRAAQQQQPPPPPLAPQAGGAAQSNDEKGPQLLLLPPTDHHRPPSGAKAGGCCR

PGELGPVGPDEKΕKGAGAGGEEKKGAGEGGIA^GEGGKEGEKKNGKYEKPPFSYNALTMM AIRQSPEK-RLTLNGLYEFIMKNFPYYPVENKQGWQ

YWMLDPSSDDVFIGGTTGIO.RRRSTTSPAKLAFKRGAALTSTGLTFMDRAGSLYWPMSPFL

SLHHPRASSTLSYNGTTSAYPSHPMPYSSVLTQNSLGNNHSFSTANGLSVDRLVNGEΓPYAT

HHLTAAALAASVPCGLSVPCSGTYSLNPCSVNLLAGQTSYFFPHVPHPSMTSQSSTSMSARA

ASSSTSPPAPRPLPCESLRPSLPSFTTGLSGGLSDYFTHQNQGSSSNPLΓH), FKBP12 (MGVEIETISPGDGRTFPKKGQTCVVHYTGMLQNGKJ^DSSRDRNKPFKFRIGKQEVIKGFE EGAAQMSLGQRAKLTCTPDVAYGATGHPGVTPPNATLΓFDVELLNLE) and calcineurin

(MTAPEPARAAPPPPPPPPPPPGADRVVKAVPFPPTHP TSEEVFDLDGΓPRVDVLKNHLVKE GRVDEEL LRHNEGAAΓLRREKTMIEVEAPITVCGDΓHGQFFDLMKLFEVGGSPANTRYLFL GDYVDRGYFSFFIHVLGTEDISINPIINNINECVLYLWVLKILYPSTLFLLRGNHECRHLTEYFT FKQECKTKYSERVYEACMEAFDSLPLAALLNQQFLCVHGGLSPEIHTLDDIRRLDRFKEPPAF GPMCDLLWSDPSEDFGNEKSQEHFSHNTVRGCSYFYNΓ^AVCEFLQNNNLLSΠRAHEAQD AGYRMYRKSQTTGFPSLITTFSAPNYLDVΎNN AAVLKYENN^ MDVFTWSLPFVGEKVTEMLVNVLSICSDDELMTEGEDQFDGSAAAIKEΠRNKΓRAIGKMA RVFSVLREESESVLTLKGLTPTGMLPSGVLAGGRQTLQSGNDVMQLAVPQMDWGTPHSFA NNSHNACREFLLFFSSCLSS; A-beta isoform). Such proteins may be used in a method of inhibiting PAI expression comprising the steps of introducing such proteins or fragments thereof into a cell. Preferred cells are those that express TGF-beta receptor or a receptor belonging to the TGF- beta receptor superfamily. Further preferable is covalent linkage of the protein or fragment thereof that inhibits TGF-beta signaling to a targeting moiety, such as a receptor binding protein. The resulting targeted inhibitor protein is thus introduced only to a specific cell type that expresses the targeted receptor. For example, vascular endothelial cells may be targeted by linking a TGF-beta inhibitor with a gp36-specific antibody fragment. Alternatively, a protein that inhibits TGF-beta signaling may be introduced into a cell by introducing a polynucleotide construct comprising polynucleotides encoding the polypeptides of the inhibitor protein. Methods for introducing a polynucleotide into a cell are known in the art and discussed herein. Further included inhibitors of PAI expression are soluble polypeptide fragments comprising the ligand-binding site of the TGF- beta receptor superfamily, antibodies and antigen binding fragments thereof that prevent binding of TGF-beta family members to their receptors. Additionally included TGF-beta antagonists include antisense polynucleotides complementary to the polynucleotide sequence of TGF-beta or TGF-beta receptor.

Angiotensin II and TV have been shown to increase PAI expression upon binding to angiotensin receptors. Therefore, preferred PAI inhibitors included in the invention are antagonists of Ang Tl and Ang TV signaling. Each antagonist of Ang II and Ang TV signaling can additionally be excluded from the invention individually or as a class. Especially preferred inhibitors of Ang II and Ang IV signaling include a class of compounds that specifically inhibit the formation of these peptides by Angiotensin Converting Enzymes. Such ACE inhibitors include: acylmercapto and mercaptoalkanoyl prolines (e.g., captopril and zofenopril); carboxyalkyl dipeptides (e.g., enalapril, lisinopril, quinapril, ramipril, and perindopril); carboxyalkyl dipeptide mimics (e.g., cilazapril and benazepril); phosphinylalkanoyl prolines (e.g., fosinopril and trandolopril); bestatin; amastatin; and L158-809. Methods of synthesizing captopril, i.e., D-3-mercapto-2-methylpropanoyl-L-proline, are included in U.S. Patents 4105776, 5972990, and 6191144, disclosures of which are hereby incoφorated by reference in their entireties. Zofenopril synthesis is detailed in U.S. Patent 4316906, disclosure of which is hereby incoφorated by reference in its entirety. Preferred dosages of these compounds range from 10 to 500 mg per day. Methods of making carboxyalkyl dipeptides are disclosed in U.S. Patents 4344949, 4374829, 4508729, 4587258, and 4837354, disclosures of which are hereby incoφorated by reference in their entireties. A PAI inhibiting dosage of enalapril is about 2.5-75 mg daily. Effective dosages of perindopril are about 0.5-5 mg/kg daily. Methods of making carboxyalkyl dipeptide mimics are disclosed in U.S. Patents 4410520 and 4512924, disclosures of which are hereby incoφorated by reference in their entireties. Preferred dosages of these compounds range from about 10 to 200 mg for a 70 kg individual. Methods of making phosphinylalkanoyl prolines are disclosed in U.S. Patent 4337201, which is hereby incoφorated by reference in its entirety. Fosinopril is preferably administered at 1-15 mg/ kg daily. Bestatin and amastatin are effective at luM in culture, with an actual IC₅₀ of about 0.2uM. Synthesis of bestatin and amastatin are described in U.S. Patents 4029547 and 4594188, disclosures of which are hereby incoφorated by reference in their entireties. L158-809 is an effective inhibitor of PAI expression in rats when added to drinking water at 80mg/L. Exemplary structures for ACE inhibitors are disclosed herein.

Additionally preferred inhibitors of PAI expression are compounds that interfere with angiotensin receptors. Each angiotensin receptor antagonist can additionally be excluded from the invention individually or as a class. Such compounds include: Dup753, WSU1291, candesartan, and valsartan. The chemical name of Dup753 is 2-butyl-4-chloro-l[(2'(lH-tetrazol-5-yl)biphenyl-4- yl)methyl]-5-(hydroxymethyl)-imidazole. Dup753 and WSU1291 are effective inhibitors of angiotensin signaling in cultured endothelial cells at luM. Methods of synthesis for Dup753 and WSU1291, approximate dosages, and pharmaceutical compositions are described in U.S. Patents 4355040, 5182264, and EP Application 0511767, disclosures of which are hereby incoφorated by reference in their entireties. Examples of this class of inhibitors are disclosed herein. Compounds that are structurally similar to angiotensin peptides are often effective competitive inhibitors of angiotensin binding to the appropriate receptor. Methods of making and using these compounds are described in U.S. Patent 5182264, disclosure of which is hereby incoφorated by reference in its entirety. Competitive inhibitors of angiotensin peptides are disclosed herein. Candesartan (i.e., 2- ethoxy-l-[[2'-(lH-tetrazol-5-yl)biphenyl-4-yl]methyl]-benzimidazole-7-carboxylate) synthesis is discussed in EP 0459136, which disclosure is hereby incoφorated by reference in its entirety.

Valsartan has an IC₅₀ of 2 InM and is usually given at a dose of 40-160mg in humans. See PCT W09749394, disclosure of which is hereby incoφorated by reference in its entirety, for methods of making and administering orally-acceptable compositions of valsartan. The structure of valsartan is disclosed herein.

Signals emanating from angiotensin receptors may also be blocked in order to decrease PAI expression. Preferred PAI inhibitors of the invention include compounds that interfere with angiotensin-mediated signaling. Each antagonist of angiotensin signaling can additionally be excluded from the invention individually or as a class. Antagonists of angiotensin signaling include: calphostin C and bisindorylmaleimide L (PKC inhibitors), BAPTA-AM (reduces intracellular calcium release), genistein (tyrosine kinase inhibitor), AG1478 (EGF receptor antagonist), PD98059 (MEK inhibitor), Y27632 (Rho inhibitor), as well as compounds that increase intracellular cAMP levels (adrenomedullin, 8-bromo-cAMP, and forskolin). These compounds are readily available commercially. The full-length sequence of adrenomedullin follows:

MK1VSVALMYLGSLAFLGADTARLDVASEFRKKWNKWALSRGKRELRMSSSYPTGLADV KAGPAQTLΓRPQDMKGASRSPEDSSPDAARΓRVKRYRQSMNNFQGLRSFGCRFGTCTVQKL AHQΓYQFTDKDKDNVAPRSKISPQGYGRRRRRSLPEAGPGRTLVSSKPQAHGAPAPPSGSAP HFL. Adrenomedullin and adrenomedullin receptor binding fragments thereof and may be synthesized or expressed in an appropriate recombinant cell type using methods common to the art (Kitamura, et al. (1993), Biochem. Biophys. Res. Comrnun. 192:553-560) and are effectively injected at a concentration of about 1-lOpmol/kg (PCT WO0058348, which disclosure is hereby incoφorated by reference in its entirety). Further included inhibitors of PAI expression are soluble polypeptide fragments comprising the ligand-binding site of the angiotensin receptor superfamily and antibodies or antigen-binding fragments thereof that prevent binding of Ang II and Ang TV to their receptors. Additionally included antagonists of angiotensin signaling include antisense polynucleotides complementary to the polynucleotide sequence of ACE, angiotensin, and angiotensin receptors. Cyclic AMP increases the expression of PAI RNA-Binding Protein (PATRBP)-l

(MPGHLQEGFGCVVTNRFDQLFDDESDPFEVLKAAENK-KKEAGGGGVGGPGAKSAAQAA AQTNSNAAGKQLT<KΈSQKTJRKNPLPPSVGVVDKΈ^

GEGKΠDT RPERRPPRERRFEKPLEEKGEGGEFSVDRPΓTDRPΓRGRGGLGRGRGGRGRGMGR GDGFDSRGKREFDRHSGSDRSGLKHEDKRGGSGSHNWGTVKDELTDLDQSNVTEETPEGE EHHPVA-DTENKENEVEEVKEEGPI^MTLDEWKAIQNIΠ)I AKVEFNIRKPNEGADGQWKK GFVLIIKSKSEEAHAEDSVMDHHFRKPANDITSQLEΓNFGDLGRPGRGGRGGRGGRGRGGRP

NRGSRTDKSSASAPDVDDPEAFPALA). PAΓRBP-1 specifically binds PAI message and targets the message for degradation. Therefore, a preferred embodiment of the invention is a method of inhibiting translation of PAI polypeptides with PAIRBP-1. This method comprises the step of introducing PATRBP-1 polypeptides into a cell. PAIRBP-1 polypeptides may be introduced into a cell by upregulating cellular cAMP levels, as discussed above. Alternatively, PATRBP-1 polynucleotides or a targeted PAIRBP-1 polypeptide chimera may be used to introduce PATRBP-1 polypeptides into a cell using techniques discussed herein.

Aldosterone increases PAI expression, thus, preferred PAI inhibitors include aldosterone antagonists. Each antagonist of aldosterone signaling can additionally be excluded from the invention individually or as a class. One such antagonist is spironolactone. Spironolactone is usually administered several times a day with a total daily dose ranging from 50- 400mg. Chemical composition, method of synthesis, and dosages are described in U.S. Patent 3013012 and U.S. Patent 5529992, disclosures of which are hereby incoφorated by reference in their entireties. The structure of Spironolactone is disclosed herein.

Estrogen inhibits PAI expression, possibly by exerting a negative effect on aldosterone secretion and angiotensin receptor expression. A preferred embodiment of the invention is a method of inhibiting PAI expression with estrogen or a physiologically acceptable derivative thereof (e.g., estradiol). An estradiol dose of 0.05mg/kg is effective to reduce PAI activity in rats. Estrogen or physiologically acceptable derivatives thereof may additionally be excluded from the invention.

Serotonin has been shown to activate PAI expression. Thus, preferred PAI inhibitors of the invention include serotonin antagonists. Each antagonist of serotonin signaling can additionally be excluded from the invention individually or as a class. Antagonists of serotonin signaling include (8beta)-N-cycloalkyl-l-alkyl-6-(substituted) ergoline-8-carboxamides, 1 l-(3,5-dimethyl-l- piperazinyl)-dibenz[b,fJ[l,4]oxazepine, ll-(2,5-dimethyl-l-piperazinyl)-dibenz[b,f][l,4]oxazepine, methiothepin, and MDL 110,907. Methods of making and using these inhibitors are described in U.S. Patents 4931447, 5824676, 5134149, and PCT WO021930, disclosures of which are hereby incoφorated by reference in their entireties. These compounds have an ED₅₀ ranging from 0.03- 3mg/kg when injected intraperitoneally. Structures of exemplary serotonin inhibitors are disclosed herein.

Vastatin/ oxysterol-type inhibitors also act to decrease PAI levels (PCT W09947136, which disclosure is hereby incoφorated by reference). Preferred vastatin compounds include simvastatin, lovastatin, and mevinoline. Preferred oxysterols include natural oxysterols such as 22(R)- hydroxycholesterol. Oxysterols are optionally combined with retinoic acid for maximal biological activity (i.e., decreasing expression or secretion of PAT). Such compounds are readily available commercially. These inhibitors are effective for decreasing PAI secretion from cultured human endothelial cells in the luM range. Inhibitors of this type may additionally be excluded individually or as a class. Agonists of the Peroxisome Proliferator-Activator Receptor (PPAR)-alpha inhibit expression of PAI. Agonists of PPAR-gamma inhibit PAI expression in insulin-resistant individuals. Thus, further preferred inhibitors of PAI expression include PPAR-alpha and gamma activators. Each agonist of PPAR-alpha and gamma can additionally be excluded from the invention individually or as an entire class.

The CRY1 protein

(MGVNAVITWFRKGLRLFΦNPALKECIQGADTTRCVYILDPWFAGSSNVGLLSU^WRFLLQCLE

DLDANLT^KLNSRLFVTRGQPADVFPRLFKEWMTKLSFFIYDSEPFGKERDAAIKKLATEAGV

EVTV SHTLYDLDKTIELNGGQPPLTYK-RFQTLISKMEPLETPVETITSEVIEKCTTPLSDDHDE KYGVPSLEELGFDTDGLSSAVWPGGETEALTRLERHLERKAWVANFERPRMNANSLLASPT GLSPYLRFGCLSCRLFYFKLTDLYKKVKKNSSPPLSLYGQLLWREFFYTAATNNPRFDKME GNPICVQΓPWDKNPEALAKWAEGRTGFPWΓDAΓMTQLRQEGWΓHHLARHAVACFLTRGDL WISWEEGMKVFEELLLDADWSΓNAGSWMWLSCSSFFQQFFHCYCPVGFGRRTDPNGDYΓR RYLPVLRGFPAKYTYOPWNAPEGIQKVAKCLIGVNYPKPMVNHAEASI^LNFFIRMKQTYQQL SRYRGLGLLASVPSNPNGNGGFMGYSAENΓPGCSSSGSCSQGSGΓLHYAHGDSQQTHLLKQ

GRSSMGTGLSGGKRPSQEEDTQSIGPKVQRQSTN) is a photoreceptor that activates the transcription factor PER2 (MNGYAEFPPSPSNPTKEPVEPQPSQVPLQEDVDMSSGSSGHETNENCSTGRDSQGSDCDDS

GKELGMLVEPPDARQSPDTFSLMMAKSEHNPSTSGCSSDQSSKVDTHKELΓKTLKELKVHL PADKKAKGKASTLATLKYALRSVKQVKANEEYYQLLMSSEGHPCGADVPSYTVEEMESVT SEHIVKNADMFAVAVSLVSGKILYISDQVASIFHCIΑΩAFSDAKFVEFLAPHDVGVFHSFTS PYKLPLWSMCSGADSFTQECMEEKSFFCRVSVRKSHENETRYHPFRMTPYLVKVRDQQGAE SQLCCLLLAERVHSGYEAPRTPPEKRΓFTTTHTPNCLFQDVDESPAVRRAAFRLFSHSVSRPE

RRVHHVGHQLVQLHQPMEQENLLHHWEAQSQGGPFE), which in turn, inhibits PAI expression. Additional transcriptional inhibitors of PAI include USF2a

(MDMLDPGLDPAASATAAAAASHDKGPEAEEGVELQEGGDGPGAEEQTAVAITSVQQAAF GDHNIQYQFRTETNGGQVTYRWQVTDGQLDGQGDTAGAVSWSTAAFAGGQQAVTQVG VDGAAQRPGPAAASVPPGPAAPFPLAVIQNPFSNGGSPAAEAVSGEARFAYFPASSVGDTTA VSVQTTDQSLQAGGQFYVMMTPQDVLQTGTQRTL\PRTHPYSPKTDGTRTPRDERRRAQHN EVERRRRDKINNWIVQLSKΠPDCNADNSKTGASKGGΓLSKACDYIRELRQTNQRMQETFKE AERLQMDNELLRQQTEELKNENALLRAQLQQHNLEMVGEGTRQ) AND E2F1 (MALAGAPAGGPCAPALEALLGAGALRLLDSSQΓVΠSAAQDASAPPAPTGPAAPAAGPCDP DLLLFATPQAPT^PTPSAPRPALGRPPVK-PJO.DLETDHQYLAESSGPARGRGRHPGKGVKSPG EKSRYETSLNLTTKRFLELLSHSADGVVDLNWAAEVLKVQKRRTYOTTNVLEGIQLIAK-KSK NHIQWLGSHTTVGVGGRLEGLTQDLRQLQESEQQLDHLMNICTTQLRLLSEDTDSQRLAYV TCQDLRSIAL PAEQMVMVΓKAPPETQLQAVDSSENFQISLKSKQGPΓDVFLCPEETVGGISPG KTPSQEVTSEEENRATDSATΓVSPPPSSPPSSLTTDPSQSLLSLEQEPLLSRMGSLRAPVDEDR LSPLVAADSLLEHVREDFSGLLPEEFISLSPPHEALDYHFGLEEGEGΓRDLFDCDFGDLTPLDF

). Preferred PAI inhibitors include transcriptional inhibitors, such as PER2, USF2a, and E2F1, and agonists of PAI transcriptional inhibitors, such as CRY1. Polypeptides may be introduced to a cell by methods common to the art and discussed herein. Transcriptional inhibitors of PAI and their agonists may additionally be excluded individually or as a class.

Dextran sulfate, select compounds with diketopiperazine, benzil, benzophenone, substituted and unsubstituted biaryl, benzyl ether, and thioether base structures, elastase, select peptides derived from vitronectin, alpha-1 acid glycoprotein, and SP40,40, antibodies specific for PAI polypeptides and antigen binding fragments thereof are competitive inhibitors of PAI. Matrix MetalloProteinase (MMP)-3 is a noncompetitive PAI inhibitor. Preferred PAI inhibitors of the invention include each member of these listed types of inhibitors. Each member of these listed types of PAI inhibitors may be excluded individually or as a group.

Dextran sulfate prevents binding of PAI to its protease substrates, thereby inhibiting PAI (JP07069901 A, which disclosure is hereby incoφorated by reference in its entirety). Select diketopiperazine based inhibitors, as well as methods to synthesize each, are disclosed in U.S. Patents 5750530, 5891877, and 5902812, disclosures of which are hereby incoφorated by reference in their entireties. Preferred substituents and functional groups are disclosed herein (Diketopiperazine Structures A-D). Additional preferred examples of diketopiperazine compounds are listed herein. Generally, these compounds are formed during condensation reactions of readily available starting materials according to methods known in the art. Select compounds with benzil, benzophenone, substituted and unsubstituted biaryl, benzyl ether, and thioether base structures are also able to prevent PAI binding to target proteases. The structures of these compounds, physiologically acceptable compositions of such, and methods of synthesis are disclosed in PCT WO0136351, which disclosure is hereby incoφorated by reference. Preferred examples of such inhibitors are disclosed herein.

Nonsubstrate proteases can inhibit PAI activity by cleaving PAI polypeptides at a site other than R346-M347 or by irreversibly binding the PAI substrate-binding site. An example of the first type of protease is Matrix MetalloProteinase (MMP)-3. The full-length sequence of MMP-3 follows: MKSLPILLLLCVAVCSAYPLDGAARGEDTSMNLVQKYLENYYDLEKDVKQFVRRKDSGPV VKOREMQK-FLGLEVTGIADSDTLEVMRKPRCGVPDVGIIFRTFPGRPKWRKTHLTYRIVNY TPDLPKDAVDSAVEKALKVWΈEVTPLTFSRLYEGEADΓMISFAVREHGDFYPFDGPGNVLA HAYAPGPGINGDAIIFDDDEQWTKDTTGTNLFLVAAHEIGHSLGLFHSANTEALMYPLYHS LTDLTRFRLSQDDTNGIQSLYGPPPDSPETPLVPTEPVPPEPGTPANCDPALSFDAVSTLRGEIL IFKDRHFWRKSLRKLEPELHLISSFWPSLPSGVDAAYEVTSKDLVFTFKGNQFWATRGNEVR AGYPRGTΗTLGFPPTVRKTDAAISDKΕKNKTYFFVEDKYWRFDEIOINSMEPGFPKQIAEDFP

GTDSKIDAVFEEFGFFYFFTGSSQLEFDPNAKKVTHTLKSNSWLNC. PAI, when cleaved at R346-M347, is an active inhibitor of target proteases. MMP-3 cleaves PAI at S337-S338 and V341-

1342 and prevents PAI from inhibiting target proteases. An example of the second type of nonsubstrate protease is elastase. Elastase irreversibly binds to the active site of PAI and prevents interaction with substrate proteases. Nonsubstrate protease polypeptides and fragments thereof that inhibit PAI activity, may be contacted with PAI in vitro or in vivo using methods discussed herein.

Preferred PAI inhibitors of the invention include nonsubstrate proteases. Each nonsubstrate protease may further be excluded from the invention individually or as a group.

Peptides derived from proteins that bind PAI are effective competitive inhibitors of PAI.

Such proteins include vitronectin, alpha-1 acid glycoprotein (Boncela, J., et al.(2001) J Biol Chem manuscript M104028200), and SP-40,40 (N. Choi-Miura (2001) Biol Pharm Bull 24:39-42). Thus, preferred PAI inhibitors of the invention include peptides derived from PAI-binding proteins. Each peptide may additionally be excluded from the invention individually or as a class. A preferred vitronectin peptide comprises amino acids 348-380 of the protein: KKQRFRHRNRKGYRSQRGHSRGRNQNSRRPSRA. More preferable are amino acids 348-370: KKQRFRHRNRKGYRSQRGHSRGR. Further preferred PAI inhibitors of this class have all optically active amino acids in the D configuration to prevent proteolytic cleavage of the peptide. Such peptides may be synthetically produced by methods common to the art. Vitronectin peptides are effective inhibitors of PAI when introduced intravenously in the 50ug/kg range (U.S. Patent 5491129, which disclosure is hereby incoφorated by reference). Pharmaceutical formulations and procedures for administration of vitronectin peptides are also disclosed in U.S. Patent 5491129.

Antibodies or antigen-binding fragments thereof that specifically bind PAI and prevent PAI activity are preferred PAI inhibitors included in the invention. Each antibody or fragment may be particularly excluded as well. A preferred monoclonal antibody is MA-124K1 (Ngo, T., et al. (2001) J Biol Chem 276:26243-8, which disclosure is hereby incoφorated by reference) and fragments thereof that inhibit PAI activity. Also preferred are polyclonal antibodies and fragments thereof that specifically inhibit PAI activity.

PAI activity may be measured using methods described in Madison, et al. (1989) Nature 339:721-3; Madison, et al. (1990) Proc. Natl. Acad. Sci. 87:3530-3; Madison, et al. (1993) Science 262:419-21; U.S. Patent 5750530, 5902812, and 5891877, which disclosures are hereby incoφorated by reference in their entireties. For instance, a compound suspected of directly inhibiting PAI activity is incubated with PAI-1 prior to addition to the tPA assay system. Inhibition of PAI-1 results in the production of plasmin from plasminogen. In turn, plasmin cleaves the chromogenic substrate S2251 (Kabi Vitrum) producing pNA (p-nitroaniline) which is detected spectrophotometrically at 405 nm. Typical IC.sub.50 values for diketopiperazine-type inhibitors are in the 1-20 uM range. IC.sub.50 values for benzil-type, benzophenone-type, substituted and unsubstituted biaryl, benzyl ether, and thioether (SUBBET)-type PAI inhibitors are on the order of 10- lOOuM. PAI expression may be measured using methods common to the art. PAI mRNA may be measured using a complementary nucleotide sequence conjugated to a detectable label (e.g., radioisotope, fluorescent or luminescent molecule). Common techniques using this method include Northern blotting and microarrays (e.g., gene chips). Alternatively, PAI mRNA may be detected using reverse transcriptase PCR (RTPCR) techniques. The level of PAI polypeptides in a biological solution (e.g., cellular extract, media, serum, or other bodily fluid) may be measured using antibody- based techniques. Such techniques include ELISAs (as discussed above), Western blotting and immunofiuorescence.

Synthesis of certain types of PAI inhibitors disclosed may be accomplished by those skilled in organic chemistry. Additional references for organic synthesis include: Morrison, et al., Organic Chemistry, second edition (1966), Allyn and Bacon, Inc., Boston and Tlie Organic Chemistry of Drug Synthesis (1992) John Wiley and Sons, Inc., New York. PAI inhibitors may be formulated into a wide variety of physiologically acceptable salts by reacting the base compounds with organic or inorganic acids or bases. Physiologically acceptable acid addition salts are typically formed by reacting a base compound with an equimolar or excess amount of acid. The reactants are generally combined in a mutual solvent such as diethyl ether or benzene. Examples of inorganic acids include hydrochloric acid, sulphuric acid and orthophosphoric acid. Examples of organic acids include p- toluenesulphonic acid, methansulphonic acid, mucic acid and succinic acid. A preferred salt is hydrochloride salt. Preferably, hydrochloride salts are made by bubbling hydrochloric acid through a solution of the compound in dry THF or DMF. Bases commonly used for formation of salts include ammonium hydroxide and alkali and alkaline earth metal hydroxides, carbonates, as well as aliphatic and primary, secondary and tertiary amines, aliphatic diamines. Examples of inorganic bases include ammonia and carbonates, hydroxides and hydrogen carbonates of group I and group Tl metals such as sodium, potassium, magnesium and calcium. Examples of organic bases include aliphatic and aromatic amines such as methylamine, triethylamine, benzylamine, dibenzylamine or alpha- or beta- phenylethylamine, and heterocyclic bases such as piperidine, 1-methylpiperidine and moφholine. Bases especially useful in the preparation of addition salts include ammonium hydroxide, potassium carbonate, methylamine, diethylamine, ethylene diamine and cyclohexylamine. PAI inhibitors may also be converted into pharmaceutically acceptable esters. Suitable esters include branched or unbranched, saturated or unsaturated Cι-C₆ alkyl esters, for example methyl, ethyl and vinyl esters. Physiologically acceptable salts and esters generally have enhanced solubility compared to the compound from which they are derived, and thus are often more amenable to formulation as liquids or emulsions.

The PAI inhibitors described herein may be used in pharmaceutical formulations as discussed in detail herein. Furthermore, delivery of PAI inhibitors may be targeted to certain cell types. Targeting may be effected by linking the compound to a targeting agent specific for the desired cells, including but not limited to, liver, skeletal muscle, adipose, or pancreatic cells. For example, a moiety that particularly targets white adipose tissue is an antibody fragment specific for lipoprotein lipase. Such moiety, when fused to a peptide of the invention or liposomal particle containing a physiological composition of the invention, will specifically target an adipose cell.

The compounds provided herein may be encapsulated in a microcapsule, liposome, or biodegradable material, including, but not limited to, waxes, cellulosic materials, and polyvinyl polymers, linked to one or more targeting particles, including, but not limited to, antibodies including monoclonal antibodies, antibody fragments, antigen fragments, ligands, ligand fragments, receptors, receptor fragments, or other molecules that bind to a specific cell type. Polar lipid groups that may be included in the encapsulating material include but are not limited to: sphingosine, ceramide, phosphatidyl choline, phsphatidyglycerol, phsphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine cardiolipin, phosphatidic acid, sphingomyelin and other sphingolipids. Other methods of targeting include entrapping a compound provided herein in an encapsulated microparticale composition that, when exposed to a selected target stimulus related to pH, temperature, radiation, or the presence of a selected ligand or ion-channel activator, decondenses to release the compound into the target site. Useful examples are included in U.S. Patent 6110490, U.S. Patent 6217886, PCT WO9704748, and PCT WO0136351, disclosures of which are hereby incoφorated by reference.

Therapeutic compositions according to the present invention may comprise advantageously one or several PAI agonist polynucleotide fragments as an antisense tool or a triple helix tool that inhibits the expression of the corresponding PAI agonist gene. Preferred PAI agonists targeted by this method include: TNF-alpha, TNF-alpha receptor, TGF-beta family members, TGF-beta receptor family members, angiotensin, angiotensin receptors, and PAI itself.

Antisense Approach

In antisense approaches, nucleic acid sequences complementary to an mRNA are hybridized to the mRNA infracellularly, thereby blocking the expression of the protein encoded by the mRNA. The antisense nucleic acid molecules to be used in gene therapy may be either DNA or RNA sequences. Preferred methods using antisense polynucleotides according to the present invention are the procedures described by Sczakiel et al, (1995) Trends Microbiol. 3(6):213-217, which disclosure is hereby incoφorated by reference in its entirety. Preferably, the antisense tools are chosen among the polynucleotides (15-200 bp long) that are antisense to PAI agonist mRNA, more preferably to the 5 'end of the PAI agonist mRNA. In another embodiment, a combination of different antisense polynucleotides complementary to different parts of the desired targeted gene are used. Preferred PAI agonists targeted by this method include: TNF-alpha, TNF-alpha receptor, TGF-beta family members, TGF-beta receptor family members, angiotensin, angiotensin receptors, and PAI itself.

The antisense nucleic acids should have a length and melting temperature sufficient to permit formation of an intracellular duplex having sufficient stability to inhibit the expression of the PAI agonist mRNA in the duplex. Strategies for designing antisense nucleic acids suitable for use in gene therapy are disclosed in Green et al, (1986) Ann. Rev. Biochem. 55:569-597 and Izant and

Weintraub, (1984) Cell 36(4):1007-15, the disclosures of which are incoφorated herein by reference. Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

The antisense oligodeoxynucleotides and oligonucleotides disclosed in International Application No. WO 92/18522, incoφorated by reference, may also be used. These molecules are stable to degradation and contain at least one transcription control recognition sequence which binds to control proteins and are effective as decoys therefor. These molecules may contain "haiφin" structures, "dumbbell" structures, "modified dumbbell" structures, "cross-linked" decoy structures and "loop" structures. In another preferred embodiment, the cyclic double-stranded oligonucleotides described in

European Patent Application No. 0572287 A2, hereby incoφorated by reference are used. These ligated oligonucleotide "dumbbells" contain the binding site for a transcription factor and inhibit expression of the gene under control of the transcription factor by sequestering the factor. Use of the closed antisense oligonucleotides disclosed in International Application No. WO 92/19732, hereby incoφorated by reference, is also contemplated. Because these molecules have no free ends, they are more resistant to degradation by exonucleases than are conventional oligonucleotides. These oligonucleotides may be multifunctional, interacting with several regions which are not adjacent to the target mRNA.

An alternative to the antisense technology that is used according to the present invention comprises using ribozymes that will bind to a target sequence via their complementary polynucleotide tail and that will cleave the corresponding RNA by hydrolyzing its target site (namely "hammerhead ribozymes"). Briefly, the simplified cycle of a hammerhead ribozyme comprises (1) sequence specific binding to the target RNA via complementary antisense sequences; (2) site-specific hydrolysis of the cleavable motif of the target strand; and (3) release of cleavage products, which gives rise to another catalytic cycle. Indeed, the use of long-chain antisense polynucleotide (at least 30 bases long) or ribozymes with long antisense arms are advantageous. A preferred delivery system for antisense ribozyme is achieved by covalently linking these antisense ribozymes to lipophilic groups or to use liposomes as a convenient vector. Preferred antisense ribozymes according to the present invention are prepared as described by Rossi et al, (1991) Pharmacol. Ther. 50:245-254 and Sczakiel et al. (1995), the specific preparation procedures being referred to in said articles being herein incoφorated by reference. The present invention also relates to recombinant vectors including the isolated polynucleotides of the present invention, and to host cells recombinant for a polynucleotide of the invention, such as the above vectors, as well as to methods of making such vectors and host cells and for using them for production of PAI inhibitor polypeptides by recombinant techniques Recombinant Vectors

The term "vector" is used herein to designate either a circular or a linear DNA or RNA molecule, which is either double-stranded or single-stranded, and which comprise at least one polynucleotide of interest that is sought to be transferred in a cell host or in a unicellular or multicellular host organism. The present invention encompasses a family of recombinant vectors that comprise a regulatory polynucleotide and/or a coding polynucleotide derived from either the PAI Inhibitor genomic sequence or the cDNA sequence. Generally, a recombinant vector of the invention may comprise any of the polynucleotides described herein, including regulatory sequences, coding sequences and polynucleotide constructs, as well as any PAI Inhibitor primer or probe as defined herein. In a first preferred embodiment, a recombinant vector of the invention is used to amplify the inserted polynucleotide derived from a PAI Inhibitor genomic sequence or a PAI Inhibitor cDNA, for example: adrenomedullin, E2F1, USF2a, PER2, CRY1, elastase, MMP-3, SP-40,40, alρha-1 acid glycoprotein, vitronectin, PATRBP-1, or biologically active fragments thereof in a suitable cell host, this polynucleotide being amplified at every time that the recombinant vector replicates. A second preferred embodiment of the recombinant vectors according to the invention comprises expression vectors comprising either a regulatory polynucleotide or a coding nucleic acid of the invention, or both. Within certain embodiments, expression vectors are employed to express a PAI Inhibitor polypeptide which can be then purified and, for example be used in ligand screening assays or as an immunogen in order to raise specific antibodies directed against the PAI Inhibitor protein. In other embodiments, the expression vectors are used for constructing transgenic animals and also for gene therapy. Expression requires that appropriate signals are provided in the vectors, said signals including various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Dominant drug selection markers for establishing permanent, stable cell clones expressing the products are generally included in the expression vectors of the invention, as they are elements that link expression of the drug selection markers to expression of the polypeptide.

More particularly, the present invention relates to expression vectors which include nucleic acids encoding a PAI Inhibitor protein, preferably a PAI Inhibitor protein such as: adrenomedullin, E2F1, USF2a, PER2, CRY1, elastase, MMP-3, SP-40,40, alpha-1 acid glycoprotein, vitronectin, PATRBP-1 and biologically active fragments thereof. The polynucleotides of the present invention may be used to express an encoded protein in a host organism to produce a beneficial effect. In such procedures, the encoded protein may be transiently expressed in the host organism or stably expressed in the host organism. The encoded protein may have any of the activities described herein. The encoded protein may be a protein which the host organism lacks or, alternatively, the encoded protein may augment the existing levels of the protein in the host organism.

In addition, the present invention relates to expression vectors which include nucleic acids complementary to a PAI agonist protein such as: TNF-alpha, TNF-alpha receptor, TGF-beta family members, TGF-beta receptor family members, angiotensin, angiotensin receptors, or PAI itself. Such antisense polynucleotides target a complementary nucleotide sequence for destruction. Methods of making such are discussed herein.

Some of the elements which can be found in the vectors of the present invention are described in further detail in the following sections.

General features of the expression vectors of the invention

A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAG (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or even a linear DNA molecule which may comprise a chromosomal, non- chromosomal, semi-synthetic and synthetic DNA. Such a recombinant vector can comprise a transcriptional unit comprising an assembly of:

(1) a genetic element or elements having a regulatory role in gene expression, for example promoters or enhancers. Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription.

(2) a structural or coding sequence which is transcribed into mRNA and eventually translated into a polypeptide, said structural or coding sequence being operably linked to the regulatory elements described in (1); and

(3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, when a recombinant protein is expressed without a leader or transport sequence, it may include a N-terminal residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.

Generally, recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium. In a specific embodiment wherein the vector is adapted for transfecting and expressing desired sequences in mammalian host cells, preferred vectors will comprise an origin of replication in the desired host, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation signals, splice donor and acceptor sites, transcriptional termination sequences, and 5 '-flanking non-transcribed sequences. DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, splice and polyadenylation signals may be used to provide the required non-transcribed genetic elements.

The in vivo expression of a PAI Inhibitor polypeptide of the present invention may be useful in order to correct a genetic defect related to the expression of the native gene in a host organism, for the treatment or prevention of any disease or condition that can be treated or prevented by increasing the level of PAI Inhibitor polypeptide expression, or to the production of a biologically inactive PAI Inhibitor protein. Consequently, the present invention also comprises recombinant expression vectors mainly designed for the in vivo production of a PAI Inhibitor polypeptide the present invention by the introduction of the appropriate genetic material in the organism or the patient to be treated. This genetic material may be introduced in vitro in a cell that has been previously extracted from the organism, the modified cell being subsequently reintroduced in the said organism, directly in vivo into the appropriate tissue.

Regulatory Elements

The suitable promoter regions used in the expression vectors according to the present invention are chosen taking into account the cell host in which the heterologous gene has to be expressed. The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell, such as, for example, a human or a viral promoter. A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed. Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted. Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors.

Preferred bacterial promoters are the Lad, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and tip promoters (EP 0036776), the polyhedrin promoter, or the plO protein promoter from baculovirus (Kit Novagen) [Smith et al, (1983) Mol. Cell. Biol. 3:2156-2165; O'Reilly et al, (1992) Baculovirus Expression Vectors: A Laboratory

Manual; W. H. Freeman and Co., New York; which disclosures are hereby incoφorated by reference in their entireties], the lambda PR promoter or also the trc promoter. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late

SV40, LTRs from retrovirus, and mouse metallothionein-L. Selection of a convenient vector and promoter is well within the level of ordinary skill in the art. The choice of a promoter is well within the ability of a person skilled in the field of genetic engineering. For example, one may refer to the book of Sambrook, et ah, (1989) or also to the procedures described by Fuller S. A. et al. (1996)

"Immunology in Current Protocols in Molecular Biology", which disclosures are hereby incoφorated by reference in their entireties.

Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

Selectable Markers Selectable markers confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. The selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, TRP1 for S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. Coli, or levan saccharase for mycobacteria, this latter marker being a negative selection marker. Preferred Vectors

As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and pGEMl (Promega Biotec, Madison, WI, USA).

Large numbers of other suitable vectors are known to those of skill in the art, and commercially available, such as the following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, ρNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).

The PI bacteriophage vector may contain large inserts ranging from about 80 to about 100 kb. The construction of PI bacteriophage vectors such as pl58 or pl58/neo8 are notably described by Sternberg (1992) Trends Genet. 8:1-16, and Sternberg (1994) Mamm. Genome. 5:397-404, which disclosure is hereby incoφorated by reference in its entirety. Recombinant PI clones comprising

PAI Inhibitor nucleotide sequences may be designed for inserting large polynucleotides of more than 40 kb [see, Linton et al, (1993) J. Clin. Invest. 92:3029-3037], which disclosure is hereby incoφorated by reference in its entirety. To generate PI DNA for transgenic experiments, a preferred protocol is the protocol described by McCormick et al, (1994) Genet. Anal. Tech. Appl.

11:158-164, which disclosure is hereby incoφorated by reference in its entirety. Briefly, E. coli

(preferably strain NS3529) harboring the PI plasmid are grown overnight in a suitable broth medium containing 25 μg/ml of kanamycin. The PI DNA is prepared from the E. coli by alkaline lysis using the Qiagen Plasmid Maxi kit (Qiagen, Chatsworth, CA, USA), according to the manufacturer's instructions. The PI DNA is purified from the bacterial lysate on two Qiagen-tip 500 columns, using the washing and elution buffers contained in the kit. A phenol/chloroform extraction is then performed before precipitating the DNA with 70% ethanol. After solubilizing the DNA in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA), the concentration of the DNA is assessed by spectrophotometry.

When the goal is to express a PI clone comprising PAI Inhibitor polypeptide-encoding nucleotide sequences in a transgenic animal, typically in transgenic mice, it is desirable to remove vector sequences from the PI DNA fragment, for example by cleaving the PI DNA at rare-cutting sites within the PI polylinker (Sfil, Noil or Salϊ). The PI insert is then purified from vector sequences on a pulsed-field agarose gel, using methods similar to those originally reported for the isolation of DNA from YACs [see, e. g., Schedl et al, (1993a), Nature, 362: 258-261; Peterson et al, (1993), Proc. Natl. Acad. Sci. USA, 90 -.7593-7597], which disclosures are hereby incoφorated by reference in their entireties. At this stage, the resulting purified insert DNA can be concentrated, if necessary, on a Millipore Ultrafree-MC Filter Unit (Millipore, Bedford, MA, USA - 30,000 molecular weight limit) and then dialyzed against microinjection buffer (10 mM Tris-HCl, pH 7.4; 250 μM EDTA) containing 100 mM NaCl, 30 μM spermine, 70 μM spermidine on a microdyalisis membrane (type VS, 0.025 μM from Millipore). The intactness of the purified PI DNA insert is assessed by electrophoresis on 1% agarose (Sea Kem GTG; FMC Bio-products) pulse-field gel and staining with ethidium bromide.

In one specific embodiment, the vector is derived from an adenovirus. Preferred adenovirus vectors according to the invention are those described by Feldman and Steg, (1996), Medecine/Sciences, 12:47-55, or Ohno et al, (1994) Science. 265:781-784, which disclosures are hereby incoφorated by reference in their entireties. Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin (French patent application No. FR-93.05954, which disclosure is hereby incoφorated by reference in its entirety).

Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery systems of choice for the transfer of exogenous polynucleotides in vivo, particularly to mammals, including humans. Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vitro gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus. Particularly preferred Murine

Leukemia Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999), Friend

(ATCC No VR-245), Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) and Moloney

Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657, VR-726,

VR-659 and VR-728). Other preferred retroviral vectors are those described in Roth et al, (1996)

Nature Medicine, 2(9):985-991, PCT Application No WO 93/25234, PCT Application No WO 94/

06920, Roux et al. (1989), Proc. Natl. Acad. Sci. U.S.A. 86:9079-9083, Julan et al. (1992), J. Gen.

Virol. 73:3251-3255, andNeda et al. (1991), J. Biol. Chem. 266:14143-14146, which disclosures are hereby incoφorated by reference in their entireties .

The bacterial artificial chromosome (BAC) cloning system [Shizuya et al. (1992), Proc. Natl. Acad. Sci. U.S.A. 89:8794-8797], which disclosure is hereby incoφorated by reference in its entirety, has been developed to stably maintain large fragments of genomic DNA (100-300 kb) in E. coli. A preferred BAC vector comprises a pBeloBACl 1 vector that has been described by Kim U-J. et al. (1996), Genomics 34:213-218, which disclosure is hereby incoφorated by reference in its entirety. BAC libraries are prepared with this vector using size-selected genomic DNA that has been partially digested using enzymes that permit ligation into either the Bam HI or HindSl sites in the vector. Flanking these cloning sites are T7 and SP6 RNA polymerase transcription initiation sites that can be used to generate end probes by either RNA transcription or PCR methods. After the construction of a BAC library in E. coli, BAC DNA is purified from the host cell as a supercoiled circle. Converting these circular molecules into a linear form precedes both size determination and introduction of the BACs into recipient cells. The cloning site is flanked by two Not I sites, permitting cloned segments to be excised from the vector by Not I digestion. Alternatively, the DNA insert contained in the pBeloBACl 1 vector may be linearized by treatment of the BAC vector with the commercially available enzyme lambda terminase that leads to the cleavage at the unique cøsN site, but this cleavage method results in a full length BAC clone containing both the insert DNA and the BAC sequences.

Another specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC No. CRL 1711) which is derived from Spodopterafrugiperda. Other suitable vectors for the expression of the PAI Inhibitor polypeptide of the present invention in a baculovirus expression system include those described by Chai et al. (1993), Biotechnol. Appl. Biochem. 18:259-273, Vlasak, et al. (1983), Eur. J. Biochem. 135:123-126, and Lenhard et al, (1996) Gene. 169:187-190, which disclosures are hereby incoφorated by reference in their entireties. Delivery Of The Recombinant Vectors

To effect expression of the polynucleotides and polynucleotide constructs of the invention, the constructs must be delivered into a cell. This delivery may be accomplished in vivo or ex vivo, as in the treatment of certain diseases states. One mechanism is viral infection where the expression construct is encapsulated in an infectious viral particle. The expression construct, preferably a recombinant viral vector as discussed herein, may transduce packaging cells through any means known in the art such as electroporation, liposomes, and CaP04 precipitation. The packaging cell generates infectious viral particles that include a polynucleotide encoding a polypeptide of the present invention. Such viral particles then may be employed to transduce eukaryotic cells in vitro, ex vivo or in vivo. The transduced eukaryotic cells will express a polypeptide of the present invention. Preferably, the viruses used in the present invention are rendered replication deficient by deletion of one or more of all or a portion of the following genes: Ela, Elb, E3, E4, E2a, or LI through L5 (U.S. Patent 6,228,844, which disclosure is hereby incoφorated by reference in its entirety). Viral delivery is discussed in more detail herein (see also, U.S. Patent 5,968,821, which disclosure is hereby incoφorated by reference in its entirety).

Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. A major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population. The development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy puφoses (for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding one of the subject CCR-proteins, rendering the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZTP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include .psi.Crip, .psi.Cre, .psi.2 and .psi.Am.

Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230: 1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson, et al. (1988) Proc. Natl.

Acad. Sci. USA 85:3014-3018; Armentano, et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145;

Huber, et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry, et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury, et al. (1991) Science 254:1802-1805; van Beusechem, et al.

(1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay, et al. (1992) Human Gene Therapy 3:641-

647; Dai, et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu, et al. (1993) J. Immunol.

150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136;

PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

In choosing retroviral vectors as a polynucleotide delivery system, it is important to note that a prerequisite for the successful infection of target cells by most retroviruses, and therefore of stable introduction of the introduced polynucleotides, is that the target cells must be dividing. In general, this requirement will not be a hindrance to use of retroviral vectors, and can be beneficial in circumstances wherein the tissue (e.g. nonfransformed cells) surrounding the target cells does not undergo extensive cell division and is therefore refractory to infection with retroviral vectors.

Furthermore, it has been shown that it is possible to limit the infection spectrum of retroviruses and consequently of retroviral-based vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example PCT publications W093/25234, WO94/06920, and W094/11524). For instance, strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux, et al. (1989) PNAS 86:9079-9083; Julan, et al. (1992) J. Gen Virol 73:3251-3255; and Goud, et al. (1983) Virology 163:251-254); or coupling cell surface ligands to the viral env proteins (Neda, et al. (1991) J Biol Chem 266:14143-14146). Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g. lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins (e.g. single-chain antibody/ env fusion proteins). This technique, while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecotropic vector in to an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences that control expression of the desired gene.

Another viral gene delivery system useful in the present invention utilitizes adenovirus- derived vectors. The genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactivate in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner, et al. (1988) BioTechniques 6:616; Rosenfeld, et al. (1991) Science 252:431-434; and Rosenfeld, et al. (1992) Cell 68: 143-155). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld, et al. (1992) cited supra), endothelial cells (Lemarchand et al.(1992) Proc. Natl. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin, et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral polynucleotides (and foreign polynucleotides contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of msertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral El and E3 genes but retain as much as 80% of the adenoviral genetic material (see, e.g., Jones, et al. (1979) Cell 16:683; Berkner, et al., supra; and Graham, et al. in Methods in Molecular Biology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp.109-127). Expression of desired polynucleotides can be under control of, for example, the E1A promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences. Yet another viral vector system useful for delivery of polynucleotides is the adeno- associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a heφes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka, et al., Curr. Topics in Micro, and Immunol. (1992) 158:97-129). It is also one of the few viruses that may integrate its nucleic acids into non- dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. , (1992) Am. J. Respir. Cell Mol. Biol. 7:349-356; Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin, et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat, et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin, et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford, et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin, et al. (1984) J. Virol. 51:611-619; and Flotte, et al. (1993) J. Biol. Chem.268:3781-3790). Other viral vector systems that may have application in gene therapy have been derived from heφes virus, vaccinia virus, and several RNA viruses. In particular, heφes virus vectors may provide a unique strategy for persistence of inserted gene expression in cells of the central nervous system and ocular tissue (Pepose, et al. (1994) Invest Ophthalmol Vis Sci 35:2662-2666).

Once the expression polynucleotide has been delivered into the cell, it may be stably integrated into the genome of the recipient cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. One specific embodiment for a method for delivering a protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect. This is particularly applicable for transfer in vitro but it may be applied to in vivo as well.

Compositions for use in vitro and in vivo comprising a "naked" polynucleotide are described in PCT application No. WO 90/11092 (Vical Inc.) and also in PCT application No. WO 95/11307 (Institut Pasteur, TNSERM, Universite d' Ottawa) as well as in the articles of Tascon et al. (1996), Nature Medicine. 2(8):888-892 and of Huygen et al, (1996) Nature Medicine. 2(8):893-898, which disclosures are hereby incoφorated by reference in their entireties.

In still another embodiment of the invention, the transfer of a naked polynucleotide of the invention, including a polynucleotide construct of the invention, into cells maybe accomplished with particle bombardment (biolistic), said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al. , (1987) Nature 327:70-73, which disclosure is hereby incoφorated by reference in its entirety. Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner, et al., Proc. Nat. Acad. Sci. USA (1987) 84:7413- 7416, which is herein incoφorated by reference); mRNA (Malone, et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which is herein incoφorated by reference); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265: 10189-10192, which is herein incoφorated by reference), in functional form. Cationic liposomes are readily available. For example,N[l-2,3-dioleyloxy)propyll-N,N,N- triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. (See, also, Feigner, et al., Proc. Nad Acad. Sci. USA (1987) 84:7413-7416, which is herein incoφorated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE

(Boehringer).

Similarly, anionic and neutral liposomes are readily available, such as from AvantiPolar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and

DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art (Straubinger, et al., Methods of Immunology (1983), 101:512-527, which is herein incoφorated by reference). For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated (U.S. Patent 5,965,421, which disclosure is hereby incoφorated by reference).

Generally, the ratio of DNA to liposomes will be from about 10: 1 to about 1: 10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ratio will be about 3: 1 to about 1: 3. Still more preferably, the ratio will be about 1: 1. Additionally, liposomes maybe targeted to specific cell types by embedding a targeting moiety such as a member of a receptor- receptor ligand pair into the lipid envelope of the vesicle. Useful targeting moieties specifically bind cell surface ligands, for example, CD48 or the SCF receptor on mast cells. Thus, anti-CD48 antibodies or SCF ligand are examples of useful mast cell-targeting moieties (U.S. Patent 6177433, U.S. Patent 6110490, and P.C.T No. WO9704748, which disclosures are hereby incoφorated by reference in their entireties). In a further embodiment, a PAI inhibitor polynucleotide of the invention may be entrapped in a liposome [Ghosh and Bacchawat, (1991), Targeting of liposomes to hepatocytes, IN: Liver Diseases, Targeted diagnosis and therapy using specific rceptors and ligands. Eds., Marcel Dekeker, New York, pp. 87-104; Wong, et al. (1980), Gene. 10:87-94; Nicolau et al, (1987), Meth. Enzymol, 149: 157-76, which disclosures are hereby incoφorated by reference in their entireties] . In a specific embodiment, the invention provides a composition for the in vivo production of the PAI Inhibitor polypeptides described herein. It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable carrier, and suitable for introduction into a tissue to cause cells of the tissue to express the said protein or polypeptide.

The amount of vector to be injected to the desired host organism varies according to the site of injection. As an indicative dose, it will be injected between 0.1 and 100 μg of the vector in an animal body, preferably a mammal body, for example a mouse body.

In another embodiment of the vector according to the invention, it may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a adipose cell. In a subsequent step, the cell that has been transformed with the vector coding for the desired PAI Inhibitor polypeptide or the desired fragment thereof is reinfroduced into the animal body in order to deliver the recombinant protein within the body either locally or systemically.

Pharmaceutical or Physiologically Acceptable Compositions of the Invention

The PAI Inhibitors of the invention can be administered to non-human animals and/or humans, alone or in pharmaceutical or physiologically acceptable compositions where they are mixed with suitable carriers or excipient(s). The pharmaceutical or physiologically acceptable composition is then provided at a therapeutically effective dose. A therapeutically effective dose refers to that amount of PAI Inhibitor sufficient to result in prevention or amelioration of symptoms or physiological status of obesity-related diseases or disorders as determined by the methods described herein. A therapeutically effective dose can also refer to the amount of PAI Inhibitor necessary for a reduction in weight or a prevention of an increase in weight or prevention of an increase in the rate of weight gain in persons desiring this affect for cosmetic reasons. A therapeutically effective dosage of a PAI Inhibitor of the invention is that dosage that is adequate to promote weight loss or weight gain with continued periodic use or administration. Techniques for formulation and administration of PAI Inhibitor may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition.

Alternatively, PAI can be administered in an amount effective to reduce the level of PAI activity to the average PAI activity of a healthy individual of normal weight. Routes of Administration. Suitable routes of administration include oral, nasal, rectal, transmucosal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, inframeduUary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocular injections using methods known in the art. A particularly useful method of administering compounds for promoting weight loss involves surgical implantation, for example into the abdominal cavity of the recipient, of a device for delivering PAI Inhibitor over an extended period of time. Other particularly preferred routes of administration are aerosol and depot formulation. Sustained release formulations, particularly depot, of the invented medicaments are expressly contemplated. Composition Formulation

Pharmaceutical or physiologically acceptable compositions and medicaments for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries. Proper formulation is dependent upon the route of administration chosen.

Certain of the medicaments described herein will include a pharmaceutically or physiologically acceptable carrier and at least one compound that is a PAI Inhibitor of the invention.

For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer such as a phosphate or bicarbonate buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Pharmaceutical or physiologically acceptable preparations that can be taken orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable gaseous propellant, e.g., carbon dioxide. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator, may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical or physiologically acceptable formulations for parenteral aαlministration include aqueous solutions of the active compounds in water-soluble form. Aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder or lyophilized form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.

Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

The pharmaceutical or physiologically acceptable compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Effective Dosage.

Pharmaceutical or physiologically acceptable compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.

Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes or encompasses a concentration point or range shown to increase leptin or lipoprotein uptake or binding in an in vitro system. Such information can be used to more accurately determine useful doses in humans. A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50, (the dose lethal to 50% of the test population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.

Compounds that exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50, with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1).

Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain or prevent weight loss or gain, depending on the particular situation. Dosages necessary to achieve these effects will depend on individual characteristics and route of administration.

Dosage intervals can also be determined using the value for the minimum effective concentration. Compounds should be administered using a regimen that maintains plasma levels above the minimum effective concentration for 10-90% of the time, preferably between 30-90%; and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

A preferred dosage range for the amount of a PAI Inhibitor of the invention, which can be administered on a daily or regular basis to achieve desired results, including a reduction in levels of circulating plasma triglyceride-rich lipoproteins, range from 0.01 - 500 mg/kg body mass. A more preferred dosage range is from 0.5 - 50 mg/kg. Of course, these daily dosages can be delivered or administered in small amounts periodically during the course of a day. It is noted that these dosage ranges are only preferred ranges and are not meant to be limiting to the invention. Other methods of administration, formulations, and preferred dosages are provided elsewhere herein, particularly those for specific compounds.

Assays for Identifying Modulators of PAI Inhibitor Activity

The invention features methods of screening for one or more compounds that modulate a PAI biological activity described herein that includes providing potential compounds to be tested to the cells, and where modulation of a PAI biological activity indicates the one or more compounds. To these assays would be added compounds to be tested for their inhibitory or stimulatory activity as compared to PAI alone or untreated cells or animals. Other assays in which an effect is observed based on the addition of PAI Inhibitor can also be used to screen for modulators of PAI activity.

The essential step is to apply an unknown compound and then to monitor an assay for a change from what is seen when only PAI or control sample is applied to the cell. A change is defined as something that is significantly different in the presence of the test compound compared to only PAI or control sample compound alone. In this case, significantly different would be an "increase" or a

"decrease" in a measurable effect of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,

70%, or 75%.

The invention features a method for identifying a potential compound to modulate body mass in individuals in need of modulating body mass comprising: a) administering to an animal a PAI Inhibitor candidate compound; b) detecting a result selected from the group consisting of LSR modulation, leptin modulation, lipoprotein modulation; FFA oxidation modulation; glucose uptake or oxidation, weight loss and c) wherein said result identifies said potential compound if said result differs from said result when said animal is administered with a control sample alone or left untreated.

EXAMPLES

The following Examples are provided for illustrative puφoses and not as a means of limitation. One of ordinary skill in the art would be able to design equivalent assays and methods based on the disclosure herein all of which form part of the instant invention. EXAMPLE 1 : Effect of PAI Inhibitors on Mice Fed a High-Fat Diet

Experiments are performed using approximately 6-week old C57B1/6 mice (8 per group). All mice are housed individually. The mice are maintained on a high fat diet throughout each experiment. The high fat diet (cafeteria diet; D 12331 from Research Diets, Inc.) has the following composition: protein kcal% 16, sucrose kcal% 26, and fat kcal% 58. The fat is primarily composed of coconut oil, hydrogenated.

After the mice are fed a high fat diet for 6 days, micro-osmotic pumps are inserted using isoflurane anesthesia, and are used to provide a PAI inhibitor, saline, and an irrelevant inhibitor to the mice subcutaneously (s.c.) for 18 days. Alternatively, PAI inhibitor, saline, or an irrelevant inhibitor is injected daily by IP. Body weight is measured on the first, third and fifth day of the high fat diet, and then daily after the start of treatment. Final blood samples are taken by cardiac puncture and are used to determine triglyceride (TG), total cholesterol (TC), free fatty acids (FFA), glucose, leptin, and insulin levels. The amount of food consumed per day is also determined for each group. EXAMPLE 2: Tests of Obesity-related Activity in Humans

Tests of the efficacy of PAI inhibitors in humans are performed in accordance with a physician's recommendations and with established guidelines. The parameters tested in mice are also tested in humans (e.g. food intake, weight, TG, TC, FFA, glucose, insulin, and leptin). It is expected that the physiological factors (TG, TC, FFA, glucose, insulin, and leptin) show changes over the short term. Changes in weight gain require a longer period of time. In addition, the diet is carefully monitored. PAI inhibitors are given in daily doses of about 6 mg per 70 kg person or about

10 mg per day. Other doses are also tested, for instance 0.1 mg or 0.5 mg per day up to 20 mg, 50 mg, or 100 mg per day. EXAMPLE 3 : Tests of Obesity-related Activity in a Murine Lipoatrophic Diabetes Model

Previously, leptin is reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodystrophy (Shimomura et al. Nature 401: 73-76 (1999); hereby incoφorated by reference in its entirety). Leptin is found to be less effective in a different lipodystrophic mouse model of lipoatrophic diabetes (Gavrilova et al. Nature 403: 850 (2000); hereby incoφorated by reference in its entirety). Weight reduction also reverses insulin resistance, glucose intolerance, as well as dyslipidemias. The instant invention encompasses the use of PIA inhibitors for reducing the insulin resistance and hyperglycaemia in this model either alone or in combination with leptin, the leptin peptide (US provisional application No 60/155,506), OBG3 (US Patent application No 09/758,055) or other compounds. Assays included are described previously in Gavrilova et al. ((2000) Diabetes Nov;49(ll):1910-6; (2000) Nature Feb 24;403(6772):850, which disclosures are hereby incoφorated by reference in their entireties) using A-ZTP/F-1 mice, except that compounds of the invention would be administered using the methods previously described in Example 1. The glucose and insulin levels of the mice are tested, and the food intake and liver weight monitored, as well as other factors, such as leptin, FFA, cholesterol and TG levels, typically measured in our experiments (see Example 5).

EXAMPLE 4: Effect of PAI inhibitors on plasma Free Fatty Acid in C57 BL/6 Mice

The effect of PAI inhibitors on postprandial lipemia (PPL) in normal C57BL6/J mice is tested. The mice are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken. All blood samples are taken from the tail using EDTA coated capillary tubes (50 μL each time point). At time 0 (8:30 AM), a standard high fat meal (6g butter, 6 g sunflower oil, 10 g nonfat dry milk, 10 g sucrose, 12 mL distilled water prepared fresh following Nb#6, JF, pg.l) is given by gavage (vol.=l% of body weight) to all animals.

PAI inhibitor is injected i.p. in 100 μL saline 1) immediately following the high fat meal; 2) 45 min following meal; 3) and at 1 hr 45 min following the meal (treated group, n=8). Control animals (n=8) are injected with saline (3xl00μL). Untreated and treated animals are handled in an alternating mode.

Blood samples are taken in hourly intervals, and are immediately put on ice. Plasma is prepared by centrifugation following each time point. Plasma is kept at -20°C and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako). Due to the limited amount of plasma available, glucose can be determined in duplicate using pooled samples (equal volumes of plasma from all animals per treatment group are pooled). Glucose, TG and FFA are determined for each group. EXAMPLE 5 : Effect of PAI inhibitors on Plasma Leptin and Insulin in C57 BL/6 Mice

The effect of PAI inhibitors on plasma leptin and insulin levels during postprandial lipemia

(PPL) in normal C57BL6/J mice is tested. The experimental procedure is the same as that described in Example 4, except that blood is drawn only at 0, 2 and 4 hours to allow for greater blood samples needed for the determination of leptin and insulin by RIA.

Briefly, 16 mice are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken. All blood samples are taken from the tail using EDTA coated capillary tubes (100 μL each time point). At time 0 (9:00AM), a standard high fat meal (see Example 4) is given by gavage (vol.=l% of body weight) to all animals. PAI inhibitor is injected i.p. in 100 μL saline 1) immediately following the high fat meal; 2) 45 min following meal; 3) and at 1 hr 45 min following the meal (treated group, n=8). Control animals (n=8) are injected with saline (3xl00μL). Untreated and treated animals are handled in an alternating mode.

Blood samples are immediately put on ice and plasma is prepared by centrifugation following each time point. Plasma is kept at -20°C and free fatty acids (FFA) are determined within 24 hours using a standard test kit (Wako). Leptin and insulin levels are determined by RIA

(ML-82K and SRI-13K, LTNCO Research, Inc., St. Charles, MO) following the manufacturer's protocol. However, only 20 μL plasma is used. Each determination is done in duplicate. Due to the limited amount of plasma available, leptin and insulin can be determined in 4 pools of 2 animals each in both treatment groups. EXAMPLE 6: Effect of PAI inhibitors on Plasma FFA. TG and Glucose in C57 BL/6 Mice

The experimental procedure is similar to that described in Example 4. Briefly, 18 mice are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken. All blood samples are taken from the tail using EDTA coated capillary tubes (50 μL each time point). At time 0 (9:00AM), a standard high fat meal (see Example 4) is given by gavage (vol.=l% of body weight) to all animals. In group 1, PAI inhibitor is injected i.p. in 100 μL saline 1) immediately following the high fat meal; 2) 45 min following meal; 3) and at 1 hr 45 min following the meal (treated group, n=8). A group 2 (n=4) received 3 times the amount of inhibitor in group 1 at the same intervals. Control animals (n=6) are injected with saline (3xl00μL). Untreated and treated animals are handled in an alternating mode. Blood samples are immediately put on ice. Plasma is prepared by centrifugation following each time point. Plasma is kept at -20 °C and free fatty acids (FFA), triglycerides (TG), cholesterol and glucose are determined within 24 hours using standard test kits (Sigma and Wako). EXAMPLE 7 : Effect of PAI inhibitors on FFA following Epinephrine Ini ection

In mice, plasma free fatty acids increase after intragastric administration of a high fat/sucrose test meal. These free fatty acids are mostly produced by the activity of lipolytic enzymes i.e. lipoprotein lipase (LPL) and hepatic lipase (HL). In this species, these enzymes are found in significant amounts both bound to endothelium and freely circulating in plasma. Another source of plasma free fatty acids is hormone sensitive lipase (HSL) that releases free fatty acids from adipose tissue after beta-adrenergic stimulation. To test whether PAI inhibitors also regulate the metabolism of free fatty acid released by HSL, mice are injected with epinephrine.

Two groups of mice (n=5 each) are given epinephrine (5μg) by intraperitoneal injection. A treated group is injected with PAI inhibitor (IP) one hour before and again together with epinephrine, while control animals receive saline. Plasma is isolated and free fatty acids and glucose are measured as described above (Example 6).

In ex vivo experiments, adipose tissue is removed from normal C57BL/6J mice and incubated in Krebs-Henseleit bicarbonate buffer. Epinephrine is added, with or without PAI inhibitor and the concentration of FFA in the medium following a 90 min incubation is determined.

EXAMPLE 8: Effect of PAI inhibitors on Triglyceride in Muscle & Liver Isolated from Mice To determine whether the effect of PAI inhibitors increases FFA delivery into muscle or liver, the hindlimb muscle and liver triglyceride content is measured after PAI inhibitor treatment of mice. Hindlimb muscles as well as liver samples are removed from treated and untreated animals and the triglyceride and free fatty acid concentration is determined following a standard lipid extraction method Shimabukuro, et al., Proc Natl Acad Sci USA 94, 4637-4641 (1997), which disclosure is hereby incoφorated by reference in its entirety, followed by TG and FFA analysis using standard test kits.

EXAMPLE 9: Longterm Effect of PAI inhibitors on Weight Gain, Weight Loss, and other physiological factors.

To test the effect of PAI inhibitors on weight gain while consuming a high calorie diet,

C57BL/6J 10 week old mice are put on a very high fat/sucrose purified diet for 19 days to promote weight gain (see Example 1), and body weight is measured. The mice are then surgically implanted with an osmotic pump (Alzet, Newark, DE) delivering either PAI inhibitors, or physiological saline. Alternately, mice are TP injected daily. The mice are continued on the high fat diet and their body weight and food intake is recorded daily over the following 10-day period. Comparison of the change in weight, food intake, plasma free fatty acids, glucose, cholesterol, insulin and triglycerides is made between groups.

Mice are then continued on a high fat diet or a standard diet without treatment of inhibitors and food intake, body weight, plasma free fatty acids, glucose, cholesterol, insulin and triglycerides are measured weekly for 4 weeks. Comparison of parameters following discontinuation of treatment is made between control mice (no inhibitor treatment) and treated mice (inhibitor discontinued after

10 days). Weight gain per calorie intake is also compared between the groups.

EXAMPLE 10: Detection of apMl by immunoprecipitation of Human Plasma after treatment with PAI inhibitors

The effect of PAI inhibitors on the plasma levels of apMl and fragments thereof is determined following treatment with a PAI inhibitor (per Example 2), blood collection and plasma separation. Immunoprecipitation of human plasma apMl followed by Western blotting is used to detect a cleavage product of apMl, the human homolog of Acφ30, using a globular head specific anti-serum for the immunoprecipitation step as well as for the detection step. Preimmune serum or serum raised against the globular head domain or human non-homologous region (HDQETTTQGPGVLLPLPKGA) are cross-linked to protein A (Sigma Chemical CO, Saint Louis,

MO) using dimethyl-pimelimidate-dihydrochloride (Sigma Chemical Co, Saint Louis, MO). After washing (0.2 M salt) proteins are eluted from protein A, separated by SDS-PAGE, transferred to

Protran® pure nitrocellulose membrane (Schleicher and Schuell, Keene, NH) using standard procedures. apMl products are visualized using globular head domain antibodies labeled with biotin; horseradish peroxidase conjugated to Streptavidin and CN/DAB substrate kit (Pierce, Rockford, IL) according to manufacturer's instructions.

The apparent molecular weight of the truncated form is 27 kDa, corresponding to about 70% of the complete form of apMl. EXAMPLE 11 : Effect of PAI inhibitor on FFA following Intralipid Injection To determine the affect of PAI inhibitors on plasma FFA levels, 6 groups of mice (n=5 each) are intravenously (tail vein) injected with 30 μL bolus of Intralipid-20% (Clintec) to generate a sudden rise in plasma FFAs, thus by-passing intestinal absoφtion. (Intralipid is an intravenous fat emulsion used in nutritional therapy). Treatment groups are injected IP with a PAI inhibitor at 30, 60, or 90 minutes before Intralipid is given, while control animals receive saline at the same time intervals. Plasma is isolated and FFAs are measured as described previously (Example 4). Levels of plasma FFA are compared between control and treated groups.

Benzothiophene Structure A Benzothiophene Structure B

wherein ^l and R³ are indcpendentiy hydrogen, — CH₃,

o o

II II

— C— (Cj-C« iltyl), or — C— Ar, or its hydrochloride salt wherein Ar is optionally substituted phenyl;

R² is selected from the group consisting of pyrrolidine, hexamethylenrimino, and pφeridino; or a phatmaceutically acceptable salt of solvate thereof.

Benzopyran Structure D

Triphenyl ethylene Structure C

* is _H, meihvl, ethyl, propyL etheπyl or ethynyh a pharmaceutically accep ble salt or solvate thereof. Daunorubicin

Atorvastatin

Simvastatin ovastatin

Pravastatin Fluvastatin

Rivastatin

ACE Inhibitor- acylmercapto and mercaptoalkanoyl prolines

T

Ri— S— (CHj),— CH— CO — N •COR wherein R is hydroxy or lower alkoxy; ] is hydrogen or lower alkyl; R₂is hydrogen, R5— CO— , R,— S— or R₇; R₃ is lower alkyl or phenyL especially the first; R« is lower alkyl; and rt is 0, 1 or 2.

ACE Inhibitor- mercaptoacyl prolines

o

R4 is hydrogen. or a physiologically acceptable salt thereof.

R₅— R

R5 is lower alkyl, phenyl, pheπyl-lower alkylene, substituted phenyl, or substituted phenyl-lower alkylene wherein said substituent is one or two groups on the phenyl ring selected from the group consisting of lower alkyl of 1 to 4 carbons, lower alkoxy of 1 to 4 carbons, lower alkylthio of 1 to 4 carbons, chloro, brorao, fluoro, iodo, trifluoromethyl, acetyloxy, and hydroxy; and n is 0, 1 or 2. ACE Inhibitor- carboxyalkyl dipeptides ACE Inhibitor- carboxyalkyl dipeptides

wherein

R and * can each independently be hydroxy, lower alkoxy, aralkyloxy, wherein A is absent, a fused 5-, 6-, or 7-raernbered P,² and BL⁷ are hydrogen, cycloaliphatic ring or a fused benzene ring which is unsubR3 is methyl, aminoloweralkyl, R* and R^s are joined through the carbon and nitrogen stituted or substituted by 1 or 2 alkoxy groups having 1 ta atoms to form proline, 4-thiaprolinc or 4-methoxy- 4 carbon atoms; n is 0 or 1, and R is hydrogen or aikyl proliαe, and having 1 to 5 carbon atoms; _ — - R¹ is alkyl having from 1-8 carbon atoms, substituted lower alkyl wherein the alkyl group has 1-5 carbon atoms »nd the substituent is amino, arylthio or aryl- oxy, aralkyl or heteroaralkyl wherein the alkyl portion has ϊ-3 carbon atoms, substituted aralkyl or heteroϊralkyl wherein the alkyl groups have 1-3 carbon atoms and the substituent(s) is halo, dihalo, amino, aminoalkyl, hydroxy, lower alkoxy or lower alkyl; and the pharmaceutically acceptable salts thereof wherein said aryl is a member selected from the group consisting of phenyl or naphthyl and said heteroaryl is a member selected from the group consisting of pyridyl, thienyl, fαryL indolyl, beπzthienyl, imidazoyl or thia- zolyl.

ACE Inhibitor- carboxyalkyl dipeptides

Preferably

wher

e R is hydrogen, lower alkyl or vheavbύkvb R. _« hydrogen, lower alkyl. or benzyl; Rj i, hvdrogen. or _^en ca y acceptable salts thereof. lower alkyl and Ar is phenyl, or snbβti ated πhαrW hav g 1 or 2 mrstiraeats selected froπ he gro_ttpcώ. ttsting of fltrariπe, chlorine, bromine, lower attyUower atkαxy, hydroxy or amino; X and Y are izideriennentlv or hydrogen, lower alkyl, lower alkoxy, lower afc_yltni_rf lower alkylsBlfinyl, lower aikylsπlfαnyl, ydroxy ar and Y together are -rtethylenediαxy; and m is 0 to 3 wherein lower alkyl, alkyl la the group phenylalkvL* and lower alkoxy has 1 to 4 straight or braiiched carbon atoms and the pharraacetrticaily acceptable sal_ts thereof.

ACE Inhibitor- carboxyalkyl dipeptide rnimics

wherein B represents an ethylene group, R³ represents a wherein B represents a methylene, ethylene or vinylene carboxyl, alkoxycarbonyl or aralkoxycarbonyl group, group, R¹ represents a hydrogen atom or an alkyl, aralRt and R⁵ each represent a hydrogen atom or R⁴ and R⁵ kyl, arni .c-a kyl, raonc-aikylamir.o-alkyl, dialkylarair-O- together represent an oxo group, R⁸ represents a alkyl, acylamino-alkyl, phthalimido-alkyl, alkoxycar- phthaloylamino group and n is 2. bonylamiπo-alkyl, aryloxycarboπylamino-alkyl, aralk- oxycarbonylamino-alkyl, alkylaminocarbonylamino- alkyl, arylaininocarbonylamino-alkyl, aralkylaminocar- bonylaraiπo-alkyl, alkylsulphonylamino-alkyl or aryl- sulphonylamino-alkyl group, R² represents a carboxyl, alkoxycarboπyl or aralkoxycarbonyl group or a group of the formula

R³ represents a carboxyl, alkoxycarbonyl or aralkoxwherein B represents an ethylene group, R³ represents a ycarbonyl group, R⁴ and R^s each represent a hydrogen carboxyl, alkoxycarbonyl or aralkoxycarbonyl group, atom or R⁴ and R⁵ together represent an oxo group, R^β R⁴ and R⁵ each represent a hydrogen atom or ⁴ and R⁵ and R⁷ each represent a hydrogen atom or an alkyl or together represent an oxo group and n is 2. aralkyl group or R⁶ and R⁷ together with the nitrogen atom to which they are attached represent a 5-mem- bered or 6-membered heteromonocyclic ring which may contain a further nitrogen atom or an oxygen or sulphur atom, and n stands for zero, 1 or 2, and pharmaceutically acceptable salts thereof have anti- hypertensive activity and can be used as medicaments in the form of pharmaceutical preparations.

ACE Inhibitor- carboxyalkyl dipeptide mimics

ACE Inhibitor- carboxyalkyl dipeptides

wherem Rl is hydrogen, lower alkyl, amino (lower) alkyl, wherein aryl, aryl (lower) alkyl, cycloalkyl (lower) alkyl;

Rl represents Iower-alkyl having 1 to 4 carbon atoms, R2 and R. represent hydrogen or lower alkyl; inclusive, R3 and R4 represent hydrogen, lower alkyl, lower

Rϊ represents hydrogen or lower-alkyl having 1 to 4 alkoxy, lower alkanoyloxy, hydroxy, halogen, tricarbon atoms, inclusive, fluoromethyl; or R3 and R+ taken together repre¬

R3 is — CH2CH₂ — CH3, in racemic form or as an sent lower alkylenedioxy; optical isomer, a salt thereof with a pharmaceuti- X represents oxo, two hydrogens or one hydroxy cally-acceptable inorganic or organic base, and an group and one hydrogen; addition salt thereof with a phaπnaceutically- Rβ and R7 independently represent hydroxy, amino, acceptable inorganic or organic acid. mono- or di-(Iower) alkylamino, lower alkoxy, aryl

(lower) alkoxy, lower alkanoyloxymethyl, (amino, mono- or di-loweralkylamino, carboxy, or lower alkoxycarbonyl) lower alkoxy; and wherein aryl represents phenyl unsubstituted or mono- or di-substttuted by lower alkyL lower alkoxy, lower alkylenedioxy, lower alkanoyloxy, hydroxy, halogen or trifluoromethyl; and cycloalkyl contains 3 to

8 carbons; or a pharmaceutically acceptable salt thereof. ACE Inhibitor" phosphinylalkanoyl prolines ACE Inhibitor - Bestatin

and thm ot er is hydrogen, alkyl, arylalkyl or

wherein X is hydrogen, ajkyl or phenyl end Y is hydrogen, alkyl, phenyl or alkoxy, or together X and Y aii'e — (CHJh—, — (CH2.3— , — CH=CH— or

Hj is hydrcigan or ftl yl; — Rs— COOΪ ia

^»N — "^■— *— COOR4 oorr »-YN X 1- •COOR4; (W 0-3

Rt it hydrøgeπ, hydroxy, alkyl, halogen, aridα, atninα, cycloalkyl, aryl, arylalkyl, carhajnoyhny, N,N-<iω«tylc8τt«wπoylQ*y, or ~-Z— Re;

R7 and V are the same sod each it halogen or — Z— Rnii, or R.7 and V- together we =0, -C-(C.!»ι)_m- - or — S-(Cllύm-&~;

Rjii hy rogen end Re' fo phenyl, 2-hydroxyphroyl or 4-hydro3(;iφhenyl or Ri and RV together, ere =rf-»

H. » alkyl, n*yU aryWkyli 1' or 2-naphthyl, or bjphe-

RlD is alkyl,, aryl qr arylalkyl; Z U owsem o fuiUur. n ii O αr li nw- rt i» 1 o 2|

Hid wherein tltw term "nryJ" refer* to ph-myl or ρ}ιcnyl substtoited wiith halogen, βlkyl, alko≠y, alkylthio, hydroxy, alkanoyl, nitro, amino, dialkyJawino or trifluoromethyl group K the term "alky." refer* to group* having I to lOcar on atoms; he term "alkoxy" refer, to groups having l to 8 carbon atom*; ffie term "cyclcMukyr refer* to groups having 3 to 7 carbon βtoma; end the teem "alkanoyl" refers to groups having 2 to 9 carbon atoms, Angiotensin Receptor Antagonist Angiotensin Receptor Antagonist

ower alkoxyl, and its salts.

Angiotensin Receptor Antagonist- Valsartan

Gui c— L— Arg-L-Vil— — Tr-L— He— -Ha- — Pro- — He. <0 CT CT (4) (ϊ) («) 0) ^' W

Sir— L-Arg— L— il-L— Tjτ— -tle— t-— Ha- L— Pτo-L-Thr(OMe). (1) CT iD (4) (5) («) (7) (t)

Str—L— Arg— — Vώ- -T r— L-Ile— — Hii— -Pro- - Thr<O e). (J) C (3) (4) <3> (6) CO rø

L-W-MethylHIe— L-Λrι— 1^-Vtl— L— Tyr— — He— L-HIt— t^-Pra— L— He. (1) * ^{m W W W OT m}

CD CT CT C4) CT C«) CO W Angiotensin Receptor Antagonist- competitive peptides

e it t e ocaton enti ed by the superscript carboxylic add. Angiotensin Receptor Antagonist- competitive peptides

S»j j.y«l.Try-Vιl-Ha.ProOH,

R"-ArK-Vil-T r-ne-Hii-Pn>*teDop-OH

S«x-Ara-taHi'-Hv»-Tιy-VtI-Hi«.Pro-πe H, or wherein ^m is H, H-Asp or CHj HCHiCO and MeDop is an L-α-methyldαpa residue. Afr -A-^-ii»<i.h<jmo-V«l-Tyr-V(J-Ha-Pio-Phe- OH, wherein aza-α'-Hva is aza-alpha'-homo-L-valme.

R.'.L-Axa- -Vώ-U-T r-1-.lIe.L-Hiι-L.Pr - .X' Zr.S«--Art(NO.>.VtI.Tyr-XMtB-Pro.X' - OCH₁C«H N0₂

(where R' is dimethylglycyl, N-methylisoleucyl, guaaidylacetyl or sarcosyl and X' is a sarcosine, O- wherein Z? is phenyl-CHiC C-; X? is Val or lie; and methylsarcosine or isoleucine residue). X'ϊis He, Ala, Leu, Thr( e) or Thr.

Aldosterone Antagonist- Spironolactone

inUibitor- (8beta)-N-cyclo_alkyl-i-alkyl-6-(_Substlt ted) erg_θline-8-c_arbαχ_amide_S

Serotonin

bcrdm R.« C|-G*«lkyl;

R* is llyl or C1- 4 ttraight chaiα alkyl; * is hydrogen of C_\O, itraight data alkyl; R« Is hydrogen, C1-C4 alkyi, hydrt∑y or Ci-C_t aJ -

<MCyj id Is Q, I_t 2 or 3; provided when JU M Ri«« nob methyl and R³ and

R,*are each hydrogen, a may not be ύ; and the phirraaceutically acccptihlβ acid addition seta thercnf-

Serotonin iπhibitor- S Seerroottoonniinn i un-hiπibmituoⁿr (2,5-di_Metlιιyl-l-piperazinyl)-tUbenzfb,fJ[l,4] xazep e π-(3

Serotonin Inhibitor MDL 110,907

Dskϋtopiperazine Structure B

Diketopiperazine Structure A

be the same o

and R_lα which may t>

a nitro group;

different, is a d-C

formula.

Diketopiperazine Structure D

Diketopiperazine Structure C

Diketopiperazine-based Examples

(3Z.6Z)-3-(5-(2-D.me-hylaπιiaoeιhylthio)-2-thieayl) irιetiιylene-6^4-{2-tMcφhcaeαLrto

2 -prperaziDedionc (3Z,6Z)-3,6-Di-(4-(2-dimethylaminoe thoxy) (3Z.6Z)-6 5-{2-DimethylamLnocthylthio)-2-thienyl) beπzylidene)-25-piperaziπcdioαe dihydrochloride; merJιyicnc-3-0-pyridylmcthyleπc)-2 S-pirκra2incdiorιe (3Z.6Z)-6-(J-(2-Dimcthyiaaώιoethylthio)-2-thieayl) (3Z,6Z)-3-Benzyhdene-6-(4- is 2-dimcthylaπιinopropyl) nιethyleGe-3^-pvddylmethylea-)-2J-pirκιa2iBedioce arabomcthylbeαzyLidene-2 -piperaziπedione; (3Z.6zV6-(5-<2-Dimethyl*iniΛoetlιyUfaio)-2-tfaicnyl) (3Z,6Z)-3-Beπzylideπe-6-(4-(2-diD.ethyiaminoethoxy) niethyler«-3-{4^yridylnicmy.eDe)-2J^^ benzylidene)-2_r5-piρerazinedione; (3Z.6Z)-6-(5-{2-Dimcthylaxπiιιo€thylthio)-2-thicnyl) (3Z,6Z)-3,6-Di-(4-(2-dimethylaminopropoxy) ιπethylene-3-(l-methyl-3-indolyl)methylenc-2.5- beπzyϋdene)-2^-piperazmed-one bis tπethan sulfoπate pirxaazLnedioαe (1:2); 02 6 )-6^B<inzyHlcac-3^5-{2-diϊsoproρylaιmDoe^ (3Z,6Z) -3-B eπzyHdeae -6.(3 ,4-di- (2-

-2-thi-myl)memyle«-2J^rxsιzinedione dime thylamπoct hoxy)benzyIideae) -2,5 - (3 ffi^Bcn2yli £nc-3^5-(2-diιw^^ prperaziπedioαe; iitoo-2-tfcieayt)iijethyleae-2J-pip««Jnnc^ (3Z,6Z)-3-(2,6-Dichlorobenzylideae)-6-(4-(2- 216Z^3-<2J-£uιyd^5- en2ofιιrMy0πBttryleπe-6 5-<2- dimethylarπinoe thoxy)be nzylide ne -2,5- dmethyl-jπinoethylthio)-2-thicnyl) ct ylcac-2^- piperazirtcdioπc; pipcrazinedione 32 £Z)-6^Beti2ylidcπe-3<5-<2-<liιn<ώyltiϊώoc^ (3Z,6Z)-3,6-Di-(4-(3-dimethylaπππoproρoxy) Me^l)mct ykMe-2^-pή)erκππc<iiooe benzylidene)-2-S-piperazinedione bis hydrogen succi(3Z.6Z)-6-(4-Ace idobenzylidette)-3-(5-<2- nate (1:2); dimethylaιninoct yltrno)-2-tbicnyI)metfayIenc-2^- (3 Z,6Z)-3-B cπzylideae -6.(4- • pφerazinedioae dime thylaminomcthylbenzylidene) -2,S- (3Z.6Z)-6-(3-Chlorobenzy!ldcne)-3-(5-(2- piperazinedione; clt ethyl*ι-uaoet-tylthio)-2-thieαy£)πtethyleιιe-2J- Methyl 4-<4-((3Z,6Z)-6-(4-Methoxybeπzylidene)-2,5- piperazLoedione dioxo-3-pipcraziaylidene)methylbenzyicarboαyl) (3Z.6Z)-6-(2-Bro obenzylidene)-3-(5-(2- butaπoate; dlmcthyliuniαoctiιylthlo)-2-tbicnyl)methyleae-2^- (3Z^6Z5-3-(4-(3 limethyIbutanoyloxy)beQzylidene)-6- pipeπ-zlαcdiooe (4-melhoxybeπzylidcne)-25-piperaziιιcdioπe; (3Z.6Z)-6-(*-Chlorobeorylldeac)-3-(5-(2- (3Z,6Z)-3-(4-Acetaπudobenzylidene)-6-(2- d methyl«-miaocthyltHo)-2-t enyl)πιetlιylcjιe-2J- fluorobcrrzylideαc)--t^-piperazinedione; pirxxaxinedione (3Z.6Z)-6-(4-Cyi.nobcazylidcπe)-3-(5-(2- (3Z,6Z)-3-B enzylideae-6-(2-cb.loro-4-(2-

(limcthyUιninoetiιylthio)-2-thienyl)methylene-2J- dimethylatαittoetboxy)beαzylideπe)-2,5- ptperazuiediooe prperazinediooe; <3Z.6Z)-6-(3.4-DtchlorobeazyUdeae)-3-(5-(2- (3Z,6Z)-3_*Beazylideae-6-(2,4-di-(2- cumcthy ιmaoethyUhio)-2-tbieayl)metlιylcne-2_τ5- ditr.ethylaπriaoρroρoxy)beazylideπe)-2,5- pfperaziaedioαe ptperazinediooe; <3Z.6Z)-p%(3-Brι>mobeazylIdeae)-3 -<5 -(2- (3Z,6Z)-3-Beazylidene-6-(2,4-di-(2- dimcthyUariaoethyltWo)~2-taieayl)methylene-2.5- dime thylaiainoe thoxy)beazylideπe -2,5 -

•piperaziaedioQB piperazinedionc; (3Z.6Z)-6-(3-Cyaaobeπzylidcαe )-3-(5-(2- (32^6Z)-3-Benzylidene-6^4-bLs(3-dύneώylarιιiαopropyl) dimeώyUmiπoethylttøo)-2-thieayl)methylene-2.5- arπiπomethylbeπzylideπe)-2^-piperazinedione pip aziαedioαe (3Z,6Z)-3-Beπzylideπe-6-(4-(2-dimethylamiπoethoxy) (3Z,6Z)-6-Cyclohexylmcthyl eae-3 -(5 -(2- beπzylidene)-25-piperazinedione; imethy ιninoethylthio)-2-thJcnyl)mcthyienc-2 5- pjrxxazjaedioae (3Z,6Z)-3-(2,4-Diftuorobenzylidene)-6-(4-(2- (3Z.6^"Z)-ό^"-(4-BeazytoxybeazyHdeae)-3-<5-<2- dirπeth lacaiaoethoxy)beπzylideπc)-2,5- dlmethyUaύαoethylthlo)-2-thleayl)methylcne-2J- piperazinedioαe; piperazincdioae (321,625-3-(4- 2-Dime lajninαethoxy)berιzylidene)-6- (3Z.6Z)-<j-(3-BeazyloxybeazylIdeae)-3-(5-<2- (4-rnethoxybeαzylidene-2_r5-pipcraziπedioπe; dimcthy ιrttαoethyithio)-2-thicayl)methylene-2-5- (3Z,6Z)-3-Benzylidene-6-(2-{2-dimethylamifloethoxy) pipcnziDecuooe berizylidenc^∑^-pipcraziaedione; (3Z.6Z)-6-(4-Bro obeazylideac)-3 -(S-(2- (3Z,6Z)-3-Benzylidene-6-(4-(2-di-aethylamiaoethoxy) d meUιylaιruaoethyltMo)-2-thJeayl)mcthylene-2J- bcrιzyu leae)-2 -piperazmedione; pipcπώjcdione (32 Z)-3-(4-AminomethylbenzyLidene)-6-benzylidene- (3 Z.6Z)-6-(9-Aatttrylmetlιyleae)-3-(5-(2- 2 -piperaziπcdionc trifluαroacctate; dimethylajπinoethylthlo)-2-thleayl)mcthylene-2^- prpermzLncdione (3Z,6Z)-3-Bcazylidene-6-(4- 02^ ^Beπz iiteBe-3-{5-<6^<liπic<hyl ιr^ dimcthylaminobcnzyUdeαe)-2_τ5-pip«raziuedionc

2-thicayl)aκttryl ιe-2^^φerazuιedione hydrochloride; and ' OZJ52^Beιιzylidωe-3-(5-C2- imcth U^ (3Z,6Z)-3-(4-Hexyloxybcazylidene)-6-(4-^ϊi furyl)rnemyleae-2J-ρipcraidnedione methoxybenzylidene)-2_r5-piperazincdione.

(32 6Z)-3-(5-<2-Dimcthyli-miaoethylthio)-2-thicayl) mcώylcae-rH«.6-diraethyl-bicydo{3.1.11 ept-2-cπyl) nictrr leac-W-pipcnziacdioBe. Di etopiperazine-based Examples

(3Z,6Z)-3-Benzylidene-6-(4-(5-methylϊmidazotyI)) tπethylene-25-piperazinedioπc; (3Z,6Z)-3-Beπzylidene-6-(4-dimethylaππno- cinnarnylideπe)-_l -piper.znιedioαe; (3Z,6Z)-3-<4-(3-Diπiethylaminopropoxy)benzylideue)-6-

(4-{l-imidazolyl)beπzylidene-2_r5-piperaziπedione; (3Z,6Z)-3-Beπzylidene-6-(4-(2-itnidazolylethoxy) benzyiidene)-2,5 -piperaziπedionc; (3Z,6Z)-3-Bcnzylidcne-6-(4-nitrocinnamylidenc-2,5- ptperaziπediofle; (3Z,6Z)-3-(4-Aπιinometh lbeazylideπe)-6-(4- πκthoxybeαzyIideπe)-2-5-piperaziπedioπe; (3Z,6Z)-3-(l-rπeιhaπcsulfoπyi-3-iπdolyl)πιcώyleαe-ό- 4- πκthoxvbcnzylideπe)-2-5-piperaziπedioπe; (3Z,6 Z)-3 -(4- c thoxy benzy lid eae)-.6-(4- phmaJirm^'doa«toxybeπzyKcinc)-2 -pipcraziπedioαc; (3Z,6Z)-3-Beαzylidene-6-(γ-ρhenylcinaamylideae)-2,5- piperazinedioπe; (3Z,6Z)-3-(l-tert-butoxycarbonyl-3-indolyl)me-hylcne-6-

(2-thenylιdeπe)-2 -pJpcraziπedioπe; (3Z,6Z)-3-(2,6-Dichlorobeazylideae)-6-(l-tert- butoxycarboayl-3-iadolyl)π.ethylene-2,5- pjpcraziπedioQc; (326Z)-3-Beπzyu^"deπe-6-(4-(2-dimethylarninoethoxy)-3- aκthoxycmnamylidene-2^-prperaziaedioαe; (32^2^-3^4-(3-Dimethylarrώoρroρoxy)benzylideae^-{4-

(l-iπιidazolyImeώyl)beazyUcfcne)-2 -prperaziaccu^'oae; (3Z-6Z)-3-Benzylidene-6-(4-N-rnethy.-N-(4-(N- methylpiperidinyl))aminornethylbenzylideae-2,5- pφeraziacdione; ((3Z,6Z)-3-Benzylidene-6-(3-indolylmethyleπe)-2,5- piperaaiπedione; (3Z,6Z)-3-(2,6-Dichlorobeazylidene)-6-(3- forylmethyfcπe)-2 -pψeraziπedioae; (3Z,6Z)-3-(3-Indolylmethyleae)-fi-(4-methoxy- beπzyIideae)-2^-prperazinedione; (3Z,6Z)-3-(2,6-Dichlorobeazy.idene)-6-(3-(l- tcrtbutoxycirboayl)iadolyl)methylene-2,5 -

(3Z,^"6Z)-3-Benzylideαe-6<4-imidazolyl)methylcπe-2-5- pxpcraziπedioπe; piperazinedione; (3Z-6Z)-3-(4-Melhoxybeazylide πe)-6-(2-(l- (3Z,6Z)-3-Beπzylidene-6-(4-(l-imidazolyl)benzylideπc)-2, tertbutoxycarbonyl)pyrrolyl)a.ethyleae-2,5-

5-piρeraziπedioπe; piperaziaediooe;

(3Z,6Z)-3-Benzyl.deπe-6-(4-(l-imidazolylmethyl) (3Z,fiZ)-3-(4-Methoxybenzylideae)-6-(3-(l- benzyiidene-2_τ5-pipcraziπedione; tertbutoxycarboayl)iado.yl)methylene-2_t5-

(3Z,6Z)-3-Benzylideae-ό-(4 2-duπethyIϊ-!iinoethoxy)-3- piperaziiiedione; methoxybenzylideαe)-2 -pipetazinedioπc hydrochloZ^Z)-3-Benzylidene-6^3-(l-tertbutoxycarbonyl)indolyl) ride; BKthyleae-Σ_jS-prperazinediooe; (3Z,fiZ)-3-(4-Methoxybeazyltdeae)-6-(2-thicnyl- mcthyIene)-2_f5-piperaziaedione; (32^2^-3-(4-Me-hoxyber-zyUdene)-6 3-rurylrneuiylene)-

2^-piperιziaedione; (3Z, 6 Z)-3 - (Ace rπido b e αz y l ide ae ) - ό - cyclohexylmethyieαe-2-5-ptperazinediooc; Z^Z)-3-(4-Acetaπιidobeπzytidene)-6-ciππaπιylidene-2^5- p crazinedione; Z-6Z)-3-BeazyUdene-6-(diethoxymeώyu3enzylider.e)-2,

5-ρiρerazmedιoae; (3Z,6Z)-3-Beazylidene.6-(4-(N-metbyl-N-(2- dimeihylamϊπoethyl)arπinomethylbenzylidene-2,5- pipcraz-acdϊoαe hydrochloride; (3Z,δZ)-3-Beazylideae-6^'-cyclohexylmethyleae-2-5- ptperaztaediααc; (3Z.6Z)-3-(4-Acctamidobenzylideαe)-6-(3,4- meihylcπedioxybenzylidcne)-2^-pipcraziπcdionc; (3Z_T6Z)-3-(2-ladolylmetfaylene)-6-(4-methox r- beazylideae)-2^-piperazincdioae; •

(3Z,6Z)-3-Beazylideae-6-(3,4-methyleaedioxy- beπzylidcae)-2^-pipcrazinedioπe; Diketopiperazine-based Examples

benzylidene)-3-{3-ώenylideπe)-2^-piρerazinedione;

⁽-ι^W!to^{ϊ |j}w^ϋι^jcnc>.2-5ii| »j»o(lioBβ ptt in_edlonc Benzil, Benzophenone, Substituted and Unsubstituted Biaryl, Benzyl Ether, and Thioether-based Examples

NAME STRUCTURE

(4-hydroxy-3-iodo-5-πitro- pheπy!)-phenyl- methanone

3,5-diiodo-4-(3-iodo- benzylαxy)- beπzoic acid

3,5-diiodo-4-(4-iodo- benzyloxy)- benzoic acid

3,5-dϋodo-4-(2-bromo- beπzyloxy)- benzoic acid

3,5-diiodo-4-(3-bromo- benzyloxy)- benzoic acid

3,5-diiodo-4-(4-bromo- benzyloxy)- benzoic acid

3 ,5-diiodo-4-(2-methyl- benzyloxy)- benzoic acid

3 ,5-diiαdo-4-(3-methyl- benzyloxy)- benzoic acid

3,5-diiodo-4-(4-roethy!- benzytaxy)- benzoic acid

4-(4-tert-butyl-benzyloxy)- 3,5- diiodo-benzoic acid

3,5-diiodo-4-(πaphthalen-

2-y(methoxy)-benzoic acid Benzil, Benzophenone, Substituted and Unsubstituted Biaryl, Benzyl Ether, and Thioether-based Examples

NAME STRUCTURE

4-(biρhenyl-2-yimethoxy}- 3,5-diiodo- benzoic acid

3 , δ-d iiodo-4-(3-methoxy- benzyloxy)- benzoic acid

3,5-diiodo-4-(3- trifluαromelhy!- benzyloxy)-benzoic acid

3,5-diiodo-4-(3- trifluoromethoxy- benzyioxy)-benzoic acid

4-(3-fluoro-benzyloxy)- 3,5-diiodo-benzoic acid

3,5-diiodo-4- pentafluoropheπylmetho xy- benzoic acid

3,5-dibromo-4-(3-iodo- Br o benzyloxy)-benzoic acid v t

Br

3,5-dichioro-4-(3-iodo- benzyloxy)-benzoic acid

3-[3,5-diiodo-4-(3-iαdo- benzyloxy)-phenyI]- propionic acid

3-[3,5-diιodo-4-(4-iodo- benzyloxy)-phenyl]- propionic acid

Benzil, Benzophenone, Substituted and Unsubstituted Biaryl, Benzyl Ether, and Thioether-based Examples

NAME STRUCTURE

[4-(3-bromo-benzyloxy)-

4-{4-nitro-benzyloxy)-3,5-

61

NAME STRUCTURE

4-hydroxy-3,5- diiodobenzoic acid 2- iodobenzyl ester

diiodo-4-(3- iodobenzyioxy)-phenyI]- propionic acid

bis-(4-hydroxy-3,5- diiodophenyl}- methanone

4-(2-fluorobenzyloxy)-3,5- diiαdobenzoic acid

-4-(4-fltιorobenzylαxy)-3,5- diiodobenzoic acid

4-(2-clhorobenzyloxy)- 3,5-diiodobenzoic acid

4-(3-chlorobenzyloxy)- 3,5-diiodobenzoic acid

[4-(4-hydroxy-3,5-ditodo- benzoyl)-2,6-diiodo- phenoxy]-acetic acid benzyl ester

4-(4-chlorob eπzy ioxy )- 0 3,5-diiodobenzoic acid

£TX

NAME STRUCTURE

4-(1 ,6-difluorobenzy!oxy)- 3,5-diiodobeπzoic acid

4-[4-(trifluorαmethoxy)- beπzyioxy]-3,-5- diiodobenzoic acid

4-[2-(trifiuoromethyl)- benzytoxy]-3_t5- diiodobenzoic acid

4-[4-(trifluoromethyl)- benzyloxyj-3,5- diiodobenzoic acid <_<cr

4-[(2-fluoro-4- bromo)benzyloxy]-3,5- diiodobenzoic acid

4-(2-iodo)benzyloxy-3,5- diiodobenzoic acid

4-(3-benzoyl)benzyloxy- 3,5-diiodobenzoic acid

[4-(4-hydroxy-3,5-diiodo- benzoyl)-2,6-diiodo- phenoxy]-acetic acid

4-(3-methy!naphthyl-2- methoxy)-3,5- diiodobenzoic acid

NAME STRUCTURE

4-[N-[(2-beπzoyl)phenyl]- methoxyamidyl]-3,5- diiodobenzoic acid

4-[4-.(trifluorαrnethyl)thio]- benzyloxy-3,5- diiodobenzoic acid X XX

4-[2-(trif!uorαmethy[)thiσ]- benzyloxy-3,5- diiodobenzoic acid X. ^'

4-(adamantyl-1- acetyloxy)-3,5- diiodobenzoic acid -

4-(4-chloro)benzyloxy- 3,5-dichlorobenzoic acid

4-(naphthyl-2-methoxy)- 3,5-dichIorobenzoic acid

4-(4-iαdo)benzyloxy-3,5- dibromobenzoic acid

4-(naphthyl-2-methoxy)- 3,5-dibromobenzoic acid

4-(9H-fluoreπ-9-yloxy)- 3,5-diiodobenzoic acid

1 ,2-bis-(4-hydroxy-3,5- diiodophenyl)-ethane-1 ,2- dione

NAME STRUCTURE

(4-fiuorophenyl)-(4- hydroxy-3,5- diiodophenyl)- methanone

4-[2-(trifluoromethoxy)- benzyloxy]-3,5- diiodobenzoic acid

4-( 10-carboxy-decyloxy)- 3,5-diiodobenzoic acid

4-(4-tert-butyl-benzyloxy)- 3.5-dichIorobenzαic acid CO*"

O

4-(2-phenyl-benzyloxy)- 3,5-dibromobenzoic acid

{4-[(4-hydroxy-3_f5- diiodophenyl)-oxo-acetyl]- 2,6-diiodopheπdxy}- acetic acid benzyl ester

{4-[(4- ydrαxy-3,5- diiodophenyi)-oxo-acetyl]- 2,6-diiodophenoxy}- acetic acid

4-(2-pheny.)benzyloxy- 3,5-dichloroberιzoic acid

NAME STRUCTURE

4-hydroxy-3,5- diiodobeπzoic add 2- (trimethylsilyl)ethoxymet hoxy ester

diiodαbenzoϊc acid

4-hexadecyIoxy- ,5- diiodobenzoic acid

[4-{4-hydroxy-3,5-diiodo- benzenesuIfonyl)-2,6- diiodo-phenoxy]-acetic acid benzyl ester

[4-(4-hydroxy-3,5-diiodo- benzenesulfonyl)-2,6- dϊiodo-phenoxy]-acetic acid phenyl ester

4-hydroxy-3,5- diiodobenzoic acid pheπoxycarbonylmethyl ester

NAME STRUCTURE

4-hydroxy-3,5- diiodobenzoic acid [4- . O chioro-2-(2-chloro- beπzoyl)- χ&. phenylcarbamoyl]-methyl ester

[3,5-diiodo-4-(3-iodo- benzyloxy)-phenyl]-(4- hydroxy-3,5- diiodophenyl)-

methanoπe

[4-(4-hydroxy-3 ,5-diiodo- benzoyt)-2,6- * diiodophenoxy]-acetic acid phenyl ester

1 -[3,5-diiodo-4-(3-iodo- beπzyloxy)-ρhenyl]-2-(4- „ ^' •y«X Λ hydroxy-3,5- diiodophenyl)-ethane-1 ,2- dione

{4-[(4-hydroxy-3,5- diiodophenyl)-oxo-acetyl]- 2,6-dϋodo-phenoxy}- acetic acid phenyl ester

carbonic acid benzyl ester 4-(4-hydroxy-3,5- diiodc~benzoyl)-2,6- diiodo-phenyl ester

4-hydroxy-3,5-diiodo- benzoic acid 3- benzyloxybenzyl ester

Benzil, Benzophenone, Substituted and Unsubstituted Biaryl, Benzyl Ether, an_d Thioether-based Examples

NAME STRUCTURE

[4-(9H-fluoreπ-9-yloxy)- 3,5-diiodo-pheπyl]-(4- hydroxy-3,5-diiodo- pheny!)-methanone

3-[3,5-diiodo-4-(3-iodo- benzyloxy)-phenyl]-3-(4- riydroxy-3,5-diiodo- phenyl)-3H-

isobenzofuran-1 -one

acid

4-hydroxy-3,5-diiodo- benzoic acid 2-(4- methoxy-phenyl)-2-oxo- elhyl ester

2-{2-[4-(4-hydroxy-3,5- diiodo-benzoyl)-2,6- diiodo-phenoxymethyi]- phenyl}-isoindole-1 ,3-

dione

4-benzyloxycarbonyloxy- 3,5-diiodo-benzoic acid

3 ,5-dibromo-4-hydroxy- benzoic acid 4 butyl- benzyl ester

4-[2-(2,3-dihydro- benzo[1 ,4]dioxin-6-yl)-2- oxo-ethoxy]-3,5-diiodo- benzoic acid

Benzi_l, Benzophenone, Substituted and Unsubstituted Biaryl, Benzyl Ether, and Thioether-based Examples

NAME STRUCTURE

4-(oxane-2-methoxy)-3,5- diiodo-benzoic acid

4-hydroxy-3,5-diiodo- benzoic acid 2-(1 ,3- dioxo-1 ,3-dihydro- isoindol-2-yl)-benzyl

ester

3,5-diiαdo-4-hydroxy-N- phenylethyl-benzamide

4-benzyl-2.6-diiodo- phenol

4-but-2-enyioxy-3,5- diiodo-benzoϊc acid

acetic acid 4-[1-(4- hydroxy-3,5-diiodo- phenyl)-3-oxo-1,3- dihydro-isobenzofuran-1 -

yl]-2,6-diiodo-phenyl ester

2,6-diiodo-4-{1 -methyl-1 ^■ phenyi-ethyl)-phenol .

4-(3,5-diiodo-4-methoxy- benzenesulfonyl}-2,6- diiodophenol

NAME STRUCTURE

4-[4-(biphenyl-3- lymethoxyJ-S.δ-diiodo- benzenesulfonyl]-2,6- diiodo-Dhenol

(3,5-diiodo-4-methoxy- phenyl )-(4-hydroxy-3 ,5- dϊiαdo-phenyl)- methanoπe

(4-hydroxy-3,5-diiodo- phenyl)-morphenoxylin-4- yl-melhanoπe

[4-(biρhenyl-3- ylmethoxy)-3,5-diiodo- phenyl]-(4-hydroxy-3,5- diiodo-phenyl)- methanαne 4-hydroxy-3,5- diiodobenzoic acid cyclohexylmethyl ester

3-(3,5-diiodo-4- methoxyphenyl)-3-(4- hydroxy-3,5- diiodophenyl)-3H-

isobenzofuran-1-one

(2-fluorophenyl)-(4- hydroxy-3,5- diiodophenyl)- met anone

BenzU, Benzophenone, Substituted and Unsubstituted Biaryl, Benzyl Ether, and Thioether-based Examples

NAME STRUCTURE

4-(2-cyclohexylethoxy)- 3,5-diiodobenzoic acid

(2,4-dimethoxyphenyl)-(4- hydroxy-3,5- diiodophenyl)- methanone

4^,-hydroxy-3',5^,-diiodo- biphenyI-4-carbαnitrile

4-hydroxy-3,5- diiodobenzoic acid 4-(N- benzyl-N-ethyl- carbamoyi)-2,6-

diiodophenyl ester

2,6-diiodo-[4-(1 -benzyl)- tetrazolyl]-pheπol

3-(4-hydroxy-3,5- diiodophenyl)-acrylic acid methyl ester

4-hydroxy-3,5- diiodobenzαic acid benzyl ester

p eny ethoxycar ony - allyl ester Benzil, Benzophenone, Substituted and Unsubstituted Biaryl, Benzyl Ether, and Thioether-based Examples

NAME STRUCTURE

4-hydroxy-3,5- diiodobeπzoic acid 1 - methylhexyl. ester

4-hydroxy-3,5- diiodobenzoic acid heptyl ester

2,6-diiodc~4-octyl-phenol

4-hydroxy-3,5- diiodobenzoic acid 2,4,4- trimethylpentyl ester

NAME STRUCTURE

4-[2-(4-bromαphenyI)-2- oxo-ethoxy]-3,5- diiodobenzoic acid.

2,6-diiodo-4-(4-nitro)- phenyl-phenol

4-(1 -hydroxynonyl)-2,6- x ^" diiodo-phenol

6-{3-iodobenzyloxy)- 2,5,7,8-tetramethyl- chromaπ-2-carboxylic « acid

6-(2-iodobenzyloxy)-

1-(3,5-diiodo-4-hydroxy)- phe yl-peπtaπ-1 -one

N,N-dibenzyl-4-hydroxy- 3,5- ϋiodo-benzamide

NAME STRUCTURE LTHYROXINE

TETRAIODOTHYRO- PROPIONIC ACID

1-(2-(BR-NAPHTHALEN-

YLOXY)-5-chloro- phenyl)-DI- E- ^

(1 ,3,5)TRIAZINE-

DIAMINE.

HYDROCHLORIDE ^"

NAME STRUCTURE

5-NITRO-2-(4-(1 ,1,3.3- TETRA ETHYLBUTYL) PHENOXY)BENZENES ULFONIC ACID

FMOC-P-BENZOYL- . PHENYLALANINE

0-(3-

CARBOXYBENZYL)-3,4-

DICHLORO

ACETOPHENONE J "

OXIME

6-(4-BENZYL-PHENYL)- HEXANOIC ACID

NAME STRUCTURE

5-CHLORO-2-(4-.

CHLORO-2-(3,4-

DICHLOROPHENYLUR - t

EIDO)-PHENOXY)-

BENZENESULFONIC

ACID

F OC-P-BENZOYL-D- PHENYLALANINE QO_yO

7-ANIUNO-4-HYDROXY- 2-

NAPHTHALENESULFO uδ NIC ACID

DIPHENYLPHOSPHINO BENZENE-3-SULFONIC ACID SODIUM SALT σ^'t ^d

NAME STRUCTURE

N-F OC-4-CHLORO-L-

PHENYLALANINE

FMOC-P-PHENYL-D- PHENYLALANINE

F OC-(3,5-DllODO)-D- TYROSINE

FMOC-N-METHYL-0- BENZYL-TYROSINE

F OC-4-BRO O-D- PHENYLALANINE

FMOC-3.4-DICHLORO- D-PHENYLALANINE

FMOC-3.4-DICHLORO- L-PHENYLALANINE

F OC-3-CHLORO- PHENYLALANINE

FMOC- NAPHTHYLALANINE BenzU, Benzophenone, Substituted and Unsubstituted Biaryl, Benzyl Ether, and Thioether-based Examples

NAME STRUCTURE

FMOC-4-ISOPROPYL- PHENYLALANINE

FMOC-L-3-

BENZOTHIENYLALANI

NE

UINOLYLIDENE)HYDR

AZIDE

CHLORO-HYDROXY- 0,,

PHENYL-AZO- i

OCTADECAN-A IDO-

OXO-PYRAZOLINYL-

PHENOXY-BENZENE-

SULFONICACID

1-(3-SULFO-4-

PHENOXY PHENYL-3-

HEPTADECYL-

PYRAZOLINE-5-ONE

NAME STRUCTURE

1-(3-SULFO-4-

PHENOXY)-PHENYL-3-

HEPTADECYL-

CARBONYL-AMINO-

PYRAZOLINE-5-ONE

2-PHENOXY-BENZENE

PHTHALIC ACID ONO- (BIPHENYL-4-YL- PHENYL-METHYL) ESTER

SODIUM DECYL DIPHENYL ETHER DISULFONATE

2-HO-5-(4'-HO- BIPHENYL-4-YLAZO)- BENZOIC ACID, SODIUM SALT ^■ MH/- CH

FMOC-O-BENZYL- ASPARTICACID

NAME STRUCTURE

2-[[(9H-FLUOREN-9-

YLMETHOXY)CARBON

YL]AMINO]-5-

[[[(2,2,5,7,8-PENTA-

METHYL-3.4-DIHYDRO-

SULFONYL)-AMINO]-

IMINO]-AMINO]-

PENTANOIC ACID

4,5,9,10-TETRAHYDRO- PYRENE-2.7-DIOL

SODIUM DODECYL DIPHENYL ETHER DISULFONATE

FMOC-(S)-3-AMINO-4- (3-BENZOTHIENYL)- BUTYRIC ACID

FMOC-D-4-TERT-

BUTYL-

PHENYLALANINE BenzU, Benzophenone, Substituted and Unsubstituted Biaryl, Benzyl Ether, and Thioether-based Examples

NAME STRUCTURE

2-TOSYL-AMINO-5-(3- CARBOXY-^TOSYL- AMINO-BENZYL)- r BENZOIC ACID ϊ h_*

NAME STRUCTURE

2-(3-ETHYNYL-

PHENYL)-1,3-DIOXO-

2,3-DIHYDRO-1H-5-(4-

CARBOXY)-BENZOYL-

ISOINDOLE

2-[4-(amido-benz-2-oic acid)]-6-methyl- 3K> Wo benzlhiazole

piperazinone

nitrobenzylideπe - 2,5- piperazinone

(4-hydroxy-3-iαdo-5-nitro- pheπyl)-pheπyl- methaπone

3,5-diiodo-4-(3-iodo- benzyioxy)- benzoic acid

3,5-diiodo-4-(4-iodo- benzyloxy)- benzoic acid

4-benzyloxy-3,5-diϊodo- benzoic aci

3,5-diiodo-4-(2-bromo- beπzyloxy}- benzoic acid

3 ,5-diiodo-4-(3-bromo- benzyloxy)- benzoic acid

NAME STRUCTURE

3,5-diiodσ-4-(4-brαmo- benzyloxy)- benzoic acid

4-{biphenyl-2-ylmethαxy)- 3,5-diiodo- benzoic acid

3,5-diiodo-4-(3-methαxy- benzyloxy)- benzoic acid

3,5-diiodα-4-(3- trifluoromethyl- benzyloxy)-beπzoic acid

3,5-diiαdo-4-(3- trifluoromethoxy- benzyloxy)-beπzoic acid

4-(3-fluoro-beπzyIoxy)- 3,5-dϋodo-benzoic acid

^■NAME STRUCTURE 3,5-diiodo-4- . pentafluorophenylmetho xy- benzoic acid

3,5-dibromo-4-(3-iodo- benzvloxy>-benzoic acid

3,5-dichloro-4-(3-iodo- benzyloxy)-beπzoic acid

3-(4-benzyloxy-3,5- diiodo-phenyl)-propionic acid benzyl ester

3-[3,5-diiodo-4-(3-iodo- beπzyloxy)-phenyl]- propionic acid

(3 ,5-diiodo-4-(3-iodo- beπzyloxy)-pheπyl]- acetic acid

[3,5-diiodo-4-(4-iodo- benzyloxy)-phenyl]- acetic acid

NAME STRUCTURE

4-(3-nitro-benzyloxy)-3,5- diiodo-benzoic acid

4-(4-nitro-benzyloxy)-3 ,5- diiodo-benzoic acid

4-(2-cyano-benzy!oxy)- 3,5-diiodo-benzoic acid

4-(3-cyano-benzyloxy)- 3,5-diiodo-benzoic acid

4-(4-cyano-beπzyloxy)- 3,5-diiodo-benzoic acid

propionic acid BenzU, Benzophenone, Substituted and Unsubstituted Biaryl, Benzyl Ether, and Thioether-based Examples

NAME STRUCTURE bis-(4-hydroxy-3,5- diiodopheπyl}- methanone

4-(2-fluorobenzyloxy)-3,5- dϋodobenzαic acid ^" . o

4-(4-fluorobenzyloxy)-3 ,5- diiodobenzoic acid

4-(2-chloro-benzyloxy}- 3,5-diiodobenzoic acid

4-(3-chtoro-benzyloxy)- 3,5-diiodobenzoic acid

[4-(4-hydroxy-3,5-diiodo- beπzoyl)-2,6-diiodo- phenoxyj-acetic acid benzyl ester

4-(4-chloro-benzyIoxy)- 3,5-diiodobenzoic acid

[4-[(6,7-dihydro-5,5,8,8- tetramethyl)naphthyl-2- methoxy]-3,5- diiodobenzoic acid

4-(1,6-dichlorσ- benzyloxy}-3,5- diiodobenzoic acid

4-(1,6-difluoro- beπzyloxy)-3,5- diiodobenzoic acid

4-[4-(trifluoromethoxy)- benzyloxy]-3,5- diiodobenzoic acid

NAME STRUCTURE

4-[2-(trifluαromethyl)- o beπzyloxy]-3,5- diiodobenzoic acid

4-[4-(trifluαromethyl)- benzyloxy]-3,5- diiodobeπzoic acid _> coY

4-[(2-fluoro-4- bromo)beπzyloxy]-3,5- diiodobenzoic acid

4-(2-iodo)benzyloxy-3,5- diiodobenzoic acid

4-(3-benzoyl)beπzyloxy- 3,5-diiodobeπzoic acid

4-[(4-hydroxy-3,5-diiodo)- benzoyl]-2,6-diiodo- phenoxy-acetic acid

4-(3-methylnaphthyl-2- methoxy)-3,5- diiodobenzαic acid

4-[N-[(2-benzαyl)phen π- MeOamidyl]-3,5- diiodobenzoic acid

4-[4-{trifluoromelhyl )thio]- benzyloxy-3,5- diiodobenzoic acid

4-[2-(triflι_oromethyl)thio]- benzyloxy-3,5- diiodobenzαic acid CO " Benzil, Benzophenone, Substituted and Unsubstituted Biaryl, Benzyl Ether, and Thioether-based Examples

NAME STRUCTURE

4-(adamantyl-1- acety!oxy)-3,5- diiodobenzoic acid

4-(4-chloro)benzy!oxy- 3 S-dichloro-beπzoic acid

4-[2-(trifiuoromethoxy)- benzyloxy]-3,5- diiodobenzoic acid

4-(10-carboxy-decyloxy)- 3,5-diiodobeπzoic acid

4-(4-tert-buty(-beπzyloxy)~ 3,5-dichloro-benzoic acid

NAME STRUCTURE

4-(2-phenyl-benzyloxy)- 3,5-dibromobenzoic acid

{4-[(4-hydroxy-3,5-diiodo- phenyl)-oxo-acetyl]-2,6- diiodo-phenylenoxy}- acetic acid benzyl ester

{4-[(4-hydroxy-3,5-diiodo- phenyl)-oxo-acetyl]-2,6- diiodo-phenylenoxy}- acetic acid

4-(2phenyl)benzyloxy- 3,5-dichloro-benzoic acid

4-hydrαxy-3,5- diiodobenzoic acid 2- trimethylsilylethoxymeth oxy ester

a.y.S.ff-tetraio o^.^-

3,5-diiodobenzoic acid

4-hexadecyloxy-3,5- diiodobenzoic acid

[4-(4-hydroxy-3,5-diiodo- benzenesulfonyl)-2,6- diiodo-phenoxy]-acetic acid benzyl ester

NAME STRUCTURE

[4-(4-hydroxy-3,5-diiodo- benzeπesulfonyl)-2,6- diiodo-pheπyleπoxy]- acetic acid phenyl ester

4-(3,5-dϋodo-4-(3-iodo- benzyloxy)- benzenesulfonyl]-2,6- diiodo-phenylenol

4-hydroxy-3,5- diiodobenzoic acid phenoxycarbonylmethyl # * ester .

4-hydroxy-3,5- diiαdobenzoic acid [4- chlofo-2-{2-chloro- ^" benzoyl)-

phenylcarbamoyl]-methyl ester

[3,5-diiodo-4-(3-iodo- beπzyloxy)-phenyl]-(4- hydroxy-3 , 5-diiodo- pheπyl)-methanone

[4-(4-hydroxy-3,5-diiodo- benzoyl)-2,6-diiodo- phenylenoxy]-acetic acid phenyl ester

1 -[3,5-diiodo-4-(3-iodo- benzyloxy)-phenyl]-2-(4- hydroxy-3,5-diiodo- phenyl)-ethane-1 ,2-dione

{4-[(4-hydroxy-3,5-diiodo- phenyl oxo-acetyl]-2,6- diiodo-phenoxyj-acetic acid phenyl ester

NAME STRUCTURE

3,3-bis-(4-hydroxy-3,5- diiodo-pheny!)-3H- isobenzofuran-1 -one

carbonic acid benzyl ester 4-(4-hydroxy-3,5- diiodo-benzoyl)-2,6-

diiodo-phenyl ester

4-hydroxy-3,5- diiodobenzαic acid 3- benzyloxy-benzyl ester

[4-(9 H-uuoren-9-yloxy )- 3,5-diiαdo-pheπyl]-(4- hydroxy-3 ,5-diiodo- phenyl)-methanone

3-[3,5-diiodo-4-(3-iodo- benzyloxy)-phenyl]-3-(4- hydroxy-3,5-diiodo- phenyl)-3H-

isobenzofuraπ-1-one

4-hydroxy-3 ,5-diiodo- benzoic acid 2-(4- methoxy-phenyl)-2-oxo- ethyl ester fx°^"

2-{2-[4-(4-hydroxy-3,5- diiodo-benzoyl)-2,6- diiodo-phenoxymethyl]- phenyl}-isσindole-1 ,3-

dioπe

4-benzyIoxycarbonyloxy- 3,5-diiodo-benzoic acid

NAME STRUCTURE

3,5-dibrσmo-4-hydroxy- benzoic acid 4-lbutyl- benzyl ester

4-[2-(2,3-dihydro- benzo[1 ,4]dioxin-6-yl)-2- oxσ-ethoxy]-3,5-diiodo- benzoic acid

4-(oxane-2-melhoxy)-3,5- diiodo-benzoic acid

4-hydrαxy-3 ,5-diiodo- benzoic acid 2-(1,3- dioxo-1,3-dihydro- .2. isoindol-2-yl)-benzyl _* ester

3,5-diiodo-4-hydroxy-N- phenylethyl-beπzamide

4-benzyl-2,6-diiodo- pheπol

4-but-2-enyloxy-3,5- diiodo-benzoic acid

acetic acid 4-[1-(4- hydroxy-3,5-diiodo- pheπyO-S-oxol.S- dihydro-isobenzofϋran-1 -

yl]-2.6-diiodo-phenyl ester

2,6*diiodo-4-(1 -methyl-1 - phenyt-ethyl)-phenol

NAME STRUCTURE

4-(3 ,5 -diiod o-4-melhoxy- benzenesu!fonyl)-2,6- diiodo-phenol

4-[4-(bipheπyl-3- lymethoxy)-3,5-diiodo- benzeπesu!fonyl]-2,6- diiodo-phenol

(3,5-diiodo-4-methoxy- phenyl)-(4-hydroxy-3 ,5- diiodo-phenyl)- methanone

(4-hydroxy-3,5-diiodo- pheπyl)-moφhenoxyIiπ-4- y!-methanone

[4-(biphenyl-3- ylmelhoxy)-3,5-diiodo- phenyl]-(4-hydroxy-3,5- diiodo-pheπyl)-

methanone

4-hydroxy-3,5- diiodobeπzoic acid cyclohexylmethyl ester

3-(3,5-diiodo-4- melhoxypheπyl)-3-(4- hydroxy-3,5- diiodophenyl)-3H-

isobenzofuran- -one

(2-fluαropheπyI)-(4- hydroxy-3,5- diiodophenyl)- methaπone

NAME STRUCTURE

4-(2-cyclohexylethoxy)- 3,5-diiodobenzoic acid

(2,4-dimethoxyphenyl)-(4- hydroxy-3,5- diiodopheπyl)- methanone

4'-hydroxy-3',5-di{odo- bipheπyl-4-carbonitrile

4-hdyroxy-3,5- diiodobenzoic acid π- butyl ester

4-hydroxy-3,5- diiodobenzoic acid 4-{N- benzyl-N-ethyl- carbamoyl)-2,6-

diiodophenyl ester

2_f6-diiαdo-[4-(1-beπzyl)- tetrazolyl]-phenol

3-(4-hydroxy-3,5- diiodophenyl)-acrylic acid methyl ester

4-hydroxy-3,5- diiodobenzoic acid benzyl ester

4-hydroxy-3,5- diiodobeπzoic acid 3-(2- oxo-2- phenylethoxycarbonyl)- r allyl ester Benzil, Benzophenone, Substituted and Unsubstituted Biaryl, Benzyl Ether, and Thioether-based Examples

NAME STRUCTURE

4-hydroxy-3,5- diiodobenzoic acid 1- methylhexyl ester

4-hydroxy-3,5- diiodobenzoic acid ncμiyi CSIGI

2,6-diiodo-4-octyl-phenol

4-hydroxy-3,5- diiodobeπzoic acid 2,4,4- trimelhylpentyl ester

3-[3,5-diiodo-4-(2- iodobenzyloxy)-phenyl]- ^• propionic acid

2,6-dich!oro-4-pheπyl- ethynyl-phenol

3-(4-hydroxy-3,5-dil- benzylidene)-1 ,3-dihydro- iπdol-2-one

2-(4-tert-butyl)-benzylαxy- 3,5-diiodαbenzoic acid

,4-dihydrα-2H- beπzo[b][1 ,4]dioxepin-7- yl)-2-oxo-ethoxy]-3,5- diiodobenzoic acid

4-[2-(4-brαmαphenyl)-2- oxo-ethoxy]-3,5- diiodobenzoic acid

NAME STRUCTURE

2,6-diiodo-4-(4-nitro}- phenyl-phenol

4-(1 -hydroxynonyl)-2,6- ^y diiodo-phenol

6-(3-iodobenzyloxy)-

4-cyclohexylmethoxy-3,5- diiodobenzoic acid

1-(3,5-dϊiodo-4-hydroxy)- phenyl-pentan-1 -one

5,7-dihydroxy-6,8-diiodo- 2-phenyl-chromen-4-one

N,N-dibenzyl-4.-rιydroxy- 3,5-diiodo-benzamide

Claims

What is claimed is:

1. A method of reducing circulating glucose levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a Plasminogen Activator Inhibitor (PAI) inhibitor.

2. A method of increasing insulin sensitivity in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a poljφeptide sequence comprising a Plasminogen Activator Inhibitor (PAI) inhibitor.

3. A method for decreasing the body weight of an individual, increasing the partitioning of lipids to the liver of an individual, or reducing the levels of plasma free fatty acids in an individual, comprising administering to said individual an effective amount of a physiologically acceptable composition comprising Plasminogen Activator Inhibitor (PAI) inhibitor.

4. The use of a Plasminogen Activator Inhibitor (PAI) inhibitor for the preparation of a medicament for reducing circulating glucose levels in an individual.

5. The use of a Plasminogen Activator Inhibitor (PAI) inhibitor for the preparation of a medicament for increasing insulin sensitivity in an individual.

6. The use of a Plasminogen Activator Inhibitor (PAI) inhibitor for the preparation of a medicament for decreasing body mass of an individual.