Title:  Azaftig, a proteoglycan for monitoring cachexia and for control of obesity

United States Patent:  6,274,550

Assignee:  Board of Supervisors of Louisiana State University and Agricultural and (Baton Rouge, LA)

Appl. No.:  340873

A proteoglycan ("azaftig") with a molecular weight of approximately 24,000 Dalton has been isolated and partially characterized from the urine of cachectic cancer and non-cancer patients. Azaftig has been shown to bind to receptors on fat cell membranes, and to cause lipolysis. Azaftig does not bind to muscle cell membranes, or cause proteolysis in muscle tissue. Azaftig detection in urine or other body fluids will allow early identification of patients in which weight loss may become a problem. Azaftig may also aid fat loss in humans in which obesity is a threat to health.

Description of the Invention

This invention pertains to the detection of a propensity for cachexia and to the control of obesity.

Cachexia is defined as significant weight loss. It occurs commonly in cancer patients and HIV-infected individuals, but can also be caused by hypercatabolism due to cardiac failure (especially, right-sided or biventricular failure), hepatic failure, renal failure, burns, inflammation (including sepsis), infection or tuberculosis. See R. B. Verdery, "Reversible and irreversible weight loss (cachexia) in the elderly," in Textbook of Internal Medicine, 2d Edition (V. T. DeVita et at. eds.), Ch. 523, pp. 2424-2425 (1992); K. I. Marton, "Approach to patient with unintentional weight loss," in Textbook of Internal Medicine, 2d Edition (V. T. DeVita et al. eds.), Ch. 444, pp. 2113-2115 (1992); R. M. Jordan et al., "Weight loss," in Internal Medicine, 4th Edition (J. H. Stein ed.), Ch. 152, pp. 1260-1262 (1994); C. P. Artz et al., "Burns: Including cold, chemical, and electrical injuries," in Textbook of Surgery, 11th Edition (D. C. Sabiston, Jr. ed.), Ch. 15, pp. 295-322 (1977); E. Braunwald, "Heart Failure," in Harrison's Principles of Internal Medicine, 13th Edition (K. J. Isselbacher ed.), Ch. 195, pp. 998-1009 (1994); and D. W. Foster, "Gain and loss in weight," in Harrison's Principles of Internal Medicine, 13th Edition (K. J. Isselbacher ed.), Ch. 40, pp. 221-223 (1994). Over 50% of cancer and HIV-infected patients experience an unintended weight loss of greater than 10% of their baseline weight. Moreover, this weight loss is associated with an increase in morbidity and mortality. Many cachectic patients manifest multiple physiological problems involving the immune system, muscular system, and hepatic function that can be directly related to loss of body weight or wasting. Therefore, understanding the mechanisms of cachexia in patients can lead to better treatment and consequently can have a substantial impact on the quality of life and survival of many cancer and HIV/AIDS patients. See G. O. Coodley et al., "The HIV Wasting Syndrome: a Review," Journal of Acquired Immune Deficiency Syndromes, vol. 7, pp. 681-694 (1994); L. M. Hecker et al., "Malnutrition in patients with AIDS," Nutrition Reviews, vol. 48, pp. 393-401 (1990); N. M. Graham et al., "Clinical factors associated with weight loss related to infection with Human Immunodeficiency Virus Type 1 in the multicenter AIDS cohort study," American Journal of Epidemiology, vol. 137, pp. 439-46 (1993); and K. A. Nelson et al., "The cancer anorexia-cachexia syndrome," Journal of Clinical Oncology, vol. 12, pp. 213-25 (1994).

Despite the prevalence of weight loss in cancer patients, the mechanisms underlying the weight loss are unknown. Current explanations for cancer or AIDS-associated weight loss are divided into two general categories--(1) mechanisms that decrease food intake (anorexia); and (2) mechanisms that increase energy expenditure through altered or increased metabolism. Hecker et al., 1990. Any mismatch between energy intake and expenditure will result in a change in weight.

Many cancer or AIDS patients have decreased oral intake and, therefore, decreased energy consumption. Accordingly, despite normal or even decreased energy expenditures in these patients, they may lose weight. Other patients experience anorexia due to the cancerous tumor itself (either by a mechanical obstruction or a change in tissue function) or due to the therapy used to treat the tumor, e.g., chemotherapy. Graham et al., 1993; Nelson et al., 1994. Similarly, many HIV/AIDS patients experience significant weight loss that correlates with decreased caloric intake. See C. Grunfeld et al., "Metabolic disturbance and wasting in the acquired immunodeficiency syndrome," The New England Journal of Medicine, vol. 327, pp. 329-337 (1992). Thus, anorexia plays a major role in weight loss for the majority of both cancer and HIV/AIDS patients.

Factors that have been identified as causing anorexia in patients include opportunistic gastrointestinal infections or tumors, side effects of treatment, enteropathy, central nervous system disease, and psychiatric disorders. In addition, numerous physiological mediators of anorexia have been reported in the literature, including tumor necrosis factor, interleukin-1, interleukin-6, .gamma.-interferon, and .alpha.-interferon. Coodley et al., 1994; Nelson et al., 1994; and Grunfeld et al., 1990. Yet the mechanisms by which these or other mediators induce anorexia remain unknown.

Another proposed mechanism contributing to the weight loss seen in cancer or AIDS patients is an increased or ineffective metabolism. It has been reported, and disputed, that resting energy expenditures in some patients rise throughout the course of the disease and increase even more at the end stage. See Coodley et al., 1994; Nelson et al., 1994; and Grunfeld et al., 1990. However, alterations in resting or total energy expenditures do not correlate with weight loss. Therefore, it is unlikely that increased energy demands alone account for wasting.

Even with decreased energy use, patients may lose weight due to ineffective metabolism. It is hypothesized that during episodes of weight loss, patients fail to switch from carbohydrate and protein oxidation to the fatty acid oxidation that would normally occur under conditions of starvation. This failure explains the observation that patients lose predominantly muscle mass rather than fat tissue. It has also been suggested that futile cycling of lipid metabolism can waste energy, thus accelerating the necessity of carbohydrate and protein breakdown, despite a decrease in total energy expenditure. See Coodley et al., 1994; Nelson et al., 1994; and Grunfeld et al., 1990.

Recently, alterations in hormone metabolism have been proposed as possible etiologies of HIV/AIDS or cancer-related weight loss, particularly due to muscle wasting. During severe or chronic infections, patients, particularly HIV/AIDS patients, demonstrate resistance to the actions of growth hormone. Because growth hormone acts to maintain muscle mass, it has been hypothesized that this resistance leads to muscle wasting and weight loss in HIV/AIDS patients. Recently, researchers demonstrated that HIV/AIDS patients with the wasting syndrome have a decreased response to exogenous growth hormone compared with a control group. In particular, the effects of growth hormone on insulin-like growth factor-I (IGF-I, a major mediator of growth hormone action) secretion was studied. When IGF-I was exogenously administered to patients with the wasting syndrome, the patients experienced a transient increase in nitrogen retention, but returned to baseline after 8-10 days. See S. A. Lieberman et al., "Anabolic effects of recombinant insulin-like growth factor-I in cachectic patients with the acquired immunodeficiency syndrome," Journal of Clinical Endocrinology and Metabolism, vol. 78, pp. 404-410 (1994). Thus, alterations in the growth hormone/IGF-I system may play an important role in HIV/AIDS cachexia.

In cancer patients, growth hormone resistance has been seen, but also other important hormones, including insulin and its antagonist glucagon, appear to be abnormally produced. Since these hormones are essential to normal metabolism, it has been postulated that abnormalities in these pathways explain the wasting syndrome in these patients. See Nelson et al., 1994. Unfortunately, the mechanisms by which cancer or HIV infection causes these alterations in hormone metabolism are poorly understood at best.

The control of caloric intake and body weight maintenance is very complex. The search for endogenous mediators over several decades has led to the identification of a variety of substances ranging from simple amino acids to large proteins and glycoproteins. However, it has been difficult to establish an unequivocal association between the amount of any one of these factors and human disease states such as anorexia/cachexia and anorexia nervosa.

Three glycoproteins or proteoglycans that modulate appetite or body weight have been identified: satietin, satiomem, and MAC16 mouse protein. A glycoprotein is a protein that contains attached carbohydrates that are not polymers of repeating units. In contrast, a proteoglycan is a protein that contains repeating units of glycosaminoglycans covalently attached to a core protein.

Satietin is a glycoprotein with a molecular weight of 50,000 Dalton that has been isolated from human and animal sera. Satietin is known to suppress food intake in mammals. See J. Knoll, "Satietin, a blood-borne, highly selective and potent anorectic glycoprotein," Biomed. Biochim. Acta, vol. 44, pp. 317-328 (1985); and J. Knoll, "Satietin: a 50,000 Dalton glycoprotein in human serum with potent, long-lasting and selective anorectic activity," J. Neural Transmission, vol. 59, pp. 163-194 (1984).

Satiomem is a proteoglycan with a molecular weight of 50,000 Dalton that has been isolated from plant and animal membranes, including human erythrocyte membrane. Satiomem has been shown to suppress food intake and cause weight loss. See R. K. Upreti et al., "A step towards developing the expertise to control hunger and satiety: Regulatory role of satiomem--A membrane proteoglycan," Neurochemical Research, vol. 20, pp. 375-384 (1995); A. M. Kidwai et al., "A Novel Plant membrane proteoglycan which causes anorexia in animals," Molecular and Cellular Biochemistry, vol. 120, pp. 111-117 (1993); and A. M. Kidwai et al., "Isolation of an anorexigenic protein from membranes," Molecular and Cellular Biochemistry, vol. 91, pp. 117-122 (1989).

The MAC16 protein is a sulfated, phosphated glycoprotein of 24 kDa initially identified from the urine of mice with the MAC16 tumor. Using a monoclonal antibody to the mice MAC16 protein, a similar protein was also found in the urine of human cachectic cancer patients. The mouse MAC16 protein causes weight loss in rodents, primarily due to a decrease in the lean body mass. The primary bioactivity of this protein is to increase muscle proteolysis and decrease protein synthesis. The MAC16 protein binds tightly to muscle cell membranes. The MAC16 protein also causes some lipolytic activity and does not affect food intake. The protein core of the mouse MAC16 protein has been identified to have at least 18 amino acids and digestion with chondroitinase AC results in a single fragment of 14 kDa. The human protein identified with the monoclonal antibody ("human MAC16") to MAC16 also increases proteolysis in muscle cells. The first 14 amino acids of "human MAC16" are identical to those of mouse MAC16 protein. The human MAC16 has been found only in the urine of cachectic cancer patients, not in patients suffering extreme weight loss from other diseases such as sepsis, burns or major surgery. See P. T. Todorov et al., "Structural Analysis of a Tumor-produced Sulfated Glycoprotein Capable of Initiating Muscle Protein Degradation," The Journal of Biological Chemistry, vol. 272, pp. 12279-88 (1997); P. Cariuk et al., "Induction of Cachexia in Mice by a Product isolated from the urine of cachectic cancer patients," British Journal of Cancer, vol. 76, pp. 606-613 (1997); M. J. Lorite et al., "Induction of muscle protein degradation by a tumour factor," British Journal of Cancer, vol. 76, pp. 1035-1040 (1997); P. Todorov et al., "Characterization of a cancer cachectic factor," Nature, vol. 379, pp. 739-742 (1996); P. T. Todorov et al., "Induction of muscle protein degradation and weight loss by a tumor product," Cancer Research, vol. 56, pp. 1256-1261 (1996); T. M. McDevitt et al., "Purification and Characterization of a Lipid-mobilizing Factor Associated with Cachexia-inducing Tumors in Mice and Humans," Cancer Research, vol. 55, pp. 1458-63 (1995); J. E. Belizario et al., "Bioactivity of skeletal muscle proteolysis-inducing factors in the plasma proteins from cancer patients with weight loss," British Journal of Cancer, vol. 63, pp. 705-710 (1991); S. A. Beck et al., "Lipid mobilising factors specifically associated with cancer cachexia," British Journal of Cancer, vol. 63, pp. 846-850 (1991); P. Groundwater et al., "Alteration of serum and urinary lipolytic activity with weight loss in cachectic cancer patients," British Journal of Cancer, vol. 62, pp. 816-821 (1990); and S. A. Beck et al., "Alterations in serum lipolytic activity of cancer patients with response to therapy," British Journal of Cancer, vol. 62, pp. 822-825 (1990).

At present there is no rational therapy for cachexia, i.e., one based on the etiology of the disease. Since common symptoms of anorexia/cachexia syndrome include loss of appetite, fat deposit, and muscle mass, all existing therapies for cachexia include agents known to increase appetite (e.g., cyproheptadine (PERIACTIN.RTM.), facilitate energy storage (e.g., megestrol acetate (MEGACE.RTM.)), or increase muscle mass (androgenic agents). While these therapies work for some patients, for many nothing works. Since time is very important for these patients, until a rational therapy can be found, a need exists to predict which patients might respond to which of the various available therapies.

Obesity plays a major role in the etiology of many chronic diseases, including cardiovascular diseases, cancer, and diabetes. Therefore, a national goal has been to reduce the prevalence of obesity in the U.S. population to no more than 20%. Unfortunately, there has been a substantial rise in obesity in U.S. during the last decade.

Obesity is generally classified into two groups based on the site of fat deposition--visceral and nonvisceral, also known as upper-body/android (apple-shaped) and lower-body/gynoid (pear-shaped) obesity, respectively. It is well-established that visceral adipose tissue is associated with greater morbidity and mortality, particularly hypertension, hyperlipidemia, and insulin resistance. Data also show that weight loss by diet, exercise, or pharmacotherapy generates a decrease in visceral adipose tissue and improvements in hypertension, hyperlipidemia, and insulin resistance. See F. X. Pi-Sunyer, "Medical Hazards of Obesity," Annals of Internal Medicine, vol. 119, pp. 655-660 (1993); and G. A. Bray, "Pathophysiology of Obesity," American Journal of Clinical Nutrition, vol. 55, pp. 488S-494S (1992).

A pharmacologic treatment to reduce body fat, particularly visceral fat, would be of great health significance. Currently there is no available pharmacotherapy that will facilitate a decrease in fat deposit. Agents like REDUX.TM. and Fen/phen have been successful in obesity treatment; however, these agents have been removed from the market due to serious side effects.

We have discovered a proteoglycan ("azaftig") with a molecular weight of approximately 24,000 Dalton that has been isolated and characterized from the urine of cachectic cancer and non-cancer patients. Azaftig has been shown to bind to receptors on fat cell membranes and to cause lipolysis. Azaftig does not bind to muscle cell membranes or cause proteolysis. Azaftig detection in urine will allow early identification of patients in whom weight loss may become a problem. Azaftig may also aid fat loss in humans in whom obesity is a threat to health.

Claim 1 of 3 Claims

We claim:

1. A substantially pure azaftig wherein said azaftig is a proteoglycan of molecular weight about 24 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; wherein said azaftig is obtained from or is identical to a proteoglycan obtained from urine of cachectic cancer patients; wherein said azaftig is a proteoglycan as determined by partial digestion with either chondroitinase ABC or chondroitinase AC; wherein said azaftig is not readily digested by neuraminidase; wherein said azaftig binds to fat cell membranes; wherein said azaftig does not bind to muscle cell membranes; and wherein said azaftig is a negatively charged molecule as determined by DEAE-Sephacel chromatography at pH 7.0.

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