WO2000000810A2 - Azaftig, a proteoglycan for monitoring cachexia and for control of obesity - Google Patents
Azaftig, a proteoglycan for monitoring cachexia and for control of obesity Download PDFInfo
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- WO2000000810A2 WO2000000810A2 PCT/US1999/014620 US9914620W WO0000810A2 WO 2000000810 A2 WO2000000810 A2 WO 2000000810A2 US 9914620 W US9914620 W US 9914620W WO 0000810 A2 WO0000810 A2 WO 0000810A2
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- azaftig
- proteoglycan
- determined
- cachexia
- urine
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4722—Proteoglycans, e.g. aggreccan
Definitions
- AZAFTIG A PROTEOGLYCAN FOR MONITORING CACHEXIA AND FOR CONTROL OF OBESITY
- This invention pertains to the detection of a propensity for cachexia and to the control of obesity.
- 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.
- numerous physiological mediators of anorexia have been reported in the literature, including tumor necrosis factor, interleukin-l , interleukin-6, ⁇ -interferon, and -interferon. Coodley et al , 1994; Nelson et al. , 1994; and
- 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.
- 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
- the MAC 16 protein is a sulfated, phosphated glycoprotein of 24 kDa initially identified from the urine of mice with the MAC 16 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 MAC 16 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 MAC 16 protein binds tightly to muscle cell membranes. The MAC 16 protein also causes some lipolytic activity and does not affect food intake.
- the protein core of the mouse MAC 16 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 MAC 16") to MAC 16 also increases proteolysis in muscle cells.
- the first 14 amino acids of "human MAC16" are identical to those of mouse MAC 16 protein.
- the human MAC 16 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.
- Figure 1 illustrates the DEAE-Sephacel elution profile of 125 I-azaftig.
- Figure 2 illustrates the decrease in the body weight of a rat due to azaftig injections.
- Figure 3 illustrates the decrease in the body weight of mice due to azaftig injections.
- Figure 4 illustrates the time course of the weight loss of mice seen in Figure 3.
- Figure 5 illustrates the time course of weight gain in mice after ceasing azaftig injections.
- Figure 8 illustrates the Sephadex G-25 elution profile of 125 I-azaftig.
- Figure 9A illustrates the specific binding of 125 I-azaftig to fat cell membrane preparations.
- Figure 9B illustrates the effect of pH on the specific binding of 125 I-azaftig to fat cell membrane preparations.
- Figure 10A illustrates the effect of concentration of l25 I-azaftig on specific binding to fat cell membrane preparations.
- Figure 10B illustrates the Scatchard analysis of the binding data from Figure 10A.
- Figure 11 illustrates the effect of azaftig on in vitro muscle cell proteolysis.
- Figure 12 illustrates the rate of blood clearance in mice of 125 I-azaftig.
- Figure 13 illustrates the DEAE-Sephacel elution profile of azaftig.
- Figure 14 illustrates the Q-Sepharose elution profile of azaftig.
- Figure 15 illustrates the binding pattern of the synthetic peptide core of MAC 16 and the MAC 16 from urine of AIDS patients.
- Azaftig has been shown to cause weight loss in mammals. It has also been shown to increase lipolysis and to bind to fat cell membrane preparations. However, unlike the MAC 16 glycoprotein, azaftig does not augment proteolysis in muscle tissue or bind to muscle cell membrane preparations.
- Urine was collected for 24 hr from a patient with a diagnosis of metastatic adenocarcinoma of unknown primary source, who had experienced a 50 lb weight loss over several months prior to diagnosis.
- the urine was treated with ammonium sulfate (80% saturation), and incubated overnight at 7°C.
- the solution was centrifuged at 6,000 x g for 1 hr, and the supernatant was removed.
- the ammonium sulfate precipitate was dissolved in 50 ml of water and centrifuged again. The supernatant was saved, and the pellet was resuspended in 5 % sodium dodecyl sulfate ("SDS").
- Both the supernatant and the SDS-dissolved precipitate were subsequently separated by SDS-polyacrylamide gel electrophoresis.
- the supernatant revealed several protein bands, with two predominant bands at 24 kilodaltons and 70 kilodaltons.
- the proteins with the molecular weight of 24 kilodaltons, or that were later determined to be its multiple (70 kilodaltons), were named azaftigs.
- Azaftig of 24 kilodaltons was isolated as described above.
- the purified protein was radiolabeled with 125 I using the chloramine-T method as described by F.C Greenwood et al. , "The preparation of l31 I-labeled growth hormone of high specific activity," Biochemical Journal, vol. 89, pp. 114-123 (1963).
- the protein was subsequently analyzed for charge using DEAE-Sephacel anion exchange chromatography.
- 125 I-azaftig was dialyzed overnight at 4°C against a solution of 8 M urea, 0.1 M Tris, 0.3% Triton X-100, and 0.15 M NaCl (pH 7.0) containing protease inhibitors. The dialyzed sample was applied to a column of
- chondroitinase ABC digestion as described by H. Saito et al. , "Enzymatic methods of the determination of small quantities of isomeric chondroitin sulfate, " J. Biol. Chem., vol. 243, pp. 1536-1542 (1968), of azaftig resulted in a decrease in the azaftig band on SDS-PAGE.
- Chondroitinase ABC is an enzyme that specifically cleaves the chondroitin sulfate or dermatan sulfate groups in proteoglycans, this loss in azaftig indicated that azaftig is a chondroitin sulfate-containing proteoglycan.
- Radiolabeled azaftig was separated by SDS-PAGE. Autoradiography demonstrated three to four distinct bands generated by purified azaftig which indicated that azaftig had a tendency to aggregate. To decrease aggregation of the sample, purified azaftig was treated with 1 % Triton X-100 and subsequently chromatographed over a Sephadex G-50 column. In addition, experiments were performed in the presence of 4 M guanidine-HCl to minimize aggregation. Both treatments resulted in decreased aggregation as seen by a single band by
- Enzymatic digestion 125 I-azaftig was digested in separate experiments by using 50 each units of neuraminidase, chondroitinase ABC, or chondroitinase AC. Each digestion product was analyzed by SDS-PAGE electrophoresis. Neuraminidase did not degrade the proteoglycan, while chondroitinases ABC and AC caused partial digestion. Chondroitinase AC produced fragments with molecular weights below 10 kDa. These data confirm that azaftig is a proteoglycan, because both chondroitinase ABC and AC specifically cleave the chondroitin sulfate or dermatan sulfate found in proteoglycans.
- Example 8 Effect of Azaftig on Food Intake
- Eighteen female NIH Swiss mice were divided into two groups, a control and treatment group of nine mice each. The mice were kept from food, but not water, for 21 hr and then fed for 3 hr on five consecutive days. On day six, 30 min before the scheduled feeding time, the mice were treated intraperitoneally either with vehicle (0.1 ml/mouse) or with azaftig (0.1 mg/kg in 0.1 ml vehicle). Thirty minutes later, food was presented. At 3 hr and 24 hr food intake for each mice was measured. The data presented in Figure 7 show that azaftig did not significantly affect food intake at either 3 hr or 24 hr.
- Azaftig was labeled using a lactoperoxidase 125 I-labeling kit purchased from ICN Radiochemicals. Briefly, 1.5 mCi neutralized carrier-free 125 I (10 ⁇ l) was added to a tube containing 30 ⁇ g azaftig (100 ⁇ l) and mixed thoroughly. Ten ⁇ of lactoperoxidase solution (1 ⁇ gl ⁇ ) in water was added to the above mixture. The reaction was initiated by adding 5 ⁇ l of 3% freshly prepared H 2 0 2 . The addition of H 2 O 2 was repeated three times at 10 min intervals until a total of 40 ⁇ l H 2 0 2 was added.
- a lactoperoxidase 125 I-labeling kit purchased from ICN Radiochemicals. Briefly, 1.5 mCi neutralized carrier-free 125 I (10 ⁇ l) was added to a tube containing 30 ⁇ g azaftig (100 ⁇ l) and mixed thoroughly. Ten ⁇ of lactoperoxidase solution (1 ⁇ g
- Binding Assay For the binding assay, 300 ⁇ l of membrane preparation, 10 ⁇ l of buffer (50mM
- proteolysis can be augmented by the addition of the glycoprotein MAC16, which also increases lipolysis. Using methods as described by P.
- Radiolabeled 125 I-azaftig was incubated with membrane preparations from a variety of tissues, including heart, muscle, adrenal, kidney, liver, and fat cells. Only the fat cells showed binding indicating a high affinity receptor. Muscle cells did not bind the 125 I-azaftig and were used as controls in later receptor assays.
- Radioactivity in the blood reached a maximum of about 1300 CPM/ 10 ⁇ l in about 20 min and remained elevated for about 30 min. Then the level of radioactivity in the blood declined slowly with a half-life of approximately 4 to 5 hr, indicating a slow clearance rate for azaftig. This slow clearance is indicative of azaftig resistance to metabolic degradation and makes azaftig a potent cachectic agent.
- the purified azaftig was used in an initial attempt to sequence the protein core of the proteoglycan. Unfortunately, the amino terminus was found to be blocked. By contrast, the amino terminus of the MAC-16 protein is not similarly blocked. See Cariuk et al. , 1997. Once 10 ⁇ g of azaftig is purified as described below in Example 13, the azaftig protein core will be sequenced by first cleaving the molecule and then sequencing the unblocked segments by methods known in the field.
- Step 1 DEAE-Sephacel chromatography
- the pooled sample of fractions 41 and 42 from Step 2 was injected into a HPLC column (Novo-pack C 18 60A 4 ⁇ m, 3.9 x 300 mm, 40° C).
- the column was eluted at a flow rate of 0.5 ml/min using a linear gradient from 0.1 % to 35% of acetonitrile in 0.1 % trifluoroacetic acid and 0.05% triethylamine.
- Each fraction was recorded for absorbance at 214 nm and tested for immunoreactivity against azaftig antibody.
- the azaftig eluted from the column in about 6 min.
- Example 14 Detection of azaftig as a Diagnostic Tool
- a detection system for azaftig will be developed to identify patients at risk of experiencing cachexia from cancer, HIV infection, or other conditions, e.g., burns, sepsis, or tuberculosis.
- cachexia a secondary condition
- a knowledge of impending cachexia would allow physicians to institute early measures to combat the condition and maintain body weight, thereby allowing continuation of therapy for the primary disease.
- detection assays can easily be developed, e.g., ELISA, RIA, and antibody-impregnated "dipsticks.”
- Biological samples appropriate for such detection include serum, saliva, and urine.
- the antibodies used in the assays may be polyclonal or monoclonal.
- azaftig will be administered by peripheral routes to normalize body weight and reduce fat deposit in obese patients at risk for hypertension, cardiovascular diseases, diabetes and other ailments associated with obesity.
- This method of reducing fat deposit is much preferable over surgical removal of fat, which is not only expensive but it also poses serious risk of infection and surgical anesthesia.
- the synthesized peptide was coupled to keyhole limpet hemocyanin (KHL) using m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBH) as the bifunctional agent.
- KHL keyhole limpet hemocyanin
- MBH m-maleimidobenzoyl-N-hydroxysuccinimide ester
- Two adult New Zealand rabbits received primary injection of peptide-KLH conjugate (0.3-0.4 mg/rabbit) emulsified in Freund's complete adjuvant. All injections were made at multiple sites by subcutaneous and intramuscular routes. Multiple booster injection was given with peptide-KLH conjugate (0.3-0.4 mg/rabbit) emulsified in Freund's incomplete adjuvant every two weeks.
- the first blood was drawn 1 week after the 5th injection, and the antibody titer was measured as described below. Thereafter, animals were injected with booster every two weeks and bled one week after each injection.
- the synthesized peptide (0.5 mg/ml) was diluted to 1.0 ug/ml in coating buffer consisting of 50 mM sodium phosphate, 145 mM NaCl, pH 7.4, and an antigen stabilizer.
- the wells of high-binding microtiter plates were coated with 0.1 ml of peptide (1.0 ug/ml) by overnight incubation at 4°C. All further operations were performed at room temperature (22-23 °C). To wash the wells of the microtiter plate or to remove its contents, the plate was rapidly inverted and the contents forcefully dashed into a tray.
- wash buffer 50 mM sodium phosphate, 145 mM NaCl, 0.05% Tween, 0.1 % NaN 3 , pH 7.4 containing an antigen stabilizer
- blocking buffer 10% bovine serum albumin, 50 mM sodium phospate, 145 mM NaCl, pH 7.4, and an antigen stabilizer
- Azaftig combined with a pharmaceutically acceptable carrier, may be administered to mammals, including humans, intravenously, subcutaneously, percutaneously, intramuscularly, or intranasally to control weight loss.
- the dosage will vary depending on the specific purpose for which azaftig is administered; appropriate dosages may readily be determined by those of skill in the art, an "effective amount" being that which increases (azaftig) weight loss by a statistically significant amount.
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002335937A CA2335937C (en) | 1998-06-30 | 1999-06-28 | Azaftig, a proteoglycan for monitoring cachexia and for control of obesity |
DE69934236T DE69934236T2 (en) | 1998-06-30 | 1999-06-28 | PROTEOGLYKAN (ADDITIONAL) FOR THE MONITORING OF KACHEXIE AND FATIBILITY CONTROL |
EP99930798A EP1108205B1 (en) | 1998-06-30 | 1999-06-28 | Azaftig, a proteoglycan for monitoring cachexia and for control of obesity |
AU47250/99A AU4725099A (en) | 1998-06-30 | 1999-06-28 | Azaftig, a proteoglycan for monitoring cachexia and for control of obesity |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15069598P | 1998-06-30 | 1998-06-30 | |
US60/150,695 | 1998-06-30 |
Publications (2)
Publication Number | Publication Date |
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WO2000000810A2 true WO2000000810A2 (en) | 2000-01-06 |
WO2000000810A3 WO2000000810A3 (en) | 2000-07-13 |
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ID=22535626
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Application Number | Title | Priority Date | Filing Date |
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PCT/US1999/014620 WO2000000810A2 (en) | 1998-06-30 | 1999-06-28 | Azaftig, a proteoglycan for monitoring cachexia and for control of obesity |
Country Status (8)
Country | Link |
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US (1) | US6274550B1 (en) |
EP (1) | EP1108205B1 (en) |
AT (1) | ATE347097T1 (en) |
AU (1) | AU4725099A (en) |
CA (1) | CA2335937C (en) |
DE (1) | DE69934236T2 (en) |
ES (1) | ES2277441T3 (en) |
WO (1) | WO2000000810A2 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
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AU4725099A (en) * | 1998-06-30 | 2000-01-17 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College, The | Azaftig, a proteoglycan for monitoring cachexia and for control of obesity |
US7479540B1 (en) | 2003-12-22 | 2009-01-20 | Chandan Prasad | Adipomodulin and related molecules and methods |
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AU4725099A (en) * | 1998-06-30 | 2000-01-17 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College, The | Azaftig, a proteoglycan for monitoring cachexia and for control of obesity |
-
1999
- 1999-06-28 AU AU47250/99A patent/AU4725099A/en not_active Abandoned
- 1999-06-28 DE DE69934236T patent/DE69934236T2/en not_active Expired - Lifetime
- 1999-06-28 US US09/340,873 patent/US6274550B1/en not_active Expired - Fee Related
- 1999-06-28 AT AT99930798T patent/ATE347097T1/en not_active IP Right Cessation
- 1999-06-28 ES ES99930798T patent/ES2277441T3/en not_active Expired - Lifetime
- 1999-06-28 EP EP99930798A patent/EP1108205B1/en not_active Expired - Lifetime
- 1999-06-28 WO PCT/US1999/014620 patent/WO2000000810A2/en active IP Right Grant
- 1999-06-28 CA CA002335937A patent/CA2335937C/en not_active Expired - Fee Related
Non-Patent Citations (3)
Title |
---|
KOLSET ET AL: 'Proteoglykaner og Patologi-Nye Aspekter (Proteoglycans and Pathology-New Aspects)' TIDSSKRIFT FOR DEN NORSKE LAEGEFORENING vol. 117, no. 7, 10 March 1997, pages 951 - 954, XP000909134 * |
See also references of EP1108205A2 * |
TODOROV P. ET AL: 'Characterization of a Cancer Cachectic Factor' NATURE vol. 379, no. 6567, 22 February 1996, pages 739 - 742, XP002927496 * |
Also Published As
Publication number | Publication date |
---|---|
CA2335937C (en) | 2009-06-02 |
US6274550B1 (en) | 2001-08-14 |
EP1108205A4 (en) | 2005-01-26 |
CA2335937A1 (en) | 2000-01-06 |
WO2000000810A3 (en) | 2000-07-13 |
EP1108205B1 (en) | 2006-11-29 |
ATE347097T1 (en) | 2006-12-15 |
ES2277441T3 (en) | 2007-07-01 |
DE69934236D1 (en) | 2007-01-11 |
AU4725099A (en) | 2000-01-17 |
EP1108205A2 (en) | 2001-06-20 |
DE69934236T2 (en) | 2007-06-21 |
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