WO2006015137A2 - A method for managing cholesterol with a serum-free and mitogen free cytokine mixture - Google Patents
A method for managing cholesterol with a serum-free and mitogen free cytokine mixture Download PDFInfo
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- WO2006015137A2 WO2006015137A2 PCT/US2005/026819 US2005026819W WO2006015137A2 WO 2006015137 A2 WO2006015137 A2 WO 2006015137A2 US 2005026819 W US2005026819 W US 2005026819W WO 2006015137 A2 WO2006015137 A2 WO 2006015137A2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/193—Colony stimulating factors [CSF]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/2006—IL-1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/2013—IL-2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
- A61K38/217—IFN-gamma
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
Definitions
- the present invention relates to a serum-free and mitogen-free mixture comprised of specific ratios of cytokines IL-I ⁇ , TNF- ⁇ , IFN- ⁇ and GM-CSF to Interleukin 2 (IL-2) such as Leukocyte Interleukin Injection (LI) or Multikine ® and methods for treating or preventing a disease or disorder, for example, cardiovascular disease, dyslipidemia; dyslipoproteinemia; hyperlipidemia; a disorder of glucose metabolism; Syndrome X; a peroxisome proliferator activated receptor-associated disorder; obesity; hypertension; and renal disease.
- IL-2 Interleukin 2
- LI Leukocyte Interleukin Injection
- Multikine ® Interleukin 2
- Circulating cholesterol carried by plasma lipoproteins which are particles of complex lipid and protein composition that transport lipids in the blood, which contributes to disease.
- Low density lipoprotein (LDL) and high density lipoprotein (HDL) are the major cholesterol-carrier proteins.
- LDL is believed to be responsible for the delivery of cholesterol from the liver, where it is synthesized or obtained from dietary sources, to extrahepatic tissues in the body.
- reverse cholesterol transport describes the transport of cholesterol from extrahepatic tissues to the liver, where it is catabolized and eliminated. It is believed that plasma HDL particles play a major role in the reverse transport process, acting as scavengers of tissue cholesterol. HDL is also responsible for the removal of non-cholesterol lipid, oxidized cholesterol and other oxidized products from the bloodstream.
- Atherosclerosis for example, is a slowly progressive disease characterized by the accumulation of cholesterol within the arterial wall. Compelling evidence supports the belief that lipids deposited in atherosclerotic lesions are derived primarily from plasma apolipoprotein B (apo B)-containing lipoproteins, which include chylomicrons, VLDL, IDL and LDL. The apo B-containing lipoprotein, and in particular LDL, has popularly become known as the "bad" cholesterol. In contrast, HDL serum levels correlate inversely with coronary heart disease. Indeed, high serum levels of HDL are regarded as a negative risk factor.
- apo B plasma apolipoprotein B
- HDL Cholesterol Transport
- the fat-transport system can be divided into two pathways: an exogenous one for cholesterol and triglycerides absorbed from the intestine and an endogenous one for cholesterol and triglycerides entering the bloodstream from the liver and other non-hepatic tissue.
- chylomicrons In the exogenous pathway, dietary fats are packaged into lipoprotein particles called chylomicrons, which enter the bloodstream and deliver their triglycerides to adipose tissue for storage and to muscle for oxidation to supply energy.
- VLDL very-low-density lipoprotein particle
- the core of VLDL consists mostly of triglycerides synthesized in the liver with a smaller amount of cholesteryl esters either synthesized in the liver or recycled from chylomicrons.
- Two predominant proteins are displayed on the surface of VLDL, apolipoprotein B-IOO (apo B-IOO) and apolipoprotein E (apo E), although other apolipoproteins are present, such as apolipoprotein CM (apo CIII) and apolipoprotein CII (apo CII).
- VLDL When a VLDL reaches the capillaries of adipose tissue or of muscle, its triglyceride is extracted. This results in the formation of a new kind of particle called intermediate-density lipoprotein (DDL) or VLDL remnant decreased in size and enriched in cholesteryl esters relative to a VLDL, but retaining its two apoproteins.
- DDL intermediate-density lipoprotein
- VLDL remnant decreased in size and enriched in cholesteryl esters relative to a VLDL, but retaining its two apoproteins.
- IDL particles In human beings, about half of the IDL particles are removed from the circulation quickly, generally within two to six hours of their formation. This is because IDL particles bind tightly to liver cells, which extract IDL cholesterol to make new VLDL and bile acids.
- the EDL not taken up by the liver is catabolized by the hepatic lipase, an enzyme bound to the proteoglycan on liver cells.
- Apo E dissociates from IDL as it is transformed to LDL.
- Apo B-IOO is the sole protein of LDL.
- the liver takes up and degrades circulating cholesterol to bile acids, which are the end products of cholesterol metabolism.
- the uptake of cholesterol- containing particles is mediated by LDL receptors, which are present in high concentrations on hepatocytes.
- the LDL receptor binds both apo E and apo B-IOO and is responsible for binding and removing both IDL and LDL from the circulation.
- remnant receptors are responsible for clearing chylomicrons and VLDL remnants (i.e., LDL).
- the affinity of apo E for the LDL receptor is greater than that of apo B- 100.
- the LDL particles have a much longer circulating life span than LDL particles; LDL circulates for an average of two and a half days before binding to the LDL receptors in the liver and other tissues.
- High serum levels of LDL, the "bad" cholesterol, are positively associated with coronary heart disease.
- cholesterol derived from circulating LDL accumulates in the walls of arteries. This accumulation forms bulky plaques that inhibit the flow of blood until a clot eventually forms, obstructing an artery and causing a heart attack or stroke.
- the amount of intracellular cholesterol liberated from the LDL controls cellular cholesterol metabolism.
- the accumulation of cellular cholesterol derived from VLDL and LDL controls three processes.
- the incoming LDL-derived cholesterol promotes storage of cholesterol by the action of ACAT, the cellular enzyme that converts cholesterol into cholesteryl esters that are deposited in storage droplets.
- ACAT the cellular enzyme that converts cholesterol into cholesteryl esters that are deposited in storage droplets.
- the accumulation of cholesterol within the cell drives a feedback mechanism that inhibits cellular synthesis of new LDL receptors. Cells, therefore, adjust their complement of LDL receptors so that enough cholesterol is brought in to meet their metabolic needs, without overloading. See Brown & Goldstein, In, The Pharmacological Basis Of Therapeutics, 8th Ed., Goodman & Gilman, Pergamon Press, New York, 1990, Ch. 36, pp. 874-896.
- LDL can also be complexed to a high molecular weight glycoprotein called apolipoprotein(a), also known as apo(a), through a disulfide bridge.
- the LDL-apo(a) complex is known as Lipoprotein(a) or Lp(a). Elevated levels of Lp(a) are detrimental having been associated with atherosclerosis, coronary heart disease, myocardial infarction, stroke, cerebral infarction, and restenosis following angioplasty.
- HMG-CoA reductase inhibitors include lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rivastatin, itavastatin, rosuvastatin, and other statins.
- Sequestrants include cholestyramine, colestipol, and dialkylaminoalkyl derivatives of a cross-linked dextran.
- Inhibitors of cholesterol absorption include ezetimibe and beta-sitosterol while acyl CoAxholesterol acyltransferase inhibitors includes avasimibe.
- Niacin is also known to effectively lower the serum concentrations of total cholesterol and triglycerides while raising HDL cholesterol. Trials indicate that niacin may reduce mortality rate in persons with CHD. It's potential adverse effects, such as flushing, itching, gastrointestinal distress, and liver toxicity, limit its use in some patients and may reduce its cost-effectiveness. Fibric acids do not reduce LDL-C substantially, although they do lower elevated triglyceride concentrations.
- HMG-CoA reductase inhibitors are preferred. They are highly effective in decreasing LDL-C and the most recommended drugs in this class shown to reduce the risk of death from CHD. Treatment with HMG-CoA reductase inhibitors improves overall survival and lower CHD risk in persons with elevated cholesterol with or without major cardiovascular disease.
- HMG-CoA reductase inhibitors The mechanism of action of HMG-CoA reductase inhibitors is the rate- limiting step in hepatic cholesterol synthesis.
- HMG-CoA is converted to mevalonic acid, a reaction catalyzed by the enzyme 3-hydroxy-3-methylglutaryl (HMG) coenzyme A reductase.
- HMG-CoA reductases exert a profound effect on blood cholesterol levels through a reduction in hepatic synthesis of VLDL cholesterol, the precursor of LDL. This action increases the synthesis of LDL receptors on the cellular membrane of both hepatic and extrahepatic tissues. Because the primary function of LDL receptors is to remove LDL from the circulation, the degree of LDL receptor increase appears to correlate with the degree of cholesterol reduction.
- Lipator or Lescol block the production of cholesterol within cells, causing the cells to increase specific receptors on their surface that will take up LDL cholesterol particles from the blood. This effect is especially prominent in the liver, which is the organ that largely controls cholesterol in the body. As the number of LDL receptors increases, the levels of total and LDL cholesterol in the blood will go down. [18] The effects on blood lipids is such that the total cholesterol and LDL cholesterol are reduced 15 to 40 percent while the triglyceride levels may decline 10 to 15 percent and the HDL cholesterol levels may increase 5 to 10 percent. Although the drug appears to be safe and well tolerated, certain side effects are associated with its use such as GI upset, headache, and dizziness and skin rashes.
- Elevation of AST and ALT has also been associated with statin use. Risk factors include preexisting elevated liver enzymes, patient history of liver disease, and significant alcohol use. Each of these factors is a contraindication to statin use. Elevation in LFTs due to statins is usually asymptomatic but anorexia, weakness, and/or abdominal pain may be present. Elevated LFTs usually fall to normal after discontinuation of the drug, but serious hepatoxicity is possible. Because of these risks, the product's labeling recommends monitoring of LFTs. After a baseline measurement, the recommendations are to check LFTs after six and 12 weeks of therapy and periodically thereafter. If persistent elevations in AST and/or ALT of greater than three times the upper limit of normal occur, the drug should be discontinued.
- HMG-CoA reductase inhibitors interfere with cholesterol synthesis and lower circulating cholesterol levels and, as such, might theoretically blunt adrenal or gonadal steroid hormone production.
- Results of clinical trials with statins in males and post ⁇ menopausal females were inconsistent with regard to possible effects of the drug on basal steroid hormone levels.
- the mean testosterone response to human chorionic gonadotropin was significantly reduced (p ⁇ 0.004) after 16 weeks of treatment with 40 mg of pravastatin.
- the percentage of patients showing a ⁇ 50% rise in plasma testosterone after human chorionic gonadotropin stimulation did not change significantly after therapy in these patients.
- HMG-CoA reductase inhibitors have not been studied in adequate numbers of patients.
- Statins may also have a negative effect on the pituitary-gonadal axis in pre-menopausal females.
- CNS vascular lesions characterized by perivascular hemorrhage and edema and mononuclear cell infiltration of perivascular spaces, were seen in dogs treated with a statin at a dose of 25 mg/kg/day, a dose that produced a plasma drug level about 50 times higher than the mean drug level in humans taking 40 mg/day. Similar CNS vascular lesions have been observed with several other drugs in this class.
- a chemically similar drug in this class produced optic nerve degeneration (Wallerian degeneration of retinogeniculate fibers) in clinically normal dogs in a dose-dependent fashion starting at 60 mg/kg/day, a dose that produced mean plasma drug levels about 30 times higher than the mean drug level in humans taking the highest recommended dose (as measured by total enzyme inhibitory activity).
- This same drug also produced vestibulocochlear Wallerian-like degeneration and retinal ganglion cell chromatolysis in dogs treated for 14 weeks at 180 mg/kg/day, a dose which resulted in a mean plasma drug level similar to that seen with the 60 mg/kg/day dose.
- a chemically similar drug in this class was administered to mice for 72 weeks at 25, 100, and 400 mg/kg body weight, which resulted in mean serum drug levels approximately 3, 15, and 33 times higher than the mean human serum drug concentration (as total inhibitory activity) after a 40 mg oral dose.
- Liver carcinomas were significantly increased in high-dose females and mid- and high-dose males, with a maximum incidence of 90 percent in males.
- the incidence of adenomas of the liver was significantly increased in mid- and high-dose females.
- Drug treatment also significantly increased the incidence of lung adenomas in mid- and high-dose males and females.
- Adenomas of the eye Harderian gland (a gland of the eye of rodents) were significantly higher in high-dose mice than in controls.
- statins may cause myopathies, including rare cases of rhabdomyolysis with resulting acute renal failure secondary to myoglobinuria.
- the risk of myopathy appears to be greater with combinations of a statin and certain other drugs, including gemfibrozil (5% incidence), niacin (2% incidence), and cyclosporin (30% incidence).
- Symptoms are any unexplained muscle pain, tenderness, especially if accompanied by malaise or fever.
- the drug should be discontinued if myopathy is suspected or if elevations in creatinine phosphokinase (CKP) greater than 10 times normal occur.
- CKP creatinine phosphokinase
- the present invention is based, in part, on methods of and methods for treating or preventing a disease or disorder, for example, cardiovascular disease, dyslipidemia; dyslipoproteinemia; hyperlipidemia; a disorder of glucose metabolism; Syndrome X; a peroxisome proliferator activated receptor-associated disorder; obesity; hypertension; and renal disease with a serum-free and mitogen-free Leukocyte Interleukin Injection (LI) mixture or Multikine® comprised of specific ratios of cytokines IL- l ⁇ to IL-2, TNF- ⁇ to IL-2, IFN- ⁇ to EL-2 and GM-CSF to IL-2.
- LI Leukocyte Interleukin Injection
- a method for a serum-free and mitogen- free cytokine mixture being administered three times a week over a two week period in a range from about 20 IU to 12000 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1 st International Standard for Human IL-2, 86/504.
- the serum-free and mitogen-free cytokine mixture is administered five times a week over a three week period in a range from about 20 IU to 12000 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1 st International Standard for Human IL-2, 86/504.
- the total daily dose can be 200, 400, 800, 1200, 1600, 2400, 3200, 4800, 8000, 9600 and 12000 IU/mL as IL-2 wherein IU represents International Units for Interleukin-2 given in World Health Organization 1 st International Standard for Human IL-2, 86/504.
- Another embodiment of the invention includes Leukocyte Interleukin,
- I cytokine to interleukin 2
- IL-l ⁇ to DL-2 at a ratio range of 0.4 - 1.5, and preferably at 0.7+/- 0.1
- TNF- ⁇ to EL-2 at a ratio range of 3.2 - 11.3, and preferably at 9.5+/- 1.8
- IFN- ⁇ to EL-2 at a ratio range of 1.5 - 10.9, and preferably at 6.0+/- 1.1 (EFN- ⁇ /IL-2)
- GM-CSF to IL-2 at a ratio range of 2.2 - 4.8, and preferably at 4.0+/- 0.5 (GM- CSF/EL-2).
- the serum-free and mitogen-free cytokine preparation or pharmaceutical composition has further different cytokines and other small biologically active molecules wherein the ratio of each of the small biologically active molecules to IL-2 is as follows: IL-3 to IL-2 in a ratio range of 0.38 - 0.68, preferably at 0.53+/- 0.15, IL-6 to IL-2 in a ratio range of 37.2 - 53.8, preferably at 46+/- 5.9, IL-8 to IL-2 in a ratio range of 261 - 561.5, preferably at 411+/- 10.6, IL-l ⁇ to IL-2 in a ratio range of 0.56 - 0.94, preferably at 0.75+/- 0.19, IL-IO to IL-2 in a ratio range of 2.82 - 3.22, preferably at 3.0+/- 0.18, IL-16 to IL-2 in a ratio range of 1.16 - 2.84, preferably at 1.84+/-0.68, G-CSF to IL-2 in
- the present invention is concerned with methods for treating or preventing a disease or disorder, for example, cardiovascular disease, dyslipidemia; dyslipoproteinemia; hyperlipidemia; a disorder of glucose metabolism; Syndrome X; a peroxisome proliferator activated receptor-associated disorder; obesity; hypertension; and renal disease with a serum-free and mitogen-free cytokine mixture comprised of specific ratios of IL-I ⁇ to IL-2, TNF- ⁇ to IL-2, IFN- ⁇ to IL-2 and GM-CSF to IL-2.
- a disease or disorder for example, cardiovascular disease, dyslipidemia; dyslipoproteinemia; hyperlipidemia; a disorder of glucose metabolism; Syndrome X; a peroxisome proliferator activated receptor-associated disorder; obesity; hypertension; and renal disease with a serum-free and mitogen-free cytokine mixture comprised of specific ratios of IL-I ⁇ to IL-2, TNF- ⁇ to IL-2, IFN- ⁇ to IL-2 and GM-CSF
- the clinical significance of the immuno-suppression in cancer patients unexpectedly impacts methods of preventing cardiovascular disease, a disorder of glucose metabolism; Syndrome X; a peroxisome proliferator activated receptor-associated disorder; obesity; hypertension and in particular dyslipidemia, dyslipoproteinemia or hyperlipidemia.
- the peritumoral dose during cancer treatment was performed by injecting, for example 100 IU/mL as IL-2 into four different sites around a tumor mass. Each of the four sites was injected with one quarter (1/4) of the entire peritumoral dose. The remaining half of the total dose was administered perilymphatically ipsilateral to the tumor site sequentially and on the same visit.
- the perilymphatic injections were given at one site at the posterior mandibular area in the area of the jugular lymphatic chain ipsilateral to the injected tumor mass.
- Leukocyte-Interleukin Injection itself, is a serum-free, mitogen-free, antibiotic-free preparation produced from human peripheral blood mononuclear cells that include T-cells, B cells and macrophages.
- cytokines there are three "families" of cytokines in LI that together are important to the unique biological activity of LI. They include direct cytotoxic/cytostatic and virocidal/virostatic cytokines such as TNF- ⁇ , and IFN- ⁇ , lympho-proliferative cytokines such as IL-I, and IL-2 and chemotactic cytokines such as IL-6, EL-8 and MIP- l ⁇ .
- the different cytokine and small biological molecules that constitute LI are all derived from the lectin (PHA) in vitro stimulation of human peripheral blood mononuclear cells that include T cells, B cells, and macrophages.
- PHA lectin
- Centrifugation on a Ficoll-Paque gradient separates the white blood cells (including T cells, B cells, and macrophages) from donor whole blood, and a series of washes (in physiologically buffered media) facilitates the isolation of lymphocytes, and the removal of red blood cells, cellular debris and other unwanted cellular components from the isolated white cell component of the whole donor blood.
- LI contains different cytokines present at specific ratios of each cytokine to
- Interleukin 2 Interleukin 2 (IL-2) as follows: IL-I ⁇ to EL-2 at a ratio range of 0.4 - 1.5, and preferably at 0.7+/- 0.1 (IL-l ⁇ /EL-2), TNF- ⁇ to IL-2 at a ratio range of 3.2 - 11.3, and preferably at 9.5+/- 1.8 (TNF- ⁇ /IL-2), IFNPy to IL-2 at a ratio range of 1.5 - 10.9, and preferably at 6.0+/- 1.1 (IFN- ⁇ /IL-2), and GM-CSF to IL-2 at a ratio range of 2.2 - 4.8, and preferably at 4.0+/- 0.5 (GM-C SF/IL-2).
- IL-I ⁇ to EL-2 at a ratio range of 0.4 - 1.5, and preferably at 0.7+/- 0.1
- TNF- ⁇ to IL-2 at a ratio range of 3.2 - 11.3, and preferably at 9.5+/- 1.8
- IL-3 to IL-2 in a ratio range of 0.38 - 0.68, preferably at 0.53+/- 0.15, IL-6 to IL-2 in a ratio range of 37.2 - 53.8, preferably at 46+/- 5.9, IL-8 to DL-2 in a ratio range of 261 - 561.5, preferably at 411+/- 10.6, DL-I ⁇ to IL-2 in a ratio range of 0.56 - 0.94, preferably at 0.75+/- 0.19, IL-10 to IL-2 in a ratio range of 2.82 - 3.22, preferably at 3.0+/- 0.18, IL-16 to IL-2 in a ratio range of 1.16 - 2.84, preferably at 1.84+/-0.68, G-CSF to IL-2 in a ratio range of 2.16 - 3.78, preferably at 2.97
- L Leukocyte-Interleukin Injection
- DL-4 IL-7
- IL-15 TfR
- sICAM cytokines and other small biologically active molecules
- PDGF-AB IFN- ⁇
- EPO EPO
- LTC 4 TGF- ⁇ 2
- FGF basic Angiogenin
- SCF SCF
- LIF LIF
- mononuclear cells are separated from human donor "buffy coats" by step-gradient centrifugation and cultured with PHA to enhance production and secretion of IL-2 and other cytokines from the donor white blood cells in culture as disclosed in U.S. Patents 5,093,479, 4,390,623, 4,388,309, 4,406,830, 4,661,447, 4,681,844 and 4,464,355, all of which are incorporated herein by reference.
- the culture supernatant is aseptically harvested, clarified and subjected to a commercial virus exclusion process. The supernatant is then further concentrated approximately 10 fold by ultrafiltration and microfiltration.
- IL-2 - Interleukin 2 (IL-2): A 15.5-kD glycoprotein synthesized by CD4+ helper T lymphocytes (Formally known as T cell Growth Factor). IL-2 has an autocrine effect acting on the CD4+ T lymphocytes that produce it and on other cells of the immune system (including B lymphocytes, CD8+ T lymphocytes, NK [Natural Killer] cells and others).
- IL-l ⁇ - Interleukin 1 beta A 17-kD cytokine synthesized by activated mononuclear phagocytes is found in free form in the circulation and mediates inflammatory responses. It acts on CD4+ T lymphocytes to help facilitate their proliferation, and acts on B-lymphocytes as a growth and differentiation factor. It also induces the synthesis of IL-6 by mononuclear phagocytes.
- TNF- ⁇ - Tumor Necrosis Factor alpha A 157 amino acid (aa) residues protein, synthesized by stimulated monocytes, macrophages, B lymphocytes, T lymphocytes, an NK cells among others, found in a trimmeric form in the circulation. TNF mediates direct anti-tumor action, causing tumor cell lysis, facilitates leukocyte recruitment, inducing angiogenesis and promotes fibroblast proliferation.
- EFN- ⁇ - Interferon Gamma A 21-24-kD glycoprotein homodimer synthesized by activated T lymphocytes and NK cells, is a powerful activator of monocytes increasing monocytes ability to destroy intracellular microorganisms and tumor cells. It has direct anti-viral and anti-proliferative activity, and causes many cell types to express Class II MHC (Major Histocompatibility Complex) cell surface molecular complex, as well as increasing the expression of Class I MHC.
- Class II MHC Major Histocompatibility Complex
- GM-CSF Granulocyte Macrophage - Colony Stimulating Factor
- a 127 aa protein found as a monomer in the circulation, produced by macrophages and T lymphocytes, fibroblast and endothelial cells. It is a growth factor for hemopoietic cells, and stimulates the growth and differentiation of myelomonocytic lineage.
- IL-3 - Interleukin - 3 (IL-3): A 20-kD lymphokine synthesized by activated
- CD4+ T helper lymphocytes acts as a colony-stimulating factor by facilitating the proliferation of some hematopoietic cells and promoting the proliferation and differentiation of T lymphocytes.
- IL-6 - Interleukin - 6 A 26-kD cytokine produced by activated T lymphocytes, mononuclear phagocytes, endothelial cells, and fibroblasts. It acts on many cells but has a special function in enabling activated B-lymphocytes to differentiate into antibody secreting plasma cells, and induces hepatocytes to form acute-phase proteins (implicated in inflammatory responses) as well as fibrinogen.
- BL-8 - Interleukin - 8 An 8-kD protein produced by macrophages and endothelial cells. Is a powerful chemotactic factor for neutrophils and T lymphocytes, and facilitates neutrophil adherence to endothelial cells.
- IL-I ⁇ - Interleukin 1 alpha (IL- l ⁇ ): A 17-kD cytokine (like IL- l ⁇ ) is cleaved from a 33-kD precursor molecule, synthesized by activated mononuclear phagocytes, is rarely found in free form in the circulation and acts as a membrane-associated substance. It assists IL-I ⁇ in mediating inflammatory responses.
- IL-IO - Interleukin - 10 An 18-kD polypeptide produced by CD4+ and CD 8+ T lymphocytes, monocytes, macrophages, activated B lymphocytes, and keratinocytes. It inhibits macrophages ability to present antigen particularly to THI- type cells, and secrete IL-6 and TNF.
- EL-16 - Interleukin - 16 (IL-16): A 14-kD tetrameric protein produced by
- CD8+ T lymphocytes eosinophils, mast cells and respiratory epithelial cells. It has strong chemoattraction properties for CD4+ T lymphocytes and monocytes.
- G-CSF Granulocyte Colony Stimulating Factor
- TNF- ⁇ - Tumor Necrosis Factor beta A 25-kD protein produced by activated lymphocytes. It can kill tumor cells in culture, and stimulates proliferation of fibroblasts. In addition it mimics most of the other actions of TNF- ⁇ .
- MIP- l ⁇ - Macrophage Inflammatory Protein - 1 alpha (MIP- l ⁇ ): A 66-aa monomeric protein produced by macrophages and other cells. It is a chemo-attractant for monocytes, T lymphocytes and eosinophils.
- RANTES - An 8-kD protein produced by T lymphocytes and is a chemo- attractant to monocytes, T lymphocytes and eosinophils, and promotes inflammation.
- EGF Epidermal Growth Factor
- PGE 2 - Prostaglandin E 2 PGE 2 belong to a family of biologically active lipids derived from arachidonic acid through the cyclooxygenase enzymatic reaction. It is released by activated monocytes and blocks MHC Class II expression on T lymphocytes and macrophages.
- TxB 2 - Thromboxane B 2 (TxB 2 ): TxB 2 is a member of biologically active compounds derived from polyunsaturated fatty acids by isomerization of prostaglandin and endoperoxidase PGH 2 via the enzyme thromboxane synthetase. TxB 2 has a physiological role in thromboembolic disease, and anaphylactic reactions.
- IU International Units
- IU International Units
- WHO 1 st International Standard for Human EL-2 86/504.
- International Units are the only recognized and standardized method to report biological activity units that are published and are derived from an international collaborative research effort.
- P - "p ⁇ 0.01" A term in mathematical statistics that denotes the level of probability of an event occurring under pre-set conditions.
- Means with 95% confidence intervals were constructed for the aggregated data and various groups of the data. Means stratified by protocol, by dose, and protocol by dose, were compared for trends over time and for differences between dose groups. Regression analyses were conducted to determine whether the slope of the change measuring the average cholesterol concentrations, as a function of LI treatment, was statistically significant from zero. Generalized Linear models (Manova) were conducted to test whether dose level differences were statistically significant, and to test whether a patients initial cholesterol level, i.e. concentration prior to treatment, had a significant relationship with the response to Leukocyte Interleukin injection.
- CIZ** *CIZ i.v. Cylophosphamide (low dose, 300 mg/M 2 ), lndomethacin (25mg, tid), and Zinc sulfate, 142 mg (po, qd, 50mg as elemental Zinc).
- CIZ was administered as follows, Cylophosphamide (iv), one time only, at three (3) days prior to the initial Multikine administration, lndomethacin (three times per day - with food, daily - staring with the first administration of Multikine to the last day of Multikine administration and Zinc Sulfate (the same as lndomethacin administration schedule)
- CIZ i.v. Cylophosphamide (low dose, 300 mg/M 2 ⁇ ), lndomethacin (25mg, tid), and Zinc sulfate, 142 mg (po, qd, 50mg as elemental Zinc).
- CIZ was administered as follows, Cylophosphamide (iv), one time only, at three (3) days prior to the initial Multikine administration, lndomethacin (three times per day - with food, daily - staring with the first administration of Multikine to 24 hours before surgical resection of the tumor, and Zinc Sulfate (the same as lndomethacin administration schedule)
- LI Leukocyte Interleukin, Injection
- a single intravenous infusion of cyclophosphamide is given, I ⁇ j., 300 mg/m 2 , three days prior to the first LI administration.
- lndomethacin 25 mg
- Zinc Sulfate 50 mg, as elemental Zinc
- multivitamin supplement once daily, is self-administered beginning 3 days after cyclophosphamide administration.
- Leukocyte Interleukin, Injection (LI) administration can also be performed in the following manner; the total daily is injected three to five times per week peri- tumorally.
- half the total LI dose can be administered peri-tumorally where 1/4 of the peritumoral dose [approximately 100 IU for 800 IU/mL total dose] is administered at each of four sites around the tumor mass.
- the remaining one half (400 IU/mL of EL-2 for 800 IU/mL total dose) is administered perilymphatically ipsilateral to the tumor site (sequentially and on the same visit) over a three week period, 5 times per week for three weeks.
- LI is administered intradermally at the circumferential margins of the visible/palpable tumor mass.
- the perilymphatic injections are given at the posterior mandibular area in the area of the jugular lymphatic chain ipsilateral to the injected tumor mass in protocols Cel-02 and Hun-02.
- Cel-01 and Cel-04 the doses of LI were only administered peri-tumorally.
- a single intravenous infusion of cyclophosphamide is given, I ⁇ j., 300 mg/m 2 , three days prior to the first LI administration.
- Indomethacin 25 mg
- Indomethacin 25 mg
- Zinc Sulfate 50 mg, as elemental Zinc
- multivitamin supplement once daily, is self-administered beginning 3 days after cyclophosphamide administration and until 24 hours prior to surgery.
- Leukocyte Interleukin Injection is prepared from selected human peripheral blood mononuclear cells obtained from the US Red Cross after passing all FDA mandated testing for blood for transfusion cultured with mitogen. No first time donors are allowed. The serum free culture supernatant is then aseptically harvested, clarified, subjected to a commercial virus exclusion process, concentrated, and microfiltered. Human Serum Albumin, Inj. USP is added to the concentrate and the resultant solution is buffered to physiological pH, brought to a target IL-2 concentration, and subjected to a second microfiltration. The formulated drug solution is aseptically dispensed into sterile serum-type vials and labeled by its content of DL-2.
- Product potency is measured by the incorporation of radiolabeled thymidine (in vitro) by the use of a cytotoxic T-lymphoid (CTLL-2), IL-2 dependent cell line.
- CTL-2 cytotoxic T-lymphoid
- the final injectable agent is further tested by ELISA or the presence of five marker cytokines: EL-2, IL-I ⁇ , GM-CSF, IFN- ⁇ , and TNF- ⁇ .
- LI is further subjected to quality control tests for sterility, bacterial endotoxins, pH, and total protein concentration, and other physical-chemical tests.
- LI is provided frozen in a borosilicate glass serum vial containing 2.2 mL of drug at the label claim as IL-2 (400 IU/ml) for peritumoral, intratumoral, perilymphatic or subcutaneous administration. LI is subjected to quality control tests for identity, sterility, bacterial endotoxins, pH, and total protein concentration. Each vial is inspected for particulate contamination and appearance. The preparation has a total protein content of 3 mg/mL wherein the material is supplied sterile and pyrogen free. LI has an assigned expiration date of 24 months from date of manufacture when the drug is stored at -2O 0 C.
- Cyclophosphamide, Inj. USP (Bristol-Myers-Squibb, UK) is supplied as a sterile powder containing 45 mg sodium chloride, 75 mg mannitol or approximately 82 mg sodium bicarbonate per 100 mg cyclophosphamide for reconstitution prior to intravenous infusion.
- Indomethacin USP (Sanofi - Synthelabo, France) is supplied as 25 mg tablets for oral self-administration with food.
- Zinc Sulfate and Multivitamins [78] Zinc sulfate (50 mg as elemental Zinc, R. P. Scherer Corporation, Clearwater,
- OTC multivitamins are supplied by the clinic to each patient for self-administration.
- Day numbers are assigned with day one defined as the first day of treatment.
- day numbers in these analyses reflect the elapsed number of days since the first day of treatment, and not actual treatment days. Thus, some patients had completed the trial by day 18, yet remained in follow, or did not. Other patients had additional or extended courses of treatment past day 18.
- baseline average (of screen & dayl)
- baseline average (of screen & dayl) &
- Day35* average (of day 35 & 40).
- Data are include all data retained for analysis from protocols 50234-01, 50234-02 and 50234-03 and 33575-01
- the regression model is statistically, highly significant, p ⁇ 0.0001. Both the durational effect of therapy as well as the dose of therapy contributed to the reduction in average cholesterol concentrations.
- the estimate slope of change due to each additional day is -0.641 as shown in Table 4. This indicates that for each day of treatment the average level of cholesterol (across all groups) decreased by -0.64. This estimate is statistically significant from zero, signifying that there is a true reduction due to treatment. Given the observational (and purposively selected) nature of the data, this equation cannot be used to make inferences to the general population. However, the data strongly suggests a relationship exists between Leukocyte Interleukin injection (LI) treatment and serum cholesterol change.
- LI Leukocyte Interleukin injection
- Source DF Type I SS Mean Square F Value Pr > F baseline 1 11.56487006 11.56487006 16.33 0.0006 dose 6 6.54895246 1.09149208 1.54 0.2136
- Example 2 [98] The following is results of a daily mean laboratory values for cholesterol aggregated across different labs & protocols, grouped by dose the dose level at 400 IU/mL as shown in Table 8.
- Example 14 The following is results of ALT values showing no statistically significant change from baseline to post treatment for patients in protocol Cel-01 (33575-01) as shown in Table 20.
- Example 17 The following is results of ALT values showing no statistically significant change from baseline to post treatment for patients in protocol Cel-04 (50234-01) as shown in Table 23.
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2005269400A AU2005269400A1 (en) | 2004-07-30 | 2005-07-29 | A method for managing cholesterol with a serum-free and mitogen free cytokine mixture |
US11/572,907 US20080063625A1 (en) | 2004-07-30 | 2005-07-29 | Method for Managing Cholesterol with a Serum-Free and Mitogen-Free Cytokine Mixture |
AT05775596T ATE513565T1 (en) | 2004-07-30 | 2005-07-29 | METHOD FOR MANAGING CHOLESTEROL USING A SERUM AND MITOGEN FREE CYTOKINE MIXTURE |
EP05775596A EP1773395B1 (en) | 2004-07-30 | 2005-07-29 | A method for managing cholesterol with a serum-free and mitogen free cytokine mixture |
CA002574970A CA2574970A1 (en) | 2004-07-30 | 2005-07-29 | A method for managing cholesterol with a serum-free and mitogen-free cytokine mixture |
Applications Claiming Priority (2)
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US59212104P | 2004-07-30 | 2004-07-30 | |
US60/592,121 | 2004-07-30 |
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WO2006015137A2 true WO2006015137A2 (en) | 2006-02-09 |
WO2006015137A3 WO2006015137A3 (en) | 2006-12-07 |
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PCT/US2005/026819 WO2006015137A2 (en) | 2004-07-30 | 2005-07-29 | A method for managing cholesterol with a serum-free and mitogen free cytokine mixture |
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Country | Link |
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US (1) | US20080063625A1 (en) |
EP (1) | EP1773395B1 (en) |
AT (1) | ATE513565T1 (en) |
AU (1) | AU2005269400A1 (en) |
CA (1) | CA2574970A1 (en) |
ES (1) | ES2367441T3 (en) |
WO (1) | WO2006015137A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2011092231A1 (en) | 2010-01-28 | 2011-08-04 | Immunservice Gmbh | Cytokines for the treatment of addictions |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0449949A1 (en) * | 1988-12-21 | 1991-10-09 | Schering Corporation | Use of interleukin-4 for lowering blood-cholesterol levels |
EP0543417A1 (en) * | 1991-11-22 | 1993-05-26 | Lipogenics, Incorporated | Tocotrienols and tocotrienol-like compounds and methods for their use |
US5632983A (en) * | 1994-11-17 | 1997-05-27 | University Of South Florida | Method for treating secondary immunodeficiency |
AU694304B2 (en) * | 1994-11-17 | 1998-07-16 | University Of South Florida | Method for making a medicament for treating secondary immunodeficiency |
US5945097A (en) * | 1996-09-06 | 1999-08-31 | Schering Corporation | Method for lowering cholesterol levels with interleukin-10 |
EP1389128A4 (en) * | 2001-04-17 | 2005-05-18 | Ludwig Inst Cancer Res | Method for inhibiting atherosclerotic plaque formation |
US6896879B2 (en) * | 2003-07-03 | 2005-05-24 | Cel-Sci Corporation | Method of pre-sensitizing cancer prior to treatment with radiation and/or chemotherapy and a novel cytokine mixture |
-
2005
- 2005-07-29 CA CA002574970A patent/CA2574970A1/en not_active Abandoned
- 2005-07-29 AT AT05775596T patent/ATE513565T1/en not_active IP Right Cessation
- 2005-07-29 WO PCT/US2005/026819 patent/WO2006015137A2/en active Application Filing
- 2005-07-29 ES ES05775596T patent/ES2367441T3/en active Active
- 2005-07-29 EP EP05775596A patent/EP1773395B1/en not_active Not-in-force
- 2005-07-29 US US11/572,907 patent/US20080063625A1/en not_active Abandoned
- 2005-07-29 AU AU2005269400A patent/AU2005269400A1/en not_active Abandoned
Non-Patent Citations (2)
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See also references of EP1773395A4 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2011092231A1 (en) | 2010-01-28 | 2011-08-04 | Immunservice Gmbh | Cytokines for the treatment of addictions |
Also Published As
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AU2005269400A1 (en) | 2006-02-09 |
US20080063625A1 (en) | 2008-03-13 |
EP1773395A4 (en) | 2009-08-05 |
ATE513565T1 (en) | 2011-07-15 |
ES2367441T3 (en) | 2011-11-03 |
EP1773395B1 (en) | 2011-06-22 |
WO2006015137A3 (en) | 2006-12-07 |
EP1773395A2 (en) | 2007-04-18 |
CA2574970A1 (en) | 2006-02-09 |
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