Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders

Definitions

• the present invention generally relates to a method of treating immune system dysfunctions with an opioid peptide, and more particularly to the use of dynorphin in either stimulating the immune system of a patient whose immune system is impaired or by suppressing the immune system of a patient whose immune system is hyperactivated.
• virus infections such as HTLV lead to the specific elimination of CD4 positive T helper cell population.
• immuno suppressed individuals show increased incidents of EBV associated ly phomas and other diseases associated with secondary infection.
• bone marrow transplant recipients whose immune systems have been affected by chemo-radiation therapy, incur a risk of developing lymphomas.
• problems associated with the immune system have also been noticed in drug addicts, who comprise a subpopulation vulnerable to infection by the AIDS virus. These all appear to involve aberrant immune system suppression.
• aberrant immune system activation examples include the autoimmune diseases, such as rheumatoid arthritis, myasthenia gravis, and the like.
• Present drug therapies for aberrant immune system activation such the widely used immuno suppressive compound Cyclosporin A, have restriction on long term use due to side effects.
• Investigators have recently begun attempts to link immuno regulation to neural opioid systems. It has become increasingly clear there are a number of opioid effects on cells of the immune system, but the mechanisms remain obscure. The authors of a recent review have concluded that the significance of opioids in immune system function remain a matter for speculation. Sibinga and Goldstein, Ann. Rev. Inanunol . , 6, pp. 219-249 (1988) .
• the endogenous opioids exist in multiple forms in the central nervous system and include the dynorphins, a series of peptides derived from the precursor prodynorphin (proenkephalin B) . Unlike either the enkephalins or the endorphins, many of the dynorphins interact with high affinity with all three major opioid receptor types ( ⁇ , ⁇ , and c) . The dynorphins are also nearly unique among endogenous opioids in that they are not analgesic in the brain, although they may be in the spinal cord.
• AA is isoleucine, leucine, or lysine
• AA 9 is arginine or proline
• AA is proline
• a carbonyl carbon at the AA 10 terminus is amidated.
• dynorphin (1-10) amide analogs do not have significant analgesic activity (unless given in huge doses where they tend to produce convulsions) , but they differ from dynorphin (1- 13) by neither potentiating nor antagonizing morphine in naive animals. In tolerant animals, on the other hand, the dynorphin (1-10) amide analogs appear to be a more potent and selective analog than dynorphin (1-13) .
• dynorphin in treating high blood pressure modifies the autonomic nervous system so as to amplify and maintain the intensity of endogenous opioid peptides.
• a mode of action may be by increasing the sensitivity of visceral afferent receptors.
• U.S. Patent 4,684,624, issued August 4, 1987, inventors Hosobuchi et al. describe the use of dynorphin-related peptides, in the acid or amidated form, to treat patients suffering from cerebral ischemia.
• the administration of these opioid peptides to patients suffering from acute focal cerebral ischemia has been found useful in prolonging survival, and appears useful in partially reversing neurologic deficits resulting from cerebral ischemia.
• Fig. 1 graphically illustrates the suppression, or inhibition, of macrophage colony forming units by morphine
• Fig. 2 graphically illustrates the reversal of morphine induced inhibition of macrophage colony forming units by dynorphin (1-10) amide;
• Fig. 3 graphically illustrates the reversal of morphine induced inhibition of thymocyte proliferation by dynorphin (1-10) amide; and Fig. 4 graphically illustrates the dose dependent effect of dynorphin on macrophage colony forming units.
• the present invention provides a method for stimulating or suppressing the immune system of a patient by administrating different amounts of a dynorphin in the acid form or amide form.
• the dynorphin administered is an opioid peptide and when in acid form is administered in less than about 200 ⁇ g/kg body weight per dose to a patient whose immune system is impaired, so that the immune system is stimulated.
• the dose in acid form is greater than about 500 ⁇ g/kg body weight to a patient whose immune system is stimulated, such as by a autoimmune disease, then the immune system is suppressed.
• a particularly preferred aspect of the inventive method is to stimulate the immune system of a patient whose immune system is impaired, such as by chronic use of a narcotic analgesic or during chemo- radiation therapy.
• dynorphin and its analogs when in the acid form, have a biphasic activity.
• the acid form of dynorphin or its analogs stimulates an immune system that is impaired.
• the effect is just the opposite and suppresses the immune system.
• Suitable compounds for practicing the invention will generally be referred to by the designation "dynorphin,” and have as the first seven amino acids:
• the naturally occurring dynorphin has seventeen amino acids, where eight through seventeen are as follows:
• Dynorphin analogs that are also suitable in practicing the invention include opioid peptides having at least ten amino acids and optionally with one or more amino acid substitutions (with respect to naturally occurring dynorphin) in the amino acid positions eight through seventeen.
• suitable compounds for practicing the inventive method also include within the "dynorphin" designation those having the following structure:
• AA 8 is TYR, ILE, LEU, or LYS
• AA 9 is ARG or PRO
• AA 10 is PRO or LYS
• AA 11 is LYS, LYS-LEU, or LYS-LEU-LYS
• w is 0 or 1.
• Either the acid or the amidated form of dynorphins can be used to practice the immune system stimulating aspect of the invention. Unlike the acid form, the amidated form of dynorphin does not appear to have the biphasic effect. Thus, in the particularly preferred method for stimulating the immune system the amidated form of dynorphin is preferred.
• dynorphin (1-10) amide or (1- 13) amide where the AA is ILE, AA is ARG, and AA 10 is PRO, and when present, AA 1 is LYS, AA 12 is LEU, and AA 13 is LYS.
• AA 1 is LYS
• AA 12 is LEU
• AA 13 is LYS.
• narcotic abuse affects all the major components of the immune system (T-cells, B-cells, and accessory cells) since macrophages play a crucial role in the presentation of antigens to T-cells and are pivotal in the phagocytosis and elimination of infectious agents.
• narcotic analgesics such as morphine, heroin, and others such as those narcotic analgesics clinically used, predisposes the users to a variety of pathological conditions due to immune system suppression.
• mice were implanted with morphine (75 mg) pellets (72 hour pellets) and at different time points, animals were sacrificed. The functional status of the thymocytes and spleen cells were investigated. Mitogenic stimulation assays were used to determine the proliferative capacity (since immunocompetent cells proliferate after activation) .
• M-CSF which is a growth factor involved in the production and functional maturation of macrophages
• dynorphin by itself, is not inhibitory to immune cell proliferation, but antagonizes the inhibitory properties of morphine.
• the use of dynorphin improves the macrophage colony formation which had been suppressed by morphine treatment. This is shown by the data of Fig. 2.
• addition of dynorphin to thymocytes prevented the morphine induced suppression of proliferation, as seen by the data of Fig. 3.
• Fig. 4 shows that similar effects can be observed in morphine tolerant animals that are injected with either 2 mg or 4 mg/kg body weight of dynorphin.
• animals were implanted with either a 75 mg morphine pellet or a placebo pellet.
• Each group then received 12 hourly injection of either 2 or 4 mg/kg body weight of dynorphin.
• Fig. 4 there was a 60% reduction in the bone marrow colony forming units in the morphine tolerant animals injected with saline when compared with the placebo animals injected with saline.
• Suitable dynorphin compounds for practice of the present invention can be by methods and apparatus known to the art for peptide synthesis.
• Doses can be administered by intravenous, subcutaneous, or intramuscular injection (I.V., S.C. or I.M) or orally.
• I.V. intravenous, subcutaneous, or intramuscular injection
• a preferred range for stimulating the immune system of patient whose immune system is impaired is the administration of between about 10 ⁇ g/kg to about 200 ⁇ g/kg of body weight, most preferably about 50 ⁇ g/kg body weight. Administration can precede the events causing immune system impairment. For example, several hours before chemo-radiation a dose of dynorphin can be administered, preferably followed subsequent to the irradiation by several spaced apart subsequent doses.
• mice are believed to be about 20 to 50 times more sensitive to the dynorphin concentrations than mice (although mice are a very good predictive model, except for this difference in dosage sensitivity) .
• Example 1 more fully describes the protocol from which the data of Fig. 1 was taken.
• Example 2 describes the protocol from which Fig. 2 taken, and similarly Example 3 for Fig. 3.
• Example 4 describes dynorphin reversal studies.
• ICR mice from Harlan were used in most studies to see the effect of chronic morphine pellet implanta- tion on stem cell proliferation. Similar studies were done on Balb C mice from Jackson Labs.
• mice were implanted with two pellets: (1) a
• the femurs from both treated and untreated animals were removed aseptically.
• the bone marrow was flushed through the cut ends by injecting with Iscoves Modified Duelbco Medium using a 30 gauge needle.
• One million nucleated cells were resuspended in 0.3% agarose prepared in IMDM containing 30% FCS and plated in a 35 mm petri dish with grids. Colony formation was assessed by treating the bone marrow cells with two different lineage specific growth factors, i.e. recombinant M-CSF (2.5 ng/ml) or mouse GM-CSF (1 ng/ml) for a period of 5 days. The colonies formed were scored using an inverted phase contrast microscope.
• ICR mice from Harlan were used in most studies to see the effect of chronic morphine pellet implanta ⁇ tion on stem cell proliferation. Similar studies were done on Balb C mice from Jackson Labs. In these studies bone marrow was obtained from untreated naive animals. The bone marrow was assayed from macrophage colonies (CFU-M) and granulocyte- macrophage colonies (CFU-GM) over a range of dynorphin concentration with 100 ⁇ M being the highest concentration and 1 nm being the lowest.
• CFU-M macrophage colonies
• CFU-GM granulocyte- macrophage colonies
• the femurs from both treated and untreated animals were removed aseptically.
• the bone marrow was flushed through the cut ends by injecting with Iscoves Modified Duelbco Medium using a 30 gauge needle.
• One million nucleated cells were resuspended in 0.3% agarose prepared in IMDM containing 30% FCS and plated in a 35 mm petri dish with grids. Colony formation was assessed by treating the bone marrow cells with two different lineage specific growth factors, i.e. recombinant M-CSF (2.5 ng/ml) or mouse GM-CSF (1 ng/ml) for a period of 5 days. The colonies formed were scored using an inverted phase contrast microscope.
• ICR mice from Harlan were used in most studies to see the effect of chronic morphine pellet implanta ⁇ tion on stem cell proliferation. Similar studies were done on Balb C mice from Jackson Labs.
• Thymus was removed aseptically from treated and untreated animals and passed through a nylon mesh to dissociate the cells. The cells were counted using the trypan blue dye exclusion method. The cells were then plated at a cell density of 1 x 10 cells per well in a 96 well plate. Thymocyte proliferation was determined by culturing the cell in the presence of 1 ⁇ g/ml IL-1.
• ICR mice from Harlan were used in most studies to see the effect of chronic morphine pellet implanta ⁇ tion on stem cell proliferation. Similar studies were done on Balb C mice from Jackson Labs. Mice were implanted with two pellets: (1) a
• mice were implanted with either a single morphine pellet (group 1) or a placebo pellet (group 2) . Each group was subdivided into 2 subgroups: A and B. Animals in group A received 4 mg/kg body weight Dynorphin (1-13) injections every 12 hours. The animals in group B received saline injections. The animals were maintained on this 12 hourly dynorphin injection regime for 72 hours after pellet implantation and then sacrificed.
• the femurs from both treated and untreated animals were removed aseptically.
• the bone marrow was flushed through the cut ends by injecting with Iscoves Modified Duelbco Medium using a 30 gauge needle.
• One million nucleated cells were resuspended in 0.3% agarose prepared in IMDM containing 30% FCS and plated in a 35 mm petri dish with grids. Colony formation was assessed by treating the bone marrow cells with two different lineage specific growth factors, i.e. recombinant M-CSF

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• 1991
  • 1991-08-02 EP EP91915040A patent/EP0541694A1/en not_active Withdrawn
  • 1991-08-02 WO PCT/US1991/005518 patent/WO1992002547A1/en not_active Ceased
  • 1991-08-02 AU AU84333/91A patent/AU660420B2/en not_active Ceased
  • 1991-08-02 JP JP3514500A patent/JPH06501930A/ja active Pending
  • 1991-08-02 CA CA002088679A patent/CA2088679A1/en not_active Abandoned

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