Title: Methods for improving brain function using leptin or analogs thereof

United States Patent:  6,518,235

Issued:  February 11, 2003

Inventors:  Oomura; Yutaka (6-14-17, Kawamo, Takarazuka-shi, Hyogo-ken, JP); Hori; Nobuaki (1-3-6-305, Chidori, Koga-shi, Fukuoka-ken, JP); Shiraishi; Takemasa (8-19-2, Kamitsuruma, Sagamihara-shi, Kanagawa-ken, JP); Sasaki; Kazuo (5-1-14, Sengoku-cho, Toyama-shi, Toyaam-ken, JP); Takeda; Hiroshi (5-2, Higashiyama-cho, Itabashi-ku, Tokyo-to, JP); Tsuji; Minoru (3-23-12-104, Kamiikedai, Ota-ku, Tokyo-to, JP); Matsumiya; Teruhiko (3-41-25, Ogikubo, Suginami-ku, Tokyo-to, JP)

Appl. No.:  200919

Filed:  November 30, 1998

The present invention is to provide a drug for improvement of brain function which is effective to prevent from or to cure dementia such as Alzheimer's disease. The drug for improvement of brain function of this invention comprises leptin of mammals as an effective component wherefore has a superior effect in improvement of learning and memory.

SUMMARY OF THE INVENTION

The inventors of this invention have found, as a result of further earnest study, that leptin of mammals has an action to improve brain function which is effective to prevent and to treat dementia such as Alzheimer's disease and cerebral apoplexy, etc., and to cure sequelae of apoplexy, in addition to the known actions to regulate food intake and to increase energy consumption.

The drug for improvement of brain function of this invention contains leptin of the mammals as an effective component. The above-mentioned leptins of the mammals are highly preserved by 84% in mouse-human and by 96% in mouse-rat in the homology of amino-acid level, and are known to be the protein with high homology, and are recognized to show the same physiological actions even when administered to the different kinds of animals. Therefore, as the leptin of mammals of this invention, leptin of any kinds of mammals is applicable, in which the amino acid sequence of the leptin of the animals is substantially homologous with human leptin and the physiological action of the leptin is recognized to be the same with that of human leptin. For example, mouse leptin indicated by sequence ID number 2 or rat leptin indicated by sequence ID number 3 is also applicable as well as the human leptin indicated by sequence ID number 1.

The leptin of this invention includes analogue peptide of the above-mentioned leptin, as well as the polypeptide which exists in vivo as indicated by the above-mentioned sequence ID numbers from 1 to 3 known as mature leptin in vivo. In other words, the essence of this invention is that the leptin of the mammals being a product of obese gene has, for the first time, been found to be useful as a drug for improvement of brain function. Further, the analogue peptide of leptin, which is substantially homologous with mature leptin existing in vivo and shows physiological actions similar to the mature leptin in vivo, is also included in this invention.

The human leptin indicated by the above-mentioned sequence ID number 1 is expressed as precursor polypeptide consisting of 167 amino acid residue in a cell as a product of obese gene, after which signal peptide consisting of 21 amino acid residue of the amino terminus is cut off, and is secreted in vivo as mature protein consisting of 146 amino acid residue.

The above-mentioned analogue peptide of leptin means the polypeptide in which one or plural amino acid residue/residues is/are added to, is/are removed from, or is/are replaced with the above-mentioned mature leptin in vivo. To be more specific, leptins indicated by sequence ID numbers from 4 to 6 in which methionine is added to the amino terminus of the above-mentioned leptins is one example.

As the drug for improvement of brain function of this invention, the above-mentioned leptin of mammals may include the pharmaceutically acceptable salts thereof, for example, salts with alkaline metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, salts with metals such as alminum and acid addition salts with hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, thiocyanic acid, boric acid, formic acid, acetic acid, propionic acid, glycolic acid, citric acid, tartaric acid, succinic acid, gluconic acid, lactic acid, malonic acid, fumaric acid, anthranilic acid, benzoic acid, cinnamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, sulfanilic acid and the like. These salts can be produced from the above-mentioned free leptin, or can be transformed to each other by the methods known to public.

The drug for improvement of brain function of this invention may contain at least one of the above-mentioned leptin of mammals and the analogue peptide of the abovementioned leptin of mammals. They can either be used independently or together. When the protein which is prepared from a different kind of mammals is applied for medical treatment of man, there are not a few cases in which it causes allograft rejection or shock symptoms on the basis of immunity defense systems, therefore human leptin, namely, human leptin and analogue peptide thereof are preferable, analogue peptide of human leptin is more preferable, and analogue peptide of human leptin indicated by the sequence ID number 4 is particularly preferable.

Processes for obtaining the leptin of this invention are, for example, a process by purification and isolation from living bodies or from cultured cells, a peptide synthesis process such as a solid-phase or a liquid-phase peptide synthesis process, and a production process by use of genetic recombination technology, etc. Since the leptin of this invention consists of many amino acid residues, the production process by use of genetic recombination technology is industrially preferable.

As expression systems (host-vector systems) for production of leptine and analogue peptide thereof by use of genetic recombination engineering, there are expression systems of bacteria, yeast, insect cell and mammal cell, for example. And it is possible to obtain analogue peptide of leptine indicated by the above-mentioned sequence ID numbers from 4 to 6 by use of genetic recombination engineering, namely for example, by ligating cDNA, coding the above-mentioned sequence ID numbers from 4 to 6, with an optional expression vector and then transfecting the vector to a proper host cell.

As to the above-mentioned cDNA which codes the leptin of this invention, human and murine leptin cDNA is mentioned in PCT Japanese publication No.9-506264, and rat cDNA is mentioned in the literature of Ogawa etc. [J. Clin. Invest., page 1647, volume 96 (year 1995] etc.

The process for genetic technological production of recombinant leptin by use of leptin gene is also described in detail in the above-mentioned publication and others. Leptin of this invention can be obtained by producing in accordance with these known processes, and some of the recombinant leptins are also available in market.

For example, it is possible to obtain human recombinant leptin indicated by sequence ID number 4, in which methionine being amino acid corresponding to initiation codon is connected to the amino terminal of polypeptide indicated by sequence ID number 1, by using cDNA, wherein the necessary initiation codon is connected to the head of the cDNA corresponding to the mature protein indicated by sequence ID number 1.

In order to ascertain whether the polypepide obtained from the above-mentioned process has the intended amino acid sequence or not, the already known processes are applied. For example, it is possible to identify the amino acid sequence of the obtained polypeptide chain by fragmentating the polypeptide chain obtained as mentioned above by using a reagent which has substrate specificity such as cyanide bromide or enzyme like trypsin, purifying by high performance liquid chromatography to isolate homogeneous peptide, and identifying amino acid sequence of these peptide fragments by automatic Edman method.

The drug for improvement of brain function of this invention is usually used by combining proper pharmaceutical carrier or diluent, and is formulated by the known process. On prescription, the drug for improvement of brain function of this invention may be used either solely or jointly, or in combination with the other drugs. As the drug for improvement of brain function of this invention is a peptide preparation, it is general to formulate as an injection. It could be prepared in a form of solution or suspension in aqueous or non-aqueous solvent such as distilled water for injection, physiological saline, Ringer solution, vegetable oil, synthetic fatty acid glyceride, higher fatty acid ester, or propylene glycol. Further, drug additives such as prevailing stabilizer, buffer, suspension agent, isotonic agent, pH modifier, preservatives may also be added. In addition to injection, sublingual tablet and nebulizer, which are absorbed through oral cavity mucus membrane and nose mucus membrane, are sometimes used for peptide drug, to which this invention can also be applied. Furthermore, it is possible to prepare the other forms of medicine, depending on diseases, which is the best for the treatment thereof like oral medicine by special preparation so that it is not decomposed even when administered orally. Advantages:

The drug for improvement of brain function of this invention has a superior effect on improvement of learning and memory, as evidenced by the tests described in detail hereinafter. The drug is active in a Passive Avoidance Learning Test. A therapeutic amount of the drug may be administered to a patient known to be in need of improvement of memory or treatment of memory deterioration. Further, the drug for improvement of brain function of this invention has high usefulness to cure diseases which cause deterioration of brain function like brain degeneration diseases such as Alzheimer's disease, senile dementia, Pick's disease, Huntington's chorea, Parkinson disease, parkinsonism dementia syndrome, progressive subcortical gliosis, progressive supranuclear palsy, thalamic degeneration syndrome, hereditary aphasia, myoclonus epilepsy; cerebrovascular disorders like celebral arteriosclerosis; cerebral infection diseases and cerebral inflammatory diseases like general paresis, various encephalitis gliomgs, Creutzfeldt-Jakob disease, subacute sclerosing panencephalitis, progressive mulifocal leukoence phalopathy, systemic lupus erythematosus; poisonous brain troubles like chronic alcoholism, carbon monoxide poisoning, heavy metal poisoning; ischemic brain diseases like head injury, epilepsy, brain tumor, intracranial hematoma, dialysis encephalopathy, brain infarction, and cerebral thrombosisa; and to improve various symptoms caused by these diseases like memory deterioration, aphasia, disturbance of consciousness, depression, apathy, delusion, and confusion.

Claim 1 of 12 Claims

What is claimed is:

1. A method for improving memory or treating memory deterioration comprising administering to a patient known to be in need of such improvement or treatment a therapeutic amount of:

at least one leptin selected from the group consisting of any one of SEQ. ID. No. 1, SEQ. ID No. 2, SEQ. ID. No. 3, SEQ. ID. No. 4, SEQ. ID. No. 5, and SEQ. ID. No. 6,

an analogue which differs by a single conservative amino acid substitution from any one of SEQ. ID. No. 1, SEQ. ID No. 2, SEQ. ID. No. 3, SEQ. ID. No. 4, SEQ. ID. No. 5, and SEQ. ID. No. 6, wherein said analogue is active in a Passive Avoidance Learning Test,

at least one leptin that is active in a Passive Avoidance Learning Test and is selected from the group consisting of a leptin having an amino acid homology of at least 99% with any one of SEQ. ID. No. 1, SEQ. ID. No. 2, SEQ. ID. No. 3, SEQ. ID. No. 4, SEQ. ID. No. 5 and SEQ. ID. No. 6, or

pharmaceutically acceptable salts thereof.

 

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