Disclosed is a physiologically active complex which comprises a proteinaceous part with TNF activity and a high molecular part bound artificially to the N-terminus of the proteinaceous part. The proteinaceous part in the complex has the amino acid sequence of SEQ ID NO:2 where Xaa is a member selected from the group consisting of asparagine, alanine, arginine, serine, threonine, proline, methionine, and leucine; while the high molecular part in the complex is a homopolymer of polyethylene glycol, copolymer of polyethylene glycol, or a derivative thereof.

SUMMARY OF THE INVENTION

Under these circumstances, the present invention was made to provide a stable physiologically active substance which has TNF activity and improved dynamics in living bodies.

After energetic studies and screenings, the present inventors found that a physiologically active substance, which comprises both a proteinaceous part having TNF activity and a high molecular part bound artificially to the N-terminus of the proteinaceous part and which has a higher stability and a longer retention time in living bodies than intact TNF with no such a high molecular part. As a result, it was found that the blood level of TNF is kept at the desired level for a relatively long period of time even when administered at a lesser dose.

The present invention solved the above object by providing a physiologically active complex comprising both a proteinaceous part having TNF activity and a high molecular part bound artificially to the N-terminus of the proteinaceous part.

Also, the present invention solved the above object by providing an agent for susceptive diseases, comprising the above physiologically active complex as an effective ingredient.

DETAILED DESCRIPTION OF THE INVENTION

Now explaining the preferred embodiments according to the present invention, the physiologically active complex as referred to as in the present invention means a physiologically active complex comprising both a proteinaceous part having TNF activity and a high molecular part bound artificially to the N-terminus of the proteinaceous part. The proteinaceous part as a constituent of the physiologically active complex of the present invention can be obtained, for example, by the protein engineering technique: Among the amino acids which constitute TNF, amino acids such as lysine having a free amino group, excluding those which are positioned at the N-terminus of TNF, can be replaced with an amino acid with no free amino group, preferably, with any of asparagine, alanine, arginine, serine, threonine, proline, methionine, and leucine.

As it is well known, TNFs vary in amino acid sequences depending on their origins; human TNF consists of 157 amino acids represented by the amino acid sequence of SEQ ID NO:1. As concrete examples of the physiologically active complex to be incorporated into the later explained agent for susceptive diseases, those which comprise, as a proteinaceous part, the amino acid sequence of SEQ ID NO:2 where Xaa is a member selected from the group consisting of asparagine, alanine, arginine, serine, threonine, proline, methionine, and leucine; preferably, those which comprises the amino acid sequence of SEQ ID NO:3 as a proteinaceous part. The above proteins, where the 11th, 65th, 90th, 98th, 112th and 128th lysines in conventionally known human TNF are replaced with any of asparagine, alanine, arginine, serine, threonine, proline, methionine, and leucine, are different from the intact human TNF, however, they exert the same or higher cytotoxic action on tumors in general as compared with the human TNF.

As described above, these proteins can be obtained by the protein engineering technique in such a manner of replacing one or more amino acids as constituents of proteins with the desired amino acid(s). For example, libraries of DNAs encoding proteins, which the amino acids with a free amino group of TNF are replaced with a random amino acid(s), are obtained by subjecting to PCR reaction an oligonucleotide obtained by replacing with a NNS sequence a codon which encodes an amino acid having a free amino group corresponding to the DNA which encodes TNF; and then in the presence of the resulting PCR products the above DNA is subjected to PCR reaction to obtain a library of DNAs which encode proteins of modified TNFs which the amino acids with free amino groups in TNF are replaced with random amino acids. Thereafter, the DNAs in the library are allowed to express the proteins which they each encode by using the phage display method, etc., followed by applying conventional sequence analysis to the expressed proteins in combination with other techniques such as a solid phase enzyme immunoassay using anti-TNF antibodies, panning method using anti-TNF antibodies or TNF-receptor-proteins, and bioassay using target cells against TNF. Thus, DNAs encoding proteins, which the amino acids with free amino groups in TNF are replaced with amino acids with no free amino group except for the N-terminal amino acid of TNF, are obtained. To select the desired DNAs from the above DNAs, the phage display method is quite useful, and the combination use of the phage display method and one or more of the above techniques facilitates to smoothly and thoroughly select a series of proteins which the amino acids with free amino groups in TNF, except for the one at the N-terminus of TNF, are replaced with amino acids with no free amino group while retaining the desired TNF activity at a relatively high level.

The protein which constitutes the physiologically active complex of the present invention can be obtained in the desired amount by introducing the DNAs thus obtained directly or after amplified by PCR reaction into appropriate hosts such as Escherichia coli via plasmid vectors to transform the host cells, selecting a clone capable of producing the desired protein from the transformed cells, and culturing the selected clone to obtain the objective protein in the desired amount. To collect the produced protein from the culture of the transformant, conventional methods used in general for purifying proteins such as dialysis, salting out, filtration, concentration, centrifugation, separatory sedimentation, gel filtration chromatography, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography, chromatofocusing, gel electrophoresis, and isoelectrophoresis can be used. These methods are appropriately used in combination.

The physiologically active complex of the present invention can be obtained by allowing to artificially bind a high molecular substance to the N-terminus of the protein having TNF activity. The high molecular substances usable in the present invention are those which are substantially water-soluble and unharmful to living bodies, more particularly, non-proteinaceous substances with lesser fear of acting as antigens in living bodies. Referring to the molecular form of the high molecular weight substances, those in a straight- or branched-form can be used, however, those in a branched form are preferable. Examples of such high molecular substances include homopolymers of polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, or polypropylene glycol; copolymers of ethylene glycol and vinyl alcohol or propylene glycol; and synthetic high molecular substances thereof; and natural high molecular substances such as elsinan, dextran, hydroxyethyl cellulose, pullulan, and methyl cellulose. Among these, homopolymers and copolymers of polyethylene glycol and derivatives thereof are preferable because they are easily available in the form of a relatively homogeneous molecular-size-distribution. The molecular weight of the high molecular substances can be usually increased or decreased within the range of 500 50,000 daltons, preferably, 1,000 10,000 daltons as an average molecular weight; and when the high molecular substances are inhomogeneous in their molecular weight, they should preferably be fractionated by conventional methods such as separation sedimentation and gel filtration chromatography prior to reaction. Depending on the kind of the high molecular substances used and the final use of the physiologically active complex, the high molecular substances with a lower molecular weight outside the above range may not substantially improve the in vivo dynamics of the complex, while those with a higher molecular weight outside the above range may lower the water solubility of the complex, and these would hinder the safe use of the complex as a pharmaceutical.

To bind the above high molecular substances to the N-terminus of the protein with TNF activity, a high molecular substance, which has been activated with a reagent that specifically acts on free amino groups to form a covalent bonding, is allowed to react with the protein; or a high molecular substance and the protein are cross-linked using a polyfunctional reagent having a functional group which specifically reacts with free amino groups. Examples of the reaction method include those which are commonly used in the art, for example, the ether bonding method as disclosed in Japanese Patent Kokai No. 289,522/87 and the amido bonding method; among these, the amido bonding method is preferable with respect to the stability of the covalent bonding formed between the proteins and the high molecular substances.

Varying depending on the reaction method used, the ratio of a protein and a high molecular substance employed in the initiation of reaction is increased or decreased within the range of 1:0.1 to 1:100, preferably, 1:0.5 to 1:50 (=(protein):(high molecular substance)) by molar ratio. In general, when the ratio is below the above range, proteins become to easily bind each other; while when the ratio is over the above range, high molecular substances become to easily bind each other. In any case, since the ratio outside the above preferable range will lower the reaction rate of the protein and the high molecular substance and decrease the purification efficiency of the reaction product, the ratio should preferably be increased or decreased within the above-identified range. The reaction temperature, pH, and time are set so as not to inactivate and decompose proteins with TNF activity and to minimize undesirable side reactions: The temperature is set to 0 100.degree. C., preferably, 20 40.degree. C.; the pH is set to 0.1 12, preferably, 5 9; and the time is set to terminate the reaction within 0.1 50 hours, preferably, within 10 hours. The physiologically active complex thus obtained can be purified by similar methods as used in purifying the proteins with TNF activity, and optionally further treated with concentration, salting out, centrifugation, lyophilization, etc., into products in a liquid or solid form, depending on final use.

The physiologically active complex of the present invention is useful as a medicament for treating and/or preventing susceptive diseases. The term "susceptive diseases" as referred to as in the present invention means diseases in general which can be treated and/or prevented by the administration of the physiologically active complex with or without other medicaments. Examples of such diseases include solid tumors such as colonic cancer, rectal cancer, gastric cancer, thyroid carcinoma, cancer of the tongue, bladder carcinoma, choriocarcinoma, hepatoma, carcinoma uteri, cancer of pharynx, lung cancer, breast cancer, malignant melanoma, neuroblastoma, pyo-ovarium, testicular tumor, osteosarcoma, pancreatic cancer, hypernephroma, goiter, brain tumor, and mycosis fungoides; hematopoietic tumors such as leukemia and lymphoma; and others such as viral diseases, bacterial diseases, and immunopathies. Thus, the agents for susceptive diseases of the present invention have a variety of uses as antitumor agents, antiviral diseases, anti-infectives, and agents for immunopathies which are used in treating and/or preventing the above diseases.

Varying depending on the types and the symptoms of susceptive diseases to be treated, the agent for susceptive diseases of the present invention is prepared to facilitate the administration of at least 0.1 ng/kg body weight per shot, preferably, 1 1,000 ng/kg body weight per shot of the physiologically active complex while varying the dose level depending on administration route; and is prepared into an extract, elixir, lower airway inhalation, capsule, granule, ophthalmic sustained-release-drug, pill, ophthalmic ointment, cataplasm for tunica mucosa oris, suspension, emulsion, plaster, suppository, powder, tablet, syrup, dipping agent, decoction, injection, tincture, eye-drop, eardrop, nasal drop, troche, ointment, cataplasm, aromatic water, nasal nebulas, liniment, limonade, fluidextract, lotion, etc.

The agent for susceptive diseases of the present invention includes those in a dosage unit form which contain, for example, an amount equal to a single dose or an integral multiple dose up to four times of the single dose, or to a division of the single dose up to 1/40 time thereof; and which are in the form of a physically separated systematic agent suitable for administration. Examples of such are capsules, granules, pills, suppositories, powders, tablets, injections, and cataplasms.

In addition to the physiologically active complex of the present invention as the effective ingredient, appropriate agents such as excipients, ointment bases, dissolving agents, corrigents, flavors, colors, a-and emulsifiers, which are commonly used in preparing medicaments, can be freely incorporated into the agent for susceptive diseases of the present invention. Within the scope of the object of the present invention, the physiologically active complex of the present invention can be used together with, as another effective ingredient, one or more other agents, for example, external dermal agents such as external dermal sterilizing and pasteurizing agents, would protecting agents, and antiphlogistics; vitamin preparations such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, and vitamin K; calcium preparations; mineral preparations; saccharide preparations; organic acid preparations; protein and amino acid preparations; revitalizers such as organ preparations; chlorophyll preparations; cell activating preparations such as dye preparations; antitumor agents such as alkylating agents, antimetabolites, antitumor antibiotic preparations, and antitumor plant-ingredient preparations; allergic agents such as antihistamines; chemotherapeutics such as antituberculosis drugs, synthetic antimicrobial agents, and antiviral agents; and others such as hormone preparations, antibiotic preparations, and biological preparations.

The physiologically active complex of the present invention can be used in combination with the following antitumor agents as adjuvants to exert a synergistically high effect which could not be easily attained by their single use: Antitumor agents such as actinomycin D, aceglatone, irosfamide, ubenimex, etoposide, enocitabin, aclarubicin hydrochloride, idarubicin hydrochloride, irinotecan hydrochloride, epirubicin hydrochloride, gemcitabine hydrochloride, daunorubicin hydrochloride, doxorubicin hydrochloride, nitrogen mustard-N-oxide hydrochloride, nimustine hydrochloride, pirarubicin hydrochloride, bleomycin hydrochloride, procarbazine hydrochloride, mitoxantrone hydrochloride, carboquone, carboplatin, carmofur, tomoxifen citrate, toremifene, krestin, medroxyprogesterone acetate, cyclophosphamide, cisplatin, schizophyllan, cirarabine, citarabine ocfosfate, zinostantin stimalamer, vinonelbine ditartrate, sobuzoxane, dacarbazine, thiotepa, tegafur, tegafur uracil, tegafur gimesutat otastat potassium, doxifluridine, docetaxel hydrate, toretinoin, neocarzinostatin, nedaplatin, paclitaxel, bicalutamido, picibanyl, hydroxycarbamide, busulfan, fluorouracil, flutamido, pentostatin, porfimer sodium, mitomycin C, methotrexate, mercaptopurine, 6-mercaptopurine riboside, bleomycin sulfate, peplomycin sulfate, and lentinan. The above-mentioned combination use will reduce the dose of antitumor agents and effectively lower their side effects.

The agent for susceptive diseases of the present invention exerts therapeutic and/or prophylactic effects on the diseases independently of its oral or parenteral administration route. Depending on the types or symptoms of susceptive diseases to be treated, the agent is administered orally or parenterally such as intradermal, subcutaneous, intramuscular, intravenous, intranasal, rectal, and intraperitoneal routes, to a subject at a dose of 0.1 to 1,000 ng/day/kg body weight, preferably, 1 to 100 ng/day/kg body weight of the physiologically active complex, where the dose is optionally divided into several portions and the administration frequency is one to seven shots per week for one week to a half year. Since the physiologically active complex of the present invention is stable and hardly decomposed by protease in the blood and stays longer in living bodies than intact TNF by two times or more depending on its administration route, the dose can be significantly minimized when administered to a subject suffering from the same susceptive disease through the same administration route, resulting in a beneficial reduction of side effects inducible by the cytotoxicity of TNF against normal cells.

 

Claim 1 of 5 Claims

1. A physiologically active complex, which comprises a proteinaceous part having a N-terminus and a water soluble polymer bound covalently to the N-terminus of the proteinaceous part, wherein said proteinaceous part comprises the amino acid sequence of SEQ ID NO: 2 where Xaa is the same or a different member selected from the group consisting of asparagine, alanine, arginine, serine, threonine, proline, methionine, and leucine, and has a TNF-.alpha. activity.
 

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