WO2011037433A2

WO2011037433A2 - Use of a quinolone derivative containing 7-(4-aminomethyl-3-oxime)pyrrolidine group that is capable of inducing granulocyte colony stimulating factor for treatment of neutropenia and recovery of hematopoiesis

Info

Publication number: WO2011037433A2
Application number: PCT/KR2010/006559
Authority: WO
Inventors: Hee Dong Park; Ju Hyun Choi; Jung Gyu Park
Original assignee: Lg Life Sciences Ltd.
Priority date: 2009-09-28
Filing date: 2010-09-27
Publication date: 2011-03-31
Also published as: TW201121965A; UY32905A; KR20110034566A; WO2011037433A3; AR078453A1

Abstract

The present invention relates to a quinolone antibiotic compound of formula (1) capable of promoting the production of G-CSF in the body, and the medicament for treatment of neutropenia and early recovery of immunocytes, comprising the same as an active ingredient.

Description

USE OF A QUINOLONE DERIVATIVE CONTAINING 7-(4-AMINOMETHYL-3-OXIME)PYRROLIDINE GROUP THAT IS CAPABLE OF INDUCING GRANULOCYTE COLONY STIMULATING FACTOR FOR TREATMENT OF NEUTROPENIA AND RECOVERY OF HEMATOPOIESIS

The present invention relates to a medicament for the treatment of neutropenia and for the early recovery of immunocytes. The medicament comprises a therapeutically effective amount of a quinolone antibiotic compound containing 7-(4-aminomethyl-3-oxime) pyrrolidine group represented by the following formula (1), a pharmaceutically acceptable salt, solvate including hydrate, ester, isomer or prodrug thereof (hereinafter, these are referred to in brief as the compound of formula (1)), which are capable of promoting the production of G-CSF (Granulocyte Colony Stimulating Factor).

wherein,

R represents hydrogen, methyl or amino,

Q represents C-H, C-F, C-Cl, C-OH, C-CH₃, C-O-CH₃ or N,

R₁ represents hydrogen, cyclopropyl, methyl or ethyl, or phenyl which is substituted with one or more fluorine atoms,

R₂ represents one of the following a) to e):

a) hydrogen, straight or branched C₁-C₄-alkyl, cyclopropyl, cyclopropylmethyl, C₃-C₆-alkynyl, 2-haloethyl, methoxymethyl, methoxycarbonylmethyl, phenyl or allyl,

b) a group of the following formula

wherein X represents hydrogen, 2-, 3- or 4-fluoro, cyano, nitro, methoxy, C₁-C₄-alkyl, or 2,4-difluoro,

c) a group of the following formulas

d) a group of the following formulas

e) a group of the following formula

wherein n denotes 0 or 1, m denotes 0, 1 or 2, X represents methylene, O or N,

and

R₃ represents -CH₂NR₅R₆, wherein R₅ and R₆ independently of one another represent hydrogen or C₁-C₃-alkyl, or R₅ and R₆ together with the nitrogen atom to which they are attached can form a ring,

R₄represents hydrogen, or

R₃ and R₄ together with the carbon atom to which they are attached represent a structure

which is combined with the pyrrolidine ring in a spiro cycle form, wherein R₇ represents hydrogen or C₁-C₄-alkyl.

The compound of formula (1) is used as a medicament for treatment of neutropenia and for recovery of hematopoiesis in this invention, and it is specifically disclosed in EP 0 688 772 B1 and KR 10-0566346 B1. The preferably used quinolone antibiotic in this invention is gemifloxacin―namely, 7-(4-aminomethyl-3-methoxyiminopyrrolidine-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid, a pharmaceutically acceptable salt, solvate including hydrate, ester, isomer or prodrug thereof (hereinafter, these are referred to in brief as gemifloxacin). The above-defined quinolone antibiotics including gemifloxacin promote and increase the production of G-CSF in organisms, thereby promoting the proliferation and differentiation of progenitor cells of neutrophil-granulocyte lineage. The gemifloxacin and the quinolone antibiotics increase the number of neutrophils and promote activities of mature neutrophils, thus they can be advantageously used as an adjuvant in anti-cancer therapy for prevention, suppression, alleviation and treatment of neutropenia and as a medicament for elevating the level of immunocytes, particularly myeloid immunocytes suppressed by immune diseases and the treatment thereof. They can be used as substitutes for G-CSF and PEGylated G-CSF derivatives.

Hematopoiesis is a major system by which the life of an organism is maintained. Several lineages of immunocytes are produced via various routes starting with bone marrow. Hematopoiesis is regulated by specific glycoproteins, which are commonly called CSFs. CSFs are so named because they promote the formation of colonies of monocytes, granulocytes or other hematopoietic cells as growth factors when hematopoietic stem cells or hematopoietic progenitor cells are cultured on semi-solid media. These factors are classified and named depending on the kinds of colonies they promote. For example, G-CSF (Granulocyte-CSF) mainly promotes the formation of colonies of neutrophils, GM-CSF mainly promotes the formation of colonies of macrophages, and multi-CSF (IL-3) mainly promotes the formation of colonies of granulocytes, macrophages, megakaryocytes and eythrocyte.

G-CSF is a cytokine which directs the division and differentiation of bone marrow stem cells and leukocyte outside the bone marrow. It also promotes the division and differentiation of neutrophil-progenitor cells and activates the mature neutrophils, thereby enhancing their phagocytosis, chemotaxis, antibody-dependent cellular cytotoxicity, superoxide-producing activity and responsiveness to chemotactic factor (Metcalf, Blood (1986) 67:257; Yan, et al., Blood (1994) 84(3): 795-799; Bensinger, et al., Blood (1993) 81(11): 3158-3163; Roberts, et al., Expt'l Hematology (1994) 22: 1156-1163; Neben, et al., Blood (1993) 81(7): 1960-1967). At present, G-CSF is usually isolated from various cells or produced by genetic recombinant technology, and it is used effectively in various anti-cancer therapies and the treatment of refractory leukemia. In anti-cancer therapy, radiation therapy and high-dose chemotherapy are typically conducted, but these treatments destroy the hematopoietic cells of bone marrow in patients and thus induce a sharp decline of leukocytes including neutrophils, thereby weakening the immune system and increasing the risk of infection. Therefore, long-term and repetitive radiation therapy and high-dose chemotherapy are limited. Among the adverse effects of these anti-cancer therapies, neutropenia is the most common and is characterized by a reduction in circulating leukocytes, particularly neutrophils. Normal healthy adults have an absolute neutrophil count (ANC) in blood of 4400/mm³ and an average normal neutrophil counts in the range of 1800-7700/mm³. When the ANC is in the range of 1000/mm³―1500/mm³, it is classified as moderate neutropenia, and when the ANC is below 500/mm³, it is classified as severe neutropenia. Severe neutropenia predisposes patients to fatal infections.

When patients receive chemotherapy or auto- or allo-transplants, a certain period is needed for recovery of hematopoiesis. During the recovery period, patients have a lower level of circular neutrophils, and thus they are easily exposed to infections by fungi or bacteria, particularly opportunistic infections by normal microorganisms such as Aspergillus fumigatus or Pseudomonas aeruginosa, and which significantly affect the survival of patients.

Lopes et. al., reported G-CSF can activate patients’ immune system and thus works as an adjuvant anti-cancer therapy to be more efficiently conducted (Lopez et al., J. Immunol. (1983) 131(6): 2983-2988; Platzer et al., J. Exp. Med. (1985) 162: 1788-1801). Especially G-CSF is efficiently used as a medicament to improve neutropenia caused as an adverse effect by anti-cancer therapies (Morstyn, G., et al., Trends Pharmacol. Sci. (1989) 10: 154-159). G-CSF is reported to be engaged in the mobilization of hematopoietic stem cells or hematopoietic progenitor cells from bone marrow to peripheral blood (Good Review article Haylock et al., Blood (1997) 89: 2233-2258).

However, G-CSF has disadvantages in that it is very unstable, and thus it can remain in body for only a very short time. To overcome these problems, binding the G-CSF with biocompatible macromolecules such as polyethyleneglycol (PEG), polyvinylalcohol and polyvinylpyrrolidone have been suggested. These methods resolve the instability problem; however, they still use exogenous G-CSF such as a recombinant G-CSF, and may induce an undesirable immune response in the administered subject. In addition, more than 20% of patients develop a tolerance against genetic recombinant G-CSF or do not respond to G-CSF, and thus G-CSF has no the therapeutic effects on them (Sada et al., J. Fermentation Bioengineering (1991) 71: 137-139, Flomenberg et al., Blood (2005) 106: 1867-1874).

The inventors have studied the methods which can promote the production and activity of endogenous G-CSF in vivo, instead of treating patients with genetically recombinant G-CSF. Through significant studies, the inventors have discovered that a quinolone antibiotics containing 7-(4-aminomethyl-3-oxime) pyrrolidine group represented by the following formula (1), pharmaceutically acceptable salt, solvate including hydrate, ester, isomers or prodrug thereof can promote the production of G-CSF in the body, and thus they can be used as medicaments for treating of neutropenia. Moreover, they promote the proliferation and differentiation of hematopoietic stem cells in bone marrow and mobilize the hematopoietic stem cells or hematopoietic progenitor cells from bone marrow to peripheral blood.

wherein,

R represents hydrogen, methyl or amino,

Q represents C-H, C-F, C-Cl, C-OH, C-CH₃, C-O-CH₃ or N,

R₂ represents one of the following a) to e):

b) a group of the following formula

c) a group of the following formulas

d) a group of the following formulas

e) a group of the following formula

wherein n denotes 0 or 1, m denotes 0, 1 or 2, X represents methylene, O or N,

and

R₄ represents hydrogen, or

The inventors also found that said quinolone antibiotics increase the number of hematopoietic stem cells in bone marrow, thereby being effective in the early recovery of immunocyte population. In the present invention, particularly gemifloxacin, a pharmaceutically acceptable salt, solvate including hydrate, ester, isomer or prodrug thereof are preferred among the compound of formula (1).

The present invention uses low-molecule synthetic compounds instead of conventional genetically recombinant high-molecule G-CSF. It thereby has outstanding advantages compared with conventional methods in respect to the administration route of drug and treatment cost. Conventional genetically recombinant G-CSF must be administered via parenteral routes such as IV, SC, continuous SC infusion or continuous IV infusion, and there are significant limits in that the patient cannot easily take it outside the hospital. Meanwhile, quinolone antibiotics according to the present invention can be administered orally so that the patient can take the drug easily without having to come to the hospital. In addition, genetically recombinant G-CSF is very expensive, and even in developed countries many patient cannot receive treatment due to its high cost. However, the present invention uses the low-cost synthetic compound so that more patients can enjoy the benefit of equivalent therapeutic effect of genetically recombinant G-CSF. Besides the above-stated merits, the compound of formula (1) is very industrially advantageous because it shows a effect identical to that of G-CSF in that when the compound of formula (1) according to present invention is treated in the artificially induced neutropenia model with an anti-cancer agent, the starting time at which the ANC is recovered from the completely diminished state to a normal level and the period required in arriving at the normal level?namely, the duration of neutropenia are almost the same compared with those when G-CSF was treated.

The object of the present invention is to provide an anti-cancer adjuvant, specifically a substitute for G-CSF, a medicament for neutropenia, a medicament for early recovery for immunocytes and a medicament for inducing the mobilization of hematopoietic stem cells or hematopoietic progenitor cells from bone marrow to peripheral blood, comprising a therapeutically effective amount of a quinolone derivative antibiotic containing 7-(4-aminemethyl-3-oxime) pyrrolidine group of formula (1), or a pharmaceutically acceptable salt, solvate including hydrate, ester, isomer or prodrug thereof.

The preferred quinolone derivative compound according to the present invention is gemifloxacin.

Another object of the present invention is to provide a method for promoting the production of G-CSF in a mammal, comprising the administration of a therapeutically effective amount of a quinolone derivative antibiotic containing 7-(4-aminemethyl-3-oxime) pyrrolidine group of formula (1), or a pharmaceutically acceptable salt, solvate including hydrate, ester, isomer or prodrug thereof to the mammal.

Another object of the present invention is to provide a method for treating conditions which are responsive to treatment with G-CSF, comprising the administration of a therapeutically effective amount of a quinolone derivative antibiotic containing 7-(4-aminemethyl-3-oxime) pyrrolidine group of formula (1), a pharmaceutically acceptable salt, solvate including hydrate, ester, isomer or prodrug thereof to the mammal. The conditions which are responsive to treatment with G-CSF include neutropenia, reduced neutrophil mobilization, reduced peripheral blood progenitor cell mobilization, sepsis, severe chronic neutropenia, bone marrow aplasia or myelosuppression, and acquired immune deficiency syndrome.

Another object of the present invention is to provide a method for increasing the immunocytes, particularly myelogenous immunocytes, and collecting hematopoietic stem cells or hematopoietic progenitor cells by inducing their mobilization from bone marrow to peripheral blood, comprising administration of a therapeutically effective amount of a quinolone derivative antibiotic containing 7-(4-aminemethyl-3-oxime) pyrrolidine group of the formula (1), or pharmaceutically acceptable salt, solvates including hydrates, ester, isomer or prodrug thereof to the mammal.

The particularly preferred quinolone derivative compound according to the present invention is gemifloxacin, and the therapeutic methods include those applied to mammals but not to humans.

Hereinafter, this invention is explained in detail focusing on gemifloxacin, one of the quinolone derivative antibiotics of the present invention; however, the scope of the present invention is not limited to gemifloxacin.

The compound of formula (1) was shown by the inventors to be capable of promoting the production of G-CSF in the body and can be used as a substitute for genetically recombinant G-CSF which has been used as an adjuvant in anti-cancer therapy. Thus, the compound of formula (1) can be used as an adjuvant in anti-cancer therapy. More specifically, in patients receiving anti-cancer chemotherapy or radiotherapy, the compound of formula (1) promotes the production of G-CSF, induces the division and differentiation of hematopoietic stem cells in bone marrow, the mobilization of hematopoietic stem cells or hematopoietic progenitor cells to peripheral blood and proliferation of myelogenous cells in peripheral blood and the spleen. Particularly, gemifloxacin was found by the inventors to promote the division of myelogenous cells systemically and the division of hematopoietic stem cells in bone marrow. Thus, the present invention comprises a method using the compound of formula (1) as a substitute for G-CSF used as an adjuvant in anti-cancer therapy such as anti-cancer chemotherapy or radiotherapy. Particularly, the invention comprises a method increasing the production of G-CSF selectively in the body, more specifically wherein the production of GM-CSF or IL-2 are not promoted, or even suppressed. Moreover, the present invention comprises a method promoting the production of G-CSF, comprising administration of the compound of formula (1) to the mammal which develops a tolerance to exogenous G-CSF, or does not respond to it for other reasons.

In the present invention, exogenous G-CSF includes the isolated G-CSF from various cells, genetically recombinant G-CSF obtained from prokaryotic cells or eukaryotic cells, and various G-CSF variants including glycosylated G-CSF. The commercially available lenograstim (Granocyte^®), filgrastim (Neupogen^®) and Pegylated filgrastim (Neulasta^®) are included.

The compound of formula (1) according to the present invention can be administered to a mammal before, during or after chemotherapy or radiation therapy. When administered before chemotherapy or radiation therapy, it can be administered at least 1 day before, preferably 2-3 days before, more preferably 4 days before. When administered after chemotherapy or radiation therapy, it can be administered on the day when chemotherapy or radiation therapy is finished, within 1 day, or within 2-3 days or at least within 4 days from the day when chemotherapy or radiation therapy is finished. When chemotherapy or radiotherapy is performed for more than 1 cycle, the compound of formula (1) can be administered as stated above after completion of the respective cycle.

The anti-cancer agents which can be used for the above anti-cancer chemotherapy include, but are not limited to, the following compounds depending on their action mechanisms, the chemical structure and the source:

Alkylating agents

The alkylating agents include, but are not limited to, mustard gas derivatives such as cyclophosphamide, chlorambucil, ifosfamide, mechlorethamine and melphalan; ethyleneimines such as hexamethylmelamine and thiotepa; alkyl sulfonates such as busulfan; hydrazine and triazenes such as altretamine, dacarbazine, procarbazine and temozolomide; nitrosoureas such as carmustine, lomustine, and streptozocin; and inorganic metal complex agents (e.g., metal complexes of platinum, palladium or ruthenium), such as cisplatin, carboplatin and oxaliplatin. The alkylating agents are very strong chemotherapy agents and are used to treat most types of cancer including hematologic malignancies and solid cancer. Unlike most types of chemotherapeutic agents, nitrosoureas can cross the blood-brain barrier and therefore may be particularly useful in treatment of brain cancers.

Plant Alkaloids

The plant alkaloids are a class of chemotherapeutic agents isolated from various plants. Taxanes (derived from the bark of certain yew trees) and the vinca alkaloids (derived from periwinkle plants) are antimicrotubule agents. Camptothecan analogs (isolated from the Camptotheca acuminata tree) and podophyllotoxin (derived from mandrake plants) are topiosomerase inhibitors. The plant alkaloids are cell-cycle specific and attack the cells during various phases of cell division. Plant alkaloids used in chemotherapy include, but are not limited to, antimicrotubule agents such as taxanes (for example, docetaxel and paclitaxel) and vinca alkaloids (for example, vinblastine, vincristine, and vinorelbine); topiosomerase inhibitors such as camptothecan analogs (for example, irinotecan and topotecan) and podophyllotoxin (etoposide and tenisopide).

Antitumor Antibiotics

Antitumor antibiotics are a class of chemotherapeutic agents produced by various species of streptomyces. Mechanisms of action of antitumor antibiotics include inhibition of topoisomerases and/or generation of free oxygen radicals which result in DNA strand breaks and inhibition of DNA synthesis. Antitumor antibiotics used in chemotherapy include, but are not limited to, anthracyclines such as daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone; cromomycin such as dactinomycin and plicamycin; and other antitumor antibiotics such as bleomycin and mitomycin.

Antimetabolites

Antimetabolites are inhibitors (antagonists) of molecules involved in cellular metabolism. Antimetabolites are generally cell-cycle specific and are classified according to the substances with which they interfere. Antimetabolites used in chemotherapy include, but are not limited to, folic acid antagonists such as methotrexate; pyrimidine antagonists such as capecitabine, cytarabine, 5-fluorouracil (5-FU), foxuridine, and gemcitabine; purine antagonists such as 6-mercaptopurine and 6-thioguanine; adenosine deaminase inhibitors such as cladribine, fludarabine, nelarabine and pentostatin; and ribonucleotide reductase inhibitors such as hydroxyurea.

Topoisomerase Inhibitors

Topoisomerase inhibitors are a class of molecules which interfere with the action of the topoisomerase enzymes topoisomerase I and II, and inhibit DNA replication. Topoisomerase inhibitors used in chemotherapy include, but are not limited to, topoisomerase I inhibitors such as ironotecan and topotecan; and topoisomerase II inhibitors such as amsacrine, etoposide, etoposide phosphate, and teniposide.

Miscellaneous Chemotherapeutic Agents

Additional types of compounds used in chemotherapy include, but are not limited to, andrenolytic agents such as the adrenocortical steroid inhibitor mitotane; enzymes such as asparaginase and pegaspargase; retinoids such as bexarotene, isotretinoin and tretinoin (all-trans-retinoic acid).

The mammals that receive chemotherapy or radiation therapy generally show the moderate or severe neutropenia. The present invention provides a method of prevention, inhibition, alleviation, or treatment of neutropenia, particularly acceleration of recovery of neutrophils, and reduction of the degree and duration of neutropenia, comprising the administration of a therapeutically effective amount of compound of formula (1) to mammals. For these methods, the compound of formula (1) may be administered according to the same method and time interval used for stimulating the production of G-CSF. When gemifloxacin as a representative compound of formula (1) was administered to the patients with reduced absolute neutrophil count (ANC) after receiving chemotherapy or radiotherapy, the time required to bring the number of neutrophils back to the normal level and the duration of neutropenia were identical to those when treating with a therapeutically effective amount of G-CSF. In addition, the present invention includes a method comprising the administration of a therapeutically effective amount of compounds of formula (1) to a patient who received chemotheraphy or radiotherapy before fever developed.

The compound of formula (1) is used as a medicament for neutropenia according to the present invention; it can be used advantageously for the following:

(1) treatment of febrile neutropenia induced in a cancer patient who received myelosuppressive chemotherapy.

(2) treatment of neutropenia induced in a cancer patient who received radiotherapy.

(3) treatment of an adult acute myeloid leukemia patient who received chemotherapy.

(4) treatment of neutropenia induced in a cancer patient who received non-myelosuppressive chemotherapy such as ganciclivir, aciclovir, famciclovir, vidarabine, cytarabine, idoxuridine, trifluridine, edoxudine, brivudine, AZT and the like.

(5) treatment of neutropenia due to infection.

(6) treatment of severe chronic neutropenia including idiopathic, congenital or cyclothymic neutropenia.

Treatment of neutropenia in this invention includes prevention, inhibition, alleviation and treatment of neutropenia. In addition, the compound of formula (1) promotes the production of G-CSF, thereby increasing the number of neutrophils and treating neutropenia. However, an increase of the glucose level is not associated with this process.

In addition, the compound of formula (1) can be used for early recovery of immunocytes in patients with loss of hematopoiesis due to chemotherapy and/or radiotherapy. For example, the survival rates of the gemifloxacin-treated groups were very high compared with those of the control group in the survival experiments who were exposed to lethal radiation (Fig. 10).

The compound of formula (1) is used as a medicament for early recovery of immunocytes according to the present invention, and this can be used advantageously for the following:

(1) early recovery of immunocytes in an auto or allogenic bone marrow transplant patient who received myeloblative chemotherapy and/or myeloablative radiotherapy, and

(2) immune-system enhancement in a patient with a metastatic cancer who received surgery or chemotherapy, thereby the spread of residual cancer can be prevented.

The compound of formula (1) can also be used

(1) to mobilize hematopoietic stem cells from bone marrow to peripheral blood in a bone marrow transplant donor, and thereby collecting them,

(2) to induce the proliferation of peripheral blood progenitor cells for collection and

(3) to treat primary bone marrow failure.

Lastly, the compound of formula (1) can be used as a medicament for transient hematopoietic reconstruction in a mammal host, and for this purpose the following (a) or (b) methods can be applied:

(a) a method comprising adding a compound of formula (1) to a medium containing a mixture of cytokines and growth factor for the culturing of cells group including hematopoietic stem cells, and thereby increasing myelogenous progenitor cells in the cells group, and

(b) a method comprising adding a compound of formula (1) to a pharmaceutically acceptable medium suitable for administering to a mammal host, and resuspending myelogenous progenitor cells.

The compound of formula 1 according to the present invention can be used as a form of free compound, or as a form of a pharmaceutically acceptable salt, solvate including hydrate, ester, isomer or prodrug form. The term “pharmaceutically acceptable salt” means a salt form of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound. Such pharmaceutically acceptable salts include acid-addition salts formed by acid having a pharmaceutically acceptable anion to form a non-toxic acid addition salt including, for example, inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, and the like; organic carboxylic acid such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, and the like; sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. In addition, the pharmaceutically acceptable salts include salts with alkaline metal or alkaline earth metal such as lithium, sodium, potassium, calcium and magnesium, and the like; salts with amino acids such as lysine, arginine and guanidine, and the like; and salts with organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline and triethylamine. The compound of formula (1) can be converted to its salts by conventional methods known in the art.

The hydrates of the compounds of formula (1) are preferred because they are very stable during the drying process associated in pharmaceutical manufacturing, and are hardly wet in the wide range of relative humidity and do not show hysteresis. For example, gemifloxacin is preferably used in its 1.5 or 3 hydrates. The hydrates of gemifloxacin are specifically disclosed in WO 98/42705, the contents of which are hereby incorporated by reference in their entirety.

The hydrates according to this invention include that of the pharmaceutically acceptable salt of formula (1) compound as well as that of the base form of formula (1) compound.

The compound of formula (1) has various pharmacological activities as stated earlier, and it may be formulated for convenient administration as a medicine or a veterinary medicine. Formulation can be accomplished according to the conventional formulation technologies and method known in the art, relating other adjuvant in anti-cancer therapy. Therefore, the present invention includes a pharmaceutical composition comprising the compound of formula (1) with a pharmaceutically acceptable carrier or an excipient.

The composition comprising the compound of formula (1) as active ingredient may be formulated for administration by any suitable route, such as oral, parenteral or topical application. The compositions may be in the form of tablets, capsules, powders, granules, lozenges, creams or liquid preparations, such as oral or sterile parenteral solutions or suspensions. Tablets and capsules for oral administration may be formulated in unit dose form and may contain conventional excipients such as binding agents, for example, hydroxypropyl methyl cellulose, hydroxypropyl celullose, syrup acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrollidone; fillers, for example, microcrystalline cellulose, lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example, magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example sodium starch glycolate, cross-linked polyvinyl pyrolidone or potato starch; or acceptable wetting agents such as sodium lauryl sulfate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or an other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example, sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, caboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example, lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, oily esters, glycerine, propylene glycol, or ethyl alcohol; preservatives, for example, methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired conventional flavoring or coloring agents.

For parenteral administration, fluid unit dosage forms are prepared utilizing the compound and a sterile vehicle, water being preferred, and they are administered, for example, via IV or IV infusion. The compounds of formula (1) can be either suspended or dissolved in the vehicle, depending on the vehicle and concentration used. In preparing solutions, the active compounds can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampoule and sealing. Advantageously, agents such as local anesthetic, preservative and buffering agents can be dissolved in the vehicle. To enhance stability, the composition can be lyophilized and the dry-lyophilized powder sealed in a vial, and an accompanying vial of water for injection may be supplied to reconstitute the powder prior to use. Parenteral suspensions are prepared in substantially the same manner except that the compounds of formula (1) are suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration. The compounds of formula (1) can be sterilized by exposure to ethylene oxide before being suspended in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compounds of formula (1).

The compounds of formula (1) may also be formulated as an intramammary composition for veterinary use.

The composition according to this invention may contain from 0.1 to 100% by weight, preferably from 10 to 99.5% by weight, more preferably from 50 to 99.5% by weight of the active ingredient measured as the free base, depending on the method of administration. Where the compositions comprise dosage units, each unit will preferably contain from 50 to 1500 mg of the active ingredient measured as the free base. The dosage as employed for adult human treatment will range from 160 to 800 mg per day for an average adult patient (body weight 70 kg), preferably from 200 to 600 mg per day, more preferably from 300 to 500 mg per day, most preferably about 320 mg per day, depending on the route and frequency of administration. The daily dosage is suitably given by administering the active ingredient once or several times in a 24-hour period e.g., up to 400 mg may be administered once a day. In practice, the dosage and frequency of administration which will be most suitable for an individual patient will vary with the age, weight and response of the patients, and there will be occasions when the physician will choose a higher or lower dosage and a different frequency of administration. Such dosage regimens are within the scope of this invention.

Conventionally used G-CSF as an adjuvant in anti-cancer therapy has disadvantages in that it is very unstable, and thus it can remain in the body only a very short time. To overcome these problems, methods comprising binding biocompatible macromolecules to G-CSF are generally suggested. According to the present invention, the quinolone antibiotic compound of formula (1) promotes stable production of G-CSF in the body, thus it is efficiently used for all diseases or disorders that can be treated or alleviated using G-CSF.

In addition, G-CSF is an expensive bio-medicine obtained from cell cultures, and the manufacturing cost is high. The substitution for G-CSF with a low-cost synthetic drug is very meaningful in that it provides the benefit of low cost and good quality of treatment.

The present invention is described in more detail by the following Examples, but the scope of the present invention is not limited thereby in any manner.

Figure 1 shows the number change of mouse peripheral blood cells by treatment with gemifloxacin;

Figure 2 shows the number change of mouse spleen cells by treatment with gemifloxacin;

Figure 3 shows the proliferation of myelogenous cells in mouse peripheral blood and spleen by treatment with gemifloxacin analyzed with flow cytometry;

Figure 4 shows the changes of various cytokines in mouse serum by treatment with gemifloxacin;

Figure 5 shows the change in the frequency of mouse bone marrow hematopoietic stem cells by treatment with gemifloxacin analyzed with flow cytometry;

Figure 6 represents the photography of histochemical analysis showing cell proliferation in mouse splenic red pulp zone by treatment with gemifloxacin (a), and the photography of histochemical analysis showing proliferation of myelogenous cells in mouse bone marrow by treatment with gemifloxacin (b);

Figure 7 shows the therapeutic efficacy of gemifloxacin for neutropenia caused by an anti-cancer agent, 5-FU, analyzed with flow cytometry;

Figure 8 shows the therapeutic efficacy of gemifloxacin for neutropenia caused by an anti-cancer agent, 5-FU, represented by the number change of myelogenous cells;

Figure 9 shows the effect of Compound A for myelogenous cells in peripheral blood, represented by the number change of neutrophils; and

Figure 10 shows the lethality of the radiation-only group treated at the lethal radiation dose and the gemifloxacin-treated group calculated by Kaplan-Meier survival analysis.

Experiment animals

Male and female C57BL/6 mice aged 6-8 weeks (body weight 20~27 g) were purchased from the Orient Bio Company and used in the experiments. All mice were kept in a standard breed environment in which temperature was controlled at 22 ± 2℃, humidity at 55-60%, and brightness and darkness were maintained for 12 hours, respectively. Solid feed and water were provided freely. Mice were injected intraperitoneally (i.p.) with PBS, or gemifloxacin at two different doses (50 mg/kg and 100 mg/kg), respectively, for 4 days, and on day 5 immunocytes were stained and serum cytokines were investigated.

Flow cytometry

The following experiments were conducted for fractional investigation of the LSK (lineage-, Sca-1+, c-Kit+) cells containing large amounts of mice hematopoietic stem cells. To obtain myelocytes, mice were anesthetized to euthanasia with 3% of halothane, and cervical spines were disarticulated. The myelocytes were isolated by washing out the bone marrow from aseptically extracted tibia, femur and ischium using syringe. For the isolated myelocytes, the antibody-staining was performed against CD4, CD8, B220, Gr-1 (Ly-6G), Mac-1 (CD11b) and Ter119 labeled antigens corresponding to lineage cells and against Sca-1, c-Kit antigens, and then the LSK cells were investigated.

To investigate the granulocytes by flow cytometry from bone marrow, spleen, and peripheral blood of mice, the isolated cells from respective organs were stained with antibodies against Gr-1 (Ly-6G) and Mac-1 (CD11b) labeled antigens of mouse.

The fractional investigation of cells such as granulocytes, lymphocytes and the like via labeled antigens was performed by the collection with FACSCalibur system and analysis with Flowjo. The numbers of total lymphocytes, granulocytes, red blood cells and platelets were counted by an animal blood counter.

Example 1

Gemifloxacin was prepared according to the method described in Example 180 of EP 688 772.

After treating the mice with 50 mg/kg or 100 mg/kg of gemifloxacin via i.p. for 4 days, the numbers of lymphocytes (CD4+, CD8+, B220+) and neutrophils (Gr-1+ CD11b+) in the peripheral blood or spleen cells of the mice were compared with those of negative control mice (PBS-treated mice). The count changes of lymphocytes and granulocytes in the peripheral blood or spleen after gemifloxacin treatment were measured by flow cytometry, the results are shown in Fig. 1 and 2. The number of neutrophils (Gr-1+ CD11b+) was increased dose-dependently in the gemifloxacin-treated group, which was verified with flow cytometry (Fig. 3). The results confirmed that the treatment of gemifloxacin in normal mice increases the number of neutrophils in peripheral blood or the spleen.

Example 2

For the mice group of Example 1, the change of cytokines in the blood was investigated with a protein array kit. The results, as shown in Fig. 4, show that the treatment with gemifloxacin induces an increases of G-CSF, MCP-5 (monocyte chemotactic factor) and TREM-1 (which is related with the activity of myelocytes), and a decrease of SDF-1 (the factor engaged in the mobilization of hematopoietic stem cells from bone marrow to peripheral blood). The results show that gemifloxacin increases the number of G-CSF in vivo and thereby plays a role in the recovery of hematopoiesis and increase of neutrophils.

Example 3

To confirm that gemifloxacin increases G-CSF in vivo and thereby promotes the self-replication of hematopoietic stem cells, the following experiment was conducted for fractional investigation of LSK (lineage-, Sca-1+, c-Kit+) cells containing a large amount of mouse hematopoietic stem cells. To obtain myelocytes, mice were anesthetized to euthanasia with 3% of halothane, and cervical spines were disarticulated. The myelocytes were isolated by washing out the bone marrow from aseptically extracted tibia, femur and ischium using syringe. For the isolated myelocytes, antibody-dying were performed for CD4-, CD8-, B220-, Gr-1 (Ly-6G)-, Mac-1 (CD11b)- and Ter119-labelled antigens corresponding to lineage cells and Sca-1, c-Kit antigens. The cells representing positive for Sca-1, c-Kit and negative for Lineage were investigated by flow cytometry. As shown in Fig. 5, the frequency of total hematopoietic stem cells of the gemifloxacin-treated group was increased about 2-fold (from 0.38% to 0.62% or 0.63%) compared with that of the negative control group (PBS-treated mice). This fact is further supported by the results shown in Fig. 6, which show the proliferation of myelocytes induced by the treatment of gemifloxacin in spleen (a) and bone marrow (b) via H&E (hematoxylin and eosin) tissue staining.

Example 4-1

The effect of gemifloxacin treatment on the proliferation of myelocytes in peripheral blood was compared with that of ciprofloxacin, the positive control drug. The myelocytes were analyzed by flow cytometry. 150 mg/kg of 5-FU, an anti-cancer drug, was administered to mice via i.p., and after 24 hours, gemifloxacin or PBS was administered in the way described in Example 1. Ciprofloxacin was given 2 times a day via i.p. in a dose of 25 mg/kg. As shown in Fig. 7, the neutrophils represented by Ly6G (Gr-1+) and CD11b+ were not increased in the peripheral blood of the negative control group (PBS-treated mice). The neutrophils in the gemifloxacin-treated group were increased more than 4-fold compared with that of negative control group (3.04% vs. 13.90%), whereas the neutrophils in the ciprofloxacin-treated group did not increase. This result obtained by flow cytometry is further supported by the actual numbers of neutrophils in shown in Fig. 8.

Example 4-2

For the compound of the following structure (hereinafter, referred to as Compound A), the effect on the proliferation of myelocytes in peripheral blood was compared with that of G-CSF, the positive control drug.

Compound A was prepared according to the method described in Example 180 of EP 688 772, except that 7-chloro-6-fluoro-1-methyl-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid was used instead of 1-cyclopropyl-7-chloro-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid as an intermediate. 7-chloro-6-fluoro-1-methyl-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid was obtained according to the method disclosed in EP 1678174 A1 using methyl amine instead of cyclopropyl amine.

Compound A (50 mg/kg), PBS and G-CSF (125 μg/kg) were given, respectively, to mice once intravenously, and after 24 hours the myelocytes in the peripheral blood were analyzed by flow cytometry. As shown in Fig. 9, the neutrophils in the Compound A-treated group were increased similar to those in the G-CSF treatment group.

Example 5

To investigate the protective effect of gemifloxacin against radiation hazards due to lethal radiation dose, mice were divided to the radiation-only group (6 mice) and gemifloxacin-treated group after radiation (6 mice). The mice were put into a radiation device and radiated 10 Gy single-radiation doses on the back side at the radiation rate of 3 Gy/min using a 6 MV linear accelerator (Siemens, PA, USA). Gemifloxacin was dissolved in saline solution, and 0.2 ml of the solution was administered to mice for 7 days via i.p. at the dose of 50 mg/kg after 24 hours of radiation. From the next day after the first mouse in the radiation-only group died, administration of gemifloxacin in the gemifloxacin-treated group was stopped and the survival rates of both groups were investigated. The lethality of the radiation-only group treated at the lethal radiation dose and the gemifloxacin-treated group was calculated by Kaplan-Meier survival analysis, and the results are shown in Fig. 10. The results show that gemifloxacin treatment protects the body from radiation hazards induced by lethal radiation, such as neutropenia, myeloablation, etc. This effect is believed to be attributed to the action of gemifloxacin which induces the early recovery of hematopoietic cells through the increase of neutrophils and hematopoietic stem cells, and thereby prevents possible infection and the like.

Claims

A medicament for the treatment of neutropenia, comprising a therapeutically effective amount of the compound represented by the following formula (1), a pharmaceutically acceptable salt, solvate including hydrate, ester, isomer or prodrug thereof as an active ingredient:

wherein,

R represents hydrogen, methyl or amino,

Q represents C-H, C-F, C-Cl, C-OH, C-CH₃, C-O-CH₃ or N,

R₁ represents hydrogen, cyclopropyl, methyl or ethyl, or phenyl which is substituted with one or more fluorine atoms,

R₂ represents one of the following a) to e):

a) hydrogen, straight or branched C₁-C₄-alkyl, cyclopropyl, cyclopropylmethyl, C₃-C₆-alkynyl, 2-haloethyl, methoxymethyl, methoxycarbonylmethyl, phenyl or allyl,

b) a group of the following formula

wherein X represents hydrogen, 2-, 3- or 4-fluoro, cyano, nitro, methoxy, C₁-C₄-alkyl, or 2,4-difluoro,

c) a group of the following formulas

d) a group of the following formulas

e) a group of the following formula

wherein n denotes 0 or 1, m denotes 0, 1 or 2, X represents methylene, O or N,

and

R₃ represents -CH₂NR₅R₆, wherein R₅ and R₆ independently of one another represent hydrogen or C₁-C₃-alkyl, or R₅ and R₆ together with the nitrogen atom to which they are attached can form a ring,

R₄ represents hydrogen, or

R₃ and R₄ together with the carbon atom to which they are attached represent a structure
which is combined with the pyrrolidine ring in a spiro cycle form, wherein R₇ represents hydrogen or C₁-C₄-alkyl.
The medicament according to claim 1, wherein the compound of formula (1) is gemifloxacin.
The medicament according to claim 1 or 2, wherein the neutropenia is febrile neutropenia induced in a cancer patient who received myelosuppressive chemotherapy.
The medicament according to claim 1 or 2, wherein neutropenia is induced in a cancer patient who received radiotherapy.
The medicament according to claim 1 or 2, wherein neutropenia is induced in an adult acute myeloid leukemia patient who received chemotherapy.
The medicament according to claim 1 or 2, wherein neutropenia is induced in a cancer patient who received non-chemotherapeutic myelosuppressive therapy.
The medicament according to claim 6, wherein the non-chemotherapeutic myelosuppressive therapy is selected from the group consisting of ganciclovir, aciclovir, valaciclovir, valganciclovir, penciclovir, famciclovir, vidarabine, cytarabine, idoxuridine, trifluridine, edoxudine and brivudine.
The medicament according to claim 1 or 2, wherein neutropenia is caused by infection.
The medicament according to claim 1 or 2, wherein the neutropenia is severe chronic neutropenia.
The medicament according to claim 9, wherein the severe chronic neutropenia is idiopathic, congenital or cyclic neutropenia.
A medicament for early recovery of immunocytes, comprising a therapeutically effective amount of the compound of formula (1) defined in claim 1, a pharmaceutically acceptable salt, solvate including hydrate, ester, isomer or prodrug thereof as an active ingredient.
The medicament according to claim 11, wherein it is for early recovery of immunocytes in an auto or allogenic bone marrow transplant patient who received myeloablative chemotherapy or myeloablative radiotherapy.
The medicament according to claim 11, wherein it is for immune enhancement in a patient with a metastatic cancer who received surgery or chemotherapy and thereby the spread of residual cancer is prevented.
A medicament for mobilization of cells from bone marrow to peripheral blood, comprising a therapeutically effective amount of the compound of formula (1) defined in claim 1, a pharmaceutically acceptable salt, solvate including hydrate, ester, isomer or prodrug thereof as an active ingredient.
The medicament according to claim 11, wherein it is for collection of hematopoietic stem cells of a bone marrow transplant donor.
A medicament for collection of cells, comprising a therapeutically effective amount of the compound of formula (1) defined in claim 1, a pharmaceutically acceptable salt, solvate including hydrate, ester, isomer or prodrug thereof as an active ingredient.
The medicament according to claim 16, wherein the collection of cells is achieved by the induction of the proliferation of peripheral blood progenitor cells.
A medicament for treatment of primary bone marrow failure, comprising a therapeutically effective amount of the compound of formula (1) defined in claim 1, a pharmaceutically acceptable salt, solvate including hydrate, ester, isomer or prodrug thereof as an active ingredient.
A medicament for a transient hematopoietic reconstruction in a mammal host, comprising a therapeutically effective amount of the compound of formula (1) defined in claim 1, a pharmaceutically acceptable salt, solvate including hydrate, ester, isomer or prodrug thereof as an active ingredient.
The medicament according to claim 11, wherein the hematopoietic reconstruction is achieved according to the following (a) or (b) method:

(a) a method comprising adding a compound of formula (1) to a medium containing a mixture of cytokines and growth factors for the culturing of cells group including hematopoietic stem cells, and therby increasing myelogenous progenitor cells in the cells group, and

(b) a method comprising adding a compound of formula (1) to a pharmaceutically acceptable medium suitable for administering to a mammal host, and resuspending myelogenous progenitor cells.
The medicament according to any one of claims 11, 14, 16, 18 and 19, wherein the compound of formula (1) is gemifloxacin.
A method of treating neutropenia or achieving the early recovery of immunocytes in a mammal, which comprises administering a therapeutically effective amount of the compound of formula (1) defined in claim 1, a pharmaceutically acceptable salt, solvate including hydrate, ester, isomer or prodrug thereof to a mammal.
A method of preventing, suppressing, alleviating or treating neutropenia caused by chemotherapy or radiotherapy, which comprises administering the compound of formula (1) defined in claim 1, a pharmaceutically acceptable salt, solvate including hydrates, ester, isomer or prodrug thereof in an amount effective in preventing, suppressing, alleviating or treating of neutropenia to a mammal that received the chemotherapy or radiotherapy.
The method according to claim 22 or 23, wherein the compound of formula (1) is gemifloxacin.
Use of a compound of formula (1) according to Claim 1 to in the manufacture a medicament for the treatment of neutropenia, preferably neutropenia caused by chemotherapy or radiotherapy, or for early recovery of immunocytes.