CN114073772A

CN114073772A - Use of an iron chelator for the treatment or prevention of polyoma viral infections

Info

Publication number: CN114073772A
Application number: CN202010831145.1A
Authority: CN
Inventors: 朱同玉; 巫国谊; 石业静; 孙佳佳; 侯玉敏; 吴楠楠; 胡春兰
Original assignee: SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Current assignee: SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Priority date: 2020-08-18
Filing date: 2020-08-18
Publication date: 2022-02-22
Also published as: WO2022037594A1

Abstract

The invention relates to the technical field of biological medicines, in particular to application of an iron chelator in preparing a medicine for treating or preventing polyoma virus infection.

Description

Use of an iron chelator for the treatment or prevention of polyoma viral infections

Technical Field

The invention relates to the field of biomedicine, in particular to application of an iron chelator in preparing a medicament for treating or preventing polyoma virus infection or inhibiting polyoma virus.

Background

Polyomaviruses are small, non-enveloped, double-stranded DNA viruses that exhibit restricted species and cell type specificity. A variety of polyomaviruses have been found to have oncogenic potential in humans and can cause chronic infections. JC virus, also known as John Cunningham Virus (JCV), is a member of the polyomaviridae family, and is also the causative agent of multifocal leukoencephalopathy (PML), a brain disease caused by viral infection that can be life threatening when severe. BK virus (BKV) is also a human specific polyomavirus, which is associated with BK viral nephropathy. Infection with JC virus and BK virus is mainly asymptotic and is often found in children with early infection. Prevalence rates in adults (seroprevalence, serorevalues) are about 70-80% (Knowls, adv. exp. med. biol.577(2006), 19-45). JC virus and BK virus mostly remain latent in the kidney cells of the host until reactivation occurs in immunosuppressed individuals. Cytomegalovirus is a common pathogen in humans, and it is often associated with asymptomatic primary infection, and subsequent viral persistence or latency stages. Primary cytomegalovirus infection and persistent reactivation of cytomegalovirus are often associated with life-threatening invasive visceral diseases in patients with congenital or acquired immunodeficiency, as well as in patients undergoing solid organ or bone marrow transplantation.

Iron chelators are commonly used to treat thalassemia, sideroblasts anemia, autoimmune hemolytic anemia, and other anemias. There are no reports of the use of iron chelators for the treatment or prevention of polyoma virus-related diseases.

Disclosure of Invention

In one aspect, the invention relates to the use of an iron chelator for the preparation of a medicament for the treatment or prevention of polyoma viral infection.

In another aspect, the present invention relates to a method of treating or preventing a polyomavirus infection comprising administering to an individual in need thereof a therapeutically effective amount of an iron chelator of the present invention. The subject is preferably a mammal, particularly a human subject.

In a further aspect, the invention relates to an iron chelator for use in the treatment or prevention of polyoma viral infection.

In one embodiment, the iron chelator is one or more selected from the group consisting of: deferasirox, deferoxamine, deferiprone, pyridoxal isonicotinyl hydrazone, Dp44mT, CGP 65015, CN128, deflazalide, Divin, VLX600, SP-420 and deferiprazole.

In another embodiment, the polyomavirus infection is selected from the group consisting of BK virus, JC virus, cytomegalovirus, SV40 virus, Merkel cell polyomavirus, echinodesis-associated polyomavirus, KI polyomavirus, WU polyomavirus, human polyomavirus 6, human polyomavirus 7, human polyomavirus 9, and human polyomavirus 12 infection.

Drawings

FIG. 1 shows the relative DNA load of the BK virus in cells.

FIG. 2 shows protein SDS gel electrophoresis of LT and VP1 of BK virus in cells.

FIG. 3 shows SDS gel electrophoresis of the proteins of VP1 of the BK virus in cells.

Fig. 4 shows the relative DNA load of the BK virus in cells.

FIG. 5 shows protein SDS gel electrophoresis of LT and VP1 of BK virus in cells.

Detailed Description

The present invention will be described in further detail below. Such description is for the purpose of illustration and not for the purpose of limitation. Other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways. Various modifications and alterations can be made by those skilled in the art without departing from the spirit of the invention.

General definitions and terms

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety if not otherwise indicated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the definitions provided herein will control.

All percentages, parts, ratios, etc., are by weight unless otherwise indicated.

When an amount, concentration, or other value or parameter is given as either a range, preferred range, or a pair of upper and lower preferable values or specific values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. When numerical ranges are recited herein, unless otherwise stated, the stated ranges are meant to include the endpoints thereof, and all integers and fractions within the ranges. The scope of the invention is not limited to the specific values recited when defining a range. For example, "1-8" encompasses 1, 2, 3, 4, 5, 6, 7, 8, as well as any subrange consisting of any two values therein, e.g., 2-6, 3-5.

The terms "about" and "approximately," when used in conjunction with a numerical variable, generally mean that the value of the variable and all values of the variable are within experimental error (e.g., within 95% confidence interval for the mean) or within ± 10% of the specified value, or more.

The terms "comprising," "including," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps. It will be understood by those skilled in the art that terms such as "including" and "comprising" encompass the meaning of "consisting of …. The expression "consisting of …" excludes any element, step or ingredient not specified. The expression "consisting essentially of …" means that the scope is limited to the specified elements, steps or components, plus optional elements, steps or components that do not materially affect the basic and novel characteristics of the claimed subject matter. It is to be understood that the expression "comprising" covers the expressions "consisting essentially of …" and "consisting of …".

The term "selected from …" means that one or more elements in the later listed groups are independently selected and may include a combination of two or more elements.

When values or range ends are described herein, it is to be understood that the disclosure includes the particular values or ends recited.

The term "one or more" or "at least one" as used herein refers to one, two, three, four, five, six, seven, eight, nine or more.

Unless otherwise indicated, the terms "combination thereof" and "mixture thereof" refer to a multi-component mixture of the elements described, such as two, three, four, and up to the maximum possible multi-component mixture.

Furthermore, no number of elements or components of the invention has been previously indicated and no limitation on the number of occurrences (or presence) of an element or component is intended. Thus, it should be read to include one or at least one and singular forms of a component or ingredient also include the plural unless the numerical value explicitly indicates the singular.

The terms "optionally" or "optionally" as used herein mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The term "iron chelator" as used herein refers to a drug that binds iron (e.g., in vivo) and is effective in increasing iron excretion, thereby reducing iron loading or abnormal deposition thereof in various organs. Examples of iron chelators that may be used include, but are not limited to, deferasirox, deferoxamine, deferiprone, Dp44mT, CGP 65015, CN128, diferusin, Divin, VLX600, SP-420, deferiprazole, and combinations thereof.

The term "iron chelator" as used herein also encompasses salts, solvates (especially hydrates), esters, prodrugs, metabolites, crystal forms, co-crystals, stereoisomers, tautomers, isotopic compounds thereof. Preferably, these forms are pharmaceutically acceptable. The iron chelator itself and forms listed or not listed above are encompassed within the scope of the present application.

The term "polyomavirus" as used herein refers to a double-stranded DNA virus that induces sarcoma or cancer in multiple sites or organs. Examples of polyomaviruses include, but are not limited to, BK virus, JC virus, cytomegalovirus, SV40 virus, Merkel cell polyomavirus, echinodesis-associated polyomavirus, KI polyomavirus, WU polyomavirus, human polyomavirus 6, human polyomavirus 7, human polyomavirus 9, human polyomavirus 12.

The term "disease associated with polyomavirus infection" or "disease associated with polyomavirus infection" as used herein refers to a disease or condition resulting from the invasion of cells of an organism by polyomavirus. These diseases or conditions may or may not exhibit significant symptoms. Examples of such diseases include, but are not limited to, BK viral nephropathy, CM viral infection, brain JC viral infection, progressive multifocal leukoencephalopathy, BK virus-associated hemorrhagic cystitis, cytomegalovirus infection after hematopoietic stem cell transplantation, cytomegalovirus infection after liver transplantation.

The term "crystalline form" or "crystalline" refers to any solid substance that exhibits a three-dimensional ordering, as opposed to an amorphous solid substance, which produces a characteristic X-ray powder diffraction pattern having well-defined peaks. The term "co-crystal" refers to a crystalline entity comprising at least two molecules in a stoichiometric or non-stoichiometric ratio. The term "solvate" refers to a complex of variable stoichiometry formed by a solute and a solvent. The term "hydrate" refers to a solvate comprising a drug and either stoichiometric or non-stoichiometric amounts of water. The term "stereoisomers" refers to compounds having the same chemical structure, but differing in the arrangement of atoms or groups in space. The term "tautomer" refers to compounds that have a particular compound structure in interchangeable forms and that vary in hydrogen atom and electron displacement. The term "isotopic compound" refers to a compound having a structure comprising one or more atomic isotopes in natural or unnatural abundance.

The term "pharmaceutically acceptable" refers to those substances which are, within the scope of normal medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit to risk ratio, and effective for their intended use.

The term "pharmaceutically acceptable salts" refers to salts formed with pharmaceutically acceptable non-toxic bases or acids, including inorganic or organic bases and inorganic or organic acids. The salt of an inorganic base may be selected, for example, from: aluminum, ammonium, calcium, copper, iron, ferrous, lithium, magnesium, manganese, manganous, potassium, sodium, and zinc salts. Further, the salt of a pharmaceutically acceptable inorganic base may be selected from ammonium, calcium, magnesium, potassium and sodium salts. One or more crystal structures may be present in the solid salt, as well as in the form of hydrates. The pharmaceutically acceptable salts of organic non-toxic bases may be selected, for example, from: primary, secondary and tertiary amine salts, the substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as arginine, betaine, caffeine, choline, N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine and tromethamine.

When the compounds disclosed herein are bases, it is desirable to prepare salts thereof with at least one pharmaceutically acceptable non-toxic acid, which may be selected from inorganic and organic acids. For example, selected from the group consisting of acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. In some embodiments, these acids may be selected, for example: citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, fumaric and tartaric acids.

The term "effective amount" means that amount of a compound or a pharmaceutically acceptable salt thereof that is effective in eliciting a biological or medical response in a tissue, system, animal or human that is observable by a researcher, veterinarian, clinician or other clinician.

The term "excipient" refers to an inactive ingredient that contains an active ingredient to increase the bulk of the formulation when the dosage form is manufactured. General classes of excipients include, for example, antiadherents, binders, coatings, disintegrants, fillers, flavors, colors, lubricants, glidants, adsorbents, preservatives, and sweeteners.

The term "sequential administration", also known as switch administration, means that when the drug is used to treat a disease, parenteral administration, such as subcutaneous injection, is used at the initial stage, and after the clinical symptoms are substantially stable and the disease condition is improved, oral drug therapy is changed.

Iron chelating agent

The term "iron chelator" as used herein has the meaning commonly understood by those of ordinary skill in the art, and is a drug that binds iron and effectively increases iron excretion, thereby reducing iron loading or abnormal deposition thereof in various organs. Iron chelators that may be used include, but are not limited to, one or more of deferasirox, deferoxamine, deferiprone, pyridoxal isoniazone, Dp44mT, CGP 65015, CN128, difrussian, Divin, VLX600, SP-420, deferiprazole.

In one embodiment, the iron chelator used in the present invention is selected from deferasirox, deferoxamine, deferiprone and combinations thereof.

Deferasirox

The deferasirox is an iron chelating agent, has a chemical name of 4- [3, 5-bis (2-hydroxyphenyl) - [1, 2,4] triazol-1-yl ] benzoic acid, and has a structural formula shown in the specification.

Deferasirox is commercially available, for example from solibao corporation.

Deferoxamine

Deferoxamine is a bacterial siderophore produced by actinomycetes and streptococci and has the following structural formula.

Deferoxamine is typically present with the acid ion to form a salt form, such as deferoxamine mesylate. Deferoxamine is commercially available, for example from solibao corporation.

Deferiprone

Deferiprone, also known as L1, CP20, Ferriprox or Kelfer, is a member of the alpha-ketohydroxypyridine class of iron chelators, useful for treating transfusional iron overload in thalassemia, and has the following structure.

Deferiprone is commercially available, for example from solibao corporation.

Dp44mT

Dp44mT is an iron chelator with selective anti-cancer activity, having the following structure.

Dp44mT is commercially available. Such as available from MedChemExpress.

CGP 65015

CGP 65015 is an orally available iron chelator that removes iron deposits. CGP 65015 has the following structure.

CGP 65015 is commercially available. Such as available from MedChemExpress.

CN128

CN128 is an orally active, selective iron chelator, commonly used in the study of beta-thalassemia, having the following structure.

CN128 is commercially available. Such as available from MedChemExpress.

Schiff base group

Deferarin (Deferatrin) is a desferthionin analogue and is also an orally active tridentate iron chelator. Deferant is often used to relieve chronic iron overload caused by blood transfusion, and also to treat severe beta thalassemia. The structure of the Schiff group is as follows.

The diferendol is commercially available. Such as available from MedChemExpress.

Divin

Divin is a potent iron chelator and is also a potent inhibitor of bacterial cell division, which has bacteriostatic effects on gram-negative and gram-positive bacteria. Divin has the following structure.

Divin is commercially available. Such as available from MedChemExpress.

Pyridoxal isonicotinyl hydrazone

Pyridoxal Isonicotinoyl Hydrazone (PIH) is a lipophilic ferric chelator that exhibits high iron chelation. Pyridoxal isonicotinyl hydrazone has the following structure.

Pyridoxal isonicotinyl hydrazone can be obtained commercially. Such as available from MedChemExpress.

VLX600

VLX600 is an iron chelation inhibitor of oxidative phosphorylation (OXPHOS). VLX600 can lead to mitochondrial dysfunction and a strong shift to glycolysis. VLX600 has the following structure.

VLX600 is commercially available. Such as available from MedChemExpress.

SP-420

SP-420 is a deferethine analog having the following structure.

SP-420 is commercially available. Such as available from MedChemExpress.

Deferiprazole

Deferiprazole (Deferitazole), also known as SPD-602 or FBS701, is a deferethiol analog having the following structure.

SPD-602 is commercially available.

In a preferred embodiment, the iron chelator is at least two selected from the group consisting of: deferasirox, deferoxamine, deferiprone, pyridoxal isonicotinyl hydrazone, Dp44mT, CGP 65015, CN128, deflazalide, Divin, VLX600, SP-420, deferiprazole.

In a particular embodiment, the iron chelator is deferasirox.

In another specific embodiment, the iron chelator is deferoxamine.

In yet another specific embodiment, the iron chelator is deferiprone.

In yet another specific embodiment, the iron chelator is VLX 600.

In yet another specific embodiment, the iron chelator is SP-420.

In yet another specific embodiment, the iron chelator is desferridazole.

In a particular embodiment, the iron chelator is a combination of deferasirox and deferoxamine.

In another specific embodiment, the iron chelator is a combination of deferasirox and deferiprone.

In yet another specific embodiment, the iron chelator is a combination of deferoxamine and deferiprone.

In a particular embodiment, the iron chelator is a combination of deferasirox, deferoxamine and deferiprone.

In a specific embodiment, the iron chelator is a combination of larospx and VLX 600.

In another specific embodiment, the iron chelator is a combination of deferasirox and SP-420.

In yet another specific embodiment, the iron chelator is a combination of deferasirox and deferiprazole.

Polyoma virus infection and related diseases

Polyoma virus

In this context, polyomavirus refers to a double-stranded DNA virus that induces sarcoma or cancer in multiple sites or organs.

In one embodiment, the iron chelator can be used to inhibit a polyoma virus selected from the group consisting of: BK virus, JC virus, cytomegalovirus, SV40 virus, Merkel cell polyomavirus, echinoderm hyperplasia-associated polyomavirus, KI polyomavirus, WU polyomavirus, human polyomavirus 6, human polyomavirus 7, human polyomavirus 9, human polyomavirus 12, in particular BK virus, JC virus or cytomegalovirus.

Accordingly, the invention also relates to the use of an iron chelator for the manufacture of a medicament for inhibiting polyoma virus replication. The iron chelator and polyoma virus are as described herein.

BK virus

The BK viral genome is a circular double-stranded DNA of approximately 5kb in length, comprising three main partitions: early coding region, late coding region and non-coding control region. The early coding regions encode three regulatory proteins (large tumor antigen [ TAg ], small tumor antigen [ tAg ], and truncated tumor antigen [ truncTAg ]), which are the first viral proteins expressed in newly infected cells and responsible for facilitating viral DNA replication and establishing a favorable cellular environment. The late coding region encodes three structural proteins (VP1, VP2, and VP3) that make up the viral capsid, as well as an unknown protein (agnoprotein) whose role during viral replication is not yet clear. The noncoding control regions contain an origin of replication, and early and late promoters that drive expression of the viral gene product.

JC virus

JC virus is a member of the polyomavirus family, whose genome consists of a double-stranded circular DNA of 5.1kb in size, which produces two types of proteins at the early stage of viral infection, i.e. before DNA replication and at the later stage of the infection cycle, a bidirectional coding sequence located between the early and late genes, is responsible for viral gene expression and contains the viral DNA origin of replication. The viral early proteins, the large T antigen (T-Ag) and the family of small T-Ag proteins, are produced by alternative splicing and have a regulatory role in coordinating the virus during its replication cycle.

Cytomegalovirus

Cytomegalovirus (CMV) is a member of the herpes virus family. In humans, Cytomegalovirus (CMV), also known as Human Cytomegalovirus (HCMV), is designated human herpes virus 5 (HHV-5). Cytomegalovirus (CMV) is the largest member of the herpes virus family, having a double-stranded DNA genome of more than 240kbp, and is capable of encoding more than 200 possible protein products. Cytomegalovirus (CMV) is also known as the sub-genus Beth herpesvirus because it is a herpesvirus that is capable of infecting monocytes as well as lymphocytes.

SV40 virus

SV40 refers to the abbreviation simian vacuolar virus 40, simian virus 40 or simian virus 40, belonging to the polyomaviridae family, an oncogenic virus found in both humans and monkeys. The genome of the SV40 virus is a circular double-stranded DNA.

Merkel polyoma cell virus

Merkel Cell Carcinoma (MCC), also known as primary neuroendocrine carcinoma of the skin, is a rare but invasive skin tumor. Merkel cell polyomavirus (MCPyV) infection, uv radiation and immunosuppression are major factors in the pathogenesis of MCC.

Polyoma virus related to acanthosis

The echinoderm hyperplasia-associated polyomavirus, also known as human polyomavirus 8, is associated with echinoderm hyperplasia.

KI polyoma virus

KI polyoma virus is a human polyoma virus, named by the discoverer unit (Karolinska institute).

WU polyoma virus

The WU polyomavirus is a human polyomavirus, named after discoverer unit (Washington University).

Diseases associated with polyoma virus

In one embodiment, the iron chelator can achieve an inhibitory effect on polyomaviruses by inhibiting their replication, thereby treating or preventing diseases associated with polyomaviruses.

Polyomavirus (infection) can cause some related diseases, such as but not limited to BK viral nephropathy, CM viral infection, brain JC viral infection, progressive multifocal leukoencephalopathy, BK virus-associated hemorrhagic cystitis, cytomegalovirus infection after hematopoietic stem cell transplantation, cytomegalovirus infection after liver transplantation. Thus, the medicament may be used for the treatment or prevention of diseases associated with polyoma virus (infection).

Thus, the invention also relates to the use of an iron chelator for the preparation of a medicament for the treatment or prevention of a polyomavirus-related disease. Wherein the iron chelator, polyoma virus and related diseases are as described herein.

In a preferred embodiment, the invention relates to the use of an iron chelator for the manufacture of a medicament for the treatment or prevention of BK viral nephropathy or hemorrhagic cystitis.

Medicaments and administration

The dose level and frequency of administration of the iron chelator will vary depending on the type of polyomavirus disease, the state of iron chelator receptor sensitivity, age and virus level and will be for individual patients. The level and frequency of dosage administered may be determined by the practitioner in view of the circumstances. In general, satisfactory results are achieved at daily dosages of from 0.001 to 100mg/kg body weight, in particular from about 0.03 to 2.5mg/kg body weight. Daily doses for larger mammals, such as humans, may be administered in a convenient form, for example in divided doses up to four times a day or in sustained release form, from about 0.5mg to about 2000mg, or more specifically, from 0.5mg to 1000 mg. Suitable unit dosage forms for oral administration contain from about 1 to 50mg of the active ingredient.

In one embodiment, the iron chelator is administered at a dose of 10-200 mg/kg/d. In a preferred embodiment, the iron chelator is administered at a dose of 20-100 mg/kg/d. For example, 20mg/kg/d, 30mg/kg/d, 40mg/kg/d, 50mg/kg/d, 60mg/kg/d, 70mg/kg/d, 80mg/kg/d, 90mg/kg/d, 100 mg/kg/d.

In one embodiment, the iron chelator is administered 1-3 times daily. In a preferred embodiment, the iron chelator is administered 3 times daily. In another preferred embodiment, the iron chelator is administered 1 time per day.

In a preferred embodiment, the deferiprone iron chelator is administered at a dose of 50-100 mg/kg/d. In a more preferred embodiment, the deferiprone iron chelator is administered at a dose of 100 mg/kg/d.

In another preferred embodiment, the deferasirox iron chelator is administered at a dose of 20-30 mg/kg/d. In a more preferred embodiment, the deferasirox iron chelator is administered at a dose of 30 mg/kg/d.

In a specific embodiment, the deferiprone iron chelator is administered at a dose of 50-100mg/kg/d, 3 times daily. In another specific embodiment, the deferasirox iron chelator is administered at a dose of 20-30mg/kg/d 1 time daily.

Iron chelators are generally suitable for administration to a variety of animals, including mammals. Examples of subjects include, but are not limited to, humans and any other primate, mammals, including commercially relevant mammals such as non-human primates, cows, pigs, horses, sheep, cats, and dogs.

The iron chelator of the present invention may be administered in the form of a pharmaceutical composition, by any conventional route; e.g., enterally, e.g., orally, e.g., in the form of tablets or capsules, parenterally, e.g., in the form of injectable solutions or suspensions; or topically, e.g., in the form of a lotion, gel, ointment, or cream, or in the form of a nasal or suppository; or sequentially, e.g., parenterally (e.g., subcutaneously) prior to treatment, or orally after the condition has improved. A particularly preferred mode is sequential administration.

In one embodiment, the drugs administered sequentially may be the same or different.

In one embodiment, the various iron chelators in the medicament of the invention are administered simultaneously or sequentially. In one embodiment, deferasirox is administered first, followed by deferoxamine. In another embodiment, deferasirox is administered first, followed by deferiprone.

In a particular embodiment, the administration form of the invention is sequential administration, first by subcutaneous injection of deferoxamine and then by oral administration of deferasirox.

When administered simultaneously, the various iron chelating agents may be present in different pharmaceutical compositions, or may be present in a single pharmaceutical composition, such as any of the many pharmaceutical compositions described herein.

Pharmaceutical composition

The iron chelators described herein may be administered in the form of a pharmaceutical composition. Thus, the present invention also relates to a pharmaceutical composition comprising an iron chelator as described herein together with one or more pharmaceutically acceptable carriers or excipients and uses related thereto.

By "pharmaceutically acceptable carrier" herein is meant a diluent, adjuvant, excipient, or vehicle that is administered with the active ingredient and which is, within the scope of sound medical judgment, suitable for contact with the tissues of humans and/or other animals without excessive toxicity, irritation, allergic response, complication, or other problem, commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, an exemplary carrier can be water. Physiological saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The composition may also optionally contain minor amounts of wetting agents, emulsifying agents, lubricating agents, stabilizing agents, or pH buffering agents, and the like. Oral formulations may contain standard carriers.

The pharmaceutical compositions comprising the iron chelator according to the invention in free base or pharmaceutically acceptable salt form together with at least one pharmaceutically acceptable carrier or diluent may be manufactured in a conventional manner by means of a mixing, granulating, coating, dissolving or lyophilizing process. For example, pharmaceutical compositions comprising a compound of the invention in combination with at least one pharmaceutically acceptable carrier or diluent may be formulated in conventional manner by admixture with a pharmaceutically acceptable carrier or diluent. Unit dosage forms for oral administration contain, for example, from about 0.1mg to about 500mg of active substance.

In one embodiment, the pharmaceutical composition is a solution, including a suspension or dispersion, such as an isotonic aqueous solution, of the active ingredient. In the case of lyophilized compositions comprising the active ingredient alone or in admixture with a carrier such as mannitol, dispersions or suspensions may be prepared prior to use. The pharmaceutical compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. Suitable preservatives include, but are not limited to, antioxidants such as ascorbic acid, microbicides such as sorbic acid or benzoic acid. The solution or suspension may also contain a thickening agent including, but not limited to, sodium carboxymethylcellulose, dextran, polyvinylpyrrolidone, gelatin, or a solubilizing agent such as tween 80 (polyoxyethylene (20) sorbitan monooleate).

Suspensions in oils may contain, as oily component, vegetable oils, synthetic or semi-synthetic oils, commonly used for injection purposes. Examples include liquid fatty acid esters containing as the acid component a long chain fatty acid having from 8 to 22 carbon atoms, or in some embodiments, from 12 to 22 carbon atoms. Suitable liquid fatty acid esters include, but are not limited to, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidic acid, behenic acid or corresponding unsaturated acids, such as oleic acid, elaidic acid, erucic acid, brassidic acid and linoleic acid, if desired, with antioxidants such as vitamin E, 3-carotene or 3, 5-di-tert-butylhydroxytoluene. The alcohol component of these fatty acid esters may have six carbon atoms and may be monovalent or polyvalent, such as mono-, di-or trivalent alcohols. Suitable alcohol components include, but are not limited to, methanol, ethanol, propanol, butanol or pentanol or isomers thereof, ethylene glycol and glycerol.

Other suitable fatty acid esters include, but are not limited to, ethyl oleate, isopropyl palmitate, isopropyl myristate, and the like,

M2375 (polyoxyethylene glycerol),

M1944CS (unsaturated polyglycolyzed glyceride prepared by alcoholysis of almond oil, containing glyceride and polyglycol ester), LABRASOL^TM(saturated PEGylated glycerides prepared by alcoholysis of TCM, containing glycerides and polyethylene glycol esters; all available from GaKefosse, France), and/or

812 (triglycerides of saturated fatty acids with chain lengths of C8 to C12, from huls AG, germany), and vegetable oils such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil or peanut oil.

Pharmaceutical compositions for oral administration may be obtained, for example, by mixing the active ingredient with one or more solid carriers, if desired granulating a resulting mixture, and processing the mixture or granules by adding further excipients, in the form of tablets or tablet cores.

Suitable carriers include, but are not limited to, fillers, for example sugars, such as lactose, sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and also binders, for example starches, such as corn, wheat, rice or potato starch, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and/or, if desired, disintegrants, such as the above-mentioned starches, carboxymethyl starch, crosslinked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate. Additional excipients include flow-regulating agents and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.

The tablet cores may be provided with a suitable, optionally enteric, coating by using, inter alia, a concentrated sugar solution, which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or a coating solution in a suitable organic solvent or solvent mixture, or, for enteric coatings, a solution of a suitable cellulose preparation, such as an acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate solution. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of the active ingredient.

Pharmaceutical compositions for oral administration may also include hard capsules, including gelatin or soft, sealed capsules containing gelatin and a plasticizer, such as glycerol or sorbitol. Hard capsules may contain the active ingredients in the form of granules, for example in admixture with fillers such as corn starch, binders and/or glidants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active ingredient may be dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oils or liquid polyethylene glycols or fatty acid esters of ethylene glycol or propylene glycol, to which stabilizers and detergents, for example of the fatty acid ester type of polyoxyethylene sorbitol, are added.

Pharmaceutical compositions suitable for rectal administration, for example suppositories, comprise a combination of the active ingredient and a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols.

Pharmaceutical compositions suitable for parenteral administration may comprise the active ingredient in water-soluble form, for example as a water-soluble salt or as an aqueous injection suspension comprising a viscosity-increasing substance, for example sodium carboxymethylcellulose, an aqueous solution of sorbitol and/or dextran, if desired, and a stabilizer. The active ingredient, optionally together with excipients, may also be in a lyophilized form and may be prepared as a solution by addition of a suitable solvent prior to parenteral administration. The solutions used, for example for parenteral administration, can also be used as infusion solutions. Injectable preparations are generally prepared under sterile conditions, and filled, for example, in ampoules or vials, and in sealed containers.

Combination drug

The iron chelating agent of the present invention may be used alone, or two or more kinds of chelating agents may be used. On this basis, the iron chelator may also be used in combination with other antiviral therapeutic agents.

For example, the therapeutic benefit of a compound of the invention may be enhanced by the use of an adjuvant drug (e.g., the therapeutic benefit of the adjuvant drug alone may be minimal, but the therapeutic benefit of the subject may be enhanced when used in combination with another drug), or, for example, the therapeutic benefit of the subject may be enhanced by the use of a compound of the invention in combination with another therapeutic agent that is also therapeutically effective. For example, the combination of an immunomodulator may enhance clinical benefit when a compound of the invention is used in the treatment of JC virus nephropathy. Alternatively, for example, if the adverse effect of using the compounds of the present invention is nausea, then an anti-nausea agent may be used in combination. Alternatively, therapies that can be combined include, but are not limited to, physical therapy, psychotherapy, radiation therapy, compression therapy of the diseased area, rest, dietary improvement, and the like. Regardless of the disease, disorder, or condition being treated, both therapies should have additive or synergistic effects to benefit the treatment of an individual.

In one embodiment, the iron chelator is used in combination with other antiviral therapeutic agents. In a preferred embodiment, the additional antiviral therapeutic agent is selected from one or more of the following: 1-O-hexadecyloxypropylcidofovir, cidofovir, leflunomide, interferon, idoxuridine, trifluorothymidine, vidarabine, ribavirin, acyclovir, propoxuridine, azidothymidine, amantadine, or moroxydine, in particular one or more of 1-O-hexadecyloxypropylcidofovir, cidofovir, leflunomide.

Where the compounds described herein are used in combination with other therapeutic agents, the route of administration of the pharmaceutical compositions of the compounds described herein may be the same as the other drugs, or the route of administration may be different due to differences in physical and chemical properties. For example, oral administration of a compound described herein may produce and maintain good blood levels, while intravenous administration of another therapeutic agent may be required. Thus, the compounds described herein and another therapeutic agent may be administered simultaneously, sequentially or separately.

Advantageous effects

The invention provides the use of an iron chelator for the manufacture of a medicament for the treatment or prevention of polyomavirus infection, and the use of an iron chelator for the manufacture of a medicament for the inhibition of polyomavirus replication. The inventors have discovered that iron chelators can effectively inhibit polyomavirus replication, thereby effectively treating or preventing polyomavirus infection and related diseases, and provide a new direction and option for treatment.

Examples

The present invention will be described in further detail with reference to specific examples.

It should be noted that the following examples are only for clearly illustrating the technical solutions of the present invention, and are not intended to limit the present invention. It will be apparent to those skilled in the art that other variations and modifications may be made in the foregoing disclosure without departing from the spirit or essential characteristics of the invention, and it is not desired to exhaustively enumerate all embodiments, but rather those obvious variations and modifications are within the scope of the invention.

Instrument for measuring the position of a moving object

Unless otherwise indicated, both instrumentation and reagent materials used herein are commercially available.

Main experimental instrument

A micropipette: purchased from EPPENDORF.

An enzyme-labeling instrument: biotek microplate reader from Applied Biosystems.

An incubator: carbon dioxide incubator, purchased from Thermo Fisher Scientific.

A PCR instrument: real-time fluorescent quantitative PCR instrument, purchased from BIO-RAD.

And (3) inverting the microscope: available from ZEISS.

Immunoblotting apparatus: purchased from BIO-RAD.

The biological safety cabinet: purchased from ESCO.

A centrifuge: model 5702R, low temperature high speed bench top centrifuge, available from EPPENDOFF.

Cell lines

Human proximal tubular epithelial cells HRPTEPiC (#4100), purchased from ScienCell, USA, and cultured using EpiCM (#4101) medium. Human bladder cancer cells TCCSUP (# ZQ0463) were purchased from Gekko Biotech, Inc. of Gekko, China and cultured in MEM medium. Vero cells of Vero (ZQ 0159) were purchased from qiao new boat biotechnology limited, china and cultured in DMEM medium. The MEM and DMEM basal media are supplemented with 10% fetal bovine serum (FBS, Lonsera) and penicillin-streptomycin double antibody to form a complete medium for cell maintenance or other experiments.

Viral strains

BK polyomavirus (BK)

VR-837^TM) Virus was amplified using Vero, purchased from the american type culture collection.

Materials and reagents

Pancreatin (0.25% Trypsin-0.1% EDTA) was purchased from Gibco.

Fetal Bovine Serum (FBS) from grandma longera.

MEM and DMEM basal media were purchased from Corning, USA.

Deferasirox, deferoxamine and deferiprone were purchased from solibao corporation.

Detection of protein expression

The invention embodies the replication condition of the BK virus in a culture system by detecting the expression of VP1 protein and/or LT protein in the BK virus. The "VP 1 protein" is a capsid protein in polyoma viruses with a molecular weight of 40KD, which is essential for the recognition of host cell receptors and for proper virion assembly. "LT" is a large tumor antigen with a molecular weight of 100KD, a key early protein necessary to drive viral replication and induce cellular transformation. Large tumor antigens play a role in viral genome replication by driving quiescent cells into the cell cycle and automatically regulating viral early mRNA synthesis. Therefore, these two proteins are often used to characterize the levels of the virus.

Example 1: study of inhibitory Effect of iron chelator on BK Virus

The purpose of this example was to investigate the inhibitory effect of iron chelators on BK virus (BKV) in cells.

1.1 Delosol (DFS), Desferrioxamine (DFA) and Deferiprone (DFP) were dissolved in DMSO or PBS respectively and prepared as 250mM, 100mM and 100mM stock solutions respectively for subsequent use.

1.2 digestion of logarithmic phase-grown HRPTEPIC and TCCSUP cells at 3X 10 using 0.25% Trypsin-0.1% EDTA⁵The number of cells per well was seeded in 6-well plates, 2ml per well. And (3) infecting the two cells in 24h by using a cell seed plate, sucking cell supernatant in a 6-well plate as dry as possible, and replacing the cell supernatant with a culture medium containing 0.5MOI BKV correspondingly. After 2h of infection, the supernatant was discarded and replaced with drug-free medium and medium containing 100. mu.M deferasirox, 100. mu.M deferoxamine and 100. mu.M deferiprone, respectively, and then placed in a cell incubator for further culture for 72 h.

1.372 h, cells were harvested and a 6-well plate cell was used to extract DNA to detect the replication of BKV in the cells. Another 6-well plate cell is used for extracting protein to carry out Western blot analysis so as to detect the protein expression condition of the BKV in the cell.

Referring to fig. 1, the results show that the expression level of BK virus DNA in cells is relatively high after BK virus infection. In contrast, a significant decrease in the expression level of BK virus DNA was observed upon addition of the iron chelator deferasirox, deferoxamine or deferiprone to the culture system.

Referring to FIG. 2, the results show that LT and VP1 of BK virus in cells show higher levels of protein expression after BK virus infection. In contrast, a significant reduction in protein expression levels of LT and VP1 of BK virus was observed in both cells, in particular in HRPTEPIC cells, after addition of the iron chelators deferasirox, deferoxamine or deferiprone to the culture system.

Therefore, after the iron chelator is added into the culture system, the replication of the virus can be effectively inhibited and the expression level of the virus-related protein can be effectively reduced. In addition, the addition of an iron chelator can also effectively reduce the DNA load of the virus in the supernatant.

Therefore, the iron chelator can effectively inhibit the replication of the polyoma virus and realize the inhibition effect on the polyoma virus.

Example 2: research on BK virus resistance and iron ion level of iron chelator

The purpose of this example was to investigate the inhibitory effect of iron chelators on BK virus in the presence of iron ions.

2.1 dissolving deferasirox, deferoxamine and deferiprone in DMSO or PBS respectively, and preparing storage solutions with concentrations of 250mM, 100mM and 100mM respectively for subsequent use.

2.2 digestion of logarithmic phase-grown HRPTEPIC and TCCSUP cells at 3X 10 using 0.25% Trypsin-0.1% EDTA⁵The number of cells per well was seeded in 6-well plates, 2ml per well. And (3) infecting the two cells in 24h by using a cell seed plate, sucking cell supernatant in a 6-well plate as dry as possible, and replacing the cell supernatant with a culture medium containing 0.5MOI BKV correspondingly. After 2h of infection, the supernatant was discarded and replaced with drug-free medium and medium containing 10. mu.M deferasirox, 25. mu.M Ferrous Citrate (FC), 10. mu.M deferasirox and 25. mu.M ferrous citrate, respectively, and then placed in a cell incubator for further culture for 72 h.

2.372 h, collecting cells, extracting DNA from a 6-well plate cell to detect the replication condition of the BKV in the cells, and extracting protein from a 6-well plate cell to perform western blot analysis to detect the expression condition of the BKV protein in the cells. Meanwhile, cell supernatant is collected, and the loading capacity of the BKV in the supernatant is detected.

Referring to fig. 3, the results show that VP1 of BK virus in cells shows higher levels of protein expression after BK virus infection. In contrast, a significant reduction in the expression level of VP1 protein of BK virus was observed after the addition of the iron chelator, deferasirox, to the culture system. And after the ferrous citrate is added into the culture system or the deferasirox and the ferrous citrate are simultaneously added into the culture system, the expression level of the VP1 protein of the BK virus does not show obvious reduction.

Referring to fig. 4, the results show that the expression level of BK virus DNA in cells is relatively high after BK virus infection. In contrast, a significant reduction in the expression level of BK virus DNA was observed with the addition of deferasirox to the culture system. And after the ferrous citrate is added into the culture system or the deferasirox and the ferrous citrate are simultaneously added into the culture system, the DNA expression level of the BK virus does not show obvious reduction.

Therefore, the addition of the iron chelator to the culture system can reduce the concentration of iron ions in the culture system, and further reduce the expression level of BK virus-related proteins in cells. Increasing the concentration of ferric ions in the culture system, no significant effect on the BK virus-associated protein expression levels in the cells was observed. Furthermore, the addition of both an iron chelator and additional iron ions to the culture system also did not have a significant effect on the expression levels of BK virus-associated proteins in the cells.

In cell culture systems (e.g., HRPTEPIC and TCCSUP cells), iron ions are usually present in minute quantities at concentrations approaching those of iron in humans. The addition of the iron chelating agent can reduce the content of iron ions, effectively inhibit the replication of viruses and realize the inhibition effect on the viruses. If an iron chelator is used with the addition of a higher concentration of iron ions, the effect of the iron chelator may be saturated or inhibited and viral replication may be restored.

Example 3: study of iron chelators for treating or preventing BK virus-related diseases

The purpose of this example was to study the inhibitory effect of iron chelators on BK virus in BKV-receptive cells by administering iron chelators separately before and after BK virus infection of the cells.

3.1 dissolving deferasirox, deferoxamine and deferiprone in DMSO or PBS respectively, and preparing storage solutions with concentrations of 250mM, 100mM and 100mM respectively for subsequent use.

3.2 digestion of log phase growing TCCSUP cells with 0.25% Trypsin-0.1% EDTA at 3X 10⁵The number of cells per well was seeded in 6-well plates and divided into 4 groups. The cells were infected in 24h of the cell plate, and the cell supernatants in 6-well plates were aspirated as dry as possible, and the 4 sets were replaced with complete medium (0.5MOI) containing BK virus, BK virus and 25. mu.M deferasirox, and BK virus, respectively. After 2h of infection, the supernatant was discarded, and the 4 fractions were replaced with complete medium, medium containing 25. mu.M deferasirox, complete medium and medium containing 25. mu.M deferasirox, respectively, and then placed in a cell culture chamber for further culture for 72 h.

3.372 h, collecting cells, extracting DNA from a 6-well plate cell to detect the replication condition of the BKV in the cells, and extracting protein from a 6-well plate cell to perform western blot analysis to detect the protein expression condition of the BKV in the cells. Meanwhile, cell supernatant is collected, and the loading capacity of the BKV in the supernatant is detected.

Referring to fig. 5, where FT indicates administration of the iron chelator throughout the viral infection, ET indicates administration of the iron chelator during viral entry into the cell, and EPT indicates administration of the iron chelator after viral entry into the cell. After infection with BK virus, both LT and VP1 of BK virus showed protein expression in TCCSUP cells. Regardless of when iron chelators were administered (e.g., FT, EPT and ET), a decrease in protein expression levels of LT and VP1 of BK virus in TCCSUP cells was observed.

It follows that administration of an iron chelator either before or after viral infection can reduce viral protein expression. Thus, the iron chelator can be used to treat and prevent polyoma viral infections.

The above description is only exemplary of the present invention and is not intended to limit the scope of the present invention, which is defined by the claims appended hereto, as well as the appended claims.

Claims

1. Use of an iron chelator in the manufacture of a medicament for the treatment or prevention of polyoma viral infection.

2. Use of an iron chelator in the manufacture of a medicament for inhibiting polyoma virus replication.

3. Use according to claim 1 or 2, wherein

The iron chelator is one or more selected from: deferasirox, deferoxamine, deferiprone, pyridoxal isonicotinyl hydrazone, Dp44mT, CGP 65015, CN128, deflazalide, Divin, VLX600, SP-420, SPD602 and FBS0701, preferably selected from one or more of the following: deferasirox, deferoxamine, deferiprone, VLX600, SP-420, deferiprazole.

4. Use according to any one of claims 1 to 3, wherein

The polyomavirus infection is selected from BK virus, JC virus, cytomegalovirus, SV40 virus, Merkel cell polyomavirus, echinodesis-associated polyomavirus, KI polyomavirus, WU polyomavirus, human polyomavirus 6, human polyomavirus 7, human polyomavirus 9 and human polyomavirus 12 infection, and preferably BK virus, JC virus or cytomegalovirus infection.

5. Use according to any one of claims 1 to 4, wherein

The medicament is for the treatment or prevention of a disease associated with polyomavirus infection selected from the group consisting of BK viral nephropathy, CM viral infection, encephalojc viral infection, progressive multifocal leukoencephalopathy, BK virus-associated hemorrhagic cystitis, cytomegalovirus infection after hematopoietic stem cell transplantation and cytomegalovirus infection after liver transplantation, in particular BK viral nephropathy or hemorrhagic cystitis.

6. Use according to any one of claims 1 to 5, wherein

The route of administration of the drug is selected from the group consisting of oral, rectal, transmucosal, nasal or enteral administration, parenteral delivery, sequential administration, and combinations thereof.

7. Use according to any one of claims 1 to 6, wherein

The iron chelator is administered simultaneously or sequentially.

8. Use according to any one of claims 1 to 7, wherein

The iron chelator is used in combination with other antiviral therapeutic agents.

9. The use of claim 8, wherein the antiviral therapeutic agent is one or more selected from the group consisting of: 1-O-hexadecyloxypropylcidofovir, cidofovir, leflunomide, interferon, idoxuridine, trifluorothymidine, vidarabine, ribavirin, acyclovir, propoxur uridine, azidothymidine, amantadine, and moroxydine.