HK1223299B - Method for preventing or treating diseases related to reduced density of interferon receptors - Google Patents

Description

Methods for preventing or treating diseases associated with decreased interferon receptor density

Technical Field

The present invention relates to medicine, in particular to the use of glutaryl histamine or a pharmaceutically acceptable salt thereof for preventing or treating diseases associated with reduced interferon receptor density.

Background Art

Type I interferons (including interferon alpha (IFNα) and interferon beta (IFNβ)) are the most important factors responsible for protecting organisms from innate immunity of viral infection and tumor growth. These cytokines perform their signaling function by interacting with a receptor on the cell surface. The receptor is a heterodimer consisting of two chains IFNAR1 and IFNAR2. The interaction of interferon with the receptor leads to the activation of a biochemical chain, which starts with the activation of Janus kinases Tyk2 and Jak1, and the activation of Janus kinases Tyk2 and Jak1 provides phosphorylation of signal transducers and transcription activators Stat1 and Stat2. The latter two molecules provide transcription of interferon-stimulated genes encoding antiviral effector proteins.

Interferon signaling is a powerful stimulus that alters the homeostasis, function, and division of a variety of cells containing interferon receptors in an organism. Uncontrolled interactions between interferon molecules and receptors can have toxic, damaging effects on the immune system and cells throughout the organism. For example, defects in the interferon signaling pathway lead to pathological conditions such as systemic lupus erythematosus. In this regard, the intensity of interferon signaling is strictly controlled, involving multiple mechanisms, and first of all, the regulation/reduction of the density of interferon receptors on the cell surface (Coccia E.M., Uze G., Pellegrini S. (2006) Negative regulation of type I interferon signaling: facts and mechanisms. Cell Mol Biol (Noisy-le-grand) 52: 77-87). This physiological mechanism includes phosphorylation, ubiquitination, endocytosis and subsequent degradation of the receptor subunit IFNAR1 in the cellular proteasome (Kumar K.G., Krolewski J.J., Fuchs S.Y. (2004) Phosphorylation and specific ubiquitin acceptor sites are required for ubiquitination and degradation of the IFNAR1 subunit of type I interferon receptor. J Biol Chem 279: 46614-46620).

Viruses exploit this physiological mechanism for their own benefit by reducing the intensity of interferon signaling, including through degradation of interferon receptors. The effects of pathogens may include targeted inhibition of interferon receptors with viral proteins. For example, hepatitis B virus uses protein X to reduce the density of interferon receptors (Cho I.R., Oh M., Koh S.S., Malilas W., Srisuttee R., Jhun B.H., Pellegrini S., Fuchs S.Y., Chung Y.H. Hepatitis B virus X protein inhibits extracellular IFN-α-mediated signal transduction by downregulation of type I IFN receptor. Int J Mol Med. 2012 Apr; 29(4): 581-6. doi: 10.3892/ijmm.2012.879. Epub 2012 Jan 3). Other viruses (including herpes viruses) achieve the effect of reducing interferon receptor density through virus-induced cellular stress (Liu J., HuangFu W.C., Kumar K.G., et al. Virus-induced unfolded protein response attenuates antiviral defenses via phosphorylation-dependent degradation of the type I interferon receptor. Cell Host Microbe 2009; 5: 72-83). For example, herpes simplex virus can effectively reduce the density of the first subunit of interferon receptor IFNAR1 by degrading it (Qian J., Zheng H., Huangfu W.C., Liu J., Carbone C.J., Leu N.A., Baker D.P., Fuchs S.Y.Pathogen recognition receptor signaling accelerates phosphorylation-dependent degradation of IFNAR1.PLoS Pathog.2011 June;7(6):e1002065.doi:10.1371/journal.ppat.1002065. electronic publication on June 9, 2001). It is known that the herpes virus family is represented by 8 types of herpes viruses that cause human diseases of varying severity. The disease is characterized in that the virus exists in the human body in its latent state.

The similarities in the genomic structures of different herpesviruses provide a common mechanism for blocking interferon signaling.

Another example of viral regulation of interferon receptors in viral infection is the disruption of the balance between interferon receptor subunits in papillomavirus tumorigenesis in women (Tirone N.R., Peghini B.C., Barcelos A.C., Murta E.F., Michelin M.A. Local expression of interferon-alpha and interferon receptors in cervical intraepithelial neoplasia. Cancer Immunol Immunother. 2009 Dec;58(12):2003-10. doi:10.1007/s00262-009-0707-6. Epub 2009 Apr 18. PubMed PMID:19381629).

Thus, acute and chronic viral infections are accompanied by suppression of the interferon system, which is caused in particular by accelerated degradation of interferon receptors (Qian J., Zheng H., Huangfu W.C., Liu J., Carbone C.J., Leu N.A., Baker D.P., Fuchs S.Y. Pathogen recognition receptor signaling accelerates phosphorylation-dependent degradation of IFNAR1. PLoS Pathog. 2011 Jun;7(6):e1002065. doi:10.1371/journal.ppat.1002065. Epub 2011 Jun 9; 2008 Jun 15;197(1):54-62).

Chronic intoxication of organisms (e.g., smoking) leads to a decrease in the density of interferon receptors in respiratory cells, thereby increasing the incidence of viral diseases in smokers and possibly leading to the risk of lung cancer (Huang Fu W.C., Liu J., Harty R.N., Fuchs S.Y. Cigarette smoking products suppress anti-viral effects of Type I interferon via phosphorylation-dependent downregulation of its receptor. FEBS Lett. 2008 Sep 22;582(21-22):3206-10; Picaud S, Bardot B, DeMaeyer E, Seif I. Enhanced tumor development in mice lacking a functional type I interferon receptor. J Interferon Cytokine Res. 2002 Apr;22(4):457-62).

Currently, recombinant (exogenous) interferon drugs are used to treat diseases associated with inhibition of the interferon system. In this case, high doses of the drug are used to compensate for the reduced sensitivity of damaged cells to exogenous interferon. This leads to toxic effects caused by interferon treatment, particularly with the occurrence of depressive syndromes (Patten S.B. Psychiatric side effects of interferon treatment. Curr Drug Saf. 2006 May; 1(2): 143-50. Review. PubMed PMID: 18690925). Resistance to this treatment is mainly determined by reduced expression of interferon receptors. For example, the reduced synthesis of interferon receptor IFNAR1 subunit mRNA observed in multiple sclerosis leads to a reduced efficacy of treatment with interferon beta (Serana F., Sottini A., Ghidini C., Zanotti C., Capra R., Cordioli C., Caimi L., Imberti L. Modulation of IFNAR1 mRNA expression in multiple sclerosis patients. J Neuroimmunol. 2008 Jun 15; 197(1): 54-62).

A similar phenomenon is observed in interferon therapy for chronic viral diseases. Virus-infected cells that lose a certain amount of interferon receptors become insensitive to interferon therapy. For example, the effects of interferon α treatment for hepatitis B are often short-lived, and the disease progresses to a worsening phase, accompanied by an increase in viral genome copies in the patient's blood. This means that a portion of the virus-infected cells in the organism do not respond to interferon therapy. For this reason, interferon drugs cannot cure chronic infections caused by various herpes viruses (Kroeker A.L., Coombs K.M. Systems biology unravels interferon responses to respiratory virus infections. World J Biol Chem. 2014 Feb 26;5(1):12-25.doi:10.4331/wjbc.v5.i1.12.Review. PubMed PMID:24600511; PubMed Central PMCID:PMC3942539); (Eron L.J., Toy C., Salsitz B., Scheer R.R., Wood D.L., Nadler P.I. Therapy of genital herpes with topically applied interferon. Antimicrob Agents Chemother. 1987 Jul;31(7):1137-9. PubMed PMID: 3310870; PubMed Central PMCID: PMC174885).

The present inventors have surprisingly discovered that an effective approach for treating diseases associated with decreased cellular sensitivity to interferon involves increasing the density of interferon receptors on the cell surface. In this case, sensitization of cells to endogenous interferon signals can produce a therapeutic effect without the need for recombinant interferon preparations or can provide for a reduction in their therapeutic dose.

Currently, no agent is known to restore or increase the density of interferon receptors in various pathological conditions, with respect to their degradation. The inventors have surprisingly discovered that glutaryl histamine leads to increased synthesis of interferon receptor messenger RNA and an increase in the density of the receptors themselves on the cell surface. Therefore, glutaryl histamine can restore and/or increase the density of interferon receptors on the cell surface, making this agent promising for the prevention or treatment of a variety of diseases. Because the density of interferon receptors on the cell surface increases, this agent could be used as a monotherapy for viral diseases to achieve the effect of increasing the sensitivity of cells to low levels of endogenous interferons. Additionally, glutaryl histamine could be used in combination with interferon drugs to enhance the response of immunosuppressed cells to exogenous interferons.

Summary of the Invention

The present inventors have demonstrated experimentally that glutaryl histamine can be used to prevent and treat diseases associated with a decrease in interferon receptor density. These diseases are particularly hepatitis B, herpes, papillomavirus infection, and multiple sclerosis. Administration of glutaryl histamine increases the density of interferon receptors on the cell surface, thereby enhancing the sensitivity of cells to the effects of endogenous interferon, thereby overcoming viral immunosuppression and providing a therapeutic effect against viral infections such as herpes types 1 to 8, hepatitis B, papillomavirus infection, and multiple sclerosis. Furthermore, resistance to interferon therapy is also overcome in these diseases.

It has been established that the therapeutic effects of glutaryl histamine are not accompanied by toxic reactions and other side effects. In addition, glutaryl histamine, being a low molecular weight substance, does not lead to the formation of neutralizing antibodies.

In view of the above, the present invention relates to an agent for preventing and/or treating a disease associated with an increased density of interferon receptors, wherein the agent is glutaryl histamine of the following formula:

The present invention also relates to a method for preventing or treating a disease associated with decreased interferon receptor density, comprising administering an effective amount of glutaryl histamine or a pharmaceutically acceptable salt thereof.

Prevention of the above-mentioned diseases includes preventing the recurrence or worsening of the diseases.

Furthermore, the present invention relates to a pharmaceutical composition for preventing or treating a disease associated with decreased interferon receptor density, wherein the composition comprises an effective amount of glutaryl histamine or a pharmaceutically acceptable salt thereof.

In addition, the present invention relates to a kit for preventing or treating a disease associated with reduced interferon receptor density, wherein the kit comprises the above composition and instructions for use thereof.

Furthermore, the present invention relates to the use of glutaryl histamine or a pharmaceutically acceptable salt thereof for preventing or treating diseases associated with decreased interferon receptor density.

In addition, the present invention relates to the use of glutaryl histamine or a pharmaceutically acceptable salt thereof in preparing a pharmaceutical composition for preventing or treating diseases associated with reduced interferon receptor density.

The disease may preferably be selected from the group comprising hepatitis B, herpes (in particular herpes caused by herpesvirus types 1, 2, 3, 4, 5, 6, 7 or 8), papillomavirus infection and multiple sclerosis.

The above-mentioned prevention or treatment is based on compensating for the reduced expression level of interferon receptors during long-term treatment with interferon. The prevention or treatment can be carried out by overcoming resistance to interferon treatment in a disease selected from the group consisting of hepatitis B, herpes, papillomavirus infection and multiple sclerosis.

In one embodiment, the prevention and treatment is performed by increasing the density of interferon receptors in multiple sclerosis treated with interferon beta. In addition, the present invention relates to preventing smoking-induced immunosuppression associated with degradation of interferon receptors in smokers, wherein the treatment may involve overcoming resistance to interferon therapy in smokers. The interferon receptors according to the present invention are interferon alpha (IFNα) and interferon beta (IFNβ) receptors.

Glutaryl histamine is preferably administered in a solid dosage form.

When administered once or more daily, glutaryl histamine or a salt thereof may be administered to a patient at a dose of 0.1 to 100 mg/kg of body weight per day, preferably 0.1 to 30 mg/kg, more preferably 0.3 to 10 mg/kg. A single dose of glutaryl histamine may be about 100 mg. The duration of administration of glutaryl histamine may be from 5 days to 12 months.

The pharmaceutically acceptable salt of glutaryl histamine according to the present invention may be a salt thereof with an alkali metal or alkaline earth metal, preferably a sodium salt, a potassium salt and a lithium salt.

Glutaryl histamine or a salt thereof is administered in an amount effective to provide the desired therapeutic result.

It should be noted that the specific dosage for a particular patient depends on many factors, such as the patient's age, weight, sex, general health and diet; the regimen and route of administration of the agent, its rate of excretion from the body; and the severity of the disease in the individual being treated.

The pharmaceutical composition of the present invention comprises an effective amount of glutaryl histamine or a pharmaceutically acceptable salt thereof required for the desired result and can be prepared as a unit dosage form (e.g., in solid, semi-solid or liquid form) comprising glutaryl histamine or a salt thereof as the active agent in a mixture with a carrier or excipient suitable for intramuscular, intravenous, oral, sublingual, inhalation, intranasal, rectal and transdermal administration. The active agent can be included in the composition together with conventional non-toxic pharmaceutically acceptable carriers suitable for preparing the following dosage forms: solutions, tablets, pills, capsules, dragees, suppositories, emulsions, suspensions, ointments, gels, patches and any other dosage forms.

Excipients can be a variety of substances, such as sugars, such as glucose, lactose, or sucrose; mannitol or sorbitol; cellulose derivatives; and/or calcium phosphates, such as tricalcium phosphate or dibasic calcium phosphate. Compounds used as binders include starch paste (e.g., corn, wheat, rice, or potato starch), gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone. If necessary, a disintegrant can be used, and can include the aforementioned starches and carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof (e.g., sodium alginate).

Additives which may optionally be used are flow control agents and lubricants, for example silicon dioxide, talc, stearic acid and its salts (for example magnesium stearate or calcium stearate) and/or propylene glycol.

Stabilizers, thickeners, colorants and aromatics may also be used as additives.

As ointment bases, there are suitable hydrocarbon ointment bases such as white petrolatum and yellow petrolatum (Vaselinum album and Vaselinum flavum, respectively), petrolatum oil (Oleum Vaselini), and white ointment and liquid ointment (Unguentum album and Unguentum flavum, respectively), in which solid paraffin or wax can be used as an additive to provide a harder texture; absorbent ointment bases such as hydrophilic petrolatum (Vaselinum hydrophylicum), lanolin (Lanolinum) and cold cream (Unguentum lenien); water-removable ointment bases such as hydrophilic ointment (Unguentum hydrophylum); water-soluble ointment bases such as polyethylene glycol ointment (Unguentum Glycolis Polyaethyleni); bentonite bases; and the like.

The base for the gel may be selected from methylcellulose, sodium carboxymethylcellulose, oxypropylcellulose, polyethylene glycol or polyethylene oxide and carbopol.

Bases for suppositories may be a water-insoluble base such as cocoa butter; a water-soluble or water-miscible base such as gelatin-glycerin or polyethylene oxide; or a combination base such as a soap-glycerin base.

The amount of active agent combined with the carrier in preparing a unit dosage form may vary depending upon the recipient being treated and the particular route of administration of the therapeutic agent.

For example, when glutaryl histamine or its salt is used in the form of an injectable solution, the amount of active agent therein is 0.1% to 5%. The diluent can be selected from 0.9% sodium chloride solution, distilled water, novocaine solution for injection, Ringer's solution, glucose solution, and specific solubilizing agents. When glutaryl histamine or its salt is administered in the form of a tablet or suppository, the amount thereof is 10 to 300 mg per unit dosage form.

The dosage forms of the present invention are prepared by conventional operations such as blending, granulating, pelleting, dissolving and lyophilizing.

It should be noted that long-term administration of glutaryl histamine or its salts at therapeutic doses or doses an order of magnitude greater than therapeutic doses has not been shown to cause adverse side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing that the synthesis of interferon receptor subunit mRNA is increased in epithelial cells A549 under the action of glutaryl histamine.

FIG. 2 is a graph showing that the amount of interferon receptor subunit protein on the surface of primary cultured human macrophages is increased under the action of glutaryl histamine.

FIG3 is a photograph of immunoblotting showing the sensitization effect of cells treated with glutaryl histamine on low-concentration interferon.

DETAILED DESCRIPTION

The present invention is described in more detail below by way of examples which support the efficacy of glutaryl histamine for preventing and treating the diseases according to the present invention, wherein the disclosed examples are not intended to limit the scope of the present invention.

Example 1

The mRNA synthesis level of interferon receptor under the action of glutaryl histamine

The continuous cell line A-549 was treated with 100 ng/mL glutaryl histamine. 16 hours after treatment, the amount of messenger RNA copies of the interferon receptor was determined by real-time PCR analysis. RNA was isolated by using an RNeasy kit (Quiagen). RNA preparations were treated with DNA enzyme (DNase I (Ambion) without RNA enzyme). Reverse transcription was performed by using a Thermoscript RT-PCR system (Invitrogen). Quantitative PCR was performed using the following primers:

Ifnar1 (forward) 5′CACTGACTGTATATTGTGTGAAAGCCAGAG 3′,

(reverse) 5′CATCTATACTGGAAGAAGGTTTAAGTGATG 3′;

Ifnar2 (forward) 5′ATTTCCGGTCCATCTTATCAT 3′,

(Reverse) 5′ACTGAACAACGTTGTGTTCC 3′.

Results As can be seen from the figure (see Figure 1), treatment of cells with glutaryl histamine resulted in at least a two-fold increase in the synthesis of mRNA for the type I interferon receptor subunit IFNAR1 and a three-fold increase in the amount of mRNA for the subunit IFNAR2.

Example 2

Effect of glutaryl histamine on the density of interferon receptors on the surface of primary human macrophages

The 10-day primary cultures of human macrophages in 96-well plates were incubated for 24 hours in the presence of or without glutaryl histamine at the following concentrations: 0.1, 1.0, 10, or 100 ng/mL. The cells were then fixed with paraformaldehyde solution (without permeabilization), washed with a detergent-containing buffer solution (PBS-Tween), blocked with a solution of bovine serum albumin, and incubated with antibodies against interferon receptor subunits IFNAR1 or IFNAR2. The prepared product was visualized by staining with a secondary antibody labeled with horseradish peroxidase and adding substrate. Optical density was measured using an ELISA spectrophotometer.

As shown in this example (see FIG2 ), treatment of macrophages with glutaryl histamine increases the density of both chains of the interferon receptor on the cell surface, as reflected by a significant increase in the signal levels stained with IFNAR2 and IFNAR1 specific antibodies.

Example 3

Effect of glutaryl histamine on cellular sensitivity to interferon

In the case where its signal conduction is inhibited by viral infection, the density of interferon receptors on the cell surface increases and the sensitivity of cells to weak endogenous interferon signals is inevitably increased. In order to prove the sensitization of cells to interferon signals, A-549 cells were treated with 100ng/mL concentration of glutaryl histamine and incubated for 8 or 24 hours, and then cells were lysed in the presence or absence of 1 IU interferon-α (IFNα: Roche Molecular Biochemicals, Mannheim). The cell lysate was subjected to immunoblotting analysis (western blotting) by using a chemiluminescent method using a substrate (chemiluminescent reagent Super Signal West Femto chemiluminescent substrate (Thermo Fisher Scientific)) by staining the membrane with a monoclonal antibody for MxA protein (sc-50509, Santa Cruz Biotechnology). MxA protein was selected as an indicator product, which indicates the expression of interferon-stimulated genes (ISGs) after treating cells with interferon.

As can be seen from this example (see Figure 3), in cells not treated with interferon, glutaryl histamine did not induce the accumulation of the antiviral MxA protein (rows 1, 2, and 3). Interferon also did not induce the synthesis of MxA protein at a low concentration of 1 IU (row 4). At the same time, cells treated with both glutaryl histamine and interferon showed an increase in MxA protein synthesis 8 hours after incubation (row 5) and 24 hours after the start of cell treatment (row 6). Thus, treatment of cells with glutaryl histamine leads to an increase in interferon signaling and, therefore, to an increase in the biosynthesis of antiviral effector molecules.

Example 4

Therapeutic efficacy of glutaryl histamine in a mouse model of herpes meningoencephalitis

Mice were infected intracerebrally with the herpes simplex viruses HSV-1/CL and HSV-2/VN in a dose of 30 μl comprising 10 LD ₅₀ .

The therapeutic effect of glutaryl histamine was studied as follows: 200 μl of the drug was orally administered once daily to infected mice at a dose of 30 mg/kg 24, 48, 72, 96, and 120 hours after infection with the virus. Under the same conditions, a placebo (200 ml of physiological solution) was administered to the mice in the control group. After infection, the animals were monitored for 14 days, and deaths due to herpes meningoencephalitis were recorded in the treated and control groups. The activity of the drug was evaluated by comparing the mortality rate of the glutaryl histamine-treated animals with that of the control animals. The reduction in mortality of the treated animals relative to the control was expressed as a percentage.

The indices of death protection and life expectancy of treated, control (infected mice not treated with glutaryl histamine) and intact (negative control) mice are provided in Table 1 .

Treatment of HSV-1/CL virus infection with glutaryl histamine showed a statistically significant decrease in mortality (p = 0.02) (from 95% to 65%) and an increase in mean life expectancy (from 4.8 days to 8.1 days). Similarly, treatment of HSV-2/VN infected mice with glutaryl histamine resulted in a statistically significant decrease in mortality (p = 0.008) (from 85% to 45%) and an increase in mean life expectancy (from 6.1 days to 10.2 days). Statistical significance was assessed by the log-rank Mantel-Cox test. Thus, glutaryl histamine has been shown to be therapeutically effective in the treatment of herpes infections in mice.

*P=0.02

**P = 0.008

Example 5

Preparation of glutaryl histamine dosage form

The dosage forms of glutaryl histamine used according to the present invention are prepared by standard methods such as mixing, granulation, pelleting, dissolution and lyophilization processes.

Tablet form

The tablet form is prepared by using the following ingredients:

Glutaryl histamine or its pharmaceutically acceptable salt 1 to 100 mg potato starch 20 to 50 mg magnesium stearate 3mg Micro-powder silica gel 1mg lactose Up to 300mg

The ingredients were mixed and compressed to form tablets weighing 300 mg.

Gelatin capsules

The above ingredients were mixed and granulated, and the resulting granules were placed in a solid gelatin capsule in an amount of 220 mg.

suppositories

Examples of suppository formulations

Glutaryl histamine or its pharmaceutically acceptable salt 1 to 100 mg Cocoa butter Amount of suppository required

Rectal, vaginal and urethral suppositories may optionally be prepared with corresponding excipients.

Solution for injection

Examples of injection solution formulations

Glutaryl histamine or its pharmaceutically acceptable salt 1 to 100 mg Water for injection 2ml

The solution for injection can be prepared by using 0.9% sodium chloride solution, distilled water or novocaine solution as a diluent. The product forms are ampoules, bottles, syringe-tubes and "inserts".

Claims (7)

1. Use of an effective amount of glutaryl histamine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for increasing the density of interferon receptors in the treatment of diseases associated with decreased interferon receptor density, wherein the interferon receptors are interferon α (IFNα) and/or interferon β (IFNβ) receptors, wherein the diseases are selected from: hepatitis B, herpes, human papillomavirus infection, multiple sclerosis, and smoking-induced immunosuppression associated with interferon receptor degradation in smokers. 2. The use according to claim 1, wherein the increased density of the interferon receptor overcomes resistance to treatment with interferon and/or compensates for reduced interferon receptor expression levels during long-term treatment with interferon. 3. The use according to claim 1, wherein glutaric acid monohydric histamine is to be applied in a solid dosage form, and the duration of said application is from 5 days to 12 months. 4. The use according to any one of claims 1 to 3, wherein when administered once or more daily, the dose of glutaric acid monohistamine or its pharmaceutically acceptable salt in the medicament is 0.1 to 100 mg/kg human body weight per day. 5. The use according to claim 4, wherein when administered once or more daily, the dose of glutaric acid monohistamine or its pharmaceutically acceptable salt in the medicament is from 0.1 to 30 mg/kg human body weight per day. 6. The use according to claim 5, wherein when administered once or more daily, the dose of glutaric acid monohistamine or its pharmaceutically acceptable salt in the medicament is 0.3 to 10 mg/kg human body weight. 7. The use according to any one of claims 1 to 3, wherein a single dose of glutaric acid monohistamine in the medicament is 100 mg.