CN117838696A

CN117838696A - Application of ruxotinib in preparation of medicine for treating influenza virus infection

Info

Publication number: CN117838696A
Application number: CN202410029475.7A
Authority: CN
Inventors: 陈思; 陈绪林; 于杨
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2024-01-09
Filing date: 2024-01-09
Publication date: 2024-04-09

Abstract

The application relates to the technical field of Lu Suoti Ni application, in particular to application of ruxotinib in preparing a medicament for treating influenza virus infection. Such uses include avoiding or reducing infection by influenza virus; inhibiting replication of influenza virus; inhibiting the inflammatory effects of influenza virus; the survival rate of the body infected by influenza virus is improved; delay the body weight reduction rate caused by infection of influenza virus; improving or avoiding cell damage of an organism caused by infection of influenza virus; improving or avoiding the alveolar structure injury of the organism caused by infection of influenza virus; improving or avoiding myocardial tissue injury of the body caused by infection of influenza virus; or eliminate, improve or avoid otitis media in the body caused by infection of influenza virus.

Description

Application of ruxotinib in preparation of medicine for treating influenza virus infection

Technical Field

The application relates to the technical field of Lu Suoti Ni application, in particular to application of ruxotinib in preparing a medicament for treating influenza virus infection.

Background

Ruxotinib (Ruxolitinib, CAS: 941678-49-5) is the first clinically effective selective JAK1/2 inhibitor to be found today. In vitro enzymatic assays found that Lu Suoti Nii had an IC50 of 3.3nM for JAK1 and 2.8nM for JAK 2. Lu Suoti Ni has a binding force with JAK1/2 which is 6 times that of TYK2 and 30 times that of JAK 3. Ruxotinib kills tumor cells by toxic mitochondrial autophagy, induces autophagy and enhances apoptosis. Lu Suoti Ni is a targeted drug for treating myelofibrosis, and has remarkable effect in reducing splenomegaly and disease-related symptoms of patients with high risk myelofibrosis. Furthermore, patients show a good tolerance to Lu Suoti ni, which can be used for a long period of time for clinical treatment and to improve the quality of life of the patient.

Disclosure of Invention

However, the inventors of the present application found that ruxotinib has application prospects against influenza virus.

Therefore, the embodiment of the application at least discloses the following technical scheme:

in a first aspect, embodiments disclose the use of ruxotinib in the manufacture of a medicament for treating influenza virus infection. The term "anti" means interfering with the progress of infection by influenza virus; inhibiting growth of influenza virus; inhibiting replication of influenza virus; inhibiting the inflammatory effects of influenza virus; the survival rate of the body infected by influenza virus is improved; delay the body weight reduction rate caused by infection of influenza virus; improving or avoiding cell damage of an organism caused by infection of influenza virus; improving or avoiding the alveolar structure injury of the organism caused by infection of influenza virus; improving or avoiding myocardial tissue injury of the body caused by infection of influenza virus; or at least one of eliminating, ameliorating or avoiding otitis media in the body caused by infection with influenza virus.

In an embodiment of the first aspect, the influenza virus is selected from one or more of influenza a, b viruses. In some embodiments, the influenza virus is selected from at least one of influenza a virus subtype H1N1, H2N2, H3N2 and avian influenza viruses of all subtypes.

In an embodiment of the first aspect, the application is selected from: avoiding or reducing the infection rate of influenza virus; inhibiting growth of influenza virus; inhibiting replication of influenza virus; inhibiting the inflammatory effects of influenza virus; the survival rate of the body infected by influenza virus is improved; delay the body weight reduction rate caused by infection of influenza virus; improving or avoiding cell damage of an organism caused by infection of influenza virus; improving or avoiding the alveolar structure injury of the organism caused by infection of influenza virus; improving or avoiding myocardial tissue injury of the body caused by infection of influenza virus; or at least one of eliminating, ameliorating or avoiding otitis media in the body caused by infection with influenza virus.

In an embodiment of the first aspect, the inhibiting an inflammatory effect produced by an influenza virus comprises: inhibit expression of at least one of IL-8, IP-10 or MCP-1.

In a second aspect, embodiments disclose a composition. The composition comprises Lu Suoti Ni with an anti-influenza virus effective amount and pharmaceutically acceptable auxiliary materials.

In some embodiments of the second aspect, the pharmaceutically acceptable adjuvant is selected from one or more of solvents, dispersants, diluents, fillers, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, excipients, flavoring agents, and carriers. In some embodiments, the dosage form of the composition comprises a tablet, capsule, granule, drop pill, liquid formulation, decoction, suppository, gel, aerosol, or patch.

In a third aspect, the embodiments disclose an anti-influenza virus composition comprising Lu Suoti ni and other antiviral or anti-inflammatory components selected from, but not limited to, one of the following: antiviral drugs (amantadine, rimantadine, balo Sha Wei, oseltamivir, zanamivir, peramivir, ribavirin, interferon, arbidol, and adefovir); non-steroidal anti-inflammatory drugs (aspirin, ibuprofen, naproxen, diclofenac, indomethacin, ketoprofen, meloxicam, celecoxib, piroxicam, shu Linda); glucocorticoids (prednisone, methylprednisolone, dexamethasone, hydrocortisone, triamcinolone acetonide, betamethasone); other anti-inflammatory agents (colchicine, allopurinol, febuxostat, methotrexate, TNF inhibitors, IL-6 inhibitors, JAK inhibitors, selective COX-2 inhibitors). In a fourth aspect, embodiments disclose an anti-influenza virus medicament. The medicine is a solution containing the ruxotinib with the concentration not lower than 0.01 mu M.

In some examples of the cell level experiments of the third aspect, the drug tested was a solution containing ruxotinib at a concentration of not less than 0.01 μm, 0.03 μm, 0.09 μm, 0.27 μm, 0.82 μm, 2.47 μm, 7.41 μm, 22.22 μm, 66.67 μm, respectively. In some embodiments of animal experiments, the drug is ruxotinib at a concentration of not less than 2 mg/mL.

One test example found that Lu Suoti Ni was not toxic to U937 cells, but was effective in inhibiting the production of three inflammatory factors IL-8, IP-10 and MCP-1 by infection of influenza virus cells U937. In U937 cells, the EC50 of Lu Suoti Ni for IL-8, IP-10 and MCP-1 was 0.12. Mu.M, 0.07. Mu.M and 0.03. Mu.M in this order. And the selection indexes of Lu Suoti Ni to the three inflammatory factors IL-8, IP-10 and MCP-1 are 833, 1429 and 3333 respectively. The test example shows that the ruxotinib has lower toxicity and good anti-inflammatory effect.

One test example shows that Lu Suoti Ni has good treatment effect in a mouse model infected by lethal dose influenza virus, can obviously reduce the weight reduction amplitude of mice, reduce the inflammatory factor level in the alveolar lavage fluid of the mice, relieve clinical pathological changes and improve the survival time and survival rate of the mice. Meanwhile, the ruxotinib can be administered three days after infection to play a role, and is complementary with the existing antiviral drugs, so that the ruxotinib has a very large clinical application prospect.

The term "pharmaceutically acceptable adjuvant" refers to a component that does not interfere with the efficacy of the biological activity of the compound ruxotinib and that is not significantly toxic to the body at the therapeutically effective concentrations at which it is administered, including any one or a combination of at least two of solvents, dispersants, diluents, fillers, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, excipients, flavoring agents, and the like, and carriers. The use of the aforementioned components for pharmaceutically active substances is well known in the art. For example, the solvents include, but are not limited to, ethanol, dimethyl sulfoxide (DMSO), and the carriers include, but are not limited to: polyethylene glycol, polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin, lactose, terra alba, sucrose, cyclodextrin, amylose, magnesium stearate, pectin, acacia, stearic acid or lower alkyl ethers of cellulose. Each component can be used alone or in combination with a plurality of kinds.

The terms "effective amount", "therapeutically effective amount" refer to the amount of a compound, agent, formulation or composition that is required to provide, after administration, a degree of relief from one or more symptoms of the disease or disorder being treated, with the intended goal being the reduction and/or alleviation of the symptoms or cause of the disease, or any other desired change in the body. For example, a therapeutically effective amount is that amount of a composition comprising a compound disclosed herein that is required to provide clinically significant relief from a condition, and an effective amount suitable in any individual case can be determined using techniques such as a dose escalation test. The therapeutically effective amount will vary depending on the activity of the compound, the condition and severity thereof caused by the viral infection, as well as the size and health of the individual to be treated, and the like. Illustratively, a therapeutically effective amount in the mouse model can be from 1mg/kg to 150mg/kg, preferably from 1mg/kg to 100mg/kg, more preferably from 5mg/kg to 30mg/kg, such as 10mg/kg or 20mg/kg,2 times/day.

The term "treatment" and the like encompasses any therapy of a human or an animal other than a human, the treatment being able to be directed against an existing disorder, or being able to be prophylactic (prophylactic treatment), including curative, palliative or prophylactic effects. Treatment can also include curing, alleviating or preventing symptoms associated with the disease rather than acting on the underlying cause of the disease. The term "preventing" and the like includes reducing the likelihood of a disease or condition from occurring or worsening in a patient, such as during the administration period for an influenza infection that has not occurred after exposure to an influenza environment, the treatment can be for an initial infection or an infection following latent virus activation.

The compositions provided herein can be adapted for any form of administration for prophylaxis and/or treatment of influenza virus, including but not limited to oral, nasal, transdermal, intravenous and parenteral administration, preferably by the oral route. The skilled artisan can select the appropriate formulation form depending on the mode of administration, e.g., for oral administration, conventional solid and liquid formulations can be prepared. In some specific embodiments, the dosage form includes, but is not limited to: tablets, capsules, granules, dripping pills, liquid preparations, soft extracts, suppositories, gels, aerosols or patches and the like.

The compositions provided herein can be in single or multiple dose form. Alternatively, the final concentration of the ruxotinib in the composition is 0.01 μm to 200 μm. The concentration refers to the final concentration of BMS-ruxotinib in a composition, such as a liquid formulation, and the amount of ruxotinib administered at the time of treatment is adjusted according to the final concentration, dosage form and individual to be treated to ensure that a therapeutically effective amount is achieved.

Drawings

Fig. 1 is a graph showing the results of the cytotoxic and anti-inflammatory activity of the ruxotinib provided in the examples.

Fig. 2 shows the results of changes in body weight of mice infected with influenza virus treated with ruxotinib as provided in the examples.

Fig. 3 is a survival result of mice infected with influenza virus treated with ruxotinib provided in the examples.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.

Influenza viruses belong to the Orthomyxoviridae (Orthomyxoviridae), the genus influenza virus. Influenza viruses can be classified into four types A, B, C, D, also called influenza a, b, c, and d, according to the antigenicity and genetic characteristics of the virion Nucleoprotein (NP) and the matrix protein (M). The genome of influenza A virus is 8-segment single-stranded negative strand RNA, the whole virus genome is about 13.6kb, and 18 virus proteins (PB 2, PB1, PA, HA, NP, NA, M1, M2, NS1, NS2, PB1-F2, PB1-N40, PA-X, M42, NS3, PA-N155, PA-N182, PB 2-S1) can be encoded. Influenza a viruses can be further divided into 18 HA subtypes and 11 NA subtypes, depending on the difference in antigenicity of the viral particle surface glycoproteins Hemagglutinin (HA) and Neuraminidase (NA). Human influenza viruses are predominantly of the H1, H2 and H3 subtypes. However, the current highly pathogenic avian influenza which is seriously damaged is mostly the subtype H5, H7 and H9, wherein the mortality rate of the subtype H5N1 is highest. Influenza B viruses typically infect humans only and do not cause influenza pandemics worldwide. Influenza C viruses exist in a scattered form, mainly attack infants, generally do not cause influenza epidemics, and can infect people and pigs. Influenza D virus infects ruminants only.

Influenza viruses have caused multiple pandemics worldwide since the discovery at the beginning of the 20 th century, resulting in tremendous loss of human life and property. Influenza pandemics cause about 5% -15% of people worldwide to be infected, with 300-500 thousands of severe cases, which can lead to death in 25-50 thousands of severe patients. Current drugs for treating influenza are mainly neuraminidase inhibitors, and representative of such drugs are oseltamivir and zanamivir. Such drugs are effective against all known human influenza viruses and highly pathogenic avian influenza viruses. However, these antiviral drugs need to be taken within 48 hours after symptoms appear to have a good therapeutic effect. Beyond this window period, the efficacy is greatly reduced. The reason for this phenomenon is that viruses cause excessive inflammatory reactions in the middle and late stages of infection, triggering "inflammatory factor storms", and inhibiting viral replication alone is insufficient to control disease progression, so that development of drugs effective for alleviating influenza inflammation is required.

Currently, drug evaluation models for treating influenza are largely divided into in vitro models (in vitro models) and in vivo models (in vivo models). The in vitro model mainly uses various influenza virus susceptibility cell lines or influenza virus pathology related cell lines to evaluate the medicine, and has the advantages of providing a large number of cells with the same genetic character, being convenient to operate, eliminating the influence of other external factors, detecting the toxicity, effective concentration and selection index of the medicine, and providing more foundation for later mechanism research. In vivo models generally use various models of animal infection models, and the overall effect of a drug in a living animal is measured by various phenotypic indicators after drug treatment. The method has the advantage that the effect of the candidate medicine in the living body can be evaluated truly and systematically.

Some of the test cases quantitatively analyze the in vitro anti-inflammatory effects of ruxotinib using the human monocyte cell line U937, which produces three important pro-inflammatory factors, and calculate its selection index. Some test cases evaluate the in vivo anti-influenza effect of ruxotinib using a mouse model infected with a lethal dose of influenza virus.

1. Test materials

U937 cells: purchased from American Type Culture Collection (ATCC) (CRL-1593.2).

Virus strain: influenza A subtype H1N 1A/Puerto Rico/8/1934 is offered by the China academy of sciences of the Wenyujin virus institute of China.

RPMI-1640 medium, fetal Bovine Serum (FBS) was purchased from GIBCO corporation; cell titer-glo Cell activity assay kit was purchased from Promega corporation (Promega, madison, wis., USA). Varioskan LUX multifunctional microplate reader available from Thermo scientific company; CO ₂ Thermostatic cell incubators were purchased from Thermo company.

2. Cell level testing

(1) Cell culture

Resuscitating frozen U937 cells, passaging for 2 times, and performing expansion culture with RPMI-1640 complete medium (RPMI-1640 medium, 10% serum, penicillin 100U/mL, streptomycin 100 μg/mL) at inoculation density not lower than 1×10 ⁴ cell/mL, passage density not higher than 2×10 ⁶ cell/mL。

(2) Preparation of test sample solution

A DMSO solution containing 10mM ruxotinib (available from TargetMol) was prepared as a test solution.

(3) Cytotoxicity detection of ruxotinib

Drug treatment wells: u937 cells were cultured according to 1.5X10 ⁴ Cells/well (volume 100. Mu.L) were inoculated into 96-well cell culture plates, and the test sample solution was diluted to 10 concentration gradients of 200. Mu.M, 66.67. Mu.M, 22.22. Mu.M, 7.41. Mu.M, 2.47. Mu.M, 0.82. Mu.M, 0.27. Mu.M, 0.09. Mu.M, 0.03. Mu.M, 0.01. Mu.M with RPMI-1640 complete medium (RPMI-1640 medium, 10% serum, penicillin 100U/mL, streptomycin 100. Mu.g/mL), and 3 wells were provided for each concentration gradient. After 48h of incubation, the cell culture plates were centrifuged at 1500rpm/min for 3min and the supernatant discarded. To the remaining cells, 100. Mu.L of Phosphate Buffer (PBS) containing 30% Celltiter-glo reagent was added, incubated at room temperature for 30min, centrifuged at 1500rpm/min for 3min and the OD450 reading was detected with a Varioskan LUX microplate reader.

Untreated control wells: the difference from the drug treatment wells is that no test solution was added.

Cell viability (%) = (OD 450 of drug treated wells/OD 450 of untreated control wells) ×100% was calculated

As shown in FIG. 1, U937 cells were treated at a maximum concentration of 200. Mu.M by Lu Suoti Ni and diluted sequentially 3-fold, for a total of 10 dilutions. Cells showed weak toxicity when treated with 200 μm for 48 hours. Cell viability was not different from the control group when treated with 66.67 μm for 48 hours, indicating that Lu Suoti ni was not toxic to cells at this concentration.

(4) Anti-inflammatory Activity test

Drug treatment wells: u937 cells were cultured according to 1.5X10 ⁴ Cells/well (volume 100 μl) were inoculated into 96-well cell culture plates, and the infected group was added with 0.1MOI (multiplicity of infection) of PR8 influenza virus. Meanwhile, the sample solution was diluted to 10 concentration gradients with RPMI-1640 complete medium (RPMI-1640 medium, 10% serum, penicillin 100U/mL, streptomycin 100. Mu.g/mL) and added to each drug treatment well, and 3 compound wells were provided for each concentration gradient, with the final concentrations of 200. Mu.M, 66.67. Mu.M, 22.22. Mu.M, 7.41. Mu.M, 2.47. Mu.M, 0.82. Mu.M, 0.27. Mu.M, 0.09. Mu.M, 0.03. Mu.M, and 0.01. Mu.M, respectively. Culturing in a cell culture incubator at 37deg.C for 48 hr, and culturing in ELISA kit such as IL-8 (CSB-E04641 hr, huamei organism) and IP-10 (CSB)E08181h, huamei organism) and MCP-1 (CSB-E04655 h, huamei organism) were processed, and supernatants from each cell well were 10-fold diluted and added to ELISA plates for detection of IL-8, IP-10 and MCP-1 content. Finally, the OD450 absorbance in each well was read using a Varioskan LUX multifunctional microplate reader.

Virus control wells: the difference from the drug treatment wells is that no sample solution was added.

Blank control wells: the difference from the drug-treated wells is that the PR8 virus solution and the test solution were not added.

The inhibition ratio (%) of the drug to inflammatory factor in each test well was calculated as =100% - (OD 450 of drug-treated wells-OD 450 of blank wells)/(OD 450 of virus control wells-OD 450 of blank wells) ×100%

As shown in FIG. 1, ruxotinib significantly inhibited the production of IL-8, IP-10 and MCP-1, and showed dose-dependent inhibition with half-effective concentrations of EC50 of 0.12. Mu.M, 0.07. Mu.M and 0.03. Mu.M, respectively.

The drug Selection Index (SI) is used to judge the safety range of the drug effect, and a selection index of more than 1 is effective, and the safety range is larger as the index is larger. The calculation formula is as follows: si=ccs 50/EC50.

In combination with the above results, the index of selection of Lu Suoti on U937 for three inflammatory factors was 833, 1429 and 3333 in order, all much greater than 1. From this, it was shown that ruxotinib has an anti-influenza virus induced inflammatory effect.

3. Animal level testing

(1) Test materials

Test animals: SPF grade 6-8 week old BALB/c female mice were purchased from the medical laboratory animal center in Guangdong province.

(2) Preparation of test sample solution

Taking a 2mg/mL sample solution as an example:

2mg of ruxotinib (purchased from TargetMol corporation) powder was dissolved in 20. Mu.L of DMSO solution (final concentration of DMSO is 2%), after the drug was completely dissolved, 300. Mu.L of PEG300 (final concentration of PEG300 is 30%) was added and mixed uniformly to clarify it, 50. Mu.L of Tween80 (final concentration of Tween80 is 5%) was added to the above system and mixed uniformly to clarify it, 630. Mu.L of ultrapure water was added to the above system to fix the volume to 1mL, and a sample solution containing 2mg/mL of ruxotinib was obtained.

(3) Test procedure

BALB/c females at 6-8 weeks of age were anesthetized with isoparaffin and then vaccinated with 50 μl of PR8 influenza virus containing 125TCID50 by nasal drip, and randomly divided into drug group (pepicitinib) and Placebo group (Placebo). In addition, normal 6-8 week old BALB/c females were inoculated with PBS of the same volume as that of the challenged mice as a negative control group (PBS). Each group contained 10 mice.

Administration of the drug group: on day 3 post-viral infection, mice were given 2mg/mL of the test solution containing ruxotinib at a dose of 7.5mg/kg/d by gavage, 2 times daily for 5 consecutive days.

Administration of placebo: placebo (0.5% (w/v) sodium carboxymethylcellulose solution) in the same dosing regimen, dosing period and dosing volume as the drug group.

Dosing of the negative control group: PBS solution at ph=7.0 for the same dosing regimen, dosing period and dosing volume as the drug group.

Weighing each group of mice at regular time every day, observing clinical pathological state, recording and scoring, and continuously observing and recording the weight and clinical scoring of the mice every day after the administration of the drug for observing the survival condition of the mice is completed, and replacing padding, adding drinking water and food at regular time, and timely taking out dead mice until the experiment is finished. And drawing a mouse weight change curve, a clinical pathology scoring curve and a survival rate curve according to the statistical result.

(4) Test results

As shown in fig. 2, when at 500TCID ₅₀ Placebo mice at the dose of influenza virus infection gradually lose weight and die on day 9 after virus infection; while the average body weight of ruxotinib-treated mice began to recover gradually after reaching a minimum on day 9 after viral infection.

As shown in fig. 3, when at 500TCID ₅₀ After challenge with PR8 influenza virus, the placebo mice all died, i.e. survival rate was 0, while ruxotinibThe survival rate of mice in the treatment group can reach 60%, which shows that the treatment of the ruxotinib can improve the survival rate of mice infected by the lethal dose of influenza virus.

The results show that Lu Suoti Ni has obvious anti-influenza virus induced inflammation effect in mice.

The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.

Claims

1. Use of ruxotinib in the manufacture of a medicament for treating influenza virus infection.

2. The use according to claim 1, wherein the influenza virus is selected from one or more of influenza a, b viruses.

3. The use according to claim 2, wherein the influenza virus is selected from at least one of influenza a virus subtype H1N1, H2N2, H3N2 and avian influenza viruses of all subtypes.

4. The use according to claim 1, wherein the use is selected from:

avoiding or reducing the infection rate of influenza virus;

inhibiting growth of influenza virus;

inhibiting replication of influenza virus;

inhibiting the inflammatory effects of influenza virus;

the survival rate of the body infected by influenza virus is improved;

delay the body weight reduction rate caused by infection of influenza virus;

improving or avoiding cell damage of an organism caused by infection of influenza virus;

improving or avoiding the alveolar structure injury of the organism caused by infection of influenza virus;

improving or avoiding myocardial tissue injury of the body caused by infection of influenza virus; or (b)

Eliminating, improving or avoiding otitis media of organisms caused by infection of influenza viruses

At least one of (a) and (b).

5. The use of claim 4, wherein the inhibition of inflammatory effects produced by influenza virus comprises: at least one of inflammatory factor expression, inflammatory cell infiltration, and inflammatory pathological injury.

6. A composition comprising: lu Suoti Ni with anti-influenza virus effective dose and pharmaceutically acceptable auxiliary materials.

7. The composition of claim 6, wherein the pharmaceutically acceptable adjuvant is selected from one or more of solvents, dispersants, diluents, fillers, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, excipients, flavoring agents, and carriers.

8. The composition of claim 6, wherein the formulation of the composition comprises a tablet, capsule, granule, drop pill, liquid, decoction, suppository, gel, aerosol or patch.

9. An anti-influenza virus composition comprising Lu Suoti ni and other antiviral or anti-inflammatory components selected from, but not limited to, one of the following: antiviral drugs (amantadine, rimantadine, balo Sha Wei, oseltamivir, zanamivir, peramivir, ribavirin, interferon, arbidol, and adefovir); non-steroidal anti-inflammatory drugs (aspirin, ibuprofen, naproxen, diclofenac, indomethacin, ketoprofen, meloxicam, celecoxib, piroxicam, shu Linda); glucocorticoids (prednisone, methylprednisolone, dexamethasone, hydrocortisone, triamcinolone acetonide, betamethasone); other anti-inflammatory agents (colchicine, allopurinol, febuxostat, methotrexate, TNF inhibitors, IL-6 inhibitors, JAK inhibitors, selective COX-2 inhibitors).