Treatment or prevention methods for coronavirus infection

Abstract

Provided herein are methods of using voclosporin for the treatment or prevention of coronavirus infection, particularly in subjects in need of immunosuppression.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 021,239, entitled “METHODS OF TREATING OR PREVENTING VIRUS INFECTION,” filed on 7 May 2020, and U.S. Provisional Application No. 63 / 022,357, entitled “METHODS OF TREATING OR PREVENTING VIRUS INFECTION,” filed on 8 May 2020. The contents of these applications are incorporated together by reference.

[0002] Provided herein are methods for treating or preventing viral infections, particularly viral infections in subjects requiring immunosuppression. [Background technology]

[0003] Viral infections can lead to fatal illnesses. For example, coronavirus infection 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), can lead to cardiopulmonary arrest as well as the rapid onset of acute respiratory distress syndrome (ARDS). The spread of COVID-19 is difficult to contain due to its high infectivity and long, often asymptomatic, incubation period.

[0004] Certain individuals face a significantly increased risk of infection and death during viral outbreaks or pandemics. For example, many patients require intermittent, long-term, or lifelong immunosuppression for medical reasons (e.g., autoimmune diseases or solid organ transplants). These patients are susceptible to viral infections due to their immunosuppressed state. Therefore, there is a need for a drug that can maintain immunosuppressed patients in a healthy state regardless of their underlying condition while simultaneously providing antiviral effects. What we offer is an embodiment that meets such needs. [Overview of the project]

[0005] Provided herein are methods for treating or preventing a viral infection in a subject, the method comprising administering to the subject a voclosporine, for example, a therapeutically effective dose of voclosporine. In some embodiments provided herein, the subject requires immunosuppression. In some embodiments provided herein, the viral infection is mitigated by inhibition of cyclophyllin A (CypA) or a CypA-related pathway.

[0006] In some embodiments provided herein, the present invention provides a method for treating or preventing a viral infection in a subject requiring immunosuppression, the method comprising administering a therapeutically effective dose of voclosporine to the subject, wherein the viral infection is ameliorated by inhibition of cyclophyllin A (CypA) or a CypA-related pathway.

[0007] In some of the embodiments provided, the viral infection is caused by a virus that is a member of Coronaviridae.

[0008] In some of the embodiments provided, the virus is an alpha coronavirus, a beta coronavirus, a delta coronavirus, or a gamma coronavirus.

[0009] In some of the embodiments provided, the virus is human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), Middle East Respiratory Syndrome-related coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), or Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2).

[0010] In some of the embodiments provided, the virus is MERS-CoV, SARS-CoV, or SARS-CoV-2.

[0011] In some of the provided embodiments, the virus is SARS-CoV-2.

[0012] In some of the provided embodiments, the therapeutically effective amount is from about 0.1 mg / kg / day to about 2 mg / kg / day.

[0013] In some of the provided embodiments, the therapeutically effective amount is about 7.9 mg BID, about 15.8 mg BID, about 23.7 mg BID, about 31.6 mg BID, about 39.5 mg BID, about 47.4 mg BID, or about 55.3 mg BID.

[0014] In some of the provided embodiments, the therapeutically effective amount is about 7.9 mg QD, about 15.8 mg QD, about 23.7 mg QD, about 31.6 mg QD, about 39.5 mg QD, about 47.4 mg QD, about 55.3 mg QD, about 63.2 mg QD, about 71.1 mg QD, about 79.0 mg QD, about 86.9 mg QD, about 94.8 mg QD, about 102.7 mg QD, or about 110.6 mg QD.

[0015] In some of the provided embodiments, the therapeutically effective amount is equivalent to or achievable at a concentration of from about 0.05 μM to about 10 μM, from about 0.1 μM to about 5 μM, from about 0.2 μM to about 2.5 μM, from about 0.3 μM to about 1.0 μM, from about 0.4 μM to about 0.9 μM, from about 0.5 μM to about 0.8 μM, from about 0.1 μM to about 0.5 μM, or from about 0.2 μM to about 0.4 μM, or about 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or about 10.0 μM or less.

[0016] In some of the provided embodiments, the method further comprises monitoring the renal function of the subject.

[0017] In some of the provided embodiments, monitoring the subject's renal function includes the following: (a) Evaluating the estimated glomerular filtration rate (eGFR) of the subject at at least a first time point and a second time point on different days, and (b) (i) If the subject's eGFR decreases by less than a predetermined value but more than a target % between the first and second time points, decreasing the daily dose of tacrolimus administered to the subject or stopping the administration, (ii) If the subject's eGFR decreases by less than the target % between the first and second time points, continuing to administer the same daily dose of tacrolimus to the subject.

[0018] In some of the provided embodiments, the predetermined value is about 50 to about 90 ml / min / 1.73m 2 [[ID= 15]]and is

[0019] In some of the provided embodiments, the predetermined value is about 60 ml / min / 1.73m 2 and is

[0020] In some of the provided embodiments, the target % is about 20% to about 45%.

[0021] In some of the provided embodiments, the target % is about 20%.

[0022] In some of the provided embodiments, the subject has a condition associated with an autoimmune disease or transplant rejection.

[0023] In some of the provided embodiments, the subject has a condition associated with transplant rejection.

[0024] In some of the provided embodiments, the condition is associated with transplant rejection of a heart, lung, liver, kidney, pancreas, skin, intestine, or cornea.

[0025] In some of the embodiments provided, the condition relates to kidney transplant rejection.

[0026] In some of the embodiments provided, the subject has an autoimmune disease.

[0027] In some of the embodiments provided, the therapeutically effective dose of voclosporine is administered without administering a therapeutically effective dose of mycophenolate mofetil (MMF) and / or a therapeutically effective dose of corticosteroid.

[0028] In some embodiments provided, the method further comprises administering a therapeutically effective amount of mycophenolate mofetil (MMF) and / or a corticosteroid.

[0029] In some of the embodiments provided, voclosporine is administered by intra-intestinal administration (e.g., oral administration, sublingual administration, or rectal administration) or parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous injection, intravenous infusion, or inhalation / blow-in).

[0030] In some of the embodiments provided, voclosporine is administered intraintestinally (e.g., orally, sublingually, or rectally).

[0031] In some of the embodiments provided, voclosporine is administered orally.

[0032] In some of the embodiments provided, voclosporine is administered parenterally (e.g., by intravenous injection, intramuscular injection, subcutaneous injection, intravenous infusion, or inhalation / blow-in).

[0033] In some of the embodiments provided, voclosporine is administered by inhalation or inhalation.

[0034] In some of the embodiments provided, voclosporine is administered in the form of an aerosol.

[0035] In some of the embodiments provided, voclosporine is administered in a pharmaceutical composition. In some of the embodiments provided, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients. In some of the embodiments provided, the pharmaceutically acceptable excipients are independently selected from one or more alcohols, D-α-tocopherol (vitamin E), polyethylene glycol succinate (TPGS), polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), medium-chain triglycerides, gelatin, sorbitol, glycerin, yellow iron oxide, red iron oxide, titanium dioxide, and water. [Brief explanation of the drawing]

[0036] [Figure 1A] The settings for the cytopathic effect (CPE) reduction assay are shown. [Figure 1B] This study demonstrates the effect of voclosporine on reducing CPE in SARS-CoV-2. [Figure 1C] This shows a comparison of the anti-SARS-CoV-2 effects of voclosporine and tacrolimus. [Figure 2A] This study demonstrates the effect of voclosporin on reducing SARS-CoV-2 viral load in Vero E6 cells. [Figure 2B] This study demonstrates the effect of voclosporin on reducing SARS-CoV-2 viral load in Calu cells. [Figure 2C] This shows the effect of voclosporine on SARS-CoV-2 infected Vero E6 cells. The numbers below the panel indicate the concentration of the test compound (voclosporine). The fluorescence signal (green fluorescence) shows the NSP4 staining of the virus (20x objective). Exposure time was the same across conditions. [Figure 3] This shows the trough serum levels of voclosporine and tacrolimus in kidney transplant recipients. [Figure 4] This document outlines a trial scheme to evaluate the antiviral effects of voclosporine in SARS-CoV-2-positive kidney transplant patients. [Figure 5]Figures A-E show the inhibition of SARS-CoV-2 replication by various immunosuppressants and their effects on the cell viability of uninfected cells (cytotoxicity) and infected cells (antiviral effect). Voclosporine (A), cyclosporine A (B), everolimus (C), tacrolimus (D), mycophenolic acid (E). [Figure 6] Figures A-D show the effects of cyclosporine A (CsA), tacrolimus (TAC), and voclosporine (VCS) treatment on the production of infectious SARS-CoV-2 progeny by human Calu-3 cells. Experiments were performed using either glass (A and C) or plastic (B and D) laboratory equipment. Cells were infected with SARS-CoV-2 in the presence of various concentrations of VCS, CsA, and TAC using stock solutions prepared from pure powders dissolved in DMSO. The viral load in the culture medium of infected cells was measured by a plaque assay in Vero E6 cells using supernatant collected 24 hours after infection. The viability of uninfected Calu-3 cells treated with the same range of compound concentrations was measured in parallel by a colorimetric viability assay (C, n=12, D, n=3). Mean ± SD figures are shown, and the statistical significance of the differences between each concentration and solvent control was evaluated by one-way ANOVA. *, p<0.1, **, p<0.01, ***, p<0.001, ****, p<0.0001. [Figure 7]A-E show the effects of various compounds on cell viability in a CPE reduction assay of Vero E6 cells infected with SARS-CoV-2. SARS-CoV-2 replication in Vero E6 cells with various drugs (colored symbols and curves) was measured by a CPE reduction assay. For each drug, 2-fold serial dilutions of the pharmaceutical formulation were examined: VCS (A), cyclosporine A / Neoral (B), TAC / Prograf (C), EVL / Certican (D), and MMF / CellCept (E). After pre-incubation with the compound, cells were infected with SARS-CoV-2 and retained in drug-containing culture medium for 3 days, after which cell viability was measured by a colorimetric assay. The cytotoxicity of the drugs was evaluated in parallel using mock-infected compound-treated cells (gray solid line). Data points represent the mean ± SD of two independent experiments. CC50 and EC50 were identified by nonlinear regression analysis, and the regression curves are plotted on the graph (solid line). [Figure 8] Figures A and B show inhibition of SARS-CoV-2 replication in Vero E6 cells treated with either the VCS drug formulation (A) or placebo (B), as measured by a CPE reduction assay. [Figure 9] This study shows the virucidal activity of VCS powder (3.2 μM), VCS pharmaceutical formulation (3.2 μM), placebo contents (equivalent to 3.2 μM VCS), and 50% ethanol (positive control) in a plaque assay. [Figure 10] A–D show the inhibition of SARS-CoV-2 replication by various immunosuppressive compounds in a CPE reduction assay using stocks prepared from pure compound powders: VCS (A), CsA (B), TAC (C), and MPA (D). [Modes for carrying out the invention]

[0037] Provided herein are methods for treating or preventing viral infection in a subject. In some embodiments, the method includes administering to the subject a voclosporine, for example, a therapeutically effective dose of voclosporine. In some embodiments provided herein, the subject requires immunosuppression.

[0038] Viral infections can have fatal consequences, especially in high-risk populations. Between December 2019 and January 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, infected more than 90 million people worldwide. More severe cases of COVID-19 are correlated with comorbidities commonly present in solid organ transplant recipients (Zhou et al., Lancet. Mar 28 2020;395(10229):1054-1062, Huang et al., Lancet. Feb 15 2020;395(10223):497-506, Guan et al., Eur Respir J. May 2020;55(5)doi:10.1183 / 13993003.00547-2020). Furthermore, initial reports indicated that the latter group was at high risk of COVID-19-related death (Williamson et al., Nature. Aug 2020;584(7821):430-436). In particular, these individuals often require intermittent, long-term, or lifelong immunosuppression for medical reasons (e.g., autoimmune disease or solid organ transplantation), which significantly increases their risk of infection and death during viral infection.

[0039] When prescribing immunosuppressive therapy to transplant recipients, a balance must be struck between preventing rejection and controlling infection. For example, kidney transplant recipients (KTRs) are at higher risk of a more severe course of COVID-19 due to their advanced age, comorbidities, and / or persistent immunosuppression. In some cases, reducing immunosuppression is recommended for KTRs infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The contribution of immunosuppression to a more severe course of COVID-19 in KTRs needs to be considered, along with the optimal treatment approach. Various reports have shown that immunosuppression does not increase the risk of severe COVID-19 illness or death (Li et al., J Heart Lung Transplant. May 2020;39(5):496-497, Zhang et al., Eur Urol. Jun 2020;77(6):742-747, Guillen et al., Am J Transplant. Jul 2020;20(7):1875-1878, Montagud-Marrahi et al., Am J Transplant. Oct 2020;20(10):2958-2959). However, an increased mortality rate has been observed in immunosuppressed COVID-19 patients. Since the effectiveness of vaccines in KTR is unknown, there is a great need for methods to treat the target population, especially those requiring immunosuppression. Generally, COVID-19 follows a triphasic course. In other words, it begins with mild, flu-like symptoms, followed by a second stage of viral replication and pneumonia, and in a small percentage of cases, a third stage of life-threatening illness, such as a cytokine storm (Siddiqi et al., J Heart Lung Transplant. May 2020). While antiviral drugs are expected to be most effective in the early stages of the disease, immunosuppressants (e.g., steroids, tocilizumab) can be considered a treatment option for later stages of the disease to reduce inflammation. Immunosuppressive therapy can ideally prevent rejection, possess antiviral properties, and reduce (excessive) inflammation, while simultaneously initiating an effective antiviral response to prevent a severe disease progression.Some recommendations state that immunosuppression should not be completely stopped but reduced, and in some cases, steroids and CNIs are recommended based on benefits observed in vitro.

[0040] The current standard of immunosuppressive therapy in most transplant centers consists of either a calcineurin inhibitor (CNI) such as tacrolimus (TAC) or cyclosporine A (CsA), antimetabolites such as mycophenolate (MPA / MPS), and, in most cases, sustained-release steroids. mTOR inhibitors such as everolimus (EVL) may also be prescribed instead of MPA or CNI. The precise effects of immunosuppression on the course of COVID-19 are not well understood. While (excessive) immunosuppression may interfere with a proper antiviral response in the early stages of the disease, some subsequent immunosuppression may prevent the overactivation of the pathological immune system, resulting in less severe disease. Consequently, in some cases, it is recommended to reduce, rather than completely abandon, immunosuppression in SARS-CoV-2-infected KTRs, depending on the risk of rejection and the severity of the disease.

[0041] Calcineurin inhibitors (CNIs) are the basic immunosuppressants of KTR, and some have been reported to possess antiviral activity against RNA viruses. In addition to MPAs, mTOR inhibitors such as CNIs and EVL have been reported to exhibit antiviral activity against human coronaviruses such as SARS-CoV and Middle East Respiratory Syndrome (MERS-)CoV. Cyclosporine A (CsA) has been shown in vitro to have antiviral effects against a wide variety of RNA viruses, including influenza (Ma et al., Antiviral Res. 2016;133:62-72), hepatitis C (Ishii et al., J Virol. 2006;80(9):4510-4520), HIV (Braaten et al., J Virol. 1996;70(8):5170-5176), norovirus (Dang et al, Antimicrobial Agents and Chemotherapy 2017;61(11):1-17), and SARS-CoV (de Wilde et al., J Gen Virol. 2011;92:2542-2548, Pfefferle et al., PLoS Pathog 2011;7(10):1-15). While not bound by theory, the antiviral effects of CsA may involve calcineurin-dependent and cyclophilin-dependent mechanisms. In the case of SARS-CoV, the virus's Nsp1 protein interacts with cyclophilin A (CypA) to enhance cytokine release by nuclear factor (NFAT) in activated T cells in a calcineurin-dependent manner (Pfefferle et al., 2011). However, all in vitro models of CsA and its effects on viral replication have shown that it may only be effective at doses far exceeding those considered safe in humans.

[0042] Voclosporine (VCS, also known as LX214 or ISA247) is a novel calcineurin inhibitor (CNI) structurally similar to CsA, except for a novel modification of the functional group at an amino acid-1 residue of the molecule, which enhances binding to calcineurin and confers good metabolic stability. Voclosporine has been tested in psoriasis and kidney transplantation and was recently approved by the FDA for the treatment of active lupus nephritis in combination with basal immunosuppressive therapy. Observations have shown that VCS is more potent and less toxic at a therapeutic level than other immunosuppressants in its class. Furthermore, VCS has been shown to inhibit norovirus replication in a CypA-dependent manner and more effectively than CsA. This modification alters voclosporine's binding to calcineurin, and its binding affinity has been shown to increase up to 5-fold compared to CsA both in vitro and in vivo (Kuglstatter et al., Acta Cryst. 2011;D67:119-23). This modification also altered the metabolic profile of voclosporine by shifting its metabolism away from amino acid-1, the primary metabolic site of CsA. This alteration of the metabolic profile results in faster excretion of metabolites and less exposure to metabolites compared to CsA. The combination of higher potency and lower metabolite exposure of voclosporine compared to CsA leads to a better PK / PD relationship, reduced dosage, and potentially improved safety profile compared to CsA. Voclosporine has the structure shown below and is disclosed in U.S. Patent No. 7,332,472, which is incorporated herein by reference in its entirety. [ka]

[0043] Similar to CsA, voclosporine also binds to CypA (Kuglstatter et al., Acta Cryst. 2011; D67: 119-23). ​​Therefore, the method disclosed herein can provide antiviral effects while maintaining a healthy state in patients requiring immunosuppression, regardless of their underlying disease.

[0044] The embodiments provided herein are based on observations described herein, which are based on a comparison of the effects of CNIs, namely tacrolimus, cyclosporine A, and vocrosporine (VCS), as well as other immunosuppressants commonly used in KTR, on SARS-CoV-2 replication in cell-based assays. Given the uncertainty of vaccine efficacy for immunosuppressed subjects like KTR and the limited options for effective (antiviral) treatment, finding alternative solutions to protect these subjects is crucial. As demonstrated herein, CNIs showed a more potent inhibitory effect on SARS-CoV-2 replication (in cell culture) than other classes of immunosuppressants. Notably, VCS showed antiviral activity at eight times lower concentrations than TAC. The concentration of VCS that reduced SARS-CoV-2 viral load may correlate with human tolerance achievable in KTR. VCS reduced viral progeny production in human Calu-3 cells at low micromolar concentrations, more effectively than cyclosporine A and tacrolimus. The observations described herein demonstrate the potential benefits of cyclophylline-dependent CNI, particularly VCS. The results described herein show that VCS exhibits potent inhibitory activity against SARS-CoV-2 replication even at low concentrations, demonstrating its usefulness in treating viral infections such as COVID-19 in subjects requiring immunosuppression. Voclosporine also offers the advantages of higher affinity for calcineurin and lower nephrotoxicity. Voclosporine may also be distributed at higher concentrations in organs such as the lungs than in the blood, and is present at high concentrations in red blood cells. As a result, high concentrations in specific organs or cells may suppress the virus. Therefore, these results support the usefulness of VCS in treating viral infections, particularly in KTRs at risk of or infected with SARS-CoV-2.

[0045] Furthermore, the results described herein present an unexpected observation: the pharmaceutical excipients in the preparation of these immunosuppressive compounds exhibited antiviral effects in cell-based assays. Surprisingly, these results were not due to the virucidal effects of surfactants that could damage the viral envelope. In the antiviral assays described herein, it was demonstrated that excipients, which improve the solubility and bioavailability of the active compounds in the pharmaceutical formulations by using high-purity powders of various immunosuppressive compounds to avoid interference caused by excipients, also influence the results of cell-based assays. Due to the lipophilicity of voclosporine and based on the results described herein, the effects of VCS and other compounds were evaluated using glass laboratory equipment (to minimize the binding of VCS to plastic materials). These results demonstrated that VCS dose-dependently and more effectively reduced the production of SARS-CoV-2 infectivity progeny in infected Calu-3 cells than CsA and TAC, as well as other classes of immunosuppressants, such as EVL and MPA.

[0046] I. Treatment method Provided herein are methods for treating or preventing a viral infection in a subject, the method comprising administering voclosporine to the subject. Also provided herein are the uses of voclosporine in the treatment or prevention of a viral infection in a subject. In some embodiments, the methods or uses provided involve the use of a therapeutically effective dose of voclosporine. In some embodiments, the subject requires immunosuppression. In some embodiments, the viral infection is ameliorated by inhibition of cyclophyllin A (CypA) or a CypA-related pathway.

[0047] In some embodiments, the provided method is for treating or preventing a viral infection in a subject requiring treatment or prevention, the method comprising administering a therapeutically effective dose of voclosporine to the subject, wherein the viral infection is ameliorated by inhibition of cyclophyllin A (CypA) or a CypA-related pathway. In some embodiments, the subject requires immunosuppression. In some embodiments, the viral infection is ameliorated by inhibition of cyclophyllin A (CypA). In some embodiments, the viral infection is ameliorated by inhibition of a CypA-related pathway.

[0048] In some embodiments, voclosporine (also known as LX214 or ISA247), a therapeutically effective dose thereof, and / or compositions comprising voclosporine are used in the compositions, methods, and uses provided herein. Uses include such methods of voclosporine or compositions comprising it, e.g., treatments, and treatments, e.g., use in treatment plans, and use of voclosporine or compositions comprising it in the preparation of agents for performing such treatments and treatments. Also provided are voclosporine or compositions comprising it for use in treating or preventing viral infections, reducing viral replication, improving symptoms associated with viral infections, or reducing disease severity or mortality. In some embodiments, such uses include performing the methods or treatments described herein, e.g., any treatment or treatment plan. In some embodiments provided herein, voclosporine or compositions comprising it are administered as monotherapy, e.g., without the administration of one or more further agents. In some embodiments provided herein, voclosporine or compositions comprising it are administered without the administration of MMF and / or corticosteroids. In some of the embodiments provided, voclosporine or a composition containing the same is administered without administering a therapeutically effective amount of MMF and / or a therapeutically effective amount of corticosteroids.

[0049] In some embodiments, the method or use is for treating a viral infection or a viral infection. In some embodiments, the method or use is for preventing a viral infection or a viral infection. In some embodiments, the method includes treating the viral infection. In some embodiments, the method includes preventing the viral infection.

[0050] In some embodiments, the viral infection is caused by a virus that is a member of Coronaviridae (e.g., alpha coronavirus, beta coronavirus, delta coronavirus, or gamma coronavirus), Orthomyxoviridae (e.g., influenza virus), Flaviviridae (e.g., flavivirus or hepacivirus), or Caliciviridae (e.g., norovirus).

[0051] In some embodiments, the viral infection is caused by a virus that is a member of the Coronaviridae family. In some embodiments, the virus is an alpha coronavirus (e.g., HCoV-229E or HCoV-NL63), a beta coronavirus (e.g., HCoV-OC43, HCoV-HKU1, MERS-CoV, SARS-CoV, or SARS-CoV-2), a delta coronavirus, or a gamma coronavirus.

[0052] In some embodiments, the virus is alphacoronavirus. In some embodiments, the virus is HCoV-229E or HCoV-NL63. In some embodiments, the virus is HCoV-229E. In some embodiments, the virus is HCoV-NL63.

[0053] In some embodiments, the virus is a beta-coronavirus. In some embodiments, the virus is HCoV-OC43, HCoV-HKU1, MERS-CoV, SARS-CoV, or SARS-CoV-2. In some embodiments, the virus is MERS-CoV, SARS-CoV, or SARS-CoV-2. In some embodiments, the virus is HCoV-OC43. In some embodiments, the virus is HCoV-HKU1. In some embodiments, the virus is HMERS-CoV. In some embodiments, the virus is SARS-CoV. In some embodiments, the virus is SARS-CoV-2.

[0054] In some embodiments, the virus is a delta coronavirus. In some embodiments, the virus is a gamma coronavirus.

[0055] In some embodiments, the viral infection is caused by a virus that is a member of the Orthomyxoviridae family (e.g., influenza virus).

[0056] In some embodiments, the viral infection is caused by a virus that is a member of the Flaviviridae family. In some embodiments, the virus is a flavivirus. In some embodiments, the virus is a hepacivirus. In some embodiments, the virus is hepacivirus C.

[0057] In some embodiments, the viral infection is caused by a virus that is a member of the Caliciviridae family. In some embodiments, the virus is norovirus.

[0058] II. Dosage In some embodiments of the methods and uses provided herein, voclosporine is administered four times a day, three times a day, twice a day, or once a day. In some embodiments, voclosporine is administered four times a day. In some embodiments, voclosporine is administered three times a day. In some embodiments, voclosporine is administered twice a day. In some embodiments, voclosporine is administered once a day.

[0059] In some embodiments, the daily dose of voclosporine is approximately 1 mg to 250 mg, approximately 5 mg to 250 mg, approximately 10 mg to 250 mg, approximately 50 mg to 250 mg, approximately 100 mg to 250 mg, approximately 150 mg to 250 mg, approximately 200 mg to 250 mg, 1 mg to 200 mg, approximately 5 mg to 200 mg, approximately 10 mg to 200 mg, approximately 50 mg to 200 mg, approximately 100 mg to The dosages are approximately 200 mg, approximately 150 mg to approximately 200 mg, approximately 1 mg to approximately 150 mg, approximately 5 mg to approximately 150 mg, approximately 10 mg to approximately 150 mg, approximately 50 mg to approximately 150 mg, approximately 100 mg to approximately 150 mg, approximately 1 mg to approximately 100 mg, approximately 5 mg to approximately 100 mg, approximately 50 mg to approximately 100 mg, approximately 1 mg to approximately 50 mg, approximately 5 mg to approximately 50 mg, or approximately 10 mg to approximately 50 mg. In some embodiments, the daily dose of voclosporine is approximately 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, or 250 mg. In some embodiments, the daily dose of voclosporine is at least about 1 mg, at least about 5 mg, at least about 10 mg, at least about 20 mg, at least about 30 mg, at least about 40 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 110 mg, at least about 120 mg, at least about 130 mg, at least about 140 mg, at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, or at least about 200 mg.

[0060] In some embodiments, the dose of voclosporine is approximately 0.1 mg / kg / day to approximately 2 mg / kg / day, approximately 0.5 mg / kg / day to approximately 2 mg / kg / day, approximately 1 mg / kg / day to approximately 2 mg / kg / day, approximately 1.5 mg / kg / day to approximately 2 mg / kg / day, approximately 0.1 mg / kg / day to approximately 1.5 mg / kg / day, approximately 0.5 mg / kg / day to approximately 1.5 mg / kg / day, approximately 1 mg / kg / day to approximately 1.5 mg / kg / day, approximately 0.1 mg / kg / day to approximately 1.0 mg / kg / day, approximately 0.5 mg / kg / day to approximately 1.0 mg / kg / day, or approximately 0.1 mg / kg / day to approximately 0.5 mg / kg / day. In some embodiments, the dose of voclosporine is about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0 mg / kg / day. In some embodiments, the dose of voclosporine is at least about 0.1, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1.0, at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, or at least about 2.0 mg / kg / day.

[0061] In some embodiments, the appropriate dose is about 7.9 mg units. In some embodiments, the dose of voclosporine is about 7.9 mg QD, about 15.8 mg QD, about 23.7 mg QD, about 31.6 mg QD, about 39.5 mg QD, about 47.4 mg QD, about 55.3 mg QD, about 63.2 mg QD, about 71.1 mg QD, about 79.0 mg QD, about 86.9 mg QD, about 94.8 mg QD, about 102.7 mg QD, or about 110.6 mg QD. In some embodiments, the dose of voclosporine is about 7.9 mg BID, about 15.8 mg BID, about 23.7 mg BID, about 31.6 mg BID, about 39.5 mg BID, about 47.4 mg BID, or about 55.3 BID.

[0062] In some embodiments, the blood trough level is about 25 to about 60 ng / mL. In some embodiments, the blood trough level is about 25, about 30, about 35, about 40, about 45, about 50, about 55, or about 60 ng / mL.

[0063] In some embodiments, the therapeutically effective dose is equivalent to, can be estimated to be, can be achieved, or is equivalent to, a concentration of approximately 0.05 μM to approximately 10 μM, approximately 0.1 μM to approximately 5 μM, approximately 0.2 μM to approximately 2.5 μM, approximately 0.3 μM to approximately 1.0 μM, approximately 0.4 μM to approximately 0.9 μM, approximately 0.5 μM to approximately 0.8 μM, approximately 0.1 μM to approximately 0.5 μM, or approximately 0.2 μM to approximately 0.4 μM. max This is an amount that can be achieved as follows. In some embodiments, the therapeutically effective dose is equivalent to, can be estimated to be, can be achieved, or can be estimated to be equivalent to, or can be estimated to be equivalent to, or can be estimated to be equivalent to, or can be estimated to be equivalent to, or can be achieved maxThis is an amount that can be achieved. In some embodiments, the therapeutically effective amount is equivalent to, can be estimated to be, can be achieved, or is equivalent to a concentration of about 0.2 μM. max This is an amount that can be achieved. In some embodiments, the therapeutically effective amount is equivalent to, can be estimated to be, can be achieved, or has a concentration of about 0.3 μM. max This is an amount that can be achieved. In some embodiments, the therapeutically effective amount is equivalent to, can be estimated to be, can be achieved, or has a concentration of about 0.4 μM. max This is an amount that can be achieved. In some embodiments, the therapeutically effective amount is equivalent to, can be estimated to be, can be achieved, or is equivalent to a concentration of about 0.5 μM. max It is a quantity that can be achieved as such.

[0064] A. Dosage adjustment In some embodiments of the methods and uses disclosed herein, the methods or treatments further include or involve monitoring the renal function of the subject. In some embodiments of the methods disclosed herein, the methods further include monitoring the renal function of the subject. One important parameter used to assess the desirability of dose reduction is estimated glomerular filtration rate (eGFR) using the CKD-EP1 formula or other suitable method. A decrease in eGFR is a negative side effect that may occur during treatment. If the decrease is too extreme, the dose should be changed.

[0065] In some embodiments, monitoring the renal function of the subject includes: (a) Evaluate the estimated glomerular filtration rate (eGFR) of the subject at at least a first and second point in time on different days, and (b)(i) If the eGFR of the subject decreases below a predetermined value but exceeds the target % between the first and second time points, the daily dosage of voriconazole administered to the subject is decreased or the administration is stopped. (ii) If the eGFR of the subject decreases less than the target % between the first and second time points, the administration of the same daily dosage of voriconazole to the subject is continued.

[0066] In some embodiments, the first time point is before the start of treatment, at the start of treatment, or during treatment. In some embodiments, the first time point is the first day of treatment before any administration of voriconazole.

[0067] In some embodiments, the predetermined value is about 50 to about 90 ml / min / 1.73m 2 . In some embodiments, the predetermined value is about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, or about 90 ml / min / 1.73m 2 .

[0068] In some embodiments, the target % is about 20% to about 45%. In some embodiments, the target % is about 20%, about 25%, about 30%, about 35%, about 40%, or about 45%.

[0069] B. Route of Administration In some embodiments of the methods and uses disclosed herein, voriconazole can be administered in any suitable form that provides a level of voriconazole sufficient to treat or prevent viral infection, by any suitable route, such as enteral administration (e.g., oral administration, sublingual administration, or rectal administration) or parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous injection, intravenous infusion, or inhalation / blowing).

[0070] In some embodiments, voclosporine is administered by intraintestinal administration. Exemplary routes of intraintestinal administration include, but are not limited to, oral administration, sublingual administration, and rectal administration (e.g., via the rectum). In some embodiments, the intraintestinal administration includes oral administration. In some embodiments, the intraintestinal administration includes sublingual administration. In some embodiments, the intraintestinal administration includes rectal administration.

[0071] In some embodiments, voclosporine is administered by parenteral administration. Exemplary routes of parenteral administration include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, intravenous infusion, and inhalation / blow-in. In some embodiments, the parenteral administration includes intravenous injection. In some embodiments, the parenteral administration includes intramuscular injection. In some embodiments, the parenteral administration includes subcutaneous injection. In some embodiments, the parenteral administration includes intravenous infusion. In some embodiments, the parenteral administration includes inhalation / blow-in.

[0072] In some embodiments, voclosporine is administered by inhalation or inhalation. Exemplary types of preparations for inhalation and / or inhalation include, but are not limited to, sprays, aerosols, mists, capsules, powders, or cartridges for use with inhalers or inhalers, and solutions / suspensions for spraying. In some embodiments, voclosporine is administered in the form of an aerosol, spray, mist, or powder. In some embodiments, voclosporine is administered in the form of an aerosol. Examples of various types of devices for administration by inhalation or inhalation include, but are not limited to, nebulizers, medium-dose inhalers (MDIs), and dry powder inhalers.

[0073] C. In some embodiments of the methods and uses provided herein, the method or treatment also includes administering a therapeutically effective amount of mycophenolate mofetil (MMF) and / or a corticosteroid. In some embodiments of the methods disclosed herein, the method also includes administering a therapeutically effective amount of MMF and / or a corticosteroid. In some embodiments, the method includes administering a therapeutically effective amount of MMF. In some embodiments, the method includes administering a therapeutically effective amount of a corticosteroid.

[0074] In some embodiments, the method includes administering voclosporine without a therapeutically effective dose of MMF and / or a therapeutically effective dose of corticosteroid.

[0075] In some embodiments of the methods and uses provided herein, the method or treatment also includes administering a therapeutically effective dose of an additional antiviral agent. In some embodiments, the method also includes administering a therapeutically effective dose of an additional antiviral agent. In some embodiments, the additional antiviral agent is remdesivir, lopinavir / ritonavir, IFN-α, lopinavir, ritonavir, penciclovir, galidesivir, disulfiram, darunavir, cobicistat, ASC09F, disulfiram, nafamostat, griffiscin, arispolivir, chloroquine, hydroxychloroquine, nitazoxanide, baloxavir marboxil, oseltamivir, zanamivir, peramivir, amantadine, rimantadine, favipiravir, laninamivir, ribavirin, umifenovir, or any combination thereof. In some embodiments, the antiviral agent is chloroquine. In some embodiments, the antiviral agent is hydroxychloroquine. In some embodiments, the antiviral agent is remdesivir.

[0076] III. Composition In some embodiments of the methods and uses provided herein, the method or treatment comprises administering a composition comprising vocrossporine, for example, a pharmaceutical composition or a therapeutic composition. In some embodiments, the method disclosed herein comprises administering a composition comprising vocrossporine. In some embodiments, the composition comprises a mixture of isomers of vocrossporine and its Z isoform. In some embodiments, the isomer mixture comprises at least about 99% by weight, about 98% by weight, about 97% by weight, about 96% by weight, about 95% by weight, about 94% by weight, about 93% by weight, about 92% by weight, about 91% by weight, about 90% by weight, about 80% by weight, about 70% by weight, about 60% by weight, about 50% by weight, about 40% by weight, about 30% by weight, about 20% by weight, or about 10% by weight of vocrossporine. In some embodiments, the isomer mixture comprises at least 95% by weight of vocrossporine.

[0077] In some embodiments of the methods and uses provided, the composition comprising the vocrossporine is a pharmaceutical formulation comprising or comprising the vocrossporine. In some embodiments, the pharmaceutical composition comprising the vocrossporine comprises one or more pharmaceutically acceptable excipients, buffers, carriers and / or media.

[0078] In some embodiments, the composition comprises conventional pharmaceutical carriers and excipients suitable for the intended type of administration.

[0079] In some embodiments, the composition containing vocrossporine may be formulated in a pharmaceutically acceptable buffer, e.g., a buffer containing a pharmaceutically acceptable carrier or medium. Generally, the pharmaceutically acceptable carrier or medium, e.g., those contained in the pharmaceutically acceptable buffer, may be any known in the art. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995) describes compositions and formulations suitable for the pharmaceutically acceptable delivery of one or more therapeutic compounds. pharmaceutically acceptable compositions are generally prepared with consideration to regulatory authority or other agency approvals, prepared in accordance with generally accepted pharmacopoeias for use in animals and humans.

[0080] A pharmaceutical composition may include a carrier administered with the compound, such as a diluent, adjuvant, excipient, or medium. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such a composition generally contains a therapeutically effective amount of the compound in a purified form, along with an appropriate amount of carrier, providing a form for appropriate administration to the patient. Such pharmaceutical carriers may be sterile solutions, such as water, and oils, including those of petroleum, animal, plant, or synthetic origin. When the pharmaceutical composition is administered intravenously, water is the usual carrier. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly for injectable formulations. The composition may also optionally contain small amounts of wetting or emulsifying agents or pH buffers. Typically, compositions containing the compound are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, for example, Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126). Generally, the formulation method is a function of the route of administration.

[0081] In some embodiments, pharmaceutically acceptable excipients, buffers, carriers and / or media include one or more of the following: alcohol, D-α-tocopherol (vitamin E), polyethylene glycol succinate (TPGS), polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), medium-chain triglycerides, gelatin, sorbitol, glycerin, yellow iron oxide, red iron oxide, titanium dioxide, and water. Various formulations of voclosporine mixtures are also described in U.S. Patents 7,060,672, 7,429,562, and 7,829,533.

[0082] IV. Patient population In some embodiments, subjects treated according to the methods and uses provided herein include subjects at risk of viral infection or who are infected with a virus. In some embodiments, subjects treated according to the methods and uses provided herein include subjects requiring immunosuppression. In some embodiments, the subject requires immunosuppression and is at risk of viral infection or is infected with a virus. In some embodiments, subjects treated according to the methods and uses provided herein include, for example, subjects requiring immunosuppression due to the risk of graft rejection. In some embodiments, the subject is a candidate for transplantation, such as organ transplantation, tissue transplantation or cell transplantation, and the subject requires immunosuppression.

[0083] In some embodiments, the subjects involved in the methods disclosed herein have an autoimmune disease or a condition related to graft rejection. In some embodiments provided, the subjects are kidney transplant recipients (KTRs).

[0084] In some embodiments, the subject has a condition related to graft rejection. In some embodiments, the condition is related to rejection of heart, lung, liver, kidney, pancreas, skin, intestine, or corneal grafts. In some embodiments, the condition is related to heart graft rejection. In some embodiments, the condition is related to lung graft rejection. In some embodiments, the condition is related to liver graft rejection. In some embodiments, the condition is related to kidney graft rejection. In some embodiments, the condition is related to pancreas graft rejection. In some embodiments, the condition is related to skin graft rejection. In some embodiments, the condition is related to intestinal graft rejection. In some embodiments, the condition is related to corneal graft rejection.

[0085] In some embodiments, the subject has an autoimmune disease. Examples of autoimmune diseases include autoimmune hematological disorders (e.g., hemolytic anemia, aplastic anemia, pure red cell aplasia and idiopathic thrombocytopenia), systemic lupus erythematosus, lupus nephritis, polychondritis, sclerosis, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Stevens-Johnson syndrome, idiopathic sprue, and (autoimmune) inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease). This includes, but is not limited to, endocrine eye disorders, Graves' disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes mellitus (type 1 diabetes mellitus), uveitis (anterior and posterior), keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, psoriatic arthritis, glomerulonephritis (with and without nephrotic syndrome, including idiopathic nephrotic syndrome or minimal change nephrotic syndrome), and juvenile dermatomyositis.

[0086] V. Definition As used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural form unless otherwise explicitly stated in the context.

[0087] Where used herein, unless otherwise specified, the terms “about” and “approximately” when used in relation to dosage or quantity intend to mean a dosage or quantity of no more than 10%, 5%, 4%, 3%, 2%, 1%, or 0.5% of the specified dosage or quantity.

[0088] As used herein, “therapeutic dose” refers to the amount that produces a desired pharmacological and / or physiological effect on the condition in question. This effect may be prophylactic, in that it completely or partially prevents the condition or its symptoms, and / or therapeutic, in that it partially or completely cures the condition and / or any side effects resulting from it.

[0089] The terms “to treat,” “to treat,” and “treatment” refer to an approach to obtain an outcome, including beneficial or desired clinical outcomes. Beneficial or desired outcomes include, but are not limited to, one or more of the following: reducing one or more symptoms of a disease or disorder; reducing the severity of a disease or disorder; stabilizing a disease or disorder (e.g., preventing or delaying the worsening of a disease or disorder); delaying the onset or recurrence of a disease or disorder; delaying or slowing the progression of a disease or disorder; improving the state of a disease or disorder; providing remission (partial or complete) of a disease or disorder; reducing the dose of one or more other medications required to treat a disease or disorder; enhancing the effect of another medication used to treat a disease or disorder; delaying the progression of a disease or disorder; improving quality of life; and / or extending the patient’s survival. Similarly, “treatment” includes mitigating the pathological consequences of a disease or disorder. The methods of this disclosure aim to achieve any one or more of these forms of treatment.

[0090] The term "subject" refers to animals including, but not limited to, primates (e.g., humans), monkeys, cattle, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, or mice. The terms "subject" and "patient" are used herein synonymously to refer to mammalian subjects such as humans.

[0091] VI. Exemplary Embodiments The embodiments provided are as follows: 1. A method for treating or preventing a viral infection in a subject, comprising administering a therapeutically effective amount of voclosporine to the subject.

[0092] 2. The method according to Embodiment 2, wherein the subject requires immunosuppression.

[0093] 3. The method according to Embodiment 1 or 2, wherein the viral infection is ameliorated by inhibition of cyclophyllin A (CypA) or a CypA-related pathway.

[0094] 4. A method for treating or preventing a viral infection in a subject requiring immunosuppression, comprising administering a therapeutically effective amount of voclosporine to the subject, wherein the viral infection is improved by inhibition of cyclophyllin A (CypA) or a CypA-related pathway.

[0095] 5. A composition comprising voclosporine for use in the treatment or prevention of a viral infection in a subject, wherein the composition comprises a therapeutically effective amount of voclosporine and is administered to the subject.

[0096] 6. The composition for use according to Embodiment 5, wherein the subject requires immunosuppression.

[0097] 7. The composition for use according to Embodiment 5 or 6, wherein the viral infection is ameliorated by inhibition of cyclophyllin A (CypA) or a CypA-related pathway.

[0098] 8. A composition comprising voclosporine for use in the treatment or prevention of viral infection in a subject requiring immunosuppression, the composition comprising a therapeutically effective amount of voclosporine, the composition administered to the subject, wherein the viral infection is improved by inhibition of cyclophyllin A (CypA) or a CypA-related pathway.

[0099] 9. The use of voclosporine in the treatment or prevention of a viral infection in a subject, wherein the subject is administered a therapeutically effective dose of voclosporine.

[0100] 10. The use of voclosporine in the manufacture of a drug for the treatment or prevention of a viral infection in a subject, wherein the drug comprises a therapeutically effective amount of voclosporine and is administered to the subject.

[0101] 11. The use according to Embodiment 9 or 10, wherein the subject requires immunosuppression.

[0102] 12. The use according to any one of embodiments 9 to 11, wherein the viral infection is ameliorated by inhibition of cyclophyllin A (CypA) or a CypA-related pathway.

[0103] 13. The use of voclosporine in the treatment or prevention of a viral infection in a subject requiring immunosuppression, wherein the subject is administered a therapeutically effective dose of voclosporine, and the viral infection is improved by inhibition of cyclophyllin A (CypA) or a CypA-related pathway.

[0104] 14. The use of voclosporine in the manufacture of a drug for the treatment or prevention of viral infection in a subject requiring immunosuppression, wherein the drug comprises a therapeutically effective amount of voclosporine and is administered to the subject, the use wherein the viral infection is improved by inhibition of cyclophyllin A (CypA) or a CypA-related pathway.

[0105] 15. The method, composition for use, or use according to any one of Embodiments 1 to 14, wherein the viral infection is caused by a virus that is a member of the Coronaviridae family.

[0106] 16. The method, composition for use, or use according to Embodiment 15, wherein the virus is an alpha coronavirus, beta coronavirus, delta coronavirus, or gamma coronavirus.

[0107] 17. The method, composition for use, or use according to Embodiment 15 or 16, wherein the virus is human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), Middle East Respiratory Syndrome-related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0108] 18. The method, composition for use, or use according to any one of Embodiments 15 to 17, wherein the virus is MERS-CoV, SARS-CoV, or SARS-CoV-2.

[0109] 19. The method, composition for use, or use according to any one of Embodiments 15 to 18, wherein the virus is SARS-CoV-2.

[0110] 20. The method, composition for use, or use according to any one of Embodiments 1 to 19, wherein the therapeutically effective dose is approximately 0.1 mg / kg / day to approximately 2 mg / kg / day.

[0111] 21. The method, composition for use, or use according to any one of Embodiments 1 to 20, wherein the therapeutically effective dose is approximately 7.9 mg BID, approximately 15.8 mg BID, approximately 23.7 mg BID, approximately 31.6 mg BID, approximately 39.5 mg BID, approximately 47.4 mg BID, or approximately 55.3 mg BID.

[0112] 22. The method, composition for use, or use according to any one of Embodiments 1 to 20, wherein the therapeutically effective dose is approximately 7.9 mg QD, approximately 15.8 mg QD, approximately 23.7 mg QD, approximately 31.6 mg QD, approximately 39.5 mg QD, approximately 47.4 mg QD, approximately 55.3 mg QD, approximately 63.2 mg QD, approximately 71.1 mg QD, approximately 79.0 mg QD, approximately 86.9 mg QD, approximately 94.8 mg QD, approximately 102.7 mg QD, or approximately 110.6 mg QD.

[0113] 23. The therapeutically effective dose is approximately 0.05 μM to 10 μM, approximately 0.1 μM to 5 μM, approximately 0.2 μM to 2.5 μM, approximately 0.3 μM to 1.0 μM, approximately 0.4 μM to 0.9 μM, approximately 0.5 μM to 0.8 μM, approximately 0.1 μM to 0.5 μM, or approximately 0.2 μM to 0.4 μM, or approximately 0.05, approximately 0.1, approximately 0.15, approximately 0.2, approximately 0.25, approximately 0.3, approximately 0.35, approximately 0.4, approximately 0.45, approximately A composition for use, or use according to any one of Embodiments 1 to 20, which is equivalent to, or can achieve, a concentration of 0.5, approximately 0.55, approximately 0.6, approximately 0.7, approximately 0.8, approximately 0.9, approximately 1.0, approximately 1.5, approximately 2.0, approximately 2.5, approximately 3.0, approximately 3.5, approximately 4.0, approximately 4.5, approximately 5.0, approximately 6.0, approximately 7.0, approximately 8.0, approximately 9.0, or approximately 10.0 μM or less.

[0114] 24. The method, composition for use, or use according to any one of Embodiments 1 to 23, wherein the renal function of the subject is monitored.

[0115] 25. Monitoring of the target renal function by the method, composition for use, or use described in Embodiment 24, which includes: (a) Evaluate the estimated glomerular filtration rate (eGFR) of the subject at at least a first and second point in time on different days, and (b)(i) If the eGFR of the subject falls below a predetermined value by more than the target % between the first and second time points, reduce the daily dose of voclosporine administered to the subject or discontinue administration. (ii) If the subject's eGFR falls below the target percentage between the first and second time points, continue administering the same daily dose of voclosporine to the subject.

[0116] 26. The method, composition for use, or use according to Embodiment 25, wherein the predetermined value is approximately 50 to approximately 90 ml / min / 1.73 m2.

[0117] 27. The method, composition for use, or use according to Embodiment 25 or 26, wherein the predetermined value is approximately 60 ml / min / 1.73 m2.

[0118] 28. The method, composition for use, or use according to any one of Embodiments 25 to 27, wherein the target percentage is approximately 20% to approximately 45%.

[0119] 29. The method, composition for use, or use according to any one of Embodiments 25 to 28, wherein the target percentage is approximately 20%.

[0120] 30. The method, composition for use, or use according to any one of Embodiments 1 to 29, wherein the subject has a condition related to an autoimmune disease or graft rejection.

[0121] 31. The method, composition for use, or use according to any one of Embodiments 1 to 30, wherein the subject has a condition related to graft rejection.

[0122] 32. The method, composition for use, or use according to Embodiment 30 or 31, relating to a rejection reaction of a heart, lung, liver, kidney, pancreas, skin, intestine, or corneal graft.

[0123] 33. The method, composition for use, or use according to any one of Embodiments 30 to 32, wherein the condition is related to kidney transplant rejection.

[0124] 34. The method, composition for use, or use described in Embodiment 30 for a subject having an autoimmune disease.

[0125] 35. The method, composition for use, or use according to any one of Embodiments 1 to 34, wherein the therapeutically effective amount of voclosporine is administered without administering a therapeutically effective amount of mycophenolate mofetil (MMF) and / or a corticosteroid.

[0126] 36. The method, composition for use, or use according to any one of Embodiments 1 to 34, further comprising administering a therapeutically effective amount of mycophenolate mofetil (MMF) and / or a therapeutically effective amount of a corticosteroid.

[0127] 37. A composition for use, or use according to any one of Embodiments 1 to 36, wherein voclosporine is administered by intestinal administration, oral administration, sublingual administration, or rectal administration, parenteral administration, intravenous injection, intramuscular injection, subcutaneous injection, intravenous infusion, or inhalation / blow-in.

[0128] 38. The method, composition for use, or use according to Embodiment 37, wherein voclosporine is administered by intraintestinal, oral, sublingual, or rectal administration.

[0129] 39. A composition for use, or use of voclosporine according to Embodiment 37 or 38, administered by oral administration.

[0130] 40. The method, composition for use, or use according to Embodiment 37, wherein voclosporine is administered by parenteral administration, intravenous injection, intramuscular injection, subcutaneous injection, intravenous infusion, or inhalation / blow-in.

[0131] 41. The method, composition for use, or use of voclosporine according to Embodiment 37 or 40, in which voclosporine is administered by inhalation or inhalation.

[0132] 42. The method, composition for use, or use according to Embodiment 41, wherein voclosporine is administered in aerosol form.

[0133] 43. A method, composition for use, or use according to any one of Embodiments 1 to 42, wherein voclosporine is administered in a pharmaceutical composition.

[0134] 44. The method, composition for use, or use according to Embodiment 43, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0135] 45. The method, composition for use, or use according to Embodiment 44, wherein the pharmaceutically acceptable excipient is independently selected from one or more comprising alcohol, D-α-tocopherol (vitamin E), polyethylene glycol succinate (TPGS), polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), medium-chain triglycerides, gelatin, sorbitol, glycerin, yellow iron oxide, red iron oxide, titanium dioxide, and water.

[0136] 46. ​​The method, composition for use, or use according to any one of Embodiments 1 to 45, wherein the viral load decreases in the subject after administration of voclosporine.

[0137] 47. A method, composition for use, or use according to any one of Embodiments 1 to 46, wherein the survival of the subject is prolonged after administration of voclosporine. [Examples]

[0138] VII. Examples The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0139] Example 1 To investigate candidate compounds that inhibit the cytopathic effects (CPE) of SARS-CoV-2, Vero E6 cells (African green monkey kidney epithelial cells) were pre-incubated with voclosporine, cyclosporine A (CsA), or tacrolimus, then infected with SARS-CoV-2, and their survival rates were subsequently evaluated.

[0140] method Vero E6 cells were grown in 96-well plates and pre-incubated for 60 minutes with culture medium, media, and either voclosporine (0.8–100 μM), CsA (0.8–100 μM), or tacrolimus (0.8–100 μM). The cells were then left uninfected for 60 minutes in the presence of the indicated concentrations of each compound, or infected with SARS-CoV-2 at a multiple of infection (MOI) of 0.015. Subsequently, the virus was removed from the medium, the cells were washed with PBS, and further incubated in fresh medium containing the respective compound until untreated infected control cells showed complete CPE (3 days). At the end of incubation, the cells were subjected to an MTS viability assay, followed by fixation and absorbance analysis using a plate reader (Figure 1A). Cell viability was assessed using the MTS assay to identify both compound cytotoxicity (uninfected cells) and viral cytotoxicity.

[0141] result Tacrolimus did not protect infected cells from virus-induced cytopathic effects. This was evident from the observation of compound-related cytotoxicity after using doses up to 25 μM. Alternatively, the protective effect of CsA occurred at 1.6–12 μM, after which the compound exhibited cytotoxicity. Treatment of infected cells with 0.8 μM voclosporine resulted in viral protection with the same level of cell viability as mock-infected cells.

[0142] As shown in Figure 1B, voclosporine did not show toxicity to uninfected cells at concentrations below 2 μM, and batch 4 voclosporine did not show toxicity to uninfected cells at concentrations up to 10 μM. On the other hand, all test batches of voclosporine promoted dose-dependent inhibition of SARS-CoV-2 CPE at concentrations from 0.01 μM to 1 μM, as indicated by increased viability of infected cells. For all batches of voclosporine, treatment at 0.8 μM was effective in maintaining cell viability to levels seen in uninfected cells.

[0143] As shown in Figure 1C, vocrosporine is found in tacrolimus (EC 50 Compared to approximately 25 μM, this is a much lower concentration (EC 50 SARS-CoV-2 was inhibited at approximately 0.4 μM. Furthermore, tacrolimus treatment induced compound-related cytotoxicity at concentrations above 25 μM (data not shown). These results indicate that voclosporine demonstrated in vitro inhibition of SARS-CoV-2 within a concentration range that did not affect the viability of the tested cells.

[0144] Example 2 To investigate the effects of voclosporin on SARS-CoV-2 in vitro, Vero E6 cells or Calu cells (human bronchial airway epithelial cells) were pre-incubated with voclosporin, infected with SARS-CoV-2, then collected, and viral load was measured by plaque assay.

[0145] method Vero E6 cells and Calu cells were grown in 96-well plates and pre-incubated for 60 minutes with 0.01 μM–10.00 μM voclosporin. Next, the cells were infected with SARS-CoV-2 at MOI1 for 60 minutes in the presence of the indicated concentrations of voclosporin. Subsequently, the virus was removed from the medium, the cells were washed with PBS, and incubated for a further 16 hours in fresh medium containing the indicated concentrations of voclosporin. At the end of incubation, the medium was collected and the viral load was measured by a plaque assay. To illustrate the amount of infection, each cell was also subjected to staining for viral NSP4, which is visualized under a fluorescence microscope.

[0146] result As shown in Figures 2A-2C, voclosporine dose-dependently inhibited the viral load of SARS-CoV-2 in Vero E6 cells and Calu cells. Batch 2 of voclosporine dose-dependently inhibited the viral load of SARS-CoV-2 in E6 cells from 0.01 to 1.00 μM, and above 1.00 μM, the viral titer decreased to the detection limit (Figure 2A). Similarly, batch 3 of voclosporine dose-dependently inhibited the viral load of SARS-CoV-2 in E6 cells and human Calu cells from 0.01 to 4.00 μM, respectively (Figure 2B). As shown in Figure 2C, voclosporine dose-dependently reduced the amount of SARS-CoV-2 infected cells, as reflected by the decrease in the fluorescence staining of viral NSP4.

[0147] Example 3 To evaluate the efficacy and safety profile of voclosporine treatment for preventing rejection in kidney transplant patients, new kidney transplant patients were administered voclosporine or tacrolimus, and rejection and other adverse outcomes were evaluated.

[0148] method Newly transplanted kidney recipients were enrolled in a 6-month phase 2b, multicenter, randomized, open-label trial. Newly transplanted kidney recipients received high-dose voclosporine (0.8 mg / kg), medium-dose voclosporine (0.6 mg / kg), low-dose voclosporine (0.4 mg / kg), or standard-dose tacrolimus (0.05 mg / kg) twice daily (BID). In addition, all subjects received induced immunosuppression with intravenous administration of daclizumab or basiliximab (administered according to product labeling) and combination therapy with MMF and corticosteroids during the trial. Trough levels of voclosporine or tacrolimus were measured over 180 days. Adverse reactions were recorded, and kidney graft rejection was assessed based on the Banff classification. Adverse reactions were analyzed at 3 and 6 months. Graft survival and patient survival were also recorded at 6 months.

[0149] result As shown in Figure 3, the trough blood levels (C0) of voclosporine for the low-dose, medium-dose, and high-dose groups were 20-30 ng / mL, 35-50 ng / mL, and 60-85 ng / mL at 0-3 months, and similarly 11-20 ng / mL, 21-30 ng / mL, and 31-40 ng / mL at 3-6 months. In comparison, the trough blood levels for the standard dose of tacrolimus were 7-20 ng / mL at 0-3 months and 5-15 ng / mL at 0-6 months.

[0150] The clinical impact of voclosporine administration was evaluated by graft rejection rates. As shown in Table 1, the medium-dose voclosporine group (0.6 mg / kg BID) showed similar rejection rates to the standard-dose tacrolimus group, as indicated by biopsy-proven acute rejection (BPAR). The incidence of new-onset diabetes mellitus (NODAT) after transplantation in the low-dose, medium-dose, and high-dose voclosporine groups was 1.6%, 5.7%, and 17.7%, respectively, compared to 16.4% in the standard-dose tacrolimus group. The Nankivell eGFR (an indicator of renal function) in the low-dose, medium-dose, and high-dose voclosporine groups was 71, 72, and 68 mL / min, respectively, compared to 69 mL / min in the standard-dose tacrolimus group. This 6-month trial showed that VCS was as effective as TAC in preventing acute rejection, and that it may be associated with similar renal function in the low-dose and medium-dose groups, as well as a reduced incidence of NODAT. [Table 1]

[0151] Example 4 Pharmacokinetic data were collected from trials in patients with active lupus nephritis. Trough concentrations for various doses are shown in Table 2. Because PK demonstrates dose-to-trough linearity, the use of therapeutic drug monitoring is not required in ongoing clinical trials. [Table 2]

[0152] Example 5 To evaluate the antiviral effects of voclosporine in SARS-CoV-2 positive kidney transplant patients, kidney transplant recipients exhibiting mild to moderate SARS-CoV-2 symptoms will be enrolled in the study, and the efficacy of combination therapy with prednisone and tacrolimus will be evaluated.

[0153] method SARS-CoV-2 positive kidney transplant patients will be enrolled in an open-label, single-center, exploratory trial for voclosporine treatment. Prior to or at enrollment, subjects' standard immunosuppressive therapy will be reduced to dual therapy with prednisone and tacrolimus in accordance with the latest center guidelines (LUMC Transplant Center treatment guidelines for COVID-positive transplant patients). Before day 1, kidney transplant recipients suspected of having COVID-19 will undergo SARS-CoV-2 diagnostic testing and be informed about this trial. After confirmation of COVID-19 infection and consent, subjects will be randomized to the study group, and the day 1 trial procedure will be performed accordingly. Specifically, 15 of 30 subjects will continue treatment with prednisone and tacrolimus throughout the trial period, while the remaining 15 subjects will switch from tacrolimus to voclosporine. Voclosporine will be administered as a BID of 6 capsules (7.9 mg each) for a maximum treatment period of 1 year (Figure 4). Safety drug monitoring will be performed during the study to maintain voclosporine trough levels at 25–60 ng / mL and tacrolimus trough levels at 3–7 ng / mL. If trough levels fall outside these ranges, the dose will be adjusted.

[0154] From day 2 to day 14, subjects will receive daily home monitoring, and from day 16 to day 28, subjects will receive home monitoring every other day. Home monitoring will be conducted via video consultation and will include self-monitoring of body temperature, blood pressure, pulse, weight, respiratory rate, and oxygen saturation as a readout for SARS-CoV-2 cytopathic effects (CPE). In addition, subjects will collect a throat swab first thing in the morning to assess viral load.

[0155] Participants are also scheduled to visit the clinic four times: on day 4 (visit 2), day 7 (visit 3), day 14 (visit 4), and day 28 (visit 5 / end of study / early termination visit) (Figure 4). A pharyngeal swab taken first thing in the morning will also be collected during the visit. From day 28 onward, participants will continue the extended safety follow-up and will visit the clinic again on days 42, 90, 180, 270, and 360 for continued evaluation. After the fifth visit, participants who choose to continue voclosporine for up to one year will receive the investigational drug.

[0156] result This study compares SARS-CoV-2 viral titers and cytopathic effects in subjects treated with prednisone and tacrolimus with those treated with prednisone and voclosporine. Subjects in the voclosporine group are expected to experience a more effective reduction in viral load and fewer severe CPEs compared to the tacrolimus group.

[0157] We will also compare graft rejection, the onset of new post-transplant diabetes, and other side effects in subjects treated with prednisone and tacrolimus with those treated with prednisone and voclosporine. Subjects in the voclosporine group are expected to have similar or lower incidences of graft rejection, diabetes, and side effects compared to the tacrolimus group.

[0158] Example 6 Stock solutions of voclosporine (Aurinia), cyclosporine (Novartis), tacrolimus (Astellas), mycophenolic acid (Roche), and everolimus (Novartis) were prepared by dissolving the pharmaceutical formulations of these drugs in DMSO (therefore, the concentrations in Figures 5A-5E are estimated concentrations). Vero E6 cells (approximately 20,000 cells / well) in 96-well cell culture plates were infected with SARS-CoV-2 (infection multiplicity 0.015), followed by incubation in 150 μl of medium containing serial dilutions of the immunosuppressant. Virus-induced cell death was quantified by MTS assay on day 3 post-infection, and absorbance at 495 nm was measured. The viability of uninfected cells was evaluated in parallel to identify the cytotoxicity of the drugs. Two independent experiments (4 series) were performed for each drug. The 50% effective concentration (EC2) is defined as the concentration that inhibits virus-induced cell death by 50%. 50 ), and the 50% cytotoxic concentration (CC), which is the concentration that reduces the viability of uninfected cells to 50% of the viability of untreated control cells. 50 The following were identified using nonlinear regression with GraphPad Prism v8.0. As shown in Figures 5A-5E, only voclosporine (Figure 5A), cyclosporine (Figure 5B), tacrolimus (Figure 5D), and mycophenolic acid (Figure 5E) were identified as having EC 50 Virus-induced cell death was inhibited at concentrations of 0.27, 3.2, 12, and 3.1 μM.

[0159] EC of tacrolimus (Figure 5D) and cyclosporine (Figure 5B) 50 The concentrations are likely toxic in vivo at the corresponding concentrations. However, as shown in Figure 5A, voclosporine maintained cell viability and inhibited SARS-CoV-2 virus replication at concentrations approximately 40 times and 10 times lower than tacrolimus and cyclosporine, respectively. (EC of voclosporine) 50 This is C, which was observed in transplant patients. max It is within the range.

[0160] Example 7: Inhibition of SARS-CoV-2 replication in Calu-3 cells by VCS, CsA, and TAC. We performed CPE reduction assays and viral production reduction assays to evaluate the effects of three calcineurin inhibitors—cyclosporine A (CsA), tacrolimus (TAC), and voclosporine (VCS)—as well as other immunosuppressants commonly used in kidney transplant recipients (KTRs), on SARS-CoV-2 replication using cell-based assays.

[0161] method Viruses and cell lines SARS-CoV-2 / Leiden-0002 (GenBank MT510999) was isolated from nasopharyngeal samples at Leiden University Medical Center (LUMC) in March 2020. Infection was performed using a viral stock that had been passaged twice in Vero E6 cells. Vero E6 cells and Calu-3 2B4 cells, referred to herein as Calu-3 cells (Tseng et al., J Virol. Aug 2005;79(15):9470-9), were cultured as previously described (Salgado-Benvindo et al., Antimicrob Agents Chemother. Jul 22 2020;64(8)doi:10.1128 / AAC.00900-20). Infection was performed using Eagle's Minimum Essential Medium (EMEM, Lonza) containing 25 mM HEPES (Lonza), 2% FCS, 2 mM L-glutamine, and an antibiotic (EMEM-2% FCS). All experiments using infectious SARS-CoV-2 were conducted at LUMC's biosafety level 3 facility.

[0162] immunosuppressive compounds Stock solutions of voclosporine (VCS, Lupkynis®), cyclosporine A (CsA, Neoral®, Novartis), tacrolimus (TAC, Prograf®, Astellas), mycophenolate mofetil (MMF, CellCept®, Roche), or everolimus (EVL, Certican®, Novartis) were prepared by dissolving the pharmaceutical formulations of these drugs in dimethyl sulfoxide (DMSO). Placebo capsules and pure VCS powder, tacrolimus (PHR1809), cyclosporine A (30024), and mycophenolate (M5255) were obtained. Remdesivir (RDV, HY-104077) was used as a control in all experiments. All compounds were dissolved in DMSO, and single-use aliquots were stored at -20°C.

[0163] Measurement of cyclosporine A, tacrolimus, and boclosporine concentrations using validated LC-MS / MS. Quantification of CsA and TAC was performed by diluting the sample with methanol followed by blank whole blood, as previously described by LC-MS / MS (Zwart et al., Br J Clin Pharmacol. Dec 2018;84(12):2889-2902). Before analysis, the sample was diluted with methanol followed by whole blood to fall within the VCS calibration curve of 0-15-600 μg / L. Human whole blood was added to 10 or 20 μl of sample until the final volume reached 200 μl, and 200 μl of 0.1 M zinc sulfate and 500 μl of internal standard solution (acetonitrile containing 32 μg / L VCS D4) were added. The sample was then vortexed at 2000 rpm for 5 minutes, centrifuged at 13000 rpm for 5 minutes, and 20 μl was injected into the LC-MS / MS system. This method was validated in accordance with the EMA Guidelines for Bioanalytical Method Validation (EMEA / CHMP / EWP / 192217 / 2009 - Guideline on bioanalytical method validation (2011)).

[0164] Cytopathic effect (CPE) reduction assay The CPE reduction assay in Vero E6 cells was generally performed as described above, except that pre-incubation of cells with the test compound was continued for 30 minutes. The plates were incubated at 37°C for 3 days, and cell viability was determined using a colorimetric assay measuring absorbance at 495 nm. EC of each compound 50 and CC 50 We identified the compounds and analyzed the resulting data using nonlinear regression. For each compound, we completed at least two independent experiments (four pairs each).

[0165] Viral production reduction assay Calu-3 cells in a 96-well plate (3 x 10⁶ cells per well). 4 Cells were seeded in 100 μl of culture medium. The following day, the cells were pre-incubated for 60 minutes with 2x serial dilutions of CsA, TAC, or VCS starting at 25 μM and RDV starting at 10 μM. Subsequently, the cells were infected with SARS-CoV-2 (MOI1, based on the titer identified in Vero E6 cells) in 50 μl of culture medium containing the compound. After incubation at 37°C for 1 hour, the cells were washed three times with PBS and 100 μl of culture medium containing the compound was added. The culture medium was collected from the wells 24 hours post-infection (h pi). Analysis of the progeny of the virus released from infected Calu-3 cells was performed by a plaque assay in Vero E6 cells. VCS concentration was measured by validated LC-MS / MS after adding 9x methanol to the collected culture medium. A cytotoxicity assay using mock-infected cells processed in the same manner as described for the CPE reduction assay was performed in parallel.

[0166] Reduced virus production in glass bottles Borosilicate glass reagent bottles (50 ml) were treated with glacial acetic acid to remove residual detergent, and then washed twice with anhydrous ethanol. Before use, the bottles were dried and UV sterilized. Using sterile glass culture tubes, 50 μl glass syringes, and glass Pasteur pipettes, 3-fold concentrated compound solutions were prepared in EMEM-2% FCS. 1 ml of each compound dilution was transferred to three different reagent bottles (triple-pack). Confluent monolayer Calu-3 cells grown in culture flasks were infected with SARS-CoV-2 / Leiden-002 at MOI1. After incubation at 37°C for 1 hour, the cells were washed three times with warm PBS, triedpsinized, and resuspended in EMEM-2% FCS. 2 ml of this cell suspension (approximately 10 ml) was used. 6 Cells were added to each reagent bottle containing 1 ml of a 3-fold concentrated compound culture medium solution. After incubation at 37°C for 24 hours, the culture medium was collected, and the infectious virus titer was identified by plaque assay in Vero E6 cells.

[0167] Identification of cytotoxicity of compounds in glass culture tubes Calu-3 cells were treated with trypsin, and 1.5 x 10⁻⁶ cells were obtained. 5 One ml of EMEM-2%FCS containing cells was divided into glass culture tubes. Two-fold dilutions of VCS, TAC, and CsA, starting at a concentration of 150 μM (three times the final concentration), were prepared in EMEM-2%FCS medium using glass laboratory equipment, and 0.5 ml of each solution was added to the corresponding cell-containing tubes (three tubes per concentration). After 24 hours of incubation, cell viability was determined as described above.

[0168] result To evaluate the effects of VCS, CsA, and TAC on SARS-CoV-2 replication, viral load reduction assays were performed using human lung epithelial cells (Calu-3) that have been shown to be tolerant to SARS-CoV-2. Because VCS is highly lipophilic and can bind to plastics, potentially reducing bioavailability in assays using plastic equipment, the effects of VCS were compared using standard cell-based assays with plastic equipment as well as custom assays using glass tubes, containers, and pipettes. RDV was included as a positive control to inhibit SARS-CoV-2 replication.

[0169] Figures 6A–6D show the effects of cyclosporine A, tacrolimus, and voclosporine on the progenitor production of infectious SARS-CoV-2 after infection (Figures 6A and 6B) and on the survival rate of Calu-3 cells after mock infection (Figures 6C and 6D). Figures 6A and 6C show data obtained from experiments using glass laboratory equipment, while Figures 6B and 6D show results from experiments using plastic laboratory equipment. Calu-3 cells in glass maintained survival and reached 1.7 × 10⁶ cells in culture medium 24 hours after infection. 6 The measured PFU / ml titer indicated support for SARS-CoV-2 replication (Figure 6A). Treatment of infected cells with 10 μM remdesivir inhibited viral replication, resulting in infectious progeny titers slightly above the detection limit of the plaque assay (data not shown). Treatment of cells with 3.2 μM voclosporine resulted in a logarithmic decrease of more than 1.5 in the titer of SARS-CoV-2 infectious progeny, while the same concentration of cyclosporine A or tacrolimus resulted in a logarithmic decrease of approximately 0.5 (Figure 6A). However, treatment with 3.2 μM voclosporine or cyclosporine A also produced cytotoxic effects, reducing cell viability to approximately 75% (Figure 6C). In summary, these results demonstrate the potent antiviral activity of voclosporine, which may be partly mediated by cytotoxicity.

[0170] The observed results were consistent with previous reports that CsA inhibited SARS-CoV-2 replication in HuH7.5 and Calu-3 cells, but not in Vero cells (Dittmar et al., bioRxiv.2020:2020.06.19.161042). However, TAC was approximately 15 μM EC 50 In contrast to observations that TAC inhibits SARS-CoV-2 replication in Vero E6 cells, previous reports have shown no TAC activity in any of these cell lines, which may be related to the use of different Vero cell subclones.

[0171] In experiments using plastic materials, treatment of cells with VCS resulted in a dose-dependent decrease in the titer of infectious progeny, reaching a logarithmic decrease of more than 1 at 6.4 μM (Figure 6B). CsA treatment produced a similar decrease at 25 μM, but the inhibition was less than that of VCS at 6.4 μM. However, at concentrations above 12.5 μM, CsA showed significant cytotoxicity, while VCS did not (Figure 6D). TAC did not show much cytotoxicity, but a concentration of 25 μM was required to logarithmically decrease the titer of infectious viral progeny more than 1. VCS showed a stronger effect in experiments conducted with glass laboratory equipment instead of plastic, which may be due to the loss of compounds that bind to plastic. The concentration of free VCS was measured after incubation with various solutions in glass containers, with and without cells. Without cells, no significant loss of compounds from the solution was observed after 24 hours of incubation in glass at 37°C (Table 3). When VCS solutions at concentrations of 0.2–3.2 μM were incubated with Calu-3 cells in glass bottles, a decrease of approximately 75% in VCS concentration was observed, indicating that the compound had bound to or been taken up by the cells. In experiments conducted using standard plastic laboratory equipment, the VCS concentration in the culture medium of infected cells after 24 hours of treatment with 25 μM VCS was also measured, and the detection concentration of voclosporine was low at 0.68 μM. [Table 3]

[0172] Even after accounting for a 75% reduction due to cell binding or uptake, these results indicate that 90% of VCS is lost due to plastic binding. Similar reductions in viral titers with 3.2 μM and 25 μM VCS on glass and plastic, respectively, support the possibility that the amount of VCS bioavailable when using plastic is likely to be only about 10% of the initially added amount. When VCS binds to plastic, more than 80% of the compound can be lost from the solution. Consequently, using stock solutions prepared from pure VCS powder with plastic laboratory equipment may underestimate the efficacy of the compound in antiviral assays. Since VCS is a highly lipophilic compound, and interactions between plastic surfaces and hydrophobic drugs can be adverse, the antiviral effect of VCS may be greater than observed in assays using plastic. These results suggest that compound loss due to plastic binding and interference from excipients in pharmaceutical formulations may underestimate the efficacy of VCS in antiviral assays involving the use of plastic. 50 This indicates that it has become difficult to determine the value.

[0173] Results using glass laboratory equipment demonstrated that VCS dose-dependently and more effectively than CsA and TAC reduced the production of SARS-CoV-2 infective progeny in infected Calu-3 cells. These results indicate that cyclophyllin-dependent CNI inhibits SARS-CoV-2 replication in cell culture more potently than other classes of immunosuppressants such as EVL and MPA. VCS inhibited SARS-CoV-2 replication at eight times lower concentrations than TAC. The TAC concentration required to inhibit SARS-CoV-2 replication may be unacceptable or toxic in humans if not taken into account that the free fraction during transport is approximately one-tenth of the total concentration (for TAC, 0.2 μM EC10). 50(This corresponds to 160 ng / ml). For CsA and VCS, 0.2 μM corresponds to concentrations of 241 ng / ml and 243 ng / ml, respectively. In particular, VCS may be distributed at higher concentrations in organs such as the lungs than in the blood, and is also found at high concentrations in red blood cells. As a result, high concentrations in specific organs or cells may suppress the virus. Therefore, these results support the usefulness of VCS as a CNI for treatment that can also inhibit SARS-CoV-2 replication at safe concentrations in humans. Since VCS is thought to be as effective as TAC in preventing rejection in KTR, VCS is useful in treating COVID-19 patients. The results described herein demonstrate the advantages of cyclophylline-dependent CNIs, especially VCS, among the immunosuppressants commonly used in transplant medicine, for immunosuppressants, such as KTR, for subjects at risk of SARS-CoV-2 infection who require immunosuppression.

[0174] Example 8: Inhibition of SARS-CoV-2 replication by immunosuppressant pharmaceutical formulations. Experiments evaluating cell viability in response to SARS-CoV-2 infection were conducted using pharmaceutical formulations of drugs commonly used to treat kidney transplant recipients. Based on the findings from Example 7, it was considered that problems related to solubility or plastic binding could be avoided, for example, by using pharmaceutical formulations that include solvents and excipients.

[0175] method The pharmaceutical formulations of VCS, CsA, TAC, EVL, and MMF (including excipients, cosolvents, and other components) were evaluated using a CPE reduction assay with Vero E6 cells as described above. Remdesivir (RDV, HY-104077) was used as a control in all experiments. After pre-incubation and SARS-CoV-2 infection, cells were kept in drug-containing culture medium for 3 days. Cytotoxicity of the test formulations was evaluated in parallel using assays with mock-infected cells as described above. In the case of VCS, the achievement of the concentration when the stock solution was dissolved at 6.4 μM was confirmed by LC-MS / MS (data not shown).

[0176] result Figures 7A-7E show the effects of various pharmaceutical formulations on the cell viability of infected cells and mock-infected cells. The CNIs VCS, CsA, and TAC affect EC levels ranging from submicromolar to low micromolar. 50 The values ​​inhibited virus-induced cell death (Figures 7A-7C). EVL (Figure 7D) did not show an inhibitory effect at the tested concentrations. The prodrug MMF (Figure 7E) was included in this comparison, but its inhibition of viral replication was not expected. This is because it is unlikely to be metabolized to its active form, mycophenolate (also known as mycophenolic acid, MPA), during this assay (Ransom, Ther Drug Monit. 1995;17(6):681-4) (Ritter and Pirofski, Transp Infect Dis. 2009;1(4):290-7 and Neyts et al., Antimicrob Agents Chemother. 1998;42(2):216-22). Therefore, the apparent antiviral effect of MMF may be due to the excipients present in the formulation.

[0177] EC of VCS, CsA and TAC 50 The values ​​were 0.22±0.01 μM, 4.3±0.6 μM, and 10±1 μM, respectively. Except for VCS, none of the compounds showed cytotoxicity, therefore their CCs were used. 50 The value was higher than 100 μM. VCS is CC 50 It showed higher cytotoxicity at approximately 4 μM, and its EC 50 Furthermore, the levels were 1 / 18 to 1 / 45 of those observed in other compounds. These results demonstrate the superior effect of VCS on inhibiting virus-mediated cell death compared to other test compounds.

[0178] Example 9: Effects of VCS pharmaceutical formulations on SARS-CoV-2 replication The antiviral effects of the contents of VCS capsules and placebo capsules were evaluated. For example, one or more excipients contained in Lupkyna™ (a pharmaceutical formulation of VCS) were found to enhance the effect of VCS, for example, the low EC of approximately 0.22 μM shown in Example 8. 50 An assay was performed to determine whether it contributed to (Figure 7A).

[0179] method The CPE reduction assay was performed as described above. Vero E6 cells infected with SARS-CoV-2 were exposed to either VCS or placebo, and the antiviral effect (infected cells) and cytotoxic effect (mock-infected cells) of each were compared. The absence of VCS in the placebo capsule was confirmed by LC-MS / MS analysis (data not shown).

[0180] result Figures 8A and 8B compare the effects of the VCS formulation and placebo on cell viability. Surprisingly, both the VCS formulation (Figure 8A) and placebo (Figure 8B) inhibited SARS-CoV-2 replication in a similar dose-dependent manner. These results demonstrate that one or more excipients in the VCS formulation can mediate antiviral activity under the experimental conditions described herein.

[0181] Example 10: Evaluation of the antiviral effect of the VCS formulation The potential antiviral activity of the placebo capsule was further evaluated.

[0182] method To identify the virucidal capacity of a compound or formulation, SARS-CoV-2 virion (5x10) 4PFU was incubated with one of the following solutions at 37°C for 2 hours: culture medium, VCS solution prepared from pure powder (3.2 μM), contents of lysed VCS capsules (3.2 μM), placebo capsule, or Tween solution (present in capsule, corresponding to 3.2 μM VCS). For antiviral activity, PBS was used as a negative control and 50% ethanol as a positive control. The test compound was incubated with SARS-CoV-2 virus stock for 2 hours. The titer of the remaining infectious virus was identified by a plaque assay in Vero E6 cells, e.g., Salgado-Benvindo et al., Antimicrob Agents Chemother. Jul 22 2020;64(8)doi:10.1128 / AAC.00900-20.

[0183] result As shown in Figure 9, only the control treatment (50% ethanol) reduced the amount of infectious SARS-CoV-2 to below the detection limit (<100 PFU / ml). None of the other treatments had a significant effect on the infectivity of the remaining virus.

[0184] These results indicate that, under the experimental conditions described herein, the excipients of these formulations do not possess antiviral effects. These results suggest that, through an uncharacterized mechanism, the presence of excipients may interfere with the readout of the CPE reduction assay.

[0185] Example 11: Preparation of immunosuppressive compounds from high-purity powder and their activity in a CPE reduction assay. The effects of excipients in pharmaceutical compositions on the efficacy of VCS and other immunosuppressive compounds were evaluated.

[0186] method The CPE reduction assay was performed as described above using high-purity powders of immunosuppressants solubilized in DMSO. The test stock was prepared from the pure powder of each compound. In the case of Neoral (CsA microemulsion), the CsA powder, the most commonly used CsA derivative in KTR treatment, was evaluated.

[0187] result Figures 10A-10D show the effect of solubilized high-purity immunosuppressive compounds on cell viability in the CPE reduction assay. As shown in Figure 10A, the VCS solution prepared from pure powder did not provide the same level of protection against SARS-CoV-2 infected cells as the solution prepared from the pharmaceutical formulation (compared to Figure 8A). However, the VCS solution from pure powder also exhibited less cytotoxicity, as observed from mock-infected cells (compared to Figure 6C). Figures 10B and 10D show similar results for CsA and MPA-treated cells, respectively. When Figure 7C is compared to Figure 10C, the TAC solution prepared from pure powder showed similar efficacy to the formulation, i.e., EC 50 SARS-CoV-2 was inhibited at approximately 15 μM.

[0188] In summary, these results indicate that excipients in certain pharmaceutical formulations contribute to the observed efficacy. In this antiviral assay, testing of high-purity powders of various immunosuppressive compounds to avoid interference caused by excipients demonstrated that EC against VCS, CsA, and TAC was effective. 50 The substantially higher values ​​demonstrated that excipients that improve the solubility and bioavailability of active compounds in pharmaceutical formulations can also influence the results of cell-based assays. However, TAC's pharmaceutical formulations did not appear to contain any excipients with antiviral effects.

[0189] These results indicate that immunosuppressive compounds may require excipients to ensure solubility and / or bioavailability for optimal activity. The preparation of the compound, e.g., pharmaceutical formulation or solubilized high-purity powder, may affect the assay results and the solubility and bioavailability of the compound when administered.

[0190] Example 12: Evaluation of the effect of plastic materials on voclosporine formulations Considering the potential interaction between VCS and plastic laboratory equipment, experiments were conducted to determine whether plastic coatings prevent VCS binding. As mentioned above, excipients in the pharmaceutical formulation of VCS may affect bioavailability, for example, by preventing VCS binding to plastics, but their nonspecific antiviral effects do not affect the true EC of VCS. 50 This could also affect the measurement. Plastic materials coated with various agents were tested to evaluate whether the coating prevented VCS from binding to the plastic, potentially allowing the use of VCS solutions prepared from pure powder in antiviral assays.

[0191] method Coating of plastic materials Plastic laboratory equipment was coated with three different coating agents: PBS (BSA, Sigma) containing 100 mg / ml bovine serum albumin, MilliQ water (PEG-3350, Sigma) containing 1% polyethylene glycol 3350, and MilliQ water (Tween40, Fluka) containing 0.2% polysorbate 40. Furthermore, this plastic material was treated with VCS by processing it with a DMSO (Sigma) solution of 500 mM VCS. All laboratory equipment, including tubes, tips, and culture plastics, was filled with blocking solution and incubated at room temperature for 2 hours, shaking to ensure a uniform surface coating. After rinsing twice with MilliQ water, these items were dried at room temperature and then used for further experiments. 0.2 and 2 μM VCS solutions were prepared in EMEM-2% FCS, and 100 μl of each VCS solution was coated onto 96-well plates for incubation. After incubation at 37°C for 2 hours, the remaining VCS concentration was measured by validated LC-MS / MS. Using a similar method, we also evaluated the binding of TAC or CsA.

[0192] result None of the coating treatments reduced the nonspecific binding and loss of VCS to the plastic (see Table 4), and only 5-7% of the original concentration was recovered after 2 hours of incubation. Even at t=0, only about 27% of the original stock concentration was recovered due to VCS loss in pipette tips and tubes during dilution preparation. Saturation of the binding sites on the plastic by treatment with 500 mM VCS prevented VCS loss from the solution but led to uncontrolled leaching of VCS from the plastic. This resulted in unpredictable concentrations higher than the concentration of the added solution; for example, when a 2 μM solution was incubated on a plastic plate saturated with VCS, VCS concentrations exceeding 15 μM were measured. [Table 4]

[0193] Binding to plastic was minimal with TAC (24% loss). In the case of CsA, the remaining concentration after 2 hours of incubation was 62% of the initial concentration (see Table 5). [Table 5]

[0194] These results indicated that the coating agent did not prevent nonspecific binding of VCS to the plastic. In some embodiments, compound loss due to plastic binding and interference from excipients in the pharmaceutical formulation resulted in EC in some assays. 50 Determining the values ​​became difficult. Since none of the coating treatments prevented nonspecific bonding to the plastic, glassware was used instead of plastic in other experiments to avoid potential problems (see, for example, Table 3).

[0195] All publications, including patents, patent applications, and scientific articles, referenced herein are incorporated herein by reference in whole for all purposes to the same extent that each individual publication, including patents, patent applications, or scientific articles, is incorporated by reference specifically and individually.

[0196] While the aforementioned inventions are described in some detail as examples and illustrations for clarity of understanding, it will be apparent to those skilled in the art that some minor changes and modifications can be made in light of the above teachings. Therefore, the description and examples should not be construed as limiting the scope of the invention. Various modifications to the compositions and methods described herein will be apparent from the description and teachings herein. Such modifications can be made without departing from the true scope and spirit of the disclosure and are intended to be included within the scope of the disclosure. The present invention encompasses the following embodiments. (Embodiment 1) A method for treating or preventing a viral infection in a subject, comprising administering a therapeutically effective amount of voclosporine to the subject. (Embodiment 2) The method according to Embodiment 1, wherein the subject requires immunosuppression. (Embodiment 3) The method according to Embodiment 1 or 2, wherein the viral infection is mitigated by inhibition of cyclophyllin A (CypA) or a CypA-related pathway. (Embodiment 4) The method according to any one of embodiments 1 to 3, wherein the viral infection is caused by a virus that is a member of Coronaviridae. (Embodiment 5) The method according to Embodiment 4, wherein the virus is an alpha coronavirus, a beta coronavirus, a delta coronavirus, or a gamma coronavirus. (Embodiment 6) The method according to Embodiment 3 or 4, wherein the virus is human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), Middle East Respiratory Syndrome-related coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), or Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). (Embodiment 7) The method according to any one of embodiments 3 to 6, wherein the virus is MERS-CoV, SARS-CoV, or SARS-CoV-2. (Embodiment 8) The method according to any one of embodiments 3 to 7, wherein the virus is SARS-CoV-2. (Embodiment 9) The method according to any one of Embodiments 1 to 8, wherein the therapeutically effective dose is approximately 0.1 mg / kg / day to approximately 2 mg / kg / day. (Embodiment 10) The effective therapeutic dose is approximately 7.9 mg BID, approximately 15.8 mg BID, approximately 23.7mg BID, approximately 31.6 mg BID, approximately 39.5mg BID, approximately 47.4 mg The method according to any one of Embodiments 1 to 8, wherein BID is approximately 55.3 BID. (Embodiment 11) The effective therapeutic dose is approximately 7.9 mg QD, approximately 15.8 mg QD, approximately 23.7mg QD, approximately 31.6 mg QD, approximately 39.5mg QD, approximately 47.4mg QD, approximately 55.3mg QD, approximately 63.2 mg QD, approximately 71.1 mg QD, approximately 79.0 mg QD, approximately 86.9 mg QD, approximately 94.8 mg QD, approximately 102.7mg QD, or approximately 110.6 mg The method according to any one of embodiments 1 to 8, which is QD. (Embodiment 12) The aforementioned therapeutically effective dose is approximately 0.05 μM to 10 μM, approximately 0.1 μM to 5 μM, approximately 0.2 μM to 2.5 μM, approximately 0.3 μM to 1.0 μM, approximately 0.4 μM to 0.9 μM, approximately 0.5 μM to 0.8 μM, approximately 0.1 μM to 0.5 μM, or approximately 0.2 μM to 0.4 μM, or approximately 0.05, approximately 0.1, approximately 0.15, approximately 0.2, approximately 0.25, approximately 0.3, approximately 0.35, approximately 0.4, The method according to any one of Embodiments 1 to 8, which is equivalent to, or can achieve, a concentration of approximately 0.45, approximately 0.5, approximately 0.55, approximately 0.6, approximately 0.7, approximately 0.8, approximately 0.9, approximately 1.0, approximately 1.5, approximately 2.0, approximately 2.5, approximately 3.0, approximately 3.5, approximately 4.0, approximately 4.5, approximately 5.0, approximately 6.0, approximately 7.0, approximately 8.0, approximately 9.0, or approximately 10.0 μM or less. (Embodiment 13) The method according to any one of Embodiments 1 to 12, wherein the renal function of the subject is monitored. (Embodiment 14) The method according to Embodiment 13, wherein monitoring of the target renal function includes the following: (a) Evaluate the estimated glomerular filtration rate (eGFR) of the subject at at least a first and second point in time on different days, and (b)(i) If the eGFR of the subject falls below a predetermined value by more than the target % between the first and second time points, reduce the daily dose of voclosporine administered to the subject or discontinue administration. (ii) If the subject's eGFR falls below the target percentage between the first and second time points, continue administering the same daily dose of voclosporine to the subject. (Embodiment 15) The aforementioned predetermined value is approximately 50 to approximately 90 ml / min / 1.73 m 2 The method according to Embodiment 14. (Embodiment 16) The aforementioned predetermined value is approximately 60 ml / min / 1.73 m 2 The method according to embodiment 14 or 15. (Embodiment 17) The method according to any one of embodiments 14 to 16, wherein the target percentage is approximately 20% to approximately 45%. (Embodiment 18) The method according to any one of embodiments 14 to 17, wherein the target percentage is approximately 20%. (Embodiment 19) The method according to any one of Embodiments 1 to 18, wherein the subject has a condition related to an autoimmune disease or graft rejection. (Embodiment 20) The method according to any one of Embodiments 1 to 19, wherein the subject has a condition related to graft rejection. (Embodiment 21) The method according to Embodiment 19 or 20, wherein the condition relates to rejection of a heart, lung, liver, kidney, pancreas, skin, intestine, or corneal graft. (Embodiment 22) The method according to any one of embodiments 19 to 21, wherein the aforementioned condition is related to kidney transplant rejection. (Embodiment 23) The method according to Embodiment 19, wherein the subject has an autoimmune disease. (Embodiment 24) The method according to any one of Embodiments 1 to 23, wherein the therapeutically effective amount of voclosporine is administered without administering a therapeutically effective amount of mycophenolate mofetil (MMF) and / or a therapeutically effective amount of corticosteroid. (Embodiment 25) The method according to any one of Embodiments 1 to 23, further comprising administering a therapeutically effective amount of mycophenolate mofetil (MMF) and / or a therapeutically effective amount of corticosteroid. (Embodiment 26) The method according to any one of Embodiments 1 to 25, wherein voclosporine is administered by intestinal administration, oral administration, sublingual administration, or rectal administration, parenteral administration, intravenous injection, intramuscular injection, subcutaneous injection, intravenous infusion, or inhalation / blow-in. (Embodiment 27) The method according to Embodiment 26, wherein voclosporine is administered by intraintestinal, oral, sublingual, or rectal administration. (Embodiment 28) The method according to embodiment 25 or 27, wherein voclosporine is administered orally. (Embodiment 29) The method according to Embodiment 26, wherein voclosporine is administered by parenteral administration, intravenous injection, intramuscular injection, subcutaneous injection, intravenous infusion, or inhalation / blow-in. (Embodiment 30) The method according to embodiment 26 or 29, wherein voclosporine is administered by inhalation or inhalation. (Embodiment 31) The method according to Embodiment 30, wherein voclosporine is administered in the form of an aerosol. (Embodiment 32) The method according to any one of Embodiments 1 to 31, wherein voclosporine is administered in a pharmaceutical composition. (Embodiment 33) The method according to Embodiment 32, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients. (Embodiment 34) The method according to Embodiment 33, wherein the pharmaceutically acceptable excipient is independently selected from one or more of the following: alcohol, D-α-tocopherol (vitamin E), polyethylene glycol succinate (TPGS), polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), medium-chain triglycerides, gelatin, sorbitol, glycerin, yellow iron oxide, red iron oxide, titanium dioxide, and water. (Embodiment 35) The method according to any one of Embodiments 1 to 34, wherein the viral load decreases in the subject after administration of voclosporine. (Embodiment 36) The method according to any one of Embodiments 1 to 35, wherein the survival of the subject is prolonged after administration of voclosporine.

Claims

1. A drug comprising a therapeutically effective dose of voclosporine for use in a method of treating or preventing a viral infection in a subject requiring immunosuppression, wherein the viral infection is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

2. The agent according to claim 1, wherein the SARS-CoV-2 virus infection is ameliorated by inhibition of cyclophyllin A (CypA) or a CypA-related pathway.

3. The agent according to claim 1 or 2, wherein the therapeutically effective dose is 0.1 mg / kg / day to 2 mg / kg / day.

4. The agent according to any one of claims 1 to 3, wherein the therapeutically effective dose is 7.9 mg BID, 15.8 mg BID, 23.7 mg BID, 31.6 mg BID, 39.5 mg BID, 47.4 mg BID, or 55.3 mg BID.

5. The drug according to any one of claims 1 to 4, wherein the therapeutically effective dose is 7.9 mg QD, 15.8 mg QD, 23.7 mg QD, 31.6 mg QD, 39.5 mg QD, 47.4 mg QD, 55.3 mg QD, 63.2 mg QD, 71.1 mg QD, 79.0 mg QD, 86.9 mg QD, 94.8 mg QD, 102.7 mg QD, or 110.6 mg QD.

6. The therapeutically effective dose is 0.05 μM to 10 μM, 0.1 μM to 5 μM, 0.2 μM to 2.5 μM, 0.3 μM to 1.0 μM, 0.4 μM to 0.9 μM, 0.5 μM to 0.8 μM, 0.1 μM to 0.5 μM, or 0.2 μM to 0.4 μM, or 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0. The agent according to any one of claims 1 to 5, which is equivalent to or can achieve a concentration of 45, 0.5, 0.55, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 μM or less.

7. The drug according to any one of claims 1 to 6, wherein the renal function of the subject is monitored.

8. The agent according to claim 7, wherein the monitoring of the renal function of the subject includes the following: (a) Evaluate the estimated glomerular filtration rate (eGFR) of the subject at at least a first and second point in time on different days, and (b) (i) The eGFR of the subject decreases between the first and second time points to a target percentage in the range of 20% to 45%, and reaches 50 to 90 ml / min / 1.73 m 2 If the value falls below a predetermined value within the specified range, the daily dose of voclosporine for the subject shall be reduced or administration shall be discontinued. (ii) If the eGFR of the subject falls below the target percentage between the first and second time points, continue administering the same daily dose of voclosporine to the subject.

9. The aforementioned predetermined value is 60 ml / min / 1.73 m 2 The drug according to claim 8.

10. The agent according to any one of claims 8 or 9, wherein the target percentage is 20%.

11. The agent according to any one of claims 1 to 10, wherein the subject has a condition related to an autoimmune disease or graft rejection.

12. The agent according to any one of claims 1 to 11, wherein the subject has a condition related to graft rejection.

13. The agent according to claim 11 or 12, wherein the condition is related to rejection of a graft of the heart, lungs, liver, kidneys, pancreas, skin, intestines, or cornea.

14. The agent according to any one of claims 11 to 13, wherein the aforementioned condition is related to kidney transplant rejection.

15. The agent according to claim 11, wherein the subject has an autoimmune disease.

16. The agent according to any one of claims 1 to 15, wherein the therapeutically effective amount of voclosporine is administered without administering a therapeutically effective amount of mycophenolate mofetil (MMF) and / or a therapeutically effective amount of corticosteroid.

17. The agent according to any one of claims 1 to 15, wherein the method comprises administering a therapeutically effective amount of voclosporine to a subject, and further comprising administering a therapeutically effective amount of mycophenolate mofetil (MMF) and / or a therapeutically effective amount of corticosteroid.

18. The agent according to any one of claims 1 to 17, wherein voclosporine is administered by intestinal administration, oral administration, sublingual administration, or rectal administration, parenteral administration, intravenous injection, intramuscular injection, subcutaneous injection, intravenous infusion, or inhalation / blow-in.

19. The agent according to claim 18, wherein voclosporine is administered by intestinal administration, oral administration, sublingual administration, or rectal administration.

20. The agent according to claim 18 or 19, wherein voclosporine is administered orally.

21. The agent according to claim 20, wherein voclosporine is administered by parenteral administration, intravenous injection, intramuscular injection, subcutaneous injection, intravenous infusion, or inhalation / blow-in.

22. The agent according to claim 18 or 21, wherein voclosporine is administered by inhalation or inhalation.

23. The agent according to claim 22, wherein voclosporine is administered in the form of an aerosol.

24. The agent according to any one of claims 1 to 23, comprising a pharmaceutical composition containing a therapeutically effective amount of voclosporine.

25. The agent according to claim 24, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

26. The agent according to claim 25, wherein the pharmaceutically acceptable excipient is independently selected from one or more of the following: alcohol, D-α-tocopherol (vitamin E), polyethylene glycol succinate (TPGS), polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), medium-chain triglycerides, gelatin, sorbitol, glycerin, yellow iron oxide, red iron oxide, titanium dioxide, and water.

27. The agent according to any one of claims 1 to 26, wherein the viral load decreases in the subject after administration of voclosporine.

28. The agent according to any one of claims 1 to 27, wherein the survival of the subject is prolonged after administration of voclosporine.

29. The use of voclosporine in the manufacture of a drug for use in a method of treating or preventing a viral infection in a subject, wherein the viral infection is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

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