Method for treating or preventing coronavirus infection bone sequelae

Abstract

Disclosed is a method of treating or preventing skeletal sequelae of coronavirus infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist, with the proviso that the endothelin receptor antagonist is not co-administered with dapagliflozin or niclosamide. The skeletal sequelae may include one or more of arthralgia, viral arthritis, osteopenia, osteochondral injury, and osteoporosis.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 448,532, filed February 27, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0002] This disclosure relates to methods and compounds for treating skeletal sequelae of coronavirus infection. Background Technology

[0003] Musculoskeletal disorders are a major acute post-traumatic sequela of COVID-19 (PASC). 1 Joint pain, or joint pain, is one of the most difficult long-term symptoms to treat. 3-6 Two to three months after SARS-CoV-2 infection, 10-23% of hospitalized COVID survivors experienced joint pain. 7,8 Without timely intervention, approximately 10% of people eventually develop debilitating chronic COVID pain syndrome within a year of infection. 1,4 .

[0004] Viral infections are a known cause of joint pain and arthritis. 9 Respiratory viral infections are associated with an increased risk of inflammatory arthritis. 10 An increasing number of clinical cases are reporting viral arthralgia following SARS-CoV-2 infection. 11 and inflammatory arthritis 12-15 SARS-CoV-2 infection can cause joint damage, possibly by triggering excessive inflammation. 16 Or autoimmune response 17 However, the exact molecular mechanism remains unknown.

[0005] Endothelial dysfunction biomarkers were detected in the plasma of hospitalized COVID patients. 18,19 (For example, endothelin-1 (ET-1) - the most effective vasoconstrictor) 20,21 ) Continues to rise. Elevated plasma ET-1 levels are associated with human... 22,23 and animals 24 Elevated ET-1 levels are related to the severity of joint damage. Elevated ET-1 levels have been shown to induce joint pain through its two G protein-coupled receptors (i.e., endothelin A and B receptors (ETAR and ETBR)). 25,26 and cartilage degeneration 27 Blocking endothelin receptors exerts analgesic and chondroprotective effects in various animal models of arthritis. 24,28-30 All evidence suggests that the endothelin system plays a plausible role in SARS-CoV-2-induced joint pain and injury.

[0006] Accordingly, there is a need to develop improved methods for treating sequelae of coronavirus infection of the skeleton. SUMMARY

[0007] Pathological manifestations of acute SARS-CoV-2 infection were reproduced in a golden Syrian hamster model 31 Pathological bone loss was observed in this model 2 However, the damage caused by SARS-CoV-2 infection to the articular cartilage covering the bone ends is still under investigation.

[0008] Here, using this well-established hamster model, the temporal changes in joint pathology following SARS-CoV-2 infection 31 were characterized. Bone-cartilage damage was observed in the acute phase of infection, accompanied by activation of endothelin signaling. We further demonstrated that timely intervention using an endothelin receptor blocker, such as macitentan, can reduce joint damage caused by viral infection. Even when endothelin receptor blocker treatment was initiated in the subacute phase of infection, it was able to restore the structural integrity of the subchondral bone.

[0009] In a first aspect, provided herein is a method of treating or preventing sequelae of coronavirus infection of the skeleton in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist, with the proviso that the endothelin receptor antagonist is not co-administered with darapladib or niclosamide.

[0010] In certain embodiments, the subject does not have pulmonary arterial hypertension.

[0011] In certain embodiments, the coronavirus infection is the result of a coronavirus selected from the group consisting of severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome-related coronavirus (MERS-CoV), human coronavirus 229E (229E-CoV-2), human coronavirus NL63 (NL63-CoV), human coronavirus OC43 (OC43-CoV), human coronavirus HKU1 (HCoV-HKU1), and variants thereof.

[0012] In certain embodiments, the coronavirus infection is the result of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and variants thereof.

[0013] In certain embodiments, the endothelin receptor antagonist is a selective ET A receptor antagonist, a selective ET B receptor antagonist, or a dual ET A receptor and ET B receptor antagonist.

[0014] In certain embodiments, the endothelin receptor antagonist is a dual ET A receptor and ET B receptor antagonist.

[0015] In certain embodiments, the endothelin receptor antagonist is selected from the group consisting of sitaxsentan, ambrisentan, atrasentan, BQ-123, sparsentan, zibotentan, abligrenatan, idonacitan, bosentan, macitentan, tezosentan, BQ-788, A192621, and mixtures thereof.

[0016] In certain embodiments, the endothelin receptor antagonist is bosentan, macitentan, ambrisentan, or mixtures thereof.

[0017] In certain embodiments, the endothelin receptor antagonist is macitentan.

[0018] In certain embodiments, the coronavirus infection is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the endothelin receptor antagonist is bosentan, macitentan, ambrisentan, or mixtures thereof.

[0019] In certain embodiments, the endothelin receptor antagonist is administered to the subject at least one of during an acute phase of the coronavirus infection or during a subacute phase of the coronavirus infection.

[0020] In certain embodiments, the skeletal sequelae is one or more of joint pain, viral arthritis, osteopenia, osteochondral injury, and osteoporosis.

[0021] In certain embodiments, the endothelin receptor antagonist is administered to the subject intra-articularly or intra-osseously.

[0022] In certain embodiments, the coronavirus infection is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the endothelin receptor antagonist is macitentan.

[0023] In certain embodiments, the macitentan is administered to the subject at least one of during an acute phase of the SARS-CoV-2 or during a subacute phase of the coronavirus infection.

[0024] In certain embodiments, the skeletal sequelae is one or more of joint pain, viral arthritis, osteopenia, osteochondral injury, and osteoporosis. BRIEF DESCRIPTION OF DRAWINGS

[0025] The above and other objects and features of the present disclosure will become apparent from the following description of the disclosure when taken in conjunction with the accompanying drawings, which

[0026] The above and other objects and features of the present disclosure will become apparent from the following description of the disclosure when taken in conjunction with the accompanying drawings, whichFigure 1 | SARS-CoV-2 infection induces osteochondral damage in hamsters. a. Experimental design for studying the temporal changes in joints after SARS-CoV-2 infection using a golden Syrian hamster model. Fifteen hamsters were used for this purpose. Ten were infected with wild-type SARS-CoV-2 via intranasal administration. Five received a simulant infection. Hind limbs were collected at designated time points. n=5 at each time point. b. Representative micro-CT images and corresponding measurements [bone mineral density (BMD) and trabecular volume fraction (BV / TV)] of the proximal tibial epiphysis of infected hamsters at each time point. n =5. c, representative TRAP staining and its corresponding quantification at 4 and 30 dpi (N. Oc / B. Pm). Scale bar, 50 m. d, Representative SP7 staining and corresponding quantitative analysis of SP7 + Temporal changes in osteoblast ratio (N.Ob / B.Pm). Scale bar, 25 m. e, f, representative (e) H&E and (f) Massons tricolor (MT) articular cartilage images showing cyst formation (arrows), fibrin exudate (arrows), and collagen deposition following SARS-CoV-2 infection. Scale bar, 100 m (H&E); 50 m (MT). g, h, quantitative assessment of (g) cyst area and (h) chondrocyte count in articular cartilage. i, representative Safranin O / Fixed Green staining and its corresponding intensity quantitatively showing the loss of proteoglycans in cartilage after viral infection. Scale bar, 100 m.jl, Simulated infection group and different time points after infection (j) p16 INK4a Representative immunostaining images and quantifications of (k) HMGB1 and (l) cysteine ​​3. Scale bar, 50 m(j,k); 100 and 20 m(l). Data are averages. Data from SEM were excluded if they fell outside the 1.5 IQR range. One-way ANOVA and Tukey's post-hoc test were performed.

[0027] Figure 2 SARS-CoV-2 infection induces endothelial dysfunction and systemic activation of endothelin signaling. a, b, (a) representative images stained with CellROX and (b) MitoTracker staining, showing the effects of pV D614G (MOI 10) and SP RBD (MOI 10). After treatment with 100 g / ml ... M hydrogen peroxide was used as a positive control. Agglomerated structures are indicated by arrows. Scale bar, (a) 50 m; (b) 10 m. c, d, relative mRNA expression of (c) EDN1, (d) endothelin receptor and RANKL in HUVECs 24 hours after pV or SP RBD stimulation. Two-tailed Student's t-test. e, simulated infection measured by ELISA. n =4), 4 dpi after SARS-CoV-2 infection ( n =5) or 30 dpi ( n Serum ET-1 levels in hamsters (=4). Single-cause ANOVA followed by Tukey post-hoc test. Data were excluded when they fell outside 1.5 IQR. f, Representative images of multichannel fluorescent staining showing temporal expression patterns of ETAR and ETBR in the knee cartilage of simulated or SARS-CoV-2 infected hamsters. Nuclei were counterstained using SN520. Scale bar, 50 μm. Quantification was performed by DAB staining. Simulated ( n =5); 4 dpi ( n =4); 30 dpi ( n =5). g, Representative immunofluorescence staining of vWF and 4HNE in the subchondral bone marrow cavity of the proximal tibia of a hamster. Co-localization of vWF and 4HNE is indicated by arrows. Cell nuclei were counterstained with DAPI. Scale bar, 10 m.

[0028] Figure 3 The viral spike protein induces oxidative stress and senescence in chondrocytes, activating endothelin signaling. a, b, representative images of (a) SARS-CoV-2 nucleocapsid protein (NP) and (b) SARS-CoV-2 spike protein (SP) in subchondral bone at different time points post-infection. (Images for each time point are not included in the provided text.) n =5. Scale, 25 m. Semi-quantitative analysis of NP and SP based on integrated optical density (IOD). c, d, at 10 24 hours after stimulation with SARS-CoV-2 spike protein RBD (SP RBD) or ET-1 (100 nM), the following were observed in the chondrocyte cell line C28 / I2: (c) EDN1 and its receptor, and (d) matrix metalloproteinases and p16. INK4a (c) Two-tailed Student's t-test; (d) One-way ANOVA and Tukey's post-hoc test. Data are averages. SEMe, p16 of the ATDC5 chondrocyte cell line, which was stimulated for 24 hours without SP RBD. INK4aRepresentative immunofluorescence staining. n =3. Scale, 50 m. f, SA- on ATDC5 cells stimulated and unstimulated by SP RBD A representative image of -gal staining. n =3. Representative images of g, h, (g) CellROX and (h) MitoTracker staining, showing SP RBD (10 Accumulation of oxidative stress and changes in mitochondrial dynamics in C28 / I2 cells after treatment with macitentan for 1 hour. C28 / I2 cells were pretreated with macitentan for 30 minutes and then stimulated with SP RBD for 1 hour. Scale bar, (g) 50 m; (h) 25 m.

[0029] Figure 4 | The viral spike protein causes joint pain and chondrocyte senescence. a, Experimental design: Demonstrating the therapeutic efficacy of intravenous injection of SARS-CoV-2 spike protein RBD (10 µg in 150 µl saline) and macitentan in a mouse model. Two doses of macitentan were initiated 2 days after RBD injection and continued for 12 days. Animals were sacrificed 14 days after RBD injection. Mediator ( n =3); SP RBD ( n =4), Low Mac ( n =3); High Mac ( n =4). b, The time-course changes in the paw withdrawal threshold in response to mechanical stimulation were assessed by the von Frey wire test in mice injected with or without SP RBD. Two-tailed Student's t-test. c, The effect of two different doses (0.3 mg / kg and 3 mg / kg) of macitentan on the paw withdrawal threshold in mice injected with SP RBD. Two-way ANOVA with Tukey post-hoc correction was used for multiple tests. d, Representative micro-CT images of the proximal tibial epiphysis showing the salvage effect of macitentan on SPRBD-induced bone loss. e, Representative Safranin O / Fixed Green staining showing the loss of proteoglycans in the articular cartilage of mice with SP RBD. Scale bar, 200 m. f, Representative immunostaining of ET-1 in articular cartilage of mice treated and untreated with macitentan. Scale bar, 50 m. g, h, in articular cartilage (g) p16 INK4a Representative immunostainings of HMGB1 in (g) and (h) show the accumulation of senescent chondrocytes in mice injected with SP RBD, both before and after macitentan treatment. Hematoxylin and DAPI were used as counterstains in (g) and (h), respectively.

[0030] Figure 5 | Early macitentan treatment partially rescues SARS-CoV-2-associated osteochondral damage. a, Experimental design showing a 4-day treatment regimen with 0.3 mg / kg macitentan starting at 1 dpi in SARS-CoV-2-infected hamsters. PBS was used as vehicle control. Animals were sacrificed at 4 dpi, hind limbs were harvested and analyzed. 4 dpi PBS ( n = 5); 4 dpi Mac ( n = 4) b, Representative immunostaining for SARS-CoV-2 spike protein (SP) in the subchondral bone marrow cavity of infected hamsters with and without macitentan treatment during the acute phase of infection. Scale bar, 25 m. c, Representative multichannel immunofluorescence staining for vWF and 4HNE in the subchondral bone marrow cavity of the proximal tibia of hamsters showing that early macitentan treatment improves vascular function. Co-localization of vWF and 4HNE is indicated with arrows. DAPI was used as counterstain. Scale bar, 25 m; 10 m (magnification). d, e, Representative TRAP and SP7 staining and corresponding quantification. Scale bar, 50 m. f, Micro-CT images of the proximal tibial epiphysis of hamsters showing changes in BMD and BV / TV after early macitentan treatment. g, h, Histopathological analysis of the knee joint cartilage of infected hamsters using (g) H&E and (h) Masson’s trichrome staining. Scale bar, (g) 100 m; (h) 50 m. i, j, Quantitative assessment of (i) cyst area and (j) chondrocyte number in the articular cartilage with or without macitentan treatment during the acute phase of infection. k, 1, Representative immunofluorescence staining and quantification of (k) p16 INK4a and (1) HMGB1 in the articular cartilage. Nuclei were stained using DAPI. Scale bar, 50 m. Two-tailed Student’s t test. Data were excluded when they fell outside 1.5 IQR.

[0031] Figure 6 | Delayed macitentan treatment alleviates SARS-CoV-2 infection subacute phase subchondral bone loss. a, Schematic representation of the experimental plan to study the effect of delayed macitentan treatment on SARS-CoV-2-infected hamsters. 0.3 mg / kg macitentan was administered intraperitoneally from 16 dpi to 30 dpi. PBS was used as vehicle control. Animals were euthanized at 30 dpi, hind limbs were collected and analyzed. n = 5 animals per group n=5. b, Representative immunostaining for SARS-CoV-2 spike protein (SP) in the subchondral bone marrow cavity of infected hamsters with and without masitinib treatment at subacute phase. Scale bar, 25 m. c, Representative multichannel immunofluorescence staining for vWF and 4HNE in the subchondral bone marrow cavity of hamster proximal tibial metaphysis showing reduced oxidative stress around blood vessels with delayed masitinib treatment. DAPI was used as counterstain. Scale bar, 25 m; 10 m (magnification). d, Micro-CT images of proximal tibial epiphysis of hamsters showing significant increase in BMD and BV / TV with delayed masitinib treatment. e, f, Representative SP7 and TRAP staining and corresponding quantification. Scale bar, 50 m. g, h, Representative images of knee joint cartilage of infected hamsters with (g) H&E and (h) Masson trichrome staining. Scale bar, (g) 100 m; (h) 50 m. i, j, Quantitative assessment of (i) cyst area and (j) chondrocyte number in articular cartilage with or without masitinib treatment at subacute phase of infection. n =5. k, I, Representative immunofluorescence staining and quantification of (k) pl6 INK4a and (I) HMGB1 in articular cartilage. DAPI was used as counterstain. Scale bar, 50 m. n =5. Unpaired two-tailed Student’s t-test. Data were excluded when falling outside 1.5 IQR.

[0032] Figure 7 | Subchondral bone microarchitecture deteriorates in SARS-CoV-2 infected hamster proximal tibial epiphysis. Measurements of trabecular thickness (Tb. Th), number (Tb. N.) and separation (Tb. S) of the proximal tibial epiphysis of golden Syrian hamsters 4 and 30 days after SARS-CoV-2 infection compared to mock infected group. Each group n =5. Data are expressed as mean ± S.E.M. One-way ANOVA with Tukey’s multiple comparison.

[0033] Figure 8 | No apparent morphological changes in meniscus and synovium before and after SARS-CoV-2 infection. Representative H&E staining showing no significant abnormal pathological changes (meniscus compression and tear, mononuclear cell infiltration, thickening of the inner synovial layer) in the meniscus and synovium of SARS-CoV-2 infected hamsters compared to mock infected group.

[0034] Figure 9| No significant changes in articular cartilage surface roughness. a, H&E images of articular cartilage were cropped from the original images for analysis. b, Images were adjusted and binarized for analysis. Articular surface contours were extracted using the findContours function in OpenCV. Articular surface roughness was then calculated using the length of the articular surface divided by its width according to a previously published protocol. c, No statistical significance in articular surface roughness after SARS-CoV-2 infection and masitinib treatment.

[0035] Figure 10 | Semi-quantification of SARS-CoV-2 viral components. a, Presence of SARS-CoV-2 nucleocapsid protein (NP) in the medullar cavity of the subchondral bone region of hamster tibia was graded based on the integrated optical density (IOD) per thousand (‰). IOD of all sections was divided into 3 categories, from “-” representing the lowest abundance to “++” representing the highest abundance. b, Table showing the number of samples in each grade based on IOD. Chi-square test. c, Presence of SARS-CoV-2 spike protein (SP) in the medullar cavity of the subchondral bone region of hamster tibia was graded based on the integrated optical density (IOD) per hundred (%). IOD of all sections was divided into 4 categories, from “-” representing the lowest abundance to “+++” representing the highest abundance. b, Table showing the number of samples in each grade based on IOD. Chi-square test. n =5); 4 dpi PBS ( n =5); 4 dpi Mac ( n =4); 30 dpi PBS ( n =5) and 30 dpi Mac ( n =5). Chi-square test.

[0036] Figure 11 | SARS-CoV-2 spike protein RBD leads to deterioration of mouse subchondral bone structure integrity. Bone volume fraction (BV / TV), trabecular thickness (Tb.Th), number (Tb.N.), separation (Tb.S) and structure model index (SMI) were measured in the epiphysis of tibia of mice receiving different treatments. Vehicle control ( n =4); SP RBD ( n =4); low dose Mac (0.3 mg / kg) ( n =4) and high dose Mac (3 mg / kg) ( n =5). Data are expressed as mean ± S.E.M. Mann-Whitney test was used in comparisons between vehicle control and SP RBD.

[0037] Figure 12| Effect of early marstil treatment on subchondral bone microarchitecture. Measurement of trabecular thickness (Tb. Th), number (Tb. N.) and separation (Tb. S) of the proximal tibial epiphysis of the golden Syrian hamsters treated or not with marstil at 4 dpi. Data are expressed as mean ± S.E.M. PBS (n=5) at 4 dpi; Mac (n=4) at 4 dpi. Unpaired two-tailed Student's t-test. n =5) at 30 dpi and Mac (n=5) at 30 dpi. Unpaired two-tailed Student's t-test. n =5) at 30 dpi and Mac (n=5) at 30 dpi. Unpaired two-tailed Student's t-test.

[0038] Figure 13 | Effect of delayed marstil treatment on subchondral bone microarchitecture. Measurement of trabecular thickness (Tb. Th), number (Tb. N.) and separation (Tb. S) of the proximal tibial epiphysis of the golden Syrian hamsters treated or not with marstil at 30 dpi. Data are expressed as mean ± S.E.M. PBS (n=5) at 30 dpi and Mac (n=5) at 30 dpi. Unpaired two-tailed Student's t-test. n =5) at 30 dpi and Mac (n=5) at 30 dpi. Unpaired two-tailed Student's t-test. n DETAILED DESCRIPTION

[0039] Definitions Throughout this disclosure, unless the context requires otherwise, the word "comprise," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also to be noted that, in this disclosure, particularly in the claims and / or paragraphs, terms such as "comprise," "comprises," "comprising," and the like can have the meaning ascribed to it in U.S. patent law; e.g., they can mean "include," "includes," "including," and the like; and that terms such as "consisting essentially of and "consists essentially of can have the meaning ascribed to it in U.S. patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the application. Furthermore, throughout this disclosure and in the claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers unless the context requires otherwise.

[0040] Unless specifically stated otherwise, as used herein, the use of the singular includes the plural (and vice versa). In addition, if use of the terms "about," "approximately," "almost," or the like before a quantity, value, or the like is stated, the present teachings also encompass the specific quantity, value, or the like itself, unless specifically stated otherwise. As used herein, unless otherwise indicated or inferred, the term "about" means a variation ±10%, ±7%, ±5%, ±3%, ±1%, or ±0% of the stated value, unless otherwise indicated or inferred.

[0041] ​

[0042] The term "therapeutically effective amount" as used herein refers to the amount of a compound or therapeutic agent that elicits the biological and / or pharmaceutical response that is being sought in a cell culture, tissue system, subject, animal, or human by a researcher, veterinarian, clinician, or medical doctor.

[0043] As used herein, the terms "treat," "treating," "treatment," and the like, refer to the reduction or amelioration of a disorder / disease and / or symptoms associated therewith. It will be understood that, although not explicitly stated, treatment of a disorder or condition does not require complete eradication of the disorder, condition, or symptoms associated therewith. In certain embodiments, treatment includes prevention of a disorder or condition and / or symptoms associated therewith. The terms "prevention" or "prevent" as used herein refer to any effect which retards or at least delays the development of a disorder, condition, or symptoms associated therewith. Prevention can include primary, secondary, and tertiary prevention, wherein: a) primary prevention avoids the development of disease; b) secondary prevention activities aim at early disease treatment, thereby increasing the chance of interventions that prevent disease progression and symptom appearance; and c) tertiary prevention reduces the negative impact of an already existing disease by restoring function and reducing complications associated with the disease.

[0044] The term "sequelae" as used herein refers to one or more pathological conditions in a subject caused by a coronavirus infection.

[0045] The term "subject" as used herein refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, and rodents.

[0046] As used herein, the term “variant” when used in connection with a virus (e.g., SARS-CoV-2) refers to a virus that is a descendant of a reference (or “parent”) virus and has one or more variations in its genome (e.g., RNA genome), or a genetically engineered virus that has one or more variations in its genome relative to a reference (or “parent”) virus, which may or may not result in changes to the proteins encoded by the RNA sequence (e.g., one or more proteins of the variant virus may include substitutions, deletions, or insertions compared to the parent strain). Known SARS-CoV-2 variants include, but are not limited to, B.1.1.7 (α variant), B.1.351 (β variant), P.1 (γ variant), B.1.617.2 (δ variant), and B.1.1.529 (ω variant). Viral variants may contain genomic sequences that share approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity or homology with a reference (or “parent”) genome sequence.

[0047] As used in this article, the term "substantially silent" EDNRB and / or EDNRA "This refers to the expression, activity, or level of the target gene or its encoded protein compared to when the inhibitory nucleic acid molecule targeting the endothelin-1 nucleic acid sequence is absent, after the introduction of a target gene." EDNRB and / or EDNRA In the presence of repressive nucleic acid molecules in the nucleic acid sequence, the expression, activity, or level of the target gene or the protein encoded by the target gene is reduced by approximately 10% to 100%, 10% to 90%, 20% to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 50% to 80%, 50% to 70%, 55% to 70%, 55% to 69.2%, 70% to 90%, or 80% to 90%. Generally, when a gene is substantially silenced, compared to the absence of repressive nucleic acid molecules, the expression, activity, or level of the target gene or the protein encoded by the target gene will be reduced by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%.

[0048] A method for treating or preventing skeletal sequelae of coronavirus infection in subjects in need, the method comprising administering a therapeutically effective amount of an endothelin receptor antagonist to the subject.

[0049] In certain embodiments, the endothelin receptor antagonist is not co-administered with a human sodium-dependent glucose transporter 2 (SGLT2) inhibitor, such as dapagliflozin. In certain embodiments, the endothelin receptor antagonist is not co-administered with an anthelmintic, such as niclosamide.

[0050] In certain embodiments, the subject does not have pulmonary arterial hypertension (PAH), cancer, cardiovascular disease, hypertension, pulmonary arterial hypertension, diabetic nephropathy, kidney disease, occlusive vascular disease, congestive heart failure, cerebral hemorrhage, cerebral aneurysm, glomerular disorder, kidney disease, congestive heart failure, or connective tissue disorder.

[0051] The endothelin receptor antagonist can be an antibody, an antibody fragment (such as Fab, Fab', F(ab')2, Fv, or single chain (ScFv)), a peptide, an inhibitory nucleic acid molecule, or a small molecule (synthetic or naturally isolated or derived), wherein the inhibitory nucleic acid molecule substantially silences EDNRB and / or EDNRA .

[0052] In certain embodiments, the endothelin receptor antagonist is a selective ET A receptor antagonist, such as sitaxentan, ambrisentan, atrasentan, zibotentan, darusentan, avosentan, and clazosentan, a selective ET B receptor antagonist, or a dual ET A receptor and ET B receptor antagonist, such as bosentan, tezosentan, and macitentan.

[0053] Exemplary endothelin receptor antagonists include, but are not limited to, sitaxentan, ambrisentan, atrasentan, BQ-123 (cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu-)), sparsentan, zibotentan, alprostadil, idonatant, bosentan, macitentan, tezosentan, BQ-788 (sodium N-{[(2R,6S)-2,6-dimethyl-l-piperidinyl]carbonyl}-4-methyl-L-leucyl-N-[(lR)-l-carboxypentyl]-l- (methoxycarbonyl)-D-tryptophanamide), A192621 ((2R,3R,4S)-4-(l,3-benzodioxol-5-yl)-l-[2-[(2,6-diethylphenyl)amino]-2- oxoethyl]-2-(4-propoxyphenyl)pyrrolidine-3-carboxylic acid), and mixtures thereof.

[0054] In certain embodiments, the endothelin receptor antagonist is macitentan.

[0055] The endothelin receptor antagonist can be administered after the subject can have been exposed to a coronavirus, after the appearance of one or more symptoms of coronavirus infection, after a positive diagnosis of coronavirus infection, during the acute phase of coronavirus infection, during the subacute phase of coronavirus infection, during the recovery phase of coronavirus infection, during the chronic phase of coronavirus infection, or a combination thereof.

[0056] The endothelin receptor antagonist can be administered parenterally or nonparenterally. Parenteral administration includes intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraarticular, intraosseous, intraspinal, and intrasternal injection and infusion. Nonparenteral administration includes oral, sublingual, topical, transdermal, ocular, aural, nasal, vaginal, rectal, mucosal, or transcutaneous. In certain embodiments, the endothelin receptor antagonist is administered by intravenous, intraarticular, or intraosseous injection.

[0057] In certain embodiments, the method further comprises co-administering a second therapeutic agent, such as an antiviral agent, an antibacterial agent, an antiparasitic agent, an analgesic agent, an anti-inflammatory agent, or a combination thereof. In certain embodiments, the antiviral agent is paroxymer, monupiravir, sotrovimab, remdesivir, lopinavir, ritonavir, or a combination thereof. The antibacterial agent can be azithromycin or ciprofloxacin. The anti-inflammatory agent can be dexamethasone.

[0058] The coronavirus infection can be caused by an existing coronavirus, such as severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome-related coronavirus (MERS-CoV), human coronavirus 229E (229E-CoV-2), human coronavirus NL63 (NL63-CoV), human coronavirus OC43 (OC43-CoV), and human coronavirus HKU1 (HCoV-HKU1), or a new variant thereof.

[0059] In certain embodiments, the SARS-CoV is wild-type SARS-CoV, B.1.1.7 (alpha variant), B.1.351 (beta variant), P.1 (gamma variant), B.1.617.2 (delta variant), B.1.1.529 (omicron variant), or a new variant thereof.

[0060] The subject can be asymptomatic or symptomatic. Where the subject is symptomatic, the subject can exhibit one or more symptoms selected from the group consisting of fever, cough, fatigue, shortness of breath, muscle pain, joint pain, sore throat, headache, and chills (e.g., fever, cough, and shortness of breath). In certain embodiments, the one or more symptoms can appear 2-14 days after the subject is exposed to the coronavirus.

[0061] Prior to the step of administering a therapeutically effective amount of an endothelin-1 antagonist, the subject can be optionally diagnosed with a coronavirus infection. The coronavirus can be diagnosed using any method known in the art, including but not limited to polymerase chain reaction (PCR), such as reverse transcription PCR (RT-PCR), real-time PCR (e.g., quantitative PCR (qPCR)), and real-time RT-PCR (rRT-PCR), or antigen testing, such as lateral flow testing.

[0062] The skeletal sequelae can include one or more of joint pain, viral arthritis, osteopenia, osteochondral injury, and osteoporosis.

[0063] The joint pain and / or osteochondral injury can involve one or more joints selected from the group consisting of sternoclavicular joint, shoulder joint, elbow joint, metacarpophalangeal joint, sacroiliac joint, knee joint, transverse tarsal joint, acromioclavicular joint, radiocarpal joint, carpometacarpal joint, hip joint, ankle joint, interphalangeal joint, temporomandibular joint, shoulder joint, zygapophyseal joint, and subtalar joint. In certain embodiments, the joint pain involves a joint selected from the group consisting of ankle joint, knee joint, sacroiliac joint, and combinations thereof.

[0064] The viral arthritis, osteopenia, and / or osteoporosis can involve one or more bones selected from the group consisting of cervical vertebra, thoracic vertebra, lumbar vertebra, sacrum, coccyx, sternum, rib, occipital bone, parietal bone, frontal bone, temporal bone, ethmoid bone, sphenoid bone, lacrimal bone, maxilla, nasal bone, palatine bone, zygomatic bone, inferior nasal concha, mandible, hyoid bone, vomer, humerus, radius, ulna, carpal bone, phalangeal bone, metacarpal bone, femur, tibia, fibula, patella, tarsal bone, phalangeal bone, metatarsal bone, clavicle, scapula, or hip bone. In certain embodiments, the viral arthritis involves one or more bones selected from the group consisting of patella, fibula, tibia, femur, vertebra, and combinations thereof.

[0065] In certain embodiments, the subject is at least 40 years old, at least 50 years old, at least 60 years old, at least 70 years old, or at least 80 years old.

[0066] Discussion Here, we report the phenotype of SARS-CoV-2-induced osteochondral injury in a hamster model, despite joint pain having been recognized as a prominent symptom in humans following acute COVID. Currently, the clinical diagnosis of viral or reactive arthritis following SARS-CoV-2 infection relies on symptoms, i.e., joint pain and laboratory examinations of case series 13-15,35,43, and lack specific imaging evidence showing structural damage to the joint after viral infection. Unlike the typical features of arthritis, we only observed osteochondral damage in infected hamsters, while other joint tissues such as synovium and meniscus were not significantly changed. Our findings suggest the urgent need to screen COVID survivors with persistent joint pain for structural damage. Furthermore, we will raise public awareness of this debilitating long COVID post-skeletal complication that requires prompt consultation and timely intervention.

[0067] The pathological mechanisms of virus-induced osteochondral damage or arthritis remain controversial. This can be the result of direct viral attack 35 , excessive inflammation 16 , or autoimmune reactions 17 . SARS-CoV-2 viral mRNA was detected in the synovial fluid of COVID patients 35 . We also found the presence of viral spike and nucleocapsid proteins in the subchondral bone marrow cavity of infected hamsters. This piece of evidence points to a direct viral attack. At the same time, we showed that elevated plasma and endothelial ET-1 is a response to SARS-CoV-2 exposure. Our data suggest that direct viral attack and excessive inflammation - the synergy of ET-1 drives osteochondral damage. Our work further substantiates this concept.

[0068] Significant bone loss in long bones and lumbar vertebrae was observed in a golden Syrian hamster model after SARS-CoV-2 infection 2 . It is postulated that SARS-CoV-2 triggers a cytokine storm, and increased pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) further induce osteoclastogenesis through upregulation of receptor activator of nuclear factor kappa-B ligand (RANKL) in lung epithelial cells. Here, our study proposes another pro-inflammatory cytokine candidate, ET-1, which is reported to stimulate osteoclast activity and bone resorption through its type A receptor 44-46 . This suggests that endothelin type A receptor is a rational therapeutic target for SARS-CoV-2-induced subchondral bone loss.

[0069] SARS-CoV-2 can induce senescence in multiple cells and tissues 47,48 , leading to organ damage 49 . Elimination of senescent cells has emerged as a promising therapeutic strategy for COVID-19-induced organ dysfunction 49,50 . The exact molecular mechanisms of SARS-CoV-2-induced cellular senescence are not fully understood. Direct induction of endothelial senescence by viral spike has recently been reported 51In our study, we also found that viral spike RBD can induce chondrocyte senescence. In addition to viral spike protein, ET-1 can accelerate cell senescence. Blocking endothelin B-type receptor effectively prevented the accumulation of senescent cells in various types of cells and animal models 24,52,53 . Therefore, we repurposed macitentan, an FDA-approved anti-hypertensive drug with dual blockade of ETAR and ETBR, to treat viral-induced organ damage, such as the osteochondral junction of the knee joint.

[0070] We demonstrated that macitentan can effectively rescue osteochondral damage in a preclinical setting, both in the acute and subacute phase of SARS-CoV-2 infection. In addition to exerting anti-inflammatory effects through endothelin receptor blockade 54 , the potential antiviral effect of endothelin receptor antagonists was also shown to reduce influenza and cytomegalovirus RNA counts in vitro 55,56 . We observed that macitentan can reduce viral spike protein in the local joint tissue of infected hamsters in vivo. Consistent with these experimental results, a clinical case series reported the unexpected recovery of SARS-CoV-2 pneumonia in patients with pulmonary arterial hypertension who received endothelin receptor antagonists, including macitentan 57 . A 2 / 3 phase clinical trial is ongoing to apply the endothelin receptor antagonist ambrisentan to COVID-19 inpatients to reduce multi-organ damage by improving vascular function (NCT04393246) 58 . It is worth further investigating the antiviral effect of endothelin receptor antagonists in the context of COVID-19.

[0071] There are two main limitations in this study, which should be carefully considered in data interpretation. First, the animals we used in this study are 10 to 12 weeks old, which corresponds to the puberty of female golden Syrian hamsters 59 . It is equivalent to the puberty of human beings when the skeletal development is ongoing. However, due to the limited healing capacity of articular cartilage 40 , the number of chondrocytes cannot be replenished after viral infection. It is well known that senescent cells are more susceptible to viral infection 51 . It is worth further studying the osteochondral damage of old hamsters rather than young hamsters. Second, we studied the wild-type SARS-CoV-2, HKU-001a. It is estimated that the severity of osteochondral damage varies greatly between different SARS-CoV-2 strain variants. This prompted further comparative studies in this direction.

[0072] In summary, we depicted the temporal changes in osteochondral functional units during the hamster's response to SARS-CoV-2 infection. SARS-CoV-2 induces cellular oxidative stress and endothelial dysfunction through systemic and local activation of endothelin signaling. ET-1 enhances vascular permeability. 60 It may also allow viral proteins to leak from the systemic bloodstream into local joint tissues. ET-1 may also trigger osteoclastogenesis via its type A receptor to induce subchondral bone loss and cystic formation at osteochondral junctions. It may promote chondrocyte senescence and shedding via its type B receptor, ultimately leading to cartilage damage. Dual blocking of endothelin type A and type B receptors, such as with macitentan, can effectively reduce subchondral bone loss and decrease virus-induced articular chondrocyte damage. The results of this study lay a solid scientific foundation for repositioning endothelin receptor antagonists for the management of long-term COVID joint injury and arthralgia.

[0073] result SARS-CoV-2 infection induces bone and cartilage damage in hamsters We examined 10-12 week old female golden Syrian hamsters ( Mesocricetus auratu Structural integrity of the knee joint Figure 1 a). Micro-computed tomography scans at 4 and 30 days post-SARS-CoV-2 infection (dpi). CT scans revealed that, compared to the simulated infection group, infected hamsters had significantly reduced subchondral bone mineral density (BMD), volume fraction (BV / TV), and trabecular thickness at 4 and 30 dpi in the proximal tibial epiphysis. Figure 1 b, 7). At 4 dpi, tartrate-resistant acid phosphatase (TRAP) was positive. + The ratio of osteoclasts to bone circumference (B.Pm) was significantly increased, while SP7 showed a significantly increased ratio. + The osteoblast count / B.Pm showed no significant change. Notably, during the subacute phase of infection, both osteoclasts and osteoblasts decreased, exhibiting unbalanced bone remodeling at 30 dpi. Figure 1 c, d).

[0074] In addition to subchondral bone loss, we also observed cyst formation at the osteochondral junction in infected animals. As shown by H&E and Masson trichrome staining, the cystic lesions were accompanied by a decrease in collagen content and the production of fibrin exudate. Figure 1 e.g., histopathological analysis further showed that chondrocytes detached as early as 4 dpi. Figure 1 h). Although cystic lesions tend to resolve over time after infection, the loss of chondrocytes and proteoglycans is still present at 30 dpi ( Figure 1g-i). In the acute phase of SARS-CoV-2 infection, i.e. 4 dpi, senescent chondrocytes accumulated in the knee joint, manifested by p16 INK4a translocation of the nucleus and increased cytoplasmic HMGB1 Figure 1 j, k).

[0075] We also examined the morphology of the menisci and synovium of infected hamsters Figure 8 ). The roughness of the articular cartilage surface was also assessed using a defined protocol 32 Figure 9 No significant changes were observed in infected animals compared to mock-infected groups. In summary, SARS-CoV-2 infection induced persistent osteochondral damage in hamsters.

[0076] SARS-CoV-2 infection triggers endothelial dysfunction with systemic activation of endothelin signaling.

[0077] SARS-CoV-2 virus and its spike protein cause endothelial damage and dysfunction 33,34 , which can be related to the disease manifestations in various tissues and organs. Using hydrogen peroxide as a positive control, we observed that both the pseudovirus expressing the D614G mutant spike protein (pVD614G) and the SARS-CoV-2 spike protein receptor binding domain (SP RBD) increased cellular oxidative stress and caused mitochondrial dysfunction in endothelial cells 。 Pseudovirus and SP RBD stimulation caused a change in mitochondrial dynamics from tubular to clumped Figure 2 Upregulation of EDN1 mRNA, the protein-coding gene for the most potent vasoconstrictor endothelin-1 (ET-1), was detected after pV or SP RBD stimulation Figure 2 Furthermore, mRNA expression of endothelin receptors as well as the receptor activator of nuclear factor kappa-B ligand (RANKL) was increased, indicating enhanced bone resorption through activated osteoclast activity.

[0078] Based on clinical observations of hospitalized COVID-19 patients 21 , we observed an upregulation of serum ET-1 levels in SARS-CoV-2 infected golden Syrian hamsters at 4 days post infection Figure 2 e). Furthermore, we observed an increase in 4HNE expression (an indicator of oxidative stress) near areas of endothelial dysfunction, as shown by von Willebrand factor (vWF) staining in the subchondral bone marrow cavity Figure 2 f), indicating that SARS-CoV-2 affects endothelial function in the joint.

[0079] Viral spike causes oxidative stress and senescence in chondrocytes with activation of endothelin signaling.

[0080] ​Clinical evidence has shown that viral RNA can be detected in synovial fluid after SARS-CoV-2 infection 35 . Recent autopsy analysis also showed that SARS-CoV-2 nucleocapsid and spike proteins were detected in chondrocytes of bronchial cartilage, despite persistent negative molecular testing for SARS-CoV-2 RNA, suggesting that accumulation of viral antigens in tissues can have a lethal impact on infected patients 36 . Consistent with this observation, we also found the presence of SARS-CoV-2 viral proteins, i.e., nucleocapsid and spike proteins, in the knee joints of infected hamsters Figure 3 a, b, 10). While the amount of nucleocapsid protein disappeared after 30 dpi, the spike protein accumulated in the joints. Interestingly, this viral antigen was mostly located around blood vessels in the subchondral bone marrow cavity. With increased vascular permeability, viral proteins can leak out from blood vessels. 37 This observation was also accompanied by upregulation of endothelin receptor subtypes (ETAR and ETBR) in chondrocytes in the joint cartilage after infection compared to mock-infected groups Figure 3 c). ETBR was recently reported to transduce ET-1 signals, leading to chondrocyte senescence and cartilage damage 24 .

[0081] Therefore, we treated C28 / I2 chondrocytes with the SARS-CoV-2 SPRBD to investigate the effects of viral components on cartilage. RBD increased the gene expression of EDN1 , EDNRA and EDNRB in chondrocytes Figure 3 d). It also upregulated matrix metalloproteinases MMP1 and MMP13 leading to cartilage degradation Figure 3 e). qPCR and immunostaining for p16 INK4a and senescence-associated β-galactosidase (SA- -gal) indicated that RBD induced chondrocyte senescence, and the synergistic effect of ET-1 and RBD promoted chondrocytes to enter the senescent state to a greater extent compared to RBD alone Figure 3 e-g).

[0082] Endothelin receptor antagonists have been shown to reduce oxidative stress and cell senescence in various in vitro models 24,38 . Here, we examined the efficacy of endothelin receptor antagonists on viral SPRBD-induced cellular oxidative stress and mitochondrial dynamics. Our data showed that macitentan effectively reduced the increase in oxidative stress induced by SARS-CoV-2 spike protein Figure 3h). Mitochondria changed from rod-like to clumps after RBD challenge, while masitinib showed a restoration of rod-like structures Figure 3 i). This suggests a potential therapeutic effect of masitinib to rescue SARS-CoV-2-induced joint damage.

[0083] Viral spike causes joint pain and chondrocyte senescence To further confirm the effect of SARS-CoV-2 spike protein on joint pain and joint damage in vivo, we injected 10 µg of viral spike RBD via the tail vein of mice. Pain behavior was longitudinally monitored for 2 weeks after spike RBD injection using the von Frey filament test 39 and animals were sacrificed for histological evaluation Figure 4 a). The effect of intraperitoneal injection of masitinib at different doses (0.3 mg / kg and 3 mg / kg) on joint pain relief starting two days after RBD injection was also evaluated.

[0084] The paw withdrawal threshold in response to mechanical stimulation decreased from three days after SP RBD injection and lasted for at least 2 weeks, indicating that hypersensitivity occurred in the acute and subacute phase after injection Figure 4 b). High doses of masitinib started to show a beneficial effect on pain sensitization, which was reflected in the increase of paw withdrawal threshold after 5 days of treatment. Low doses of masitinib were also able to achieve the same pain perception tolerance as high doses after 12 days of treatment regimen Figure 4 c).

[0085] We further investigated the structural and cellular changes in joints after RBD challenge and the therapeutic effect of masitinib. Similar to hamsters infected with SARS-CoV-2 virus, viral spike RBD could induce subchondral bone loss in mice injected with spike protein RBD Figure 4 d, 10). Loss of proteoglycans and surface fibrillation were observed in the articular cartilage of RBD-treated mice Figure 4 e). RBD also led to upregulation of ET-1, nuclear p16 INK4a translocation and cytoplasmic HMGB1 translocation in articular cartilage Figure 4 f-h), indicating that spike protein RBD alone could cause chondrocyte senescence in a mouse model. Masitinib at 0.3 mg / kg performed better in preserving proteoglycans and preventing accumulation of senescent chondrocytes, although the analgesic effect was faster at higher doses Figure 4 e-h). Therefore, we selected a low dose of masitinib for further investigation.

[0086] Early masitinib treatment partially rescues SARS-CoV-2-induced osteochondral damage To investigate the efficacy of macitentan against true SARS-CoV-2 infection, infected hamsters were intraperitoneally injected daily at a dose of 0.3 mg / kg body weight, one day after viral exposure. The therapeutic dose was selected based on our experimental use in a viral spike protein RBD mouse model. Figure 4 Animals were euthanized at 4 dpi to investigate the early therapeutic effect on acute joint injury. Figure 5 a). Macitentan tends to reduce the amount of viral spike protein in local joint tissue ( Figure 5 b). Early treatment also reduced the expression levels of vWF and 4HNE in the medullary cavity of subchondral bone, demonstrating that masitantan improved vascular function and reduced oxidative stress (b). Figure 5 c). Compared with the propagation agent (PBS) group, TRAP in the subchondral bone of animals treated with macitentan was significantly lower. + The number of osteoclasts was significantly reduced, but SP7 was not affected. + The number of osteoblast progenitor cells ( Figure 5 d, e). As a result, short-term macitentan treatment during the acute phase could restore subchondral BMD, although there was no significant difference in BV / TV at 4 dpi, which may be due to the short duration of treatment, i.e., 4 days ( Figure 5 f, 12).

[0087] More interestingly, macitentan treatment alleviated SARS-CoV-2-induced cartilage damage by effectively reducing the area of ​​cysts and decreasing fibrin exudate at the osteochondral junction. Figure 5 It also prevents virus-induced collagen loss, as shown by Masson's trichrome staining (gi). Figure 5 h). The number of chondrocytes is a key factor in cartilage homeostasis and disease, indicating the ability to synthesize and replenish the matrix. 40 Compared to the caustic agent treatment group, more chondrocytes were observed in the articular cartilage of hamsters treated with macitentan. Figure 5 j). We also found that early macitentan treatment effectively prevented the accumulation of senescent chondrocytes, which was reflected in the p16 nucleus in articular cartilage. INK4a and a decrease in the number of HMGB1 cells in the cytoplasm ( Figure 5 (k, l). In general, early macitentan treatment can effectively alleviate SARS-CoV-2-induced acute joint injury.

[0088] Delayed macitentan treatment alleviates subacute subchondral bone loss during SARS-CoV-2 infection. Due to the limited healing capacity of articular cartilage, SARS-CoV-2-induced osteochondral damage persisted for up to 30 days post-infection (dpi) in infected hamsters. Figure 1). This would help to understand the possible causes of fluctuating joint pain in clinical reports lasting for weeks or months 41,42 . Therefore, it is worth exploring whether delayed mascimonan treatment after the acute phase of SARS-CoV-2 infection is still effective in rescuing the bone and cartilage damage.

[0089] Mascimonan treatment was initiated at 16 dpi and lasted for 2 weeks until 30 dpi, which was considered as the subacute phase of SARS-CoV-2 infection ( Figure 6 a). We found that delayed mascimonan treatment could reduce the amount of retained viral spike protein and restore the virus-induced joint structural damage ( Figure 6 b). Oxidative stress in the subchondral bone, as shown by 4HNE immunostaining, subsided at 30 dpi. However, the marker of endothelial dysfunction, vWF, was still present in the bone marrow but at a lower level compared to the acute phase. Mascimonan treatment in the subacute phase could effectively reduce the expression level of vWF. This suggests that delayed treatment could also improve vascular function in the joint ( Figure 6 c).

[0090] Micro-CT analysis showed that the subchondral bone mass and microstructure of the tibial epiphysis, including BMD, BV / TV, trabecular thickness, number, and separation, were improved after mascimonan treatment in infected animals ( Figure 6 d, 13). In contrast to the acute phase, treatment in the subacute phase of viral infection increased the number of SP7 + osteoblast progenitor cells, but did not affect the number of TRAP + osteoclasts ( Figure 6 e, f). The slight but insignificant improvement in cyst formation and chondrocyte detachment after one month of infection could be due to the active growth of adolescent hamsters. However, since the articular cartilage cannot self-repair, delayed mascimonan treatment in the subacute phase would not lead to further improvement in the articular cartilage structure ( Figure 6 g-j). However, delayed treatment could effectively reduce the number of senescent chondrocytes in the articular cartilage, as shown by pl6 INK4a and HMGB1 staining ( Figure 6 k, l). In summary, delayed mascimonan treatment could still alleviate the virus-induced bone and cartilage damage.

[0091] Methods SARS-CoV-2 virus and biosafety SARS-CoV-2 (HKU-001a strain, GenBank accession number: MT230904) was isolated from a nasopharyngeal aspirate sample of a COVID-19 patient in Hong Kong, China 61 . The virus was amplified in VeroE6 cells to prepare the working stock as previously described 62All experiments involving the use of authentic SARS-CoV-2 virus followed the standard operating procedures of the Biosafety Level 3 facility at the University of Hong Kong (HKU).

[0092] SARS-CoV-2 Golden Syrian hamster infection model All procedures were approved by the Committee on the Use of Live Animals for Teaching and Research at the University of Hong Kong (CULATR). On day 0 post-infection (dpi), 50 l of 10 5 PFU SARS-CoV-2 was used to intranasally infect 10-12-week-old female Golden Syrian hamsters ( Mesocricetus auratus ). 63,64 Fifty microliters of PBS was used for mock-infected hamsters. Hamsters were sacrificed at 0, 4, or 30 dpi, and sera and hind limbs were collected for subsequent analyses.

[0093] SARS-CoV-2 Spike protein RBD-treated mouse model All experimental procedures were approved by the Animal Subjects Ethics Sub-committee of the Hong Kong Polytechnic University (ASESC) (22-23 / 363-BME-R-GRF). Six-month-old female Balb / c mice were used in the study. Briefly, mice were anesthetized using isoflurane (3-4% for induction and 1-2% for maintenance). 10 g of recombinant wild-type SARS-CoV-2 Spike protein receptor binding domain (RBD) was injected via the tail vein. The control group was injected with the same volume of PBS. Animals were sacrificed 14 days post-injection. Hind limbs were collected for micro-CT and histological analyses.

[0094] In vivo maraviroc treatment For the RBD mouse model, we used 2 different doses of maraviroc (Actelion Pharmaceuticals Ltd, Allschwil, Switzerland) (0.3 mg / kg and 3 mg / kg). Intraperitoneal injections were performed daily starting from 2 days post-RBD injection. For the hamster infection model with authentic SARS-CoV-2 virus, 0.3 mg / kg of maraviroc was used. The dose is equivalent to 2.34 mg 65 Two treatment regimens were adopted to investigate different treatment timeframes. For early treatment, maraviroc was administered intraperitoneally at 1 dpi and continued for 4 days. For delayed treatment, the drug was given in the same manner only at 16 dpi and continued for 2 weeks. Animals were sacrificed at the end of treatment.

[0095] Von Frey silk experiment Nociceptive tolerance in mice was assessed using an electronic von Frey sensory analyzer (IITC, USA) with a detection range of 0 to 74 g, based on a previously published simplified up-down approach. 39 Before formal measurements, the animals were trained for 3 days. In short, the animal was placed on an elevated grid where it remained for at least 15 minutes to acclimatize. The entire scenario was conducted in a black box to keep the animal calm. Briefly, after the trial, intermediate silk #3 was selected as the starting silk. The silk was applied to the midfoot surface of the hind paw, and the withdrawal response was recorded. The next silk was selected based on the mouse's response. The precise force was recorded after 5 stimuli, with an adjustment factor of [missing value]. 0.5. Von Frey filament tests were performed before, and 3, 7, and 14 days after injection of the spike protein RBD. Improvement in nociceptive tolerance was assessed by comparing readings between the RBD and macitentan treatment groups.

[0096] ELISA method for measuring serum ET-1 levels Blood was collected when the hamster was euthanized and left to stand at room temperature for 20 minutes, then... Serum was obtained by centrifugation at 1500 × g for 10 minutes at C. ET-1 in the serum was quantified using an endothelin-1 ELISA kit (ab133030, Abcam) according to the manufacturer's protocol. The heat-inactivated serum sample was added to an antibody-coated plate for assay. Absorbance was measured at 450 nm to determine the concentration.

[0097] Microcomputed tomography (MCM) CT analysis If necessary, bone samples from hamsters and mice were fixed in 4% paraformaldehyde for 48 hours, followed by fixation in 70% ethanol for 24 hours to inactivate pathogens in a biosafety level 3 facility. High-resolution micro-CT systems (1276, SkyScan, Kontich, Belgium) were then used at 13.0 pixels per second. Samples were scanned at a resolution of m. A voltage of 90 kV and a current of 200 μA were used during the scanning process. Standard densities of 0.25 and 0.75 g / cm³ were used to calibrate bone mineral density (BMD). 3The model was reconstructed and analyzed using NRecon and CTAn software (Skyscan Company), respectively. One hundred images covering the trabecular bone of the proximal epiphysis region above the tibial growth plate were selected as the region of interest (ROI) for measuring the trabecular parameters, including bone volume fraction (BV / TV) and bone mineral density (BMD), trabecular number (Tb.N.), thickness (Tb.Th.), and separation (Tb.Sp.). Three-dimensional structures were generated using 20 images in the middle of the joint using CTvol (Skyscan Company). Measurements of mouse samples were the same, except that the resolution was 8.67 m per pixel, and the binning was 1.

[0098] Histological analysis Bone samples from hamsters and mice were harvested and fixed. Tibiae were decalcified using 10% ethylenediaminetetraacetic acid (EDTA) (pH 7.4). The samples were dehydrated with ethanol and cleared with xylene. The samples were then embedded in paraffin. Five-micrometer sections were made for histological analysis. The sections were stained with hematoxylin and eosin (H&E) (ab245880, Abeam), Safranin O / fast green (Alfa Aesar), Masson’s trichrome (Abeam), and tartrate-resistant acid phosphatase (TRAP) (Amizona Scientific, US) according to standard staining protocols for histopathological evaluation. Articular cartilage roughness was determined according to a published protocol 32 . The severity of the cystic lesions was quantified by the percentage of cyst area divided by the total area of the articular cartilage.

[0099] Immunohistochemistry and immunocytochemistry Sections were stained with different antibodies to detect specific proteins in our samples. Briefly, antigen retrieval was performed using citrate buffer or Tris / ETDA according to the recommendations on the product sheet. Quenching of endogenous peroxidase activity was performed using hydrogen peroxide. After blocking, the sections were incubated with primary antibodies overnight at 4°C. Antibodies used: anti-SARS-CoV-2 spike protein RBD (MA5-36247), anti-SARS-CoV-2 nucleocapsid protein (MA17404, Invitrogen), anti-endothelin-1 (ab117757, Abeam), anti-ETAR (ab117521, Abeam), anti-ETBR (ab117529, Abeam), anti-p16 INK4a(ab189034 and ab54210, Abcam) and anti-HMGB1 (ab18256, Abcam), anti-SP7 (ab209484, Abcam), and anti-cysteine ​​3 (43-7800, Invitrogen). Positive signals were visualized using diaminobenzidine (DAB) staining (Vector Lab, US) or fluorescent secondary antibody (Invitrogen). Semi-quantitative analysis of NPs and SPs was performed by measuring the integrated optical density (IOD) of DAB staining. Precise grading methods were employed. Figure 10 As shown in the image.

[0100] For multichannel staining of ETAR and ETBR, Dendron Fluor multichannel was used. IHC assay kit (Neon, Histova, China). Primary antibody was incubated overnight at 4°C, followed by tyramine signal amplification (TSA) the next day, and then a new antibody was used daily. Positive signals were visualized using horseradish peroxidase (HRP) secondary antibody and different fluorophores NEON520 and NEON570. SN520 was used as a nuclear staining agent. Images were captured using an OLYMPUS BX51WI and Leica TCS SPE confocal microscope.

[0101] For the immunocytochemistry of chondrocytes, cells grown on coverslips were fixed, permeabilized, and blocked before being treated with anti-p16. INK4a (ab189034, Abcam) was incubated overnight at 4°C. Secondary antibody was applied the next day to visualize the positive signal, and images were captured using a Leica TCS SPE confocal microscope. All image analyses were performed using Image-Pro Plus 6.0 and ImageJ.

[0102] Preparation of pseudoviruses and recombinant SARS-CoV-2S1 receptor-binding domains (RBDs) According to the manufacturer's protocol, pseudoviruses carrying the D614G mutant spike protein were prepared using Lenti-X lentiviral packaging plasmid (632673, Takara). Briefly, the plasmid was first amplified and diluted, then mixed with transfection reagents. The resulting DNA packaging mixture was then added to 293T cells. The supernatant was then harvested and the viral titer was determined. HUVEC, C28 / I2, and ATDC cells (MOI 10) were infected with the pseudovirus, and samples were collected for analysis after 24 hours of incubation. The recombinant receptor-binding domain (RBD) (residues 306-543) of the SARS-CoV-2 spike protein was synthesized from a reference sequence shown in GenBank ID YP_009724390.1. This was based on previous publications. 66The modified protocol was used to express and purify the protein in E. coli.

[0103] Cell culture The chondrocyte cell lines C28 / I2 (human), ATDC5 (mouse), and human umbilical vein endothelial cells (HUVEC) were donated by Professor Danny Chan of the University of Hong Kong, Professor Limin Rong of Sun Yat-sen University, and Professor Mo Yang of the Hong Kong Polytechnic University, respectively. Both chondrocyte cell lines were prepared in a solution containing 5% heat-inactivated FBS at 50 U / ml. −1 Penicillin and 50 μg ml −1 HUVECs were cultured in DMEM / F12 medium containing streptomycin. HUVECs were cultured in endothelial cell culture medium containing 1% endothelial cell growth supplement and 1% antibiotic solution (ScienCell).

[0104] In vitro cell model To investigate the direct effects of spike protein RBD and endothelin-1 (ET-1) on chondrocytes, 10 g / ml RBD or pV was used. ATDC5, C28 / I2, or HUVEC cells were treated with D614G (MOI 10) and / or 100 nM ET-1 for 24 hours. Cell lysates were collected for subsequent RNA extraction and real-time PCR for gene expression studies. The aging marker p16 was analyzed. INK4a and β-galactosidase ( -gal) staining. To investigate the rescue effect of macitentan on RBD-induced oxidative stress accumulation and mitochondrial dynamics changes, cells seeded on coverslips were stained with 10 Pretreatment with methylmacitenitan for 30 minutes, followed by treatment with RBD for one hour.

[0105] Assay for senescent β-galactosidase activity According to the manufacturer's instructions, chondrocytes were stained for β-galactosidase (β-gal) staining using the Senescence-Associated β-galactosidase (SA-β-gal) Staining Kit (CST). Briefly, the cells were washed and fixed in PBS, and then stained at 37°C. Staining with β-galactosidase staining solution overnight in a C-type drying incubator. Images were captured using a Nikon Eclipse TS100.

[0106] Evaluation of cellular oxidative stress and mitochondrial morphology To assess cellular oxidative stress, we used CellROX® Deep Red reagent solution (Invitrogen) according to the manufacturer’s instructions. Positive signals were visualized by excitation / emission at 640 / 665 nm. To visualize the morphology of mitochondria, we stained cells with MitoTracker® Red CMXRos (Invitrogen) with an excitation / emission wavelength of 579 / 599 nm. Cells were then counterstained and mounted with ProLong® Gold antifade reagent containing 4',6-diamidino-2-phenylindole (DAPI). All images were captured using a Leica TCS SPE confocal microscope.

[0107] Real-time quantitative polymerase chain reaction (RT-qPCR) assay of cultured cells Total RNA was extracted from cultured cells using E.Z.N.A.® TOTAL RNA KIT (Omega) according to the manufacturer’s instructions. RNA was quantified and reverse-transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermofisher). RT-PCR was then performed by setting up reaction mixtures using the QuantiNova SYBR Green PCR Kit (Qiagen) and specific primers (Supplementary Table 1) and then running in a BIORAD CFX96 Touch™ Real-Time PCR Detection System. The expression of GAPDH was used to normalize the expression levels of other genes.

[0108] Statistical analysis All data are expressed as mean ± S.E.M. unless otherwise stated. The exact sample size for each group is indicated in the figure legends. Each data point is represented by a dot on each graph. When deemed appropriate, one-way ANOVA or Kruskal-Wallis test was used for comparison between time courses differences (0, 4, 30 dpi). To compare infected and treated groups at different time points, unpaired two-tailed Student’s t-test or Mann-Whitney test was performed depending on the distribution of the data sets. When overall significance was detected between groups, the corresponding post-hoc test was performed, such as Tukey’s multiple comparison post-hoc test or Bonferroni’s post-hoc test. Two-way ANOVA and Tukey’s post-correction for multiple testing were used to compare paw withdrawal thresholds at different time points for different groups. The level of significance was set at p < 0.05. Prism 8 (GraphPad) was used to generate analyses and graphs.

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Claims

1. A method for treating or preventing skeletal sequelae of coronavirus infection in subjects in need, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist, provided that the endothelin receptor antagonist is not co-administered with dapagliflozin or niclosamide.

2. The method of claim 1, wherein the subject does not suffer from pulmonary hypertension.

3. The method of claim 1, wherein the coronavirus infection is caused by a coronavirus selected from the group consisting of: severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome-associated coronavirus (MERS-CoV), human coronavirus 229E (229E-CoV-2), human coronavirus NL63 (NL63-CoV), human coronavirus OC43 (OC43-CoV), human coronavirus HKU1 (HCoV-HKU1), and variants thereof.

4. The method of claim 1, wherein the coronavirus infection is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants.

5. The method of claim 1, wherein the endothelin receptor antagonist is a selective ET receptor. A Receptor antagonists, selective ET B receptor antagonists, or dual ET A receptors and ET B Receptor antagonists.

6. The method of claim 1, wherein the endothelin receptor antagonist is a dual ET. A receptors and ET B Receptor antagonists.

7. The method of claim 1, wherein the endothelin receptor antagonist is selected from the group consisting of: sitassentan, ambesentan, atrasentan, BQ-123, sparsentan, zipostentan, alpracitentan, edonantan, bosentan, macitentan, tezosentan, BQ-788, A192621, and mixtures thereof.

8. The method of claim 1, wherein the endothelin receptor antagonist is bosentan, macitentan, ambesentan, or a mixture thereof.

9. The method of claim 1, wherein the endothelin receptor antagonist is macitentan.

10. The method of claim 1, wherein the coronavirus infection is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the endothelin receptor antagonist is bosentan, macitentan, ambesentan, or a mixture thereof.

11. The method of claim 1, wherein the endothelin receptor antagonist is administered to the subject during at least one acute or subacute phase of coronavirus infection.

12. The method of claim 1, wherein the skeletal sequelae are one or more of arthralgia, viral arthritis, osteopenia, osteochondral injury, and osteoporosis.

13. The method of claim 1, wherein the endothelin receptor antagonist is administered to the subject via intra-articular or intraosseous injection.

14. The method of claim 1, wherein the coronavirus infection is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the endothelin receptor antagonist is macitentan.

15. The method of claim 14, wherein macitentan is administered to the subject during at least one acute phase of SARS-CoV-2 or subacute phase of coronavirus infection.

16. The method of claim 15, wherein the skeletal sequelae are one or more of arthralgia, viral arthritis, osteopenia, osteochondral lesions, and osteoporosis.

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