Treatment of the Antiphospholipid Syndrome with S-Hydroxychloroquine

Abstract

A method of treating antiphospholipid syndrome by administering to a patient a pharmaceutical composition containing S-(+)-hydroxychloroquine and a pharma- ceutical acceptable excipient, wherein the pharmaceutical composition is substantially free of (R)-(-)-hydroxychloroquine.

Description

[Background technology]

[0001] background Antiphospholipid syndrome (APS) is an autoimmune disease associated with thrombosis and miscarriage that can lead to life-threatening blood clots in the lungs and brain. APS is the leading cause of stroke in people under the age of 50. In pregnant women, it often results in spontaneous abortion and stillbirth.

[0002] APS reportedly affects 0.3-1% of the population. See McDonnell et al., Blood Review 39, 1-14 (2020). There is currently no cure. Warfarin, a long-term anticoagulant, is the standard treatment for APS-associated thrombosis to reduce the risk of blood clots; however, it carries an increased risk of bleeding complications. See Rand et al., Blood 112, 1687-95 (2008).

[0003] For additional references regarding APS, see Agar et al., Blood 116, 1336-43 (2010); Rand et al., Blood 115, 2292-99 (2010); Rand et al., Lupus 17, 922-30 (2008); and Conti et al., Clin Exp Immunol 132, 509-16 (2003).

[0004] Hydroxychloroquine (HCQ), an antimalarial compound, has been suggested to play a role in reducing the extent of thrombosis and reversing antiphospholipid ("aPL") antibody-induced platelet activation in animal models of injury-induced thrombosis. See Rand et al. (2008). Its efficacy and safety have yet to be determined in large-scale clinical trials.

[0005] HCQ has two optical isomers, the (R)-(-)-isomer ("R-HCQ") and the (S)-(+) isomer ("S-HCQ"). A racemic mixture containing 50:50 R-HCQ and S-HCQ was used in all of the above studies.

[0006] Long-term and high-dose administration of HCQ can cause blurred vision and potentially damage the retina, cornea, or macula, and can lead to vision loss in some patients due to accumulation in ocular tissues.

[0007] HCQ is also known to be cardiotoxic: it can cause interventricular conduction delays, Q to T wave interval prolongation, torsades de pointes, ventricular arrhythmias, hypokalemia, and hypotension. See U.S. Patent Application Serial No. 17 / 176,679. Summary of the Invention [Problem to be solved by the invention]

[0008] There is a need to develop safe and effective methods to treat APS. [Means for solving the problem]

[0009] To meet the above needs, methods of treating APS with a pharmaceutical composition containing S-(+)-hydroxychloroquine ("S-HCQ") and a pharma- ceutically acceptable excipient are provided. Effect of the Invention

[0010] Accordingly, the present invention provides a method of treating APS, comprising: The present invention relates to a method comprising the steps of: (i) identifying a subject suffering from APS, and (ii) administering to the subject an effective amount of a pharmaceutical composition comprising S-HCQ and a pharma- ceutically acceptable excipient, thereby treating APS. The pharmaceutical composition is substantially free of (R)-(-)-hydroxychloroquine ("R-HCQ").

[0011] The methods of the present invention are suitable for treating all types of APS, including primary APS, secondary APS, and catastrophic APS.

[0012] The pharmaceutical composition may be administered in any form, including granules, tablets, capsules, pills, powders, liquids, suspensions, or syrups. Preferably, the pharmaceutical composition is administered to a patient at a dose of 100 mg to 800 mg (e.g., 120 mg to 600 mg, 150 mg to 500 mg, and 180 mg to 450 mg) of S-HCQ per day.

[0013] S-HCQ refers to the compound itself and its pharma- ceutically acceptable salts, examples of which include hydrochloride, sulfate, and phosphate.

[0014] The details of several embodiments of the invention are set forth in both the following description and in the drawings. Other features, objects, and advantages of the invention will be apparent from the specification and the appended claims. Finally, all publications and patent documents cited herein are incorporated by reference in their entirety.

[0015] The following description refers to the accompanying drawings. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 includes a photograph showing HCQ (S-HCQ or R-HCQ) molecule bound to β2-glycoprotein I, which has an a-helical structure containing four binding sites, obtained from molecular docking studies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description As summarized above, methods are provided for treating APS by administering to an APS patient a pharmaceutical composition containing highly pure S-HCQ and a pharma- ceutical acceptable excipient, and which is substantially free of R-HCQ.

[0018] The purity of S-HCQ is measured by the enantiomeric excess, defined as the difference between the molar percentages of S-HCQ and R-HCQ, where the combined molar percentage of S-HCQ and R-HCQ is 100%. For example, a purity of S-HCQ with an enantiomeric excess of 99% contains 99.5 mol% S-HCQ and 0.5 mol% R-HCQ. A pharmaceutical composition is considered to be substantially free of R-HCQ when it contains an enantiomeric excess of S-HCQ of 99% or more (e.g., 99.2% or more, 99.5% or more, and 99.8% or more).

[0019] Such preparations of S-HCQ with high enantiomeric excess are described in U.S. Patent Application Serial No. 17 / 176,679 and U.S. Patent No. 5,314,894.

[0020] The S-HCQ in the pharmaceutical composition is either a free base or a pharma- ceutically acceptable salt. The pharma-ceutically acceptable salt may be, but is not limited to, a sulfate, a phosphate, and a hydrochloride. Preferably, it is a sulfate.

[0021] The pharmaceutical composition contains S-HCQ in the range of 5% to 95% by weight, for example, 30% to 80% by weight, 40% to 70% by weight, 40% to 55% by weight, and 60% to 70% by weight.

[0022] The pharmaceutical composition is substantially free of R-HCQ, for example, containing 2% by weight or less of R-HCQ (eg, 1% or less and 0.5% or less).

[0023] An exemplary pharmaceutical composition contains 50%-70% S-HCQ and 30%-50% of one or more pharmaceutical excipients.

[0024] To practice the methods of the invention, a subject suffering from APS is typically administered an effective amount of a pharmaceutical composition corresponding to a daily dose of 100 mg to 800 mg (e.g., 200 mg and 400 mg) of S-HCQ.

[0025] Administration of S-HCQ is associated with fewer side effects, particularly with regard to cardiotoxicity, compared to R-HCQ and racemic HCQ (i.e., an equimolar mixture of S-HCQ and R-HCQ).

[0026] In addition to S-HCQ, the pharmaceutical composition also contains a pharma- ceutically acceptable excipient, which may be any physiologically inert excipient used in the pharmaceutical field, including, but not limited to, binders, diluents, surfactants, disintegrants, lubricants, flow agents, and colorants. For examples of excipients, see U.S. Patent Application Publication No. 2008 / 020634.

[0027] The pharmaceutical composition can be provided in any form, such as granules, tablets, capsules, pills, powders, liquids, suspensions, and syrups. It can be prepared according to conventional methods described in many publications, see, for example, US Patent Application Publication No. 2018 / 0194719.

[0028] The methods of the present invention are surprisingly effective in treating subjects suffering from primary APS (thrombotic APS and obstetric APS), secondary APS, and fulminant APS.

[0029] Primary APS is a thrombotic condition characterized by recurrent arterial and venous thrombosis, recurrent miscarriage, and the presence of circulating aPL antibodies responsible for thrombophilia and pregnancy morbidity in the absence of comorbidities. In patients with secondary APS, there is a pre-existing autoimmune condition. Fulminant APS, the most severe form of APS, is a multisystem autoimmune state associated with aPL antibodies, characterized by vascular thrombosis or miscarriage in the presence of simultaneous multiorgan failure with small vessel occlusion.

[0030] Symptoms of APS vary from patient to patient and can include signs such as blood clots, miscarriage, rashes, chronic headaches, dementia, seizures, arterial thrombosis, autoimmune thrombocytopenia, autosomal dominant inheritance, blurred vision, central retinal artery occlusion, iritis, keratitis, lupus anticoagulants, retinal detachment, retinal vasculitis, scleritis, venous thrombosis, vision loss, and vitritis.

[0031] Without being bound by theory, HCQ is believed to treat APS by binding to β2-glycoprotein I ("β2-GP1"), a blood protein associated with APS. β2-GP1 circulates in the blood at high concentrations, i.e., 0.2 mg / mL, that can regulate blood clotting. See McDonnell et al.

[0032] β2-GP1 exists in two conformations, a closed cyclic form and an open linear form. The trigger that causes β2-GP1 to change conformation between these two forms is unknown. Among them, 90% of β2-GP1 travels in the blood in the cyclic form. See Agar et al., Blood 116, 1336-43 (2010). In its linear form, β2-GP1 exposes two domains, the N-terminal domain I ("DI") and the C-terminal domain V ("DV"). DI is the main region for receiving antibodies, e.g., aPL antibodies. DV is involved in binding to blood cell membranes. The change of β2-GP1 from the cyclic form to the linear form promotes the binding of antibodies to blood cell membranes and initiates the coagulation reaction. This is accomplished by forming β2-GP1-antibody complexes and is a key pathogenic pathway to APS.

[0033] It is recognized that β2-GP1-antibody complexes exert their blood coagulation effects by, among other mechanisms, disrupting the anticoagulant annexin V shield on blood cells. See McDonnell et al.

[0034] Annexin V, a cellular protein, inhibits clot formation by binding to phospholipids in blood cell membranes, forming a shield that blocks antibodies from attacking the phospholipids. In APS patients, the annexin V shield is disrupted by antibodies via β2-GP1.

[0035] HCQ is thought to disrupt the above pathogenic pathways by preventing β2-GP1 from changing its three-dimensional conformation to a linear form.

[0036] Without further elaboration, it is believed that one skilled in the art can utilize the present disclosure to its fullest extent based on the disclosure herein.

[0037] The following specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

[0038] Working Example Example 1: Molecular docking of S-HCQ and R-HCQ on β2-glycoprotein I To evaluate the binding stability of S-HCQ and R-HCQ with β2-GP1, structures were obtained from the Protein Data Bank (PDB:1AV1), a global open access archive of biopolymer structure data, and molecular docking was simulated. Protein structures and amino acid sequences were aligned and edited using BIOVIA® Discovery Studio software (Dassault Systemes, San Diego, California). Computational analysis was performed to predict the hydrophobic regions of β2-GP1 using SwissDock, a docking service provided by the Swiss Institute of Bioinformatics. The molecular structures were visualized and analyzed using software from UCSF Chimeram (University of California, San Francisco).

[0039] Molecular docking helps us understand how HCQ binds to β2-GP1 and inhibits its conformational change. Separate molecular docking simulations with β2-GP1 were performed for S-HCQ and R-HCQ. Upon binding to antibodies, β2-GP1 undergoes a conformational change from a closed form (cyclic) to an open form (linear). The latter promotes the formation of β2-GP1-antibody complexes and leads to thrombosis. Therefore, it is of great importance to know whether the binding of HCQ can interfere with the conformational change of β2-GP1.

[0040] The results of molecular docking are shown in Figure 1. As shown, β2-GP1 contains a pseudocontinuous amphipathic a-helix with five domains and four junctions (shown as 1, 2, 3, and 4 in the figure). Two adjacent a-helical domains are connected by a junction. The four junctions, together or separately, allow the movement of the a-helix, thereby changing the β2-GP1 conformation between the cyclic and linear forms.

[0041] Molecular docking showed that HCQ molecule can bind to any one of four binding sites (1, 2, 3 and 4). Among them, binding to binding site 3 is effective for inhibiting the conformational change. The strength of binding is measured by the affinity energy calculated in the molecular docking study. The higher the affinity energy, the stronger the binding.

[0042] S-HCQ binds to bond 3 with an affinity energy of 12.6 kcal / mol, which is very high. By fixing bond 3 of the a-helix of β2-GP1, S-HCQ inhibits the conformational change from cyclic to linear form required for antibodies to bind and form a complex with β2-GP1, which is necessary for the APS pathogenic pathway. Therefore, S-HCQ blocks this pathway, thereby effectively treating APS.

[0043] In contrast, R-HCQ binds to bond 3 with an affinity energy of only 10.5 kcal / mol, an energy level that is less effective at preventing the conformational change of the a-helix of β2-GP1. Furthermore, the bond angle of R-HCQ on the a-helix of β2-GP1 is different from that of S-HCQ, making R-HCQ less effective at suppressing the conformational change.

[0044] Example 2: Inhibition of β2-GP1-antibody formation by HCQ In vitro studies tested S-HCQ and R-HCQ and demonstrated their efficacy in reducing the binding of aPL antibodies to β2-GP1 using THP-1, a human monocytic cell line derived from a patient with monocytic leukemia.

[0045] THP-1 is a human peripheral blood monocyte that highly expresses β2-GP1 and is associated with increased APS in patients. Before treatment with HCQ, THP-1 cells were fixed with 3.7% paraformaldehyde solution, blocked with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA), and incubated with mouse aPL antibody (A500-006A, Bethyl Laboratories, Montgomery, Texas). Cell immunofluorescence staining was used to measure the expression of β2-GP1.

[0046] For dot blot assays, separate samples were prepared as follows: THP-1 total lysates were loaded onto polyvinylidene fluoride membranes, blocked with PBS containing 1% BSA, and incubated with mouse anti-β2-GP1 A500-006A and S-HCQ, R-HCQ, or racemic HCQ at a concentration of 10 mg / mL each. Control samples were obtained following the same procedure as above, except that no HCQ was added.

[0047] An ELISA assay was then performed to determine whether S-HCQ or R-HCQ inhibited the binding of β2-GP1-antibody complexes to THP-1 membranes. THP-1 cells thus treated were cultured at a density of 3.6 × 10 in medium containing 80% RPMI-1640 (Thermo Fisher Scientific, Waltham, Massachusetts), 20% anti-β2-GP1 immunoglobulin G ("IgG") (0.2 mg / mL) in HEPES-buffered saline (HBS, pH 7.45), and S-HCQ. 5 The cells were resuspended to a density of 1000 cells / mL. Three samples were prepared from the medium with different S-HCQ concentrations, i.e., 1 μg / mL, 2.5 μg / mL, or 5 μg / mL. The control sample was obtained following the same procedure as above, except that no S-HCQ was added. The optical absorbance was measured at 450 nm.

[0048] All assays above were performed in triplicate. Reported results are expressed as mean ± SEM. All statistical analyses were performed using GraphPad Prism® (Version 8.0 GraphPad Software Inc, San Diego, California) branded software. Student's test was used for comparison between two groups. A p-value <0.05 was considered statistically significant.

[0049] Using cellular immunofluorescence staining, the THP-1 cells in this study were found to express high levels of β2-GP1, which was confirmed by the overlap of green round dots on the THP-1 cells, which appeared as round single-cell morphology with nuclear blue dots.

[0050] Dot blot analysis showed that S-HCQ inhibited the binding of β2-GP1 to the antibody by a remarkable 97% (±2%, p<0.05). In comparison, R-HCQ inhibited the binding of β2-GP1 to the antibody by only 23% (±17%), and racemic HCQ inhibited the binding by 80% (±4%).

[0051] ELISA assay demonstrated that S-HCQ inhibited the binding of β2-GP1 to the antibody in a dose-dependent manner, i.e., 25% at 1 μg / mL S-HCQ, 60% at 2.5 μg / mL S-HCQ, and 90% at 5 μg / mL S-HCQ (p<0.05).

[0052] S-HCQ was found to effectively inhibit the binding of antibodies to β2-GP1, and was surprisingly more effective than R-HCQ and racemic HCQ.

[0053] Example 3: Restoration of the Annexin A5 Anticoagulant Shield on Cell Membranes by HCQ As noted above, the endogenous protein annexin A5 forms a shield on the surface of blood cells that inhibits blood clotting, thus reducing the risk of APS. In this example, S-HCQ significantly restored the annexin A5 anticoagulant shield that was destroyed by aPL antibodies.

[0054] THP-1 cells were first maintained in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, Massachusetts) containing 10% fetal bovine serum, 2 mM L-glutamine, and 50 U / mL penicillin-streptomycin antibiotics. They were then plated in 96-well culture plates at 8 × 10 4 The cells were seeded at a density of 1000 cells / well and allowed to reach confluence. THP-1 cells were then treated with anti-β2-GP1 IgG in the presence of 0.5 μg / mL HCQ. The IgG-treated THP-1 cells were rinsed with HBS-CaCl2 solution to remove free annexin V, leaving only annexin V attached to the cell surface, after which the level of annexin V on the cell surface was measured by optical absorbance.

[0055] Three samples were prepared each with one of the three HCQ (i.e., S-HCQ, R-HCQ, and racemic HCQ) solutions. A comparative sample was obtained following the same procedure as above, except that no HCQ was added. Patient serum containing β2-GP1-antibody was used as a control sample.

[0056] Cell immunofluorescence staining confirmed high expression levels of Annexin V as green dots in immunofluorescence images. In addition, the expression of Annexin V in THP-1 was confirmed by Western blot.

[0057] S-HCQ-treated THP-1 cells had annexin V at a relative level of 3.45, compared to a relative level of 2.54 seen in R-HCQ-treated THP-1 cells and a relative level of 3.09 seen in racemic HCQ-treated THP-1 cells. Control samples showed a relative level of annexin V of only 1.

[0058] The above results indicate that S-HCQ shows surprising promise over R-HCQ and racemic HCQ in the treatment of APS.

[0059] Example 4: Reduction of in vivo thrombus formation by HCQ The therapeutic effect of HCQ on inhibiting thrombosis was evaluated in an animal model of APS-associated thrombosis.

[0060] Isolation of anti-β2-glycoprotein I Serum and plasma from six APS patients (i.e., patients 1-6) were selected to participate in this study. For APS confirmation, anticardiolipin (aCL), anti-β2GPI, and lupus anticoagulant (LA) activities were measured. APS-derived anti-β2GPI samples were obtained by purification of serum samples using rProtein A / Protein G GraviTrap™ (Cytiva™, Merck KGaA, Darmstadt, Germany). The concentration of anti-β2GPI antibodies in each sample was tested using binding to β2GPI by enzyme-linked immunosorbent assay (ELISA) (Eagle Biosciences, Inc., Amherst, New Hampshire).

[0061] In vitro endothelial cell activation was induced by the anti-β2GPI antibody obtained above. Endothelial cells (i.e., HUVEC) were seeded and incubated with the anti-β2GPI antibody from APS. As a positive control, some HUVEC were treated with lipopolysaccharide (LPS, 3 mg / mL). Surface expression of E-selectin, cell-cell adhesion, and vascular cell adhesion molecule 1 (VCAM-1) was detected and found to be positively correlated with the amount of β2GPI IgG.

[0062] APS-associated thrombosis mouse model C57BL / 6 male mice aged 8–12 weeks (purchased from BioLasco, Taiwan) were used in this study. All procedures were approved by the Institutional Animal Care Committee of Taipei Medical University.

[0063] Venous thrombosis was induced in mice using an endothelial injury model. Mice were intravenously (IV) injected with 7.5% FeCl3 (positive control), saline (negative control), or APS-derived anti-β2GP1 antibody (from patient 2) at doses of 100, 200, or 300 AU (test group). Anesthesia was administered 72 hours after injection. The right femoral vein was exposed and 1500 g / mm 2 The researchers pinched the area with a pressure of 1000 to induce the formation of a blood clot.

[0064] It was determined that anti-β2GP1 antibodies effectively induced thrombosis at all three concentrations, i.e., 100, 200, and 300 AU, and therefore, in all following studies, anti-β2GP1 antibodies were injected into mice at 100 AU.

[0065] Reduction of thrombus formation by racemic HCQ in a mouse model of APS-associated thrombosis Following the procedure described above, mice (i.e., HCQ-treated mice) were injected with 100 AU of anti-β2GP1 antibody from patient 5 and 2000 μg of racemic HCQ (200 μl at 10 mg / ml). A control group of mice (n=4; APS-induced mice) was injected with 100 AU of anti-β2GP1 antibody only. After 72 hours, the right femoral vein of each mouse was exposed and 1500 g / mm 2 The mice were pinched with a pressure of 0.01 mmHg to induce thrombus formation. The thrombosis time (in minutes) was recorded. The thrombosis time in the healthy mice group (n=2) was 5 minutes, whereas the mean thrombosis time in the APS-induced mice was 2 minutes. In contrast, the thrombosis time in the HCQ-treated mice was 5 minutes, the same as in the healthy mice. The results showed that anti-β2GP1 antibodies accelerated thrombosis in APS-induced mice, while racemic HCQ inhibited the function of anti-β2GP1 antibodies.

[0066] After pinching for 5 min, the thrombus was removed from the femoral vein. Racemic HCQ significantly reduced the thrombus size compared to the group of mice that did not receive HCQ treatment.

[0067] The results indicate that racemic HCQ, including both S-HCQ and R-HCQ, is useful in the treatment of APS by reducing thrombus formation in APS patients.

[0068] S-HCQ reduces thrombus formation in a mouse model of APS-associated thrombosis Risk factors for APS-associated thrombosis and venous thromboembolism (VTE) overlap and are primarily related to endothelial dysfunction (ED). E-selectin and VCAM-1 are associated with a high risk of APS-associated thrombosis.

[0069] Based on the above APS animal model, R-HCQ and S-HCQ were administered to mice to reduce thrombosis. The mice were divided into 6 groups, each of which was injected with (1) IgG as a control group, (2) 100 AU of anti-β2GPI-antibody as a comparison group, (3) a combination of 100 AU of anti-β2GPI-antibody and 300 μg of S-HCQ as treatment group 3, (4) a combination of 100 AU of anti-β2GPI-antibody and 200 μg of S-HCQ as treatment group 4, (5) a combination of 100 AU of anti-β2GPI-antibody and 200 μg of R-HCQ as treatment group 5, or (6) a combination of 100 AU of anti-β2GP1-antibody and 100 μg of R-HCQ as treatment group 6.

[0070] Serum expression levels of E-selectin and VCAM-1, two biomarkers of APS-associated thrombosis, were measured. The percentage of inhibition in treatment groups 3-6 was calculated based on the expression levels of E-selectin and VCAM-1 compared to the control group. See Table 1 below for the results. A lower expression level of E-selectin or VCAM-1 indicates a higher inhibition of thrombosis.

[0071] As shown in Table 1, mice treated with 300 μg or 200 μg S-HCQ had much lower expression of both E-selectin and VCAM-1 compared to mice treated with R-HCQ. Surprisingly, mice treated with 200 μg S-HCQ were found to inhibit thrombosis by 51.4%, compared to only 26.5% in mice treated with 200 μg R-HCQ. [Table 1]

[0072] Other embodiments All of the features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced with an alternative feature serving the same, equivalent, or similar purpose. Unless expressly stated otherwise, each feature disclosed is only one example of a generic series of equivalent or similar features.

[0073] From the above, those skilled in the art can easily grasp the essential features of the present invention, and can make various changes and modifications to adapt the present invention to various applications and conditions without departing from the spirit and scope of the present invention. Therefore, other embodiments are also included in the scope of the following claims.

Claims

1. A pharmaceutical composition for treating antiphospholipid syndrome (APS), comprising: A pharmaceutical composition comprising (S)-(+)-hydroxychloroquine and a pharma- ceutically acceptable excipient, said pharmaceutical composition being substantially free of (R)-(-)-hydroxychloroquine.

2. The pharmaceutical composition of claim 1, wherein S-hydroxychloroquine is in the form of a pharma- ceutically acceptable salt.

3. The pharmaceutical composition of claim 2 , wherein the pharma- ceutically acceptable salt is a hydrochloride, sulfate, or phosphate salt.

4. A pharmaceutical composition described in any one of claims 1 to 3, wherein S-hydroxychloroquine is administered at 100 mg to 800 mg per day.

5. The pharmaceutical composition of claim 4, wherein the dosage is 150 mg to 500 mg per day.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is in the form of granules, tablets, capsules, pills, powders, liquids, suspensions, or syrups.

7. The pharmaceutical composition according to any one of claims 1 to 3, wherein the APS is primary APS, secondary APS, or fulminant APS.

8. The pharmaceutical composition according to any one of claims 1 to 3, wherein the APS is a thrombotic APS or an obstetric APS.

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