WO2023144353A1

WO2023144353A1 - Entospletinib for use in the treatment of covid19 patients

Info

Publication number: WO2023144353A1
Application number: PCT/EP2023/052094
Authority: WO
Inventors: Menno DE WINTHER
Original assignee: Academisch Medisch Centrum
Priority date: 2022-01-27
Filing date: 2023-01-27
Publication date: 2023-08-03

Abstract

The invention relates to entospletinib (GS-9973) or a pharmaceutically acceptable salt, ester or derivative thereof for use in the treatment of a viral infection.

Description

ENTOSPLETINIB FOR USE IN THE TREATMENT OF C0VID19 PATIENTS

FIELD

The present invention relates to methods of treatment of viral infections by entospletinib. In particular, the invention relates to the treatment of coronavirus, more in particular COVID-19

BACKGROUND

Patients diagnosed with coronavirus disease 2019 (COVID-19) become critically ill primarily around the time of activation of the adaptive immune response. We have previously shown that antibodies play a role in the worsening of disease at the time of seroconversion (Hoepel et al., Sc Transl Med, 2021). We have shown that early-phase severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) spike protein-specific immunoglobulin G (IgG) in serum of critically ill COVID-19 patients induces excessive inflammatory responses by human alveolar macrophages. We identified that this excessive inflammatory response is dependent on two antibody features that are specific for patients with severe COVID-19. First, inflammation is driven by high titers of anti-spike IgG, a hallmark of severe disease. Second, we found that anti-spike IgG from patients with severe COVID-19 is intrinsically more proinflammatory because of different glycosylation, particularly low fucosylation, of the antibody Fc tail. Low fucosylation of anti-spike IgG was normalized in a few weeks after initial infection with SARS- CoV-2, indicating that the increased antibody-dependent inflammation mainly occurs at the time of seroconversion. We also identified Fcgamma receptor (FcgammaR) Ila and FcgammaRIII as the two primary IgG receptors that are responsible for the induction of key COVID-19-associated cytokines such as interleukin-6 (IL-6) and tumor necrosis factor (TNF). In addition, we could show that anti-spike IgG-activated human macrophages can subsequently break pulmonary endothelial barrier integrity and induce microvascular thrombosis in vitro. Last, we could demonstrate that the inflammatory response induced by anti-spike IgG can be specifically counteracted by fostamatinib, an FDA- and EMA-approved therapeutic small-molecule inhibitor of Syk kinase (from Rigel Pharmaceuticals). Of note, in our study we also observed suppression of anti-viral mechanisms by immune complexes, potentially leading to less capacity to fight SARS-CoV-2 infection.

Current standard of care involves dexamethasone treatment which is a broad-acting inflammatory inhibitor. This has the major drawback that protective inflammatory mechanisms, such as the anti-viral response of patients, is also blocked, potentially hampering improvement of patients. Based on our and others data Rigel Pharmaceuticals has initiated phase 2 and phase 3 trials (see their website for details and outcome of phase 2). The phase 2 trial suggests safety and efficacy in COVID19 patients. Unfortunately, also Fostamatinib shows broad-ranging anti-inflammatory effects also impacting on anti-viral responses.

It is therefore an object of the invention to provide a treatment which suppresses the inflammatory response induced by SARS-CoV-2 immune complexes but leaves the response to viral mimics intact.

DESCRIPTION OF THE FIGURES

Figure 1 shows the experimental setup of macrophage generation and activation.

Figure 2 demonstrates the effect of dexamethasone. Macrophages were activated with either polylC (grey) or polylC+immune complexes (black) in the presence of increasing concentrations of dexamethasone and indicated cytokines were measured.

Figure 3 demonstrates the Effect of R406. Macrophages were activated with either polylC (grey) or polylC+immune complexes (black) in the presence of increasing concentrations of R406 and indicated cytokines were measured.

Figure 4 demonstrates the effect of entospletinib. Macrophages were activated with either polylC (grey) or polylC+immune complexes (black) in the presence of increasing concentrations of entospletinib and indicated cytokines were measured.

Figure 5(A-D) shows the immunoregulatory activities of dexamethasone and SYK inhibitors R406 and entospletinib on IL-6 production by stimulated macrophages. (A) Schematic overview of the experimental setup. Monocyte-derived alveolar macrophage-like macrophages (MDAMs) were generated by differentiating peripheral monocytes with M-CSF and IL-10. The generated MDAMs were then treated with inhibitors in increasing concentration or DMSO 30 min prior to stimulation with viral stimulus poly( I :C) with or without the presence of immune complexes. Immune complex is formed by plate-bounded SARS-CoV-2 spike proteins and monoclonal anti-spike IgGs. All conditions are with SARS-CoV-2 spike proteins. (B-D) IL-6 production was used as the pro-inflammatory activation readout. Representative data of macrophage activation assay for (B) dexamethasone(C) R406 and (D) entospletinib, with the left Y axis and black curves showing the concentration measured from poly( I :C) and anti-spike immune complex conditions and right Y axis and grey curves activation with poly( l:C) alone. Half maximal inhibitory concentrations (IC50) from different macrophage donors (dexamethasone (n = 6), R406 (n = 5), entospletinib (n = 14)) per stimulation condition are plotted as box plots indicating 10-90 percentile and median. Significant differences were calculated with a paired t test. *P < 0.05.

SUMMARY OF THE INVENTION The present invention is based on the surprising finding that entospletinib has better specificity than Fostamatinib. It suppresses the inflammatory response induced by SARS-CoV-2 immune complexes but leaves the response to viral mimics intact. Moreover, entospletinib restores the anti-viral response which is normally suppressed by immune complexes alone.

The invention therefore provides entospletinib (GS-9973) or a pharmaceutically acceptable salt, ester or derivative thereof for use in the treatment of a viral infection.

Said entospletinib or a pharmaceutically acceptable salt, ester or derivative thereof for use according to the invention, is wherein said viral infection is caused by a coronavirus.

Preferably, said viral infection is caused by SARS-CoV-2.

Preferably said treatment is a treatment of COVID-19 patients at risk of developing severe disease caused by SARS-CoV-2. Le. hospitalized COVID-19 subjects or subjects in need thereof.

The invention further provides a pharmaceutical composition for use according to the invention comprising Entospletinib and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

The invention therefore provides entospletinib (GS-9973) or a pharmaceutically acceptable salt, ester or derivative thereof for use in the treatment of a viral infection. Entospletinib (GS-9973) is an oral, selective inhibitor of spleen tyrosine kinase (Syk).

Some embodiments provide methods for treating the treatment according to the invention comprising the step of administering a therapeutically effective amount of entospletinib or a pharmaceutically acceptable salt, ester or derivative thereof.

The invention further provides a pharmaceutical composition for use according to the invention comprising entospletinib and a pharmaceutically acceptable carrier. Such compositions are prepared in a manner well known in the pharmaceutical art. See, e.g. W02018053190A1. The pharmaceutical compositions may be administered in either single or multiple doses. The pharmaceutical composition may be administered by various methods including, for example, rectal, buccal, intranasal and transdermal routes. In certain embodiments, the pharmaceutical composition may be administered by intraarterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, or as an inhalant. In some embodiments, the pharmaceutical composition is administered orally.

Dosing

The specific dose level of compounds described herein for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease in the subj ect undergoing therapy. For example, a dosage may be expressed as a number of milligrams of a compound of the formula per kilogram of the subject's body weight (mg/kg). Dosages of between about 0.01 and 200 mg/kg may be appropriate. In some embodiments, about 0.01 and 150 mg/kg may be appropriate. In other embodiments a dosage of between 0.05 and 100 mg/kg may be appropriate. Normalizing according to the subject's body weight is particularly useful when adjusting dosages between subjects of widely disparate size, such as occurs when using the drug in both children and adult humans or when converting an effective dosage in a non-human subject such as dog to a dosage suitable for a human subject.

The daily dosage may also be described as a total amount of a compound of the formulae administered per dose or per day. Daily dosage of a compound may be between about 1 mg and 2,000 mg, between about 1,000 to 2,000 mg/day, between about 1 to 1 ,000 mg/day, between about 1 to 500 mg/day, between about 100 to 150 mg/day, between about 1 to 100 mg/day, between about 1 to 50 mg/day, between about 50 to 100 mg/day, between about 100 to 125 mg/day, between about 100 to 150 mg/day, between about 100 to 175 mg/day, between about 100 to 200 mg/day, between about 100 to 225 mg/day, between about 100 to 250 mg/day, between about 100 to 350 mg/day, between about 100 to 400 mg/day, between about 100 to 450 mg/day, or between about 100 to 500 mg/day.

When administered orally, the total daily dosage for a human subject may be between 1 mg and 1 ,000 mg/day, between about 1 to 100 mg/day, between about 1 to 50 mg/day, between about 50 to 100 mg/day, between 50 to 300 mg/day, between 50 to 200 mg/day, between 75 to 200 mg/day, between 75 to 150 mg/day, between 100 to 200 mg/day, between about 200 to 300 mg/day, between about 300 to 400 mg/day, between about 400 to 500 mg/day, between about 100 to 150 mg/day, between about 150 to 200 mg/day, between about 200 to 250 mg/day, between about 75 to 150 mg/day, or between about 150 to 300 mg/day.

The compounds of the present application or the compositions thereof may be administered once, twice, three, or four times daily, using any suitable mode described above.

EXAMPLE

We compared the effects of dose-ranges of three types of treatment (i.e. inhibitors) on the activation of IL-10 induced MCSF-differentiated macrophages (as a model for alveolar macrophages, Chen et al., Frontiers in Imm 2019) by either polylC (i.e. viral mimic) or a combination of polylC and immune complexes (ICs) of IgG and SARS-CoV-2 spike protein (as described before, Figure 1). We have previously shown (Hoepel et al., Sc Transl Med 2021) that poly IC+ICs stimulates macrophage activation, which can be inhibited by Syk-inhibition. The three types of treatment were dexamethasone (general anti-inflammatory, currently part of SOC for severe COVID-19 patients), R406 (the active metabolite of Fostamatinib) and entospletinib. As a readout for activation, we focused on two established COVID-19 associated pro-inflammatory cytokines, TNF and IL-6, and also analyzed the effect on I PIO (CXCL10), an interferon response gene and thereby a marker for interferon activation which is part of the anti-viral response. High plasma IL-6 and TNF have been shown to be independent predictive markers of disease severity and death (Del Valle et al., Nat Med 2020) and low interferon responses have been shown to associate with disease severity (Yao et al., Cell reports, 2021, Zhang et al., Science 2020). Our previous data (Hoepel et al., ScTransI Med 2021) shows that polylC+ICs strongly induces TNF and IL-6 and at the same time suppresses IFN responses by macrophages.

We found that dexamethasone reduced TNF and IL-6 production (Figure 2), either induced by polylC alone of polylC+ICs. Moreover, dexamethasone had an inhibitory effect on I P10 secretion when induced by polylC alone while it did not affect the IP10 secretion when induced by polylC+ICs. Fostamatinib, similarly suppressed IL-6 and TNF secretion by macrophages and only impacted on the polylC induced I P10 secretion (Figure 3).

Most interestingly, Entospletinib reduced polylC+ICs induced IL6 and TNF secretion but only very mildly suppressed these cytokines when induced by polylC alone (Figure 4). Moreover, IP10 production induced by polylC was not affected while it did restore (enhance) the I P10 secretion after polylC+ICs.

Our data show that Entospletinib is very effective and specific in reducing the inflammatory activation of macrophages by polylC+ICs while at the same time leaving the induction of these cytokines by a viral mimic largely intact. Moreover, entospletinib restores the anti-viral response as read-out by I P10. These data indicate that entospletinib is an improvement over current SOC or Fostamatinib for the treatment of severe COVID19 patients.

Cells

Buffy coats from healthy anonymous donors were acquired from the Sanquin blood supply in Amsterdam, the Netherlands. All the subjects provided written informed consent prior to donation to Sanquin. Monocytes were isolated from the Buffy coats through density centrifugation using Lymphoprep (Axis-Shield) followed by human CD14 magnetic beads purification with the MACS cell separation columns (Miltenyi Biotec). The resulting monocytes were seeded on tissue culture plates and subsequently differentiated to macrophages for 6 days in the presence of 50ng/mL human M-CSF (Miltenyi Biotec) with Iscove's Modified Dulbecco's Medium (IMDM, Lonza) containing 5% fetal bovine serum (FBS, Biowest) and 86pg/mL gentamicin (Gibco). The medium was renewed on the third day. After a 6 day differentiation period, the medium was replaced by culture medium without M-CSF and supplemented with 50 ng/mL IL-10 (R&D Systems) for 24 hours to generate alveolar macrophage-like monocyte-derived macrophages. These macrophages were then detached with TrypLE Select (Gibco) for further treatment and stimulation.

Coating

To mimic spike protein specific immune complexes, 2pg/mL soluble prefusion-stabilized spike proteins of SARS-CoV-2 was coated overnight on a 96-well high-affinity plate (Nunc). Plate were blocked with 10% fetal calf serum (FCS) in phosphate buffered saline (PBS) for 1 hour at 37°C. Then diluted serum or 2 pg/mL anti-SARS-CoV-2 monoclonal antibodies were added and incubated for 1 hour at 37°C.

Compound treatment

Dexamethasone, R406 (the active metabolite of Fostamatinib; Selleckchem) and entospletinib (Selleckchem) were prepared in dimethyl sulfoxide (DMSO) (Sigma-Aldrich). For the non-compound control groups, DMSO/ethanol concentration were corrected after serial dilution with a final concentration smaller than 0.1% (v/v) for all samples. Cells were incubated with inhibitors or control 30 minutes before addition to the coated wells.

Meso Scale Discovery multiplex assay

V-PLEX Custom Human CytokinelO-plex kits for Proinflammatory Panel 1 and Chemokine Panel 1 (IL- ip, IL-6, IL-8, IL-10, TNF, CXCL10) were purchased from Mesoscale Discovery (MSD). The lyophilized cocktail mix calibrators for Proinflammatory Panel 1 and Chemokine Panel 1 were reconstituted in provided assay diluents respectively. Proinflammatory cytokines and chemokines in supernatant collected at 16 hours after stimulation were detected with pre-coated V-PLEX. The assays were performed according to manufacturer's protocol with overnight incubation of the diluted samples and standards at 4°C. The electrochemiluminescence signal (ECL) were detected by MESO QuickPlex SO. 120 plate reader (MSD) and analyzed with Discovery Workbench Software (v4.0, MSD). The concentration of each sample was calculated based on the four-parameter logistic fitting model generated with the standards (concentration was determined according to the certificate of analysis provided by MSD).

A full description of the data presented deposited at BioRxiv (https://www.biorxiv.org/content/10.1101/2022.12.20.521247yl). The study further shows that entospletinib is effective in protecting against disruption of endothelial cell barrier function and induced platelet adhesion to activated endothelial cells (figure 4 from the preprint), suppresses immune-complex induced platelet activation itself (figure 5 from the preprint) and works against different variants of concerns (VOCs; figure 6 of the preprint).

Claims

1. Entospletinib (GS-9973) or a pharmaceutically acceptable salt, ester or derivative thereof for use in the treatment of a viral infection caused by a coronavirus.

2. Entospletinib or a pharmaceutically acceptable salt, ester or derivative thereof for use according to claim 1, wherein said viral infection is caused by SARS-CoV-2.

3. Entospletinib or a pharmaceutically acceptable salt, ester or derivative thereof for use according to any of the claims above, wherein said treatment is a treatment of subjects at risk of developing severe disease caused by SARS-CoV-2.

4. Pharmaceutical composition for use according to any one of the above claims comprising Entospletinib or a pharmaceutically acceptable salt, ester or derivative thereof and a pharmaceutically acceptable carrier.