key: cord-0963824-079c0i5d
authors: Schumann, S.; Kaiser, A.; Nicoletti, F.; Mangano, K.; Fagone, P.; van Wijk, E.; Yan, Y.; Schulz, P.; Ludescher, B.; Niedermaier, M.; von Wegerer, J.; Rauch, P.; Setz, C.; Schubert, U.; Brysch, W.
title: Immune modulating drug MP1032 with SARS-CoV-2 antiviral activity in vitro: A potential multi-target approach for prevention and early intervention treatment of COVID-19.
date: 2020-11-04
journal: nan
DOI: 10.1101/2020.11.03.20216580
sha: 77365fcb00e9c95ae0e321d2d71f9cf6bcd1d6d0
doc_id: 963824
cord_uid: 079c0i5d

At least since March 2020, the multiorgan disease COVID-19 has a firm grip on the world. Although most of the cases are mild, patients from risk populations could develop a cytokine storm, which is characterized by a systemic inflammatory response leading to acute respiratory distress syndrome and organ failure. The present paper will introduce the small molecule MP1032, describe its mode of action, and give rationale why it is a promising option for prevention/treatment of SARS-CoV-2-induced cytokine storm. MP1032 is a phase-pure anhydrous polymorph of 5-amino-2,3-dihydro-1,4-pthalazinedione sodium salt that exhibits good stability and bioavailability. The physiological action of MP1032 is based on a multi-target mechanism including localized, self- limiting antioxidant activities that were demonstrated in a model of lipopolysaccharide (LPS)-induced joint inflammation. Furthermore, immune-regulatory and PARP-1 modulating properties, coupled with antiviral effects against SARS-CoV-2 were shown in various cell models. Efficacy has been preclinically elucidated in LPS-induced endotoxemia, a model with excessively activated immune responses that shares many similarities to COVID-19. So far, during oral clinical development with three-months daily administrations, no serious adverse drug reactions occurred highlighting the outstanding safety profile of MP1032.

respiratory distress syndrome and organ failure. The present paper will introduce the small 23 molecule MP1032, describe its mode of action, and give rationale why it is a promising option 24 for prevention/treatment of SARS-CoV-2-induced cytokine storm. MP1032 is a phase-pure 25 anhydrous polymorph of 5-amino-2,3-dihydro-1,4-pthalazinedione sodium salt that exhibits 26 good stability and bioavailability. The physiological action of MP1032 is based on a multi-27 target mechanism including localized, self-limiting antioxidant activities that were 28 demonstrated in a model of lipopolysaccharide (LPS)-induced joint inflammation. 29

Furthermore, immune-regulatory and PARP-1 modulating properties, coupled with antiviral 30 effects against SARS-CoV-2 were shown in various cell models. Efficacy has been 31 preclinically elucidated in LPS-induced endotoxemia, a model with excessively activated 32 immune responses that shares many similarities to COVID-19. So far, during oral clinical 33 development with three-months daily administrations, no serious adverse drug reactions 34 occurred highlighting the outstanding safety profile of MP1032. 35

The multiorgan disease COVID-19 (Corona virus disease 2019) is caused by infection with 37 SARS-CoV-2. It may result in the acute respiratory distress syndrome (ARDS) and multiple 38 organ failure that have been ascribed to a cytokine storm (also called cytokine release 39 syndrome or macrophage overactivation syndrome), a systemic inflammatory response 40 associated with an overstimulated immune system [1, 2] . However, not all people exposed to 41 SARS-CoV-2 are infected and not all infected patients develop severe COVID-19. Among 42 1099 patients analyzed in Wuhan, 16% progress to the severe phase showing respiratory 43 failure that requires mechanical ventilation [3] . 44

One of the biggest unanswered questions is why some people develop severe disease, whilst 45 others do not. More and more data support the notion that there is a skewed mortality towards 46 elderly men with underlying diseases [3, 4] . Elderly persons often show a diminished/impaired 47 pulmonary immunity with inadequate innate and adaptive immune responses. The so called 48 inflammaging is characterized by elevated reactive oxygen species (ROS), inflammatory 49 cytokines and acute phase reactants (e.g. C-reactive protein) that lead to continuous low-grade 50 inflammation accompanied by an overall decline in innate immune responses [5] . The process 51 of inflammaging together with the presence of other age-related diseases, such as diabetes and 52 cardiovascular diseases, is thought to be associated with increased severity and mortality of 53 viral infections in elderly people [6] . 54 SARS-CoV-2 infects epithelial cells of the upper respiratory tract via the receptor for 55 angiotensin-converting enzyme 2 (ACE2) triggering innate response mechanisms [7] ; at later 56 PARP-1. We therefore investigated whether MP1032 can affect PARP-1 activity. At first, a 114 coupled enzyme assay with fluorometric read-out was used to determine the effect on human 115 PARP-1 activity in a cell-free system. In principle, PARP activity is indicated by NAD + 116 depletion and reduced fluorescence. Hence, reduced PARP-1 activity due to PARP inhibition 117 rescues NAD + and leads to higher fluorescence values. For MP1032 an inhibitory activity on 118 human PARP-1 was detected in the low micromolar range (IC 50 = 1.55 μ M), while the 119 established PARP inhibitor 4-amino-1,8-naphthalimide (4-ANI) showed an IC 50 of 0.13 μ M 120 ( Figure 2A ). In a second experiment it was evaluated whether MP1032 is also able to inhibit 121 PARP-1 activity in differentiated HL-60 cells. By measuring the amount of poly-ADP-122 ribosylated proteins, it was shown that differentiated but untreated HL60 showed already high 123 levels of PARP-1 activity ( Figure 2B ). Further, it indicates that poly-ADP-ribosylation is not 124 massively increased by LPS stimulation, an effect that was previously described [19] . 125

Nevertheless, upon treatment with 1 mM MP1032 or 50 μ M 4-ANI, the amount of poly-ADP-126 ribosylation proteins decreased dramatically by over 90%. 127

Overall, these studies demonstrate that MP1032 works as ROS scavenger acting directly and 128 exclusively at the site of inflammation. Furthermore, MP1032 seems to modulate PARP-1 129 activity directly and independently from its ROS scavenging capacities. 130

During bacterial as well SARS-CoV-2-induced inflammation, oxidative stress and PARP-1 133 activation are tightly linked to the induction of proinflammatory cytokine production. The 134 potential effect of MP1032's ROS scavenging and PARP-1-inhibiting properties on the 135 downstream activation of pro-inflammatory cytokines was tested in HL-60 and murine/human 136 primary immune cells. 137 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint macrophage/monocyte linage and LPS stimulation with either 0.1 µg/ml or 1 µg/ml LPS led 139 to a dose-dependent TNFα and IL-6 induction ( Figure 3A ). Pre-treatment with 1 mM MP1032 140 reduced the secretion of both cytokines. Using 1 µg/ml LPS, MP1032 reduced the TNFα and 141 IL-6 levels about 39% and 52%, respectively. When 0.1 µg/ml LPS was used, MP1032 led 142 mainly to a decrease of TNFα (about 53%). 143

Using LPS-stimulated murine peritoneal macrophages, we assessed the effects of MP1032 in 144 a primary cell model. While MP1032 did not affect cytokine secretion of unstimulated murine 145

PMs (data not shown), LPS-induced proinflammatory cytokine concentrations were reduced 146 by MP1032 pre-treatment ( Figure 3B ). In detail, 1 mM MP1032 reduced levels of TNFα, IL-147 6, IL-12, and IL1-β by about 40%, 80%, 60%, and 50%, respectively. Interestingly, for all 148 cytokines similar MP1032 effects were observed after 48 h and 72 h of culture. Furthermore, 149 these effects were validated in PMs of a second mouse strain (Balb/c mice) and a clear dose-150 dependency was observed with six different MP1032 concentrations ranging from 1 mM to 151 0.25 mM (Supplementary Figure 1) . 152

Using human PBMCs, MP1032 at concentrations of 0.5 mM and 1 mM reduced LPS-induced 153

TNFα and IL-6 secretion in a dose-dependent manner ( Figure 3C ). IL-1β levels were reduced 154 to a much lower extent, which did not reach statistical significance. Notably, the oxidized 155 form of MP1032 (3-aminophthalic acid sodium salt) did not have any effect on LPS-induced 156

TNFα and IL-6 secretion indicating that the ROS scavenging properties described under 2.1 157 and an intact structure are mandatory to achieve suppression of proinflammatory cytokine 158 production. 159

The efficacy of MP1032 to inhibit the secretion of proinflammatory cytokines in vitro 160 prompted us to evaluate its in vivo efficacy. The used sublethal LPS-challenged model 161 represents a model of endotoxic shock that is pathophysiologically characterized by 162 dysregulated secretion of proinflammatory cytokines [20] . As depicted in Figure 4 , MP1032 163 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint pretreatment significantly reduced plasma cytokine levels compared to vehicle-treated mice 164 by 50% (TNFα) and 25% (IL-6). The positive control dexamethasone was administered two 165 times (instead of once as for MP1032) and reduced TNFα as well as IL-6 concentrations by 166 70% and 45%, respectively. Thus, these cumulative data implicate that MP1032 exhibits strong antiviral activity against 180 SARS-CoV-2, while having no impact on Vero B4 cell viability. 181

The data presented above clearly indicate that MP1032 is a promising treatment option for 183 SARS-CoV-2-infection and the prevention of the COVID-19 stage. To further emphasize this, 184 clinical safety data obtained from a randomized, double-blind, placebo-controlled phase II 185 study in psoriasis patients are presented here. In total, 155 patients were randomized receiving 186 either 300 mg MP1032, 150 mg MP1032, or placebo twice daily over a study period of 12 187 weeks. Safety evaluation revealed that MP1032 at both doses was safe and generally well-188 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint tolerated with no clinically important safety issues being identified. No deaths occurred and 189 only three treatment-emergent serious adverse events (SAEs) occurred in the placebo group. 190

Most of the treatment-emergent adverse events (TEAEs) were 'unlikely' or 'not related' to 191 MP1032 and patients receiving 300 mg MP1032 showed a statistically significant reduction in 192 the incidence of adverse events compared to patients receiving placebo ( Figure 6) . Table 1) . 212 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint development in the field of autoimmune inflammatory diseases (like psoriasis or rheumatoid 214 arthritis) and age-related degenerative diseases caused by chronic low-grade inflammation 215 (inflammaging). Similarly, alveolar inflammation and acute lung injury in COVID-19 are 216 driven by cellular pathologies, particularly with respect to oxidative stress and the pattern of 217 cytokines involved in the cytokine release syndrome [23, 24] . Several antibodies directed 218 against these pro-inflammatory cytokines or their respective receptors have already been 219 evaluated in the clinical setting, so far with moderate success only. This may be due to the 220 fact that these biologics suppress the host immune system, which is detrimental in the early 221 non-severe stages where the adaptive immune response helps to eliminate the virus and 222 protects against disease progression to severe stages [25] . The importance of treatment timing 223 with immune suppressants was demonstrated by the RECOVERY trial. In this study, 224 dexamethasone resulted in lower mortality among severe COVID-19 cases who were 225 receiving respiratory support, while in patients who were not receiving invasive mechanical 226 ventilation, dexamethasone treatment was harmful and resulted in a higher mortality rate [26] . 227

In contrast to immune-suppressing biologics or corticosteroids, MP1032 acts as an immune 228 regulator since it does not completely suppress cytokine induction, which serves an important 229 role during anti-infective response. The redox-balancing mechanism of MP1032 rather 230 curtails the overshoot of the immune response. Such a pharmacodynamic profile lends itself 231 for early intervention treatment, where preserving a physiologic immune response and 232 inhibiting an overshoot are essential for preventing disease progression. 233 Similar to chronic inflammatory diseases, several respiratory viruses induce a dysregulated 234 ROS formation by disrupting antioxidant mechanisms leading to an unbalanced oxidant : 235 antioxidant ratio and subsequent oxidative cell damage [27] . Early use of antioxidants, such as 236 vitamin C and N-acetyl cysteine is therefore discussed as potential option to prevent/treat 237 COVID-19 [28, 29] and several studies are currently ongoing (e.g. ClinicalTrials.gov 238 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint these antioxidants, which at high concentrations tend to scavenge even physiologically 240 necessary ROS, MP1032 action is self-regulating and highly localized. This unique 241 mechanism is based on the fact that excessive ROS production creates intracellularly a highly 242 localized alkaline environment [18] . Due to its chemical properties, circulating MP1032 is 243 activated by this pH shift only at the inflammatory site. As MP1032 scavenges excessive 244 ROS, the pH normalizes and MP1032 activation shuts down. Hence, excessive ROS are 245 scavenged, but physiologically necessary ROS levels are spared, ensuring that there is no 246 impairment of cellular signaling. 247

In vitro evaluation of antiviral activity directed against SARS-CoV-2 is preferably performed 248 in Vero cells [30] . Using this model, we could show that MP1032 suppresses virus replication 249 in a dose-dependent manner while the drug has no effect on cell viability. The molecular basis 250 for this anti-viral effect still needs to be evaluated. It is conceivable that MP1032 inhibits 251 virus entry via acting on ACE2 receptor as it was proposed for N-acetyl cysteine [31] . 252 MP1032 could further attenuate prolonged virus replication by preventing oxidative stress 253

[32] or by limiting ADP-ribosylation of the viral nucleocapsid protein via PARP-1 inhibition 254 [33] . Additional studies should be conducted to evaluate this in more detail. 255 was formed in the last reaction step and its polymorphic purity was confirmed by X-ray 260 powder diffraction as previously described [17] . 261 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint PBMCs were isolated from buffy coats of three different healthy blood donors (Institute for  289 transfusion medicine, Suhl, Germany) by density gradient centrifugation (Ficoll Hypaque, 290 density 1.077 g/ml, Biochrome). One hour prior to stimulation with 0.1 µg/ml LPS (from 291 The study was performed at the animal facility of the Changchun University of Chinese 310

Medicine. The animals were kept under standard laboratory conditions (water and food ad 311 libitum). Animals were single housed according to the Chinese legislation; animal handling 312 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2020. Pietro al Natisone, Udine, Italy). They were allowed to adapt to the animal facility for at least 337 one week before commencing the study. Fifteen minutes prior to i.p. LPS challenge 338 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint treated via i.p. injection with 0.5 mg/kg MP1032. As control, dexamethasone (Dex; 0.3 340 mg/kg, Soldesam, Labotatorio Biologico Milanese, Varese, Italy) was dosed twice, 24 h and 1 341 h before LPS application. As vehicle, water for injection was used. Mice were sacrificed 2 h 342 after LPS challenge and plasma TNFα and IL-6 concentrations were determined by ELISA. 343

In a randomized, double-blind, placebo-controlled study, two oral doses of MP1032 were 345 evaluated over a period of 12 weeks in patients with moderate to severe chronic plaque 346 psoriasis (PASI 10-20). In total, 155 patients were randomized receiving either 300 mg 347 MP1032, 150 mg MP1032, or placebo twice daily (EudratCT-No:2017-003484-36). Adverse 348 events were coded according to the Medical Dictionary for Regulatory Activities. Poly-ADP-ribosylation was normalized to the protein content measured in the cell lysates. 360

Cell-free human PARP-1 enzyme activity was determined in a coupled enzyme assay with 362 fluorometric read-out. In a first step, 200 nM NAD + were mixed with the PARP mix 363 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint consisting of 1.25 μ g activated (partly cleaved) DNA (Sigma#D4522) and 1.0 U/well hPARP-364 1 (Trevigen #4668-100-01) in assay buffer (50 mM Tris and 2 mM MgCl 2 (pH 8.0)). 365

Subsequently, MP1032 or 4-ANI prepared in assay buffer were added to the reaction mixture. 366

Each assay was performed with a NAD + standard curve (0 -100 nM NAD + ) and a PARP 367 minus control for data analysis. After 90 min, alcohol dehydrogenase/diaphorase mix 368 consisting of 50 mU alcohol dehydrogenase, 5 mU diaphorase, 50 μ M resazurin and 2% 369

EtOH (v/v) was added. Fluorescence kinetic (5 min steps for 40 min) was measured using 370

Synergy2 plate reader (BioTek, Germany) with λ exc 540/25 nm, λ em 590/20 nm and 550 nm 371 mirror. Data were calculated using NAD + standard curve and expressed as percent hPARP-1 372 inhibition. 373

Protein samples were separated by SDS-PAGE, transferred onto nitrocellulose membranes, 375 blocked with 3% bovine serum albumin, and incubated with the appropriate primary antibody. 376

The SARS-CoV-2 Nucleocapsid antibody (Genetex, GTX135357) and the anti-rabbit 377 secondary antibody coupled to horseradish peroxidase (Dianova, Germany) were used. 378

All data are presented as mean ± SEM. Statistical calculation was performed using GraphPad 380 Prism (GraphPad Software, La Jolla, USA). Normal distribution was tested using the 381 Kolmogorov-Smirnov test. Unpaired T-Test (with or without Welch's correction depending 382 on the comparability of variances) or Mann-Whitney Test were used to assess the direct 383 treatment effects vs. the vehicle group. To assess the incidence of treatment-emergent adverse 384 events in the clinical phase II study, two-sided Fisher's exact test was used. Differences with 385 p<0.05 were considered statistically significant. 386 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint

No clinically proven specific antiviral agent is currently available for SARS-CoV-2 infection 388 and thus the medical need for safe and easily distributable drugs is high. Here, we show that 389 MP1032, a phase-pure anhydrous polymorph of 5-amino-2,3-dihydro-1,4-pthalazinedione 390 sodium salt exerts potent immune-modulatory, self-regulated ROS scavenging, and SARS-391

CoV-2 specific antiviral properties. This pharmacodynamic profile which simultaneously 392 addresses several crucial pathophysiological processes of a SARS-CoV-2 infection renders 393 MP1032 a promising candidate for the treatment and possibly prevention of COVID-19. This 394 assumption should be tested in a clinical trial. 395 The funders sponsored all the research but had no impact on the study results. 411 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint

This research did not receive any specific grant from funding agencies. 413 414 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2020. Tumor necrosis factor-alpha UPE Ultra-weak photon emission WST-1

Water-soluble tetrazolium salt 1 assay 416 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2020. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint were isolated from female C57Bl/6 mice four days after i.p. injection of 3% thioglycolate 566 medium. One hour prior to LPS stimulation (0.1 µg/ml), isolated cells were pre-treated with 567 0.5 or 1 mM MP1032. (C) human peripheral blood mononuclear cells were isolated from 568 blood buffy coats by density gradient centrifugation. One hour prior to stimulation with 0.1 569 µg/ml LPS, cells were treated with 0.5 or 1 mM MP1032. Cell-free supernatants were 570 collected from all cell types after 24 h and secreted cytokine levels were detected by ELISA. 571

Data are expressed as mean + SEM relative to the LPS-treated vehicle group that was 572 normalized to 100%. (A/B) n= 2, (C) n=3. Statistical analyses were performed using unpaired 573

T Test with or without Welch's correction; *p<0.05, **p<0.01. 574 575 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint assessed by the water-soluble tetrazolium salt 1 assay; staurosporine (1 µM) served as 597 positive control. Data are expressed as mean ± SEM, n= 3/group. Statistical analyses were 598 performed using unpaired T Test with Welch's correction; **p<0.01, *p<0.05. 599 600 601 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint Figure 6 : Incidence of treatment-emergent adverse events in a clinical phase II study. 603 In total, 155 patients with moderate to severe chronic plaque psoriasis were randomized 604 receiving either 300 mg MP1032, 150 mg MP1032, or placebo twice daily of a period of 12 605 weeks (EudratCT-No:2017-003484-36). Adverse events were coded according to the Medical 606

Dictionary for Regulatory Activities. Statistical analyses were performed using two-sided 607

Fisher's exact test; *p<0.05. 608 609 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.03.20216580 doi: medRxiv preprint

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