Randomized Controlled Trial of DDS for Covid-19 ARDS

Background Clinicians considered DDS administration to treat SARS-CoV-2 inammasome. DDS is helpful in the molecular regulation of Nod-like receptor family pyrin domain-containing 3 (NLRP3). Objective To study the targeting of NLRP3 itself or up-/downstream factors of the NLRP3 inammasome by DDS must be responsible for its observed preventive effects, functioning as a competitor. Methods This is a randomized controlled trial (RCT). We set out to use objective criteria of improvement. We treated the patients with standard Covid-19 acute respiratory distress syndrome (ARDS) treatment with DDS. The RCT results were analyzed. Results ARDS progression was blocked in 17 of 19 total patients at the rst period. The 44 subjects were analyzed during the second period. It is signicant at the ARDS onset stage. The mortality of ARDS-onset patients was 0% with DDS and 40% without DDS among the total of 65 cases. The t-value of RCT is -1.5, and the p-value is .075475. The result is signicant at p < 0.10. ARDS onset with DDS prescribed group survived more 90% than ARDS onset without DDS group. The chi-square is 5.8108. The p-value is .015928. (Signicant at p < .05) (RR 0.15, OR 0.09) Conclusion There was a signicant difference in DDS treatment results in the ARDS-onset group. We conrmed that DDS clinically treated the onset of ARDS by targeting SARS-CoV-2-activated inammasomes. Like chemically reacting substances, inammasome and DDS compete, proving that it is effective in early ARDS.

NLRP3 is a critical component of the innate immune system that mediates caspase-1 activation and the secretion of the proin ammatory cytokines IL-1β/IL-18 in response to microbial infection and cellular damage. However, aberrant activation of the NLRP3 in ammasome has been linked with several in ammatory disorders, including cryopyrin-associated periodic syndromes, AD, type 2 diabetes, and atherosclerosis. Diverse stimuli activate NLRP3. Multiple molecular and cellular events have been shown to trigger its activation. NLRP3 responds to signalling events, including ionic ux, mitochondrial dysfunction, reactive oxygen species production, and lysosomal damage [10]. Although ARDS is a complication of SARS-CoV-2 infection, it is not a viral-replication-associated condition or a condition that causes tissue injury [11]. Instead, ARDS results from dysregulated hyperin ammation in response to viral infection [12].
A Covid-19 committee at Hunt Regional Hospital reviewed the use of DDS as an off-label medication based upon treatment adjuncts and in ammasome theory [1,5]. After RNA virus infection, NLRP3 in ammasomes are activated by the mitochondrial protein mitofusin 2 [13]. Mitochondrial antiviral signalling protein (MAVS) with NLRP3 and SAMHD1 (SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1) are linked to the NF-kB pathway [14] and NLRP3 to regulate in ammasome activity [15].
DDS is a well-known drug for treating leprosy, a chronic, progressive bacterial infection caused by the bacterium Mycobacterium leprae. As a therapeutic and prophylactic drug for in ammasomes caused by SARS-CoV-2, it was administered to patients with worsening ARDS despite standard Covid-19 treatment. Those who do not take DDS are 2.55% and those who take DDS are 1.05% [16], so this is epidemiological data consistent with the theory. South Korea's Sorokdo National Hospital reported that about 10,000 Hansen's disease patients were not infected from 1 January 2020 to 15 April 2021.
(Supplement S1) These pathologic characteristics of SARS-CoV-2-activated in ammasomes are intense, rapid stimulation of the innate immune response that triggers the activation of the NLRP3 pathway. Affected patients develop ARDS, such as refractory hypoxia, which is invariably fatal. Hypoxia's leading cause is severe in ammation leading to pulmonary microangiopathy, with relentless clotting unchallenged due to a state of brinolysis [17].

Methods
This study was proposed by an observer who developed the theory of the in ammasome competitor [1], and the clinical study was conducted in collaboration with medical staff at Hunt Regional Hospital when 100% of patients died from Covid-19 ARDS. It was a case series for medical staff at Hunt Regional Hospital. Still, a cross-sectional study was conducted to treat Covid-19 ARDS by the medical staffs, and it was also a randomized controlled trial of Covid-19 ARDS onset patients to the observer. Statistical analysis was performed according to the observer's point of view.

Hunt Regional Hospital ethically approved this clinical patient treatment based on the World Medical
Association's Declaration of Helsinki. The patients provided written informed consent (or their parents or guardians). We administered medicines in compliance with medical and pharmacy laws with informed consent. For every patient admitted with Covid-19 ARDS, the medical doctor treated the patient with standard Covid-19 treatment. We started oral DDS 100 mg to target NLRP3 in ammasomes.
We set out to use objective criteria of improvement, such as A. a reduction in the FIO 2 requirement and B. a decrease in the progression of hypoxia. This is case series with or without intervention; cross-sectional study. The criterion for ARDS onset was the requirement of FIO 2 via simple nasal cannulation of up to 15 L/min. The criteria for aggravated cases of ARDS were FIO 2 administered via an HFNC of 95-100% and/or BiPAP. The criterion for severe cases of ARDS was the need for mechanical ventilation. The crosssectional study observed the result of medical treatment from 21 December to 29 December 2020.
In the Hunt Regional Hospital ICU, the doctors treating Covid-19 patients change approximately every eight days. The hospital stopped prescribing DDS after the rst period for two weeks (standard Covid-19 treatment group without DDS (19)) and rigorously revalidated its effectiveness. Furthermore, during the second period, 44 patients with ARDS in the intensive care unit were re-examined. The trial group was 22 subjects who treated with standard Covid-19 treatment and DDS. The control group was 22 subjects who treated only with standard Covid-19. After the second period, two medical staffs suffered from Covid-19 ARDS, which was the third period. Since the medical staff did not administer DDS according to the ARDS period, the ARDS mortality study corresponds to a single-blind study. This study was performed to nd the most suitable time for DDS treatment. J.B. classi ed the ARDS onset stage after completing a randomized controlled trial (RCT). Therefore, we used the statistical effectiveness for survival at the Pandemic period and analyzed the data for RCT.

Protocol for administration
Written informed consent should be obtained, and potential side effects should be explained. The common side effects are haemolytic anaemia (in patients with G6PD de ciency) [18], methemoglobinemia, and allergic reaction. The patients should also be informed that currently, G6PD is a send-out test and can take up to 5-7 days. Cimetidine 400 mg orally TID will now be administered to counter DDS methemoglobinemia side effects [19]. The venous methemoglobin level should be checked every day, and a mild methemoglobin level of 2-10% [20] is tolerated well. DDS should be discontinued if the level reaches 15 or above.

Results
An off-label medication at the rst period Patients started to feel better within 24 hours after the administration of DDS with standard Covid-19 therapy. Our most prominent cases in the rst period were in two patients on an HFNC with 50 LPM ow and 100% fraction of inspired oxygen (FIO 2 ), intermittently requiring bilevel positive airway pressure (BiPAP) and barely having an O 2 saturation (SaO 2 ) in the 87-91% range for several days. These patients' clinical conditions worsened slowly every day, nearly requiring intubation. With DDS administration, one patient had his/her FIO 2 requirement reduced to 50% within 48 hours, and the other patient had his/her FIO 2 need reduced to 80%. Encouraged by an objective response, we started to administer DDS to other patients. We did not observe any improvement in mechanically ventilated patients. Nineteen patients with severe, worsening ARDS and imminent death due to SARS-CoV-2 infection were treated. According to our rst results, ARDS progression was blocked in 17 of 19 total patients, and two patients' conditions progressed to requiring mechanical ventilation. ARDS improved in 10 patients (decreased FIO 2 ). (Table 1) Cross-sectional study at the second period We stopped prescribing DDS for two weeks, determined the prescribing guidelines for DDS [21], and started treating again. We prescribed DDS in the second period, including the hiatus (22 cases) after the rst period. The results of the rst (off-label medication) and second (cross-sectional) period were almost identical.
Cross-sectional study: The results of treating ARDS-onset cases were as follows: decreased FIO 2 in 7 patients and no worsening in 1 patient. The results of treating aggravated ARDS cases were as follows: decreased FiO 2 in 6 patients and no worsening in 3 patients. The results of treating severe ARDS cases were as follows: no response in 2 patients. There were three deaths in the aggravated ARDS group treated with DDS. At the same time, there were no deaths in the ARDS-onset DDS-treated group. (Table 2) Simultaneously, in the same hospital ICU, we examined the results of standard Covid-19 treatment of ARDS without DDS. There were eight deaths in the ARDS-onset group not treated with DDS, with progressing hypoxia, and almost all of these patients died before reaching the criteria for aggravated ARDS. (Table 3 There was a signi cant difference in the ARDS-onset group when DDS was prescribed (DDS group) compared with when DDS was not prescribed. DDS must be applicable at ARDS-onset stage. Since ARDS has a direct correlation with mortality, we decided to conduct randomized controlled trial (RCT).
RCT Study: Mortality of the ARDS-onset stage at the third period Because a signi cant difference in ARDS treatment results was observed in the onset group when DDS was prescribed and when DDS was not prescribed, all data from all periods were collected (Fig. 1). The ARDS-onset mortality rates were 0% (with DDS) and 40% (without DDS). One patient died without DDS treatment after hospital discharge. The mortality rate was 0% because all studied patients were survived at the onset stage in the DDS group. (Table 6) The observer compared the 17 participants who received the DDS intervention (M = 1.

Discussion
The focus of this study is a period when treatment effects change dramatically, so patients with Covid-19 ARDS onset must be carefully analyzed. Only Covid-19 ARDS onset patients meet random allocation criteria for the observer. Here is why: For 19 cases in the rst period, the DDS trial did not classify the patient's ARDS condition as mild or severe. The observer observed the treatment progress, and medical staffs reported the results, so it was a random allocation for ARDS onset. In the second cross-sectional study, patients were randomly allocated because the hospital manager determined when the administration was prohibited or when it could be administered. For 22 cases in the second period, the DDS trial did not also classify the patient's ARDS condition as mild or severe. In the third period, two medical staffs were infected while treating, corresponding to random allocations. After the trial was completed, 65 subjects were classi ed by the observer.
In In our t-test study ( t-value is -1.5, and the p-value is 0.075475 and signi cant at p < 0.10.), out of 378 leprosy patients, a minimum of 68 were actually infected with Covid-19 and four were dead. The Covid-19 prevalence of leprosy patients may be above 18% and maximum 5.8% mortality. The sixty-four persons have survived because of the metronomic DDS prescription. It is only the dada interpretation of the report based on our study [16,22]. Patients co-infected with M. leprae and SARS-CoV-2 may experience a highergrade proin ammatory state with increased IL-6 (P = 0.043) and IL-12B (P = 0.017) expression [23]. Since the dosage of DDS should be increased from 100mg to 200mg, the treatment guidelines recommend 100-300mg per day. This is because it competes with the in ammasome.
Pathophysiology of SARS-CoV-2-associated ARDS Refractory progressive respiratory failure has been the primary cause of death in the Covid-19 pandemic.
In patients who died from Covid-19-associated or in uenza-associated respiratory failure, the histologic pattern of the peripheral lung was diffuse alveolar damage with perivascular T-cell in ltration. The lungs from patients with Covid-19 also showed distinctive vascular features, consisting of severe endothelial injury associated with the intracellular virus and disrupted cell membranes. Histologic analysis of pulmonary vessels in patients with Covid-19 showed widespread thrombosis with microangiopathy [24]. These observations indicated pulmonary vascular disease in patients with Covid-19 ARDS [25].
By studying moderately and severely ill Covid-19 patients, we found active NLRP3 in peripheral blood mononuclear cells and postmortem patient tissue. In ammasome-derived products, such as active caspase-1 and IL-18, were observed in the sera and interleukin-6 (IL-6) and LDH, markers of Covid-19 severity. Higher IL-18 and caspase-1 are associated with disease severity and poor clinical outcome [5]. It appears that IL-6 levels in Covid-19 patients are not exceptionally high in the broader context of ARDS [26].
Covid-19 ARDS onset can present with relatively preserved aeration on chest CT imaging despite severe respiratory hypoxemia. In some patients, this early, high-compliance phenotype evolves into a lowcompliance phenotype with poor aeration. We described the rst clinical presentations as low elastance, high compliance, and preserved aeration (L-type) and the second as high elastance, low compliance, and poor aeration (H-type) [27]. vomiting, and anorexia [25]. With the data on these diverse complications, the relationship of the virus, its direct cytotoxic effects, its effects on the renin-angiotensin-aldosterone systems, its impact on the endothelium, and the various manifestations of Covid-19 disease was explored [28].
In vitro human brain organoid experiments and in vivo SARS-CoV-2 mouse model hybrid experiments revealed possible evidence for the neuroinvasive capacity of SARS-CoV-2 and an unexpected consequence of direct infection of neurons by SARS-CoV-2 [29].
Brainstem involvement, especially pre-Bötzinger complex involvement [30,31], could explain the respiratory failure and sudden high death rate of Covid-19 ARDS patients. It could also explain the sudden recovery of cases 1, 2, 3, and 4 (within 24-48 hours) after taking DDS in the ARDS-onset and aggravated stages in the second phase. DDS treats and prevents transient bulbar palsy through the medulla oblongata. DDS also has a therapeutic effect on Covid-19-associated ARDS, as evidenced by the recovery of several patients within 24 -48 hours of taking DDS. Therefore, the causal relationship is clear.
The solubility of DDS varies over an extensive range depending on the solvent used (e.g., water, 0.2 mg/mL, methanol, 52 mg/mL). DDS has been considered a di cult-to-handle compound for experimental investigations, especially using living cell assays [32]. According to in ammasome competitor theory [1], the treatment's preventive effect will not appear if DDS does not reach the proper concentration. After the ingestion of a single 50-to a 300-mg dose of DDS, maximal serum concentrations are reached between 0.63 and 4.82 mg/L [33]. A doctor should administer DDS after thoroughly testing it.

An in ammasome competitor thesis
After progressive dyspnoea and worsening desaturation, we tend to see a pattern portending fatal outcome, with no further standard treatments to offer. According to an in ammasome competitor theory, we started oral DDS 100 mg to target the NLRP3 in ammasome [1].
Furthermore, the DDS dose was increased to 200 mg PO daily because the theory is that SARS-CoV-2activated in ammasomes and DDS compete for DNA binding at the molecular level. We have used DDS so far in a total of 19 patients. We established objective criteria for improvement, such as reducing the FIO 2 requirement and a decrease in the progression of hypoxia.
As in the debate between the in ammatory hypothesis [34,35] and the regression hypothesis [36] for Alzheimer's disease, in 17 out of 19 patients receiving treatment targeting the NLRP3 in ammasome, the progression of ARDS was blocked, whereas in 2 patients, no response to DDS treatment was observed, and their conditions progressed to requiring mechanical ventilation. DDS passes the BBB very well. DDS is a newly proven drug for the treatment and prevention of AD [3].
The ndings for DDS are essential. SARS-CoV-2 might gain entry to the CNS through the olfactory bulb to invade the brainstem [37]. Moreover, SARS-CoV-2 CNS access might also occur from the peripheral circulation through BBB compromise. Another possible SARS-CoV-2 CNS entry route could be dispersal from the lungs into the vagus nerve via pulmonary stretch receptors, eventually reaching the brainstem [38,39]. Brainstem involvement, especially pre-Bötzinger complex involvement, could explain the respiratory failure and high death rate of Covid-19 patients and the sudden recovery of patients after taking DDS. (Fig. 2) Suggested in ammasome treatment mechanism The receptor-binding domain of the S protein on the surface of SARS-CoV-2 interacts with the ACE2 receptor (ACE2) in host cells [40]. It is now well-established that the entry of SARS-CoV-2 into host cells is facilitated by its neuropilin-1 (NRP1), a transmembrane receptor that lacks a cytosolic protein kinase domain [41]. NRP1 is also expressed in the CNS, including olfactory-related regions such as the olfactory tubercles and paraolfactory gyri [42]. Furthermore, NRP1 is a host factor for the entry of SARS-CoV-2 into the brain through the olfactory epithelium [43]. SARS-CoV-2 evokes a response that needs strong induction of a subclass of cytokines, including type I and, obviously, type III interferons and a few chemokines, such as the response to in uenza A virus, speci cally respiratory syncytial virus [14,44]. SAMHD1 links to the NF-kB pathway [14]. DDS is a small molecule with anti-in ammatory and immunosuppressive properties as well as antibacterial and antibiotic properties. DDS passes through the BBB [45,46], and high-dose sulfadiazine results in an effective CSF concentration in humans [47]. DDS binds to myeloperoxidase and regulates the production of hypochlorite. It reduces the in ammatory response of cells [1]. The nucleophilic/electrophilic region of DDS interacts with amino acids by molecular bonding. Neurotoxicity, aggregation, and free radical formation are initiated by the methionine (Met) residue at position 35 in the Aβ C-terminal domain [48][49][50]. Two-electron oxidation of bicarbonate is mediated by hydrogen peroxide after the generation of peroxymonocarbonate (HCO4 − ). The bicarbonate/carbon dioxide pair stimulated one-electron oxidation. Carbonate radical anions (CO3 • ) mediate one-electron reactions to promote oneelectron oxidation to e ciently oxidize Met residue thioester sulfur to sulfur radical cations (MetS •+ ) [51].
DDS has a structure that can competitively reduce the positively charged sulfur radical production rate because it has a similar structure to methionine sulfoxide [1]. Control for reversing protein ubiquitylation was the subject of our study. The reversibility of ubiquitination by deubiquitinating enzymes (DUBs) serves as a signi cant regulatory layer within the ubiquitin system. The human genome encodes approximately 100 DUBs, and DUBs have implicated pathologies, including neurodegeneration and cancer [52]. The conjugation of ubiquitin can be reversed by DUBs, which re ect additional regulation of ubiquitin [53]. The covalent attachment of ubiquitin to substrates has generated a repurposed drug (DDS) capable of competing with pathogenic targets in Ub-conjugating targeting chimaeras [1,54]. The nucleophilic properties of DDS compete with ubiquitin (Ub), similar to DUBs. Before loading Ub onto the substrate, the Ub-activating (E1)/Ub-conjugating (E2)/E3 ligase acts at each stage of the ubiquitination process. Enzymes can carry Ub via a thioester linkage, which allows vigorous favourable attack of the substrate nucleophile. DDS can compete with the ubiquitination cascade. The identical mechanism can potentially ubiquitinate cysteine thiols and hydroxyls on serines, threonines, leucines, and tyrosines [1,52]. DDS noncovalently binds/interacts with the minor groove of DNA. The DDS-DNA interaction/binding relative binding energy is −6.22 kcal mol-1, estimated using in silico studies. Docking analysis further revealed that DDS preferentially binds to the AT-rich region of DNA [55]. The nucleophilic properties of DDS also compete with NLRP3. ORF8b activates NLRP3 through the interaction of the AT-rich repeat domain of NLRP3 [1].
DDS was added in the desperate attempt to stop the decline in these patients' conditions. Excluding the patients whose conditions progressed to requiring mechanical ventilation, the rapid recovery of ARDS patients within 24 hours of being treated with DDS provides evidence supporting the use of this very new, meaningful clinical treatment in the ICU. Activated microglia were found adjacent to neurons by pathologic studies, suggesting neuronophagia in the olfactory bulb, substantia nigra, a dorsal motor nucleus of the vagal nerve, and the pre-Bötzinger complex in the medulla [56]. Transient global amnesia is a rare clinical syndrome in which a sudden onset of anterograde amnesia recovers within 24 hours. Although the underlying pathophysiology is uncertain, focal hippocampal ischaemia, venous congestion, migraine-related mechanisms, hypoxic-ischaemic events, epilepsy, and metabolic stress may be involved [57]. Serum neuro lament light chain (NFL) is a speci c biomarker of neuronal injury. NFL was higher in patients with Covid-19 than in the comparator groups. Higher NFL levels were associated with short-term outcomes, indicating that neuronal injury is common in critically ill patients [58]. Brainstem involvement could explain sudden respiratory failure [56]. The molecular properties of DDS, including electron density and its Laplacian delocalization index, have provided a speci c mechanism for chemical bonding and atomic and molecular details [59]. The redox properties of DDS dependent on amine and sulfone moieties explain the oxidation mechanism of DDS by electron transfer [60]. As it applies to electrons, we can understand the various neuropathologic ndings of Covid-19, including its mysterious sensory manifestations. Early indications of DDS's effects show that it may have the potential to alleviate the course of Covid-19 [22]. DDS has the following potential: DDS can be used along with oral therapeutics for physically obstructing the replication of DNA in in ammasomes. Proteins covalently attached to DNA are common and impose physical obstacles to DNA replication, repair, transcription, and recombination[61] and large DNA-protein crosslinks can be cleaved into DNA-peptide crosslinks; these smaller fragments also disrupt normal replication [62]. (Fig. 3) COVID-19 patients present high serum levels of free DNA, myeloperoxidase-DNA (MPO-DNA) complexes, and citrunylated histone H3 (Cit-H3).
Those who do not take DDS are 2.55%, and those who take DDS are 1.05% [16], so this is epidemiological data consistent with the in ammasome competitor thesis. South Korea's Sorokdo National Hospital reported that about 10,000 Hansen's disease patients were not infected from 1 January 2020 to 15     Fisher's exact test statistic value is 0.0433. The result is significant at p < .05.  Figure 1 ARDS onset data from periods 1, 2, and 3. The mortality rate was 0% because all studied patients were survived at the onset stage in the DDS prescribed group. Because of a signi cant difference in ARDS treatment results: 0% (with DDS) and 40% (without DDS), all data from periods 1, 2, and 3 were collected.
In the 2nd period, eight patients who did not use DDS died by the progression of hypoxia. The total number of deaths is eight.

Figure 2
The perivascular space in the brain. The perivascular space in the brain consists of a single or double layer of invaginated pia, forming an interstitial uid-lled space representing an extension of the extracellular uid space around the intracranial vessels as they descend into the brain parenchyma.
Human sensory stimuli affect the breathing sensation via the cerebral cortex and hypothalamus. The abnormal muscular sensation is also a contributor to dyspnoea. The respiratory muscles are not intentionally activated in healthy breathing [68][69][70]. Nucleophilic properties of DDS compete with NLRP3. DDS binds to myeloperoxidase and regulates the production of hypochlorite, thereby reducing the cellular circumstance. The topological properties of DDS, such as electron density and its Laplacian delocalization index, the negative potential of the vicinity of O and O atoms is susceptible to severe electrophilic attack. The nucleophilic/electrophilic region of DDS interacts with amino acids by molecular bonding. DDS has a structure that can reduce the sulfur radical production rate by electron charge transfer because they are structurally similar to methionine sulfoxide. Proteins contain many nucleophilic sites capable of attacking a ubiquitin (Ub)-conjugating enzyme (E2)-