WO2021254582A1

WO2021254582A1 - Efficacy of (sofosbuvir plus ledipasvir) in egyptian patients with covid-19compared to standard of care treatment.

Info

Publication number: WO2021254582A1
Application number: PCT/EG2020/000013
Authority: WO
Inventors: Mohamed Abdel-Salam EL-GOHARY
Original assignee: Almaza Military Fever Hospital
Priority date: 2020-06-15
Filing date: 2020-06-15
Publication date: 2021-12-23

Abstract

The antivirals ledipasvir is particularly attractive as therapeutics to combat the new corona virus with minimal side effects, commonly fatigue and headache. The drugs (ledipasvir/sofosbuvir) could be very effective owing to their dual inhibitory actions on two viral enzymes. The present study aimed to test and suggest possible inhibitors, DAA drugs, currently in the market stop the infection immediately. Sofosbuvir, ledipasvir can be used against the new strain of coronavirus that emerged with promising results.

Description

Efficacy of (Sofosbuvir plus ledipasvir) in Egyptian patients with COVID-19compared to standard of care treatment.

Technical Field:

Over the past two decades, coronaviruses (CoVs) have been associated with significant disease outbreaks in East Asia and the Middle East. The severe acute respiratory syndrome (SARS) and the Middle East respiratory syndromes (MERS) began to emerge in 2002 and 2012, respectively. At present, a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the Coronavirus Disease 2019 (COVID-19), has emerged in late 2019, which has posed a global health threat with its ongoing pandemic in many countries and territories .

CoVs belong to the family Coronaviridae (subfamily Coronavirinae), the members of which infect a broad range of hosts, producing symptoms and diseases ranging from a common cold to severe and ultimately fatal illnesses such as SARS, MERS, and, as of present, COVID-19. The SARS-CoV-2 (formerly 2019-nCoV) is considered as one of the seven members of the CoV family that infect humans (3), and it belongs to the same lineage of CoVs that causes SARS; however, this novel virus is genetically distinct. Until 2020, six CoVs were known to infect humans include HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKUl, SARS-CoV, and MERS-CoV. Though SARS-CoV and MERS-CoV have resulted in outbreaks with high mortality, others remain associated with mild upper- respiratory tract illnesses.

The COVID-19 that emerged in China spread rapidly throughout the country and subsequently to other countries. Due to the severity of this outbreak and the potential of spreading on an international scale, the WHO declared a “global health emergency” on January 31st, 2020. Subsequently, on March 11th, 2020, a pandemic situation was declared. At present, we are not in a position to effectively treat COVID-19 since neither approved vaccines nor specific antiviral drugs for treating human CoV infections are available .

Comparing the genome of SARS-CoV-2 with that of the closely related SARS/SARS- like CoV revealed that the sequence coding for the spike protein with a total length of 1,273 amino acids showed 27 amino acid substitutions. Six of these substitutions are in the region of the receptor-binding domain, and another six substitutions are in the underpinning subdomain (SD) (16). Phylogenetic analyses have revealed that the SARS-CoV-2 is closely related (88% similarity) to two SARS-like CoVs derived from bats (bat-SL-CoVZC45 and bat-SL-CoVZXC21). Furthermore, the SARS-CoV-2 is genetically distinct from SARS-CoV (79% similarity) and MERS-CoV (50%) .

THE VIRUS (SARS-CoV-2)

Coronaviruses are positive-sense RNA viruses having an extensive and promiscuous wide range of natural hosts and affect multiple systems (23, 24). Coronaviruses can cause clinical diseases in humans that may extend from the common cold to more severe respiratory diseases like SARS and MERS.

For the time being, earlier, the WHO named this currently emerging virus as a 2019-novel coronavirus (2019-nCoV) and later the disease as COVID-19. Later, this virus has been proposed to be designated/named as "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2) by the International Committee on Taxonomy of Viruses (ICTV) Coronaviridae Study Group that determined the virus belongs to the existing species, Severe acute respiratory syndrome-related coronavirus, and found this virus related to SARS-CoVs (26). The SARS-CoV-2 is a member of the order Nidovirales, family Coronaviridae, subfamily Orthocoronavirinae, which is sub-divided into four genera, viz. Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. The genera Alphacoronavirus and Betacoronavirus originate from bats, while the Gammacoronavirus and Deltacoronavirus have evolved from birds and swine gene pools.

Coronaviruses possess an unsegmented, single-stranded (ss) positive-sense RNA genome of around 30 kb, enclosed by a 5’-cap and 3’-poly-A tail (30). The genome of SARS- CoV-2 is 29.891 kb long, with a G + C content of 38%. These viruses are encircled with an envelope containing viral nucleocapsid. The nucleocapsids in CoVs are arranged in helical symmetry, which reflects an atypical attribute in positive-sense RNA viruses.

The electron micrographs of SARS-CoV-2 revealed a divulging spherical outline with some degree of pleomorphism, virion diameter varying from 60 to 140 nm, and distinct spikes of 9 to 12 nm, giving the virus an appearance of a solar corona (3). The CoVs genome is arranged linearly as 5'-leader-UTR-replicase-structural genes-(S-E-M-N)-3' UTR-poly (A). Accessory genes such as 3a/b, 4a/b, hemagglutinin-esterase gene (HE) are also seen intermingled within the structural genes (30). The SARS-CoV-2 has also been found to be arranged similarly and encodes several accessory proteins, although it lacks the HE, which is characteristic of some Betacoronaviruses. The positive-sense genome of CoVs serves as mRNA and is translated to polyprotein la/lab (pp la/lab). A replication-transcription complex (RTC) is formed in double-membrane vesicles (DMVs) by non-structural proteins (nsps), encoded by the polyprotein. Subsequently, the RTC synthesizes a nested set of subgenomic RNAs (sgRNAs) via discontinuous transcription .

Based on molecular characterization, the SARS-CoV-2 is considered as a new Betacoronavirus belonging to the subgenus Sarbecovirus. Few other critical zoonotic viruses (MERS-related-CoV and SARS-related-CoV) also belong to the same genus. However, the SARS-CoV-2 was identified as a distinct virus based on the percent identity with other Betacoronavirus; conserved ORF lab is below 90%. An overall 80% nucleotide identity was observed between SARS-CoV-2 and original SARS-CoV along with 89% identity with ZC45 and ZXC21 SARS related CoVsof bats. In addition to this, 82% identity has been observed between SARS-CoV-2 and human SARS-CoV Tor2 and human SARS-CoV BJ01 2003 (31). A sequence identity of only 51.8% was observed between MERS-related-CoV and the recently emerged SARS-CoV-2. Phylogenetic analysis of the structural genes also made known that SARS-CoV-2 is closer to bat SARS- related-CoV. Therefore, SARS-CoV-2 might have originated from bats, while other amplifier hosts might have played a possible role for this disease transmission to humans. Of note, the other two zoonotic Co Vs (MERS- related-CoV and SARS-related-CoV) have also originated from bats. Nevertheless, for SARS and MERS, civet cat and camels act as amplifier hosts, respectively.

Coronaviruses genome and subgenome encode six open reading frames (ORFs). The majority of the 5’ end is occupied by ORFla/b, which produces 16 nsps. The two polyproteins, ppla and pplab, are initially produced from ORFla/b by a -1 frameshift between ORFla and ORFlb. The viral encoded proteases cleave polyproteins into individual nsps [Main protease (Mpro), chymotrypsin-like protease (3CLpro), and papain- like protease (PLPs)]. The SARS-CoV-2 also encodes these nsps, and their functions have been elucidated recently. Remarkably, a difference between SARS-CoV-2 and other CoVsis the identification of a novel short putative protein within ORF3 band a secreted protein with an alpha helix and beta-sheet with six strands encoded by the ORF8.

Coronaviruses encode four major structural proteins, namely Spike (S), Membrane (M), Envelope (E), and Nucleocapsid (N), which are described in detail as below.

Spike glycoprotein ‘S’

Coronavirus S protein is a large multifunctional class I viral transmembrane protein. The size of this abundant S protein varies from 1160 amino acids (IBV, Infectious Bronchitis Virus in poultry) to 1400 amino acids (FCoV, Feline Coronavirus). It lies as a trimer on the virion surface, giving the virion a ‘corona’ or crown-like appearance. Functionally it is required for the entry of the infectious virion particles inside the cell through interaction with various host cellular receptors.

Furthermore, it acts as a critical factor for tissue tropism and the determination of host range. Notably, S protein is one of the vital immunodominant proteins of CoVs capable of inducing host immune response. The ectodomain in all Co Vs S protein shows a similar domain organization, divided into two domains. The first one, SI, helps in host receptor binding while the latter one, S2, accounts for the fusion. The former (SI) is further divided into two subdomains, namely the N- terminal domain (NTD) and C- terminal domain (CTD). Both these subdomains act as the receptor-binding domains interacting efficiently with various host receptors. The SI CTD contains the receptor-binding motif (RBM). In each coronavirus spike protein, the trimeric SI locates itself on top of the trimeric S2 stalk. Lately, the structural analyses of the S proteins of COVID-19have revealed 27 amino acid substitutions within a length of 1273 amino acid stretch). Among the six substitutions, located in the RBD (aa 357-528) while four substitutions in RBM at the CTD of the SI domain. To the note, no amino acid change is seen in the RBM, which binds directly to the Angiotensin-converting enzyme-2 (ACE2) receptor in SARS-CoV (16, 46). At present, the main emphasis is to know about how many differences would be required to change the host tropism. Sequence comparison revealed 17 non-synonymous changes in the early sequence of SARS-CoV-2 than the later isolates of SARS-CoV. The changes were found scattered over the genome of the virus with nine substitutions in the open reading frame (ORF) lab, ORF8 (4 substitutions), spike gene (3 substitutions), and ORF7a (single substitution). Notably, the same non-synonymous changes were found in a familial cluster, indicating that the viral evolution might have happened during person-to-person transmission. Such adaptive evolutions are frequent and constitute a constantly ongoing process once the virus spreads among new hosts. Even though no functional changes occur in the virus associated with this adaptive evolution, close monitoring of the viral mutations that occurs during subsequent human-to-human transmission is warranted.

M protein

The M protein is the most abundant viral protein present in the virion particle, gives a definite shape to the viral envelope. It binds to nucleocapsid and acts as a central organizer of the coronavirus assembly. Coronaviruses M proteins are highly diverse concerning amino acid contents but maintain overall structural similarity within different general. The M protein has three transmembrane domains, flanked by short amino- terminal outside the virion, and a long carboxy-terminal inside the virion. Overall, the viral scaffold is maintained by M-M interaction. To the note, the M protein of SARS-CoV-2 does not have an amino acid substitution in comparison to the SARS-CoV.

E protein

The coronaviruses E protein is the most enigmatic and smallest among the major structural proteins . It plays a multifunctional role in the pathogenesis, assembly, and release of the virus . It is a small integral membrane polypeptide that acts as viroporin (ion-channel) . Inactivation or absence of this protein is related to altered virulence of coronaviruses due to changes in morphology and tropism . The E protein consists of three domains, namely short hydrophilic amino-terminal, a large hydrophobic transmembrane domain, and an excellent C terminal domain. The SARS-CoV-2 E protein reveals a similar amino acid constitution without any substitution .

N protein

The N protein of coronavirus is multipurpose. Among several functions, it plays a role in complex formation with viral genome, facilitates M protein interaction needed during virion assembly, and enhances transcription efficiency of the virus (55, 56). It contains three highly conserved and distinct domains, namely an N-terminal domain (NTD), RNA-binding domain or a linker region (LKR), and a C-terminal domain (CTD) . The NTD binds with the 3' end of the viral genome, perhaps via electrostatic interactions, and is highly diverged both in length as well as sequence . The charged LKR is serine and arginine-rich and also known as SR (Serine and Arginine) domain . The LKR region is capable of direct interaction with in vitro RNA interaction and is also responsible for cell signaling. It also modulates the antiviral response of the host by working as an antagonist for interferon and RNA interference. In comparison to SARS-CoV, the N protein of SARS-CoV-2 possess five amino acid mutations, where the two are in the intrinsically dispersed region (IDR, 25 & 26 positions), one each in the NTD (103 position), LKR (217 position) and CTD (334 position). NSPs and accessory proteins

Besides the important structural proteins, SARS-CoV-2genome contains 15 nsps, nspl-nsplO and nspl2-16 and 8 accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b, and orf 14) . All these proteins play a specific role in viral replication. The difference in respect to the accessory proteins with SARS-CoV, SARS-CoV-2 does not contain 8a protein and longer 8b, and shorter 3b proteins . The nsp7, nspl3, envelope, matrix, or accessory proteins p6 and 8b have not been detected with any amino acid substitutions in comparison to coronaviruses.

The virus structure of SARS-CoV-2 is depicted in Fig. 1.

SARS-CoV-2 spike glycoprotein gene analysis i. Sequence percent similarity analysis

We assessed the nucleotide (NT) percent similarity using the MegAlign software program, where the similarity in between the current novel SARS-CoV-2 isolates was found in the range of 99.4 to 100 %. Among the other Serbecovirus CoV sequences, the novel SARS- CoV-2 sequences showed the highest similarity with Bat-SL-CoV with the NT percent identity ranges between 78.2 to 78.8%. Meanwhile, earlier reported SARS-CoVs showed

70.6 to 74.9 % similarity at NT levels with SARS-CoV-2. Further, the NT percent similarity was 55.4%, 45.5% to 47.9%, 46.2% to 46.6%, and 45.0% to 46.3% with the other four subgenera, namely Hibecovirus, Nobecovirus, Merbecovirus, and Embecovirus, respectively. The percent similarity index of current outbreak isolates signposts a close relationship of SARS-CoV-2 isolates to Bat-SL-CoV, indicating a common origin. However, particular pieces of evidence based on further complete genomic analysis of current isolates are necessary to draw any supposition. Though, it was ascertained that the current novel SARS- CoV-2 isolates belong to the subgenus of Sarbecovirus falling inside the diverse range of Betacoronaviruses. Their possible ancestor was hypothesized to be of bat CoV strains wherein bats might have played a crucial part in harboring this class of viruses. ii.Splits-Tree phytogeny analysis

In the unrooted phylogenetic tree of different betacoronaviruses based on the S protein, virus sequences from different subgenera grouped into separate clusters. SARS-CoV- 2 sequences from Wuhan and other countries exhibited a close relationship and appeared in a single cluster (Fig. 2). The CoVs from the subgenus Sarbecovirus appeared jointly in the splits-tree and divided into three sub-clusters, namely SARS-CoV-2, Bat-SARS-like-CoV (Bat-SL- CoV) and SARS-CoVs (Fig. 2). In the case of other subgenera like Merbecoviruses, all the sequences grouped in a single cluster whereas in Embecovirus different species comprising of canine respiratory CoVs, bovine CoVs, equine CoVs, and human CoV strain (OC43) grouped inside a common cluster. Isolates in the subgenus Nobecovorus and Hibecovirus were found placed separately away from other reported SARS-CoVs but share a common origin from bats.

CLINICAL PATHOLOGY OF SARS-CoV-2 (COVID-19)

Regardless of the coronavirus type, immune cells like mast cells that are present in the submucosa of the respiratory tract and nasal cavity are considered as the primary barrier against this virus (92). Advanced in-depth analysis of the genome has identified 380 amino acid substitutions between the amino acid sequences of SARS-CoV-2 and the SARS/SARS- like coronaviruses. This difference in the amino acid sequence might have contributed to the difference in the pathogenic divergence of SARS-CoV-2.

1 The 2019-n-CoV invades the lung parenchyma resulting in severe interstitial inflammation of the lungs. This will be evident on CT images as ground-glass opacity in the lungs. This lesion, even though initially, involves a single lobe but later expands to multiple lung lobes . The histological examination of lung biopsy samples obtained from COVID-19 infected patient showed diffuse alveolar damage, cellular fibromyxoid exudates, hyaline membrane formation, and desquamation of pneumocytes, indicative of acute respiratory distress syndrome . It has also been found that the SARS-CoV-2 infected patients often have lymphocytopenia along with/ without leukocyte abnormalities. The degree of lymphocytopenia gives an idea about the disease prognosis as it is found positively correlated with the disease severity. Pregnant women are considered to be having a higher risk of getting infected by COVID-19. The coronaviruses can cause adverse outcomes for the fetus, such as intrauterine growth restriction, spontaneous abortion, preterm delivery, and perinatal death.

Nevertheless, the possibility of intrauterine maternal-fetal transmission (vertical transmission) of Co Vs is low, and it is not reported in either SARS or MERS . Researchers have mentioned the probability of in utero transmission of novel SARS- CoV-2 from COVID-19 infected mothers to their neonates in China based upon the rise in IgM, IgG antibody levels and cytokine values in the blood obtained from newly borne infants immediately post-birth; however, reverse transcriptase-polymerase chain reation (RT-PCR) did not confirm the presence of SARS-CoV-2 genetic material in the infants . Recent studies show that at least, in some cases, preterm delivery and its consequences, are associated. Nevertheless, some cases have raised doubts regarding the possibility of vertical transmission

The COVID-19 infection was associated with pneumonia, and some developed acute respiratory distress syndrome (ARDS). The blood biochemistry indexes such as albumin, lactate dehydrogenase, C-reactive protein, lymphocytes (%), and neutrophils (%) gives an idea about the disease severity in COVID-19 infection. During COVID-19, patients may present leukocytosis, leukopenia with lymphopenia , also hypoalbuminemia, an increase of LDH, AST, ALT, bilirubin, and especially D-dime . Middle-aged and elderly patients with primary chronic diseases, especially high blood pressure and diabetes, were found to be more susceptible to respiratory failure and thereby having poorer prognosis. Providing respiratory support at early stages improved the disease prognosis and facilitated recovery . The ARDS in COVID-19 is due to the occurrence of cytokine storms that results in exaggerated immune response, immune regulatory network imbalance, and, finally, can even lead to multiple organ failure . In addition to the exaggerated inflammatory response seen in patients with COVID-19 pneumonia, the bile duct epithelial cell-derived hepatocytes up-regulate the ACE2 expression in liver tissue by compensatory proliferation that might result in hepatic tissue injury.

Therapeutics and drugs

There is no currently licensed specific anti-viral treatment for the MERS and SARS- CoV infections, and the primary measure in the clinical management is focused on alleviating clinical symptoms and supportive cares. The first therapeutic drugs that might be effective in managing COVID-19 include remdesivir, lopinavir/ritonavir alone or in combination with interferon-β, convalescent plasma, and mAbs. Nevertheless, before utilizing these drugs for COVID-19 pneumonia patients, efficacy and safety studies should be conducted by further clinical trials. Although a controlled trial of ritonavir-boosted lopinavir and interferon-alpha 2b therapy has been registered for hospitalized patients with COVID-19 (ChiCTR2000029308). Besides, the use of hydroxychloroquine and tocilizumab and their potential role in modulating inflammatory response in the lungs.

Furthermore, RNA synthesis inhibitors (like 3TC, TDF), remdesivir, neuraminidase inhibitors, peptide (EK1), anti-inflammatory drugs, abidol, Chinese traditional medicine, such as Lianhuaqingwen and ShuFengJieDu Capsules, could be the promising drug treatment for COVID-19. However, further clinical trials are required for confirming their safety and efficacy in managing the COVID-19 infection.

However, therapeutic agents that are having anti-SARS-CoV-2 activity can be broadly classified into three categories; drugs that block the entry of the virus into the host cell, drugs that block viral replication as well as its survival. within the host cell, and drugs that attenuate the exaggerated host immune response. Inflammatory cytokine storm is commonly seen in critically ill COVID-19 patients. Hence, they may be benefited from the use of timely anti- inflammation treatment. Anti-inflammatory therapy using drug-like glucocorticoids, cytokine inhibitors.

Another option is to repurpose broadly acting anti-viral drugs that have already been used for other viral infections. Such drugs have the advantage of easy availability, known pharmacokinetic and pharmacodynamic properties, solubility, stability, side effects, and also well-established dosing regimens. The same two protease inhibitors lopinavir and ritonavir, when combined with another drug ribavirin, were found to be associated with favorable clinical response in SARS patients indicating therapeutic efficacy. However, in the present scenario, due to the lack of specific therapeutic agents against SARS-CoV-2, the hospitalized patients confirmed for the disease will receive supportive care like oxygen therapy and fluid therapy along with the antibiotic therapy for managing secondary bacterial infections.

Antiviral drugs

Several classes of routinely used antiviral drugs like oseltamivir (neuraminidase inhibitors), acyclovir, ganciclovir, and ribavirin does not have any effect on COVID-19 and hence not recommended. Oral administration of neuraminidase inhibitors such as oseltamivir has been widely used as an experimental drug for COVID-19 suspected cases in the hospitals of China even though there is no evidence of its efficacy. Recently, the in vitro antiviral efficacy of FAD-approved drugs such as ribavirin, penciclovir, nitazoxanide, nafamostat, and chloroquine were compared with that of the two broad-spectrum antiviral drugs remdesivir and favipiravir against the SARS-CoV-2. Among the evaluated drugs, both remdesivir and chloroquine were found to be highly effective in controlling COVID-19 infection in vitro.

The molecular docking study conducted in the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 using different commercially available anti-polymerase drugs identified that the drugs such as Ribavirin, Remdesivir, Galidesivir, Tenofovir, and Sofosbuvir were found to bind RdRp tightly indicating a great potential to be used as a therapeutic agent against COVID-19.

Chloroquine is an anti-malarial drug known to possess antiviral activity due to its ability to block virus-cell fusion by raising the endosomal pH necessary for fusion. It also interferes with the virus-receptor binding by interfering with the terminal glycosylation of SARS-CoV cellular receptors, angiotensin-converting enzyme 2 (ACE2). In a recent multicentre clinical trial that was conducted in China, chloroquine phosphate was found to exhibit both efficacy and safety in the therapeutic management of SARS-CoV-2 associated pneumonia. This drug is already included in the treatment guidelines issued by the National Health Commission of the People's Republic of China. The preliminary clinical trials using hydroxychloroquine, another aminoquinoline drug, gave promising results. The COVID-19 patients received 600 mg of hydroxychloroquine daily along with azithromycin as a single-arm protocol. This protocol was found to be associated with a significant reduction in the viral load. Finally, it resulted in a complete cure — however, the study comprised a small population and hence the possibility of a misinterpretation. However, in another case study, the authors had raised concerns over the efficacy of hydroxychloroquine-azithromycin in the treatment of COVID- 19 patients since no observable effect was seen when they were used. In some cases, the treatment was discontinued due to the prolongation of the QT interval.

Recently, another FDA approved drug ivermectin was found to inhibit the in vitro replication of SARS-CoV-2. The findings from this study indicate that a single treatment of this drug was able to induce a ~5000-fold reduction in the viral RNA at 48h in cell culture.. In the coming days, further, in vivo studies will give an insight into the clinical utility of this wonder drug.

Sequence analysis, modeling, and docking are used to build a model for Wuhan COVID-19 RNA dependent RNA polymerase . Additionally, the newly emerged Wuhan HCoVRdRp model is targeted by anti-polymerase drugs, including the approved drugs Sofosbuvir and Ribavirin (Elfiky, A 2020). Also , The 3C-like cleavage sites on the corona viral polyproteins are highly conserved.

Based on the hypothesis that coronavirus and influenza viruses are both RNA virus which depend on mRNA-dependent RNA polymerase (RdRp) to replicate ; favipiravir, ( an antiviral drug targeting RdRP,7 approved in Japan for influenza ) was assessed for the clinical efficacy and safety as treatment for COVID-19 .

Based on the near-identical substrate specificities and high sequence identities, we are of the opinion that some of the previous progress of specific inhibitors development for the SARS- CoV enzyme can be conferred on its SARS CoV-2 counterpart. With the 3CL molecular model, we performed virtual screening for purchasable drugs and proposed 16 candidates for consideration. Among these, the antivirals ledipasvir or velpatasvir are particularly attractive as therapeutics to combat the new coronavirus with minimal side effects, commonly fatigue and headache .

The chymotrypsin-like protease of SARS-CoV-2 shares structure similarity with HCV and HIV proteases. The therapeutic effects of danoprevir, boosted by ritonavir, on treatment na'ive and experienced COVID-19 patients were evaluated . The data from this small-sample clinical Chinese study showed that danoprevir boosted by ritonavir was safe and well tolerated in all patients. After 4 to 12-day treatment of danoprevir boosted by ritonavir, all eleven patients enrolled, two na^'ive and nine experienced, were discharged from the hospital as they met all four criteria as follows: normal body temperature for at least 3 days; significantly improved respiratory symptoms; lung imaging shows obvious absorption and recovery of acute exudative lesion; and two consecutive RT-PCR negative tests of SARS- CoV-2 nucleotide acid (respiratory track sampling with interval at least one day) . Background Art

HARVONI^® (ledipasvir and sofosbuvir) tablets, for oral use HARVONI Initial U.S. Approval: 2014

WARNING: RISK OF HEPATITIS B VIRUS REACTIVATION IN PATIENTS

COINFECTED WITH HCV AND HBV

See full prescribing information for complete boxed -warning.

INDICATIONS AND USAGE

HARVONI is a fixed-dose combination of ledipasvir, a hepatitis C virus (HCV) NS5A inhibitor, and sofosbuvir, an HCV nucleotide analog NS5B polymerase inhibitor, and is indicated for the treatment of chronic hepatitis C virus (HCV) in adults and pediatric patients 3 years of age and older.

DOSAGE AND ADMINISTRATION

. Recommended dosage in adults: One tablet (90 mg of ledipasvir and 400 mg of sofosbuvir) taken orally once daily with or without food. (2.3)

DOSAGE FORMS AND STRENGTHS

• Tablets: 90 mg of ledipasvir and 400 mg of sofosbuvir;

CONTRAINDICATIONS

If used in combination with ribavirin, all contraindications to ribavirin also apply to HARVONI combination therapy.

WARNINGS AND PRECAUTIONS

Bradycardia with amiodarone coadministration: Serious symptomatic bradycardia may occur in patients taking amiodarone, particularly in patients also receiving beta blockers, or those with underlying cardiac comorbidities and/or advanced liver disease. Coadministration of amiodarone with HARVONI is not recommended.

. ADVERSE REACTIONS

_• The most common adverse reactions observed with treatment with HARVONI were fatigue, headache, and asthenia.

• Clearance of HCV infection with direct acting antivirals may lead to changes in hepatic function, which may impact safe and effective use of concomitant medications. Frequent monitoring of relevant laboratory parameters (INR or blood glucose) and dose a4justments of certain concomitant medications may be necessary.

Dosing for Pediatric Patients 3 Years and Older Using HARVONI Tablets or Oral

Pellets

Recommended Dosing for Ribavirin in Combination Therapy with HARVONI for

Pediatric Patients 3 Years and Older

a. The daily dosage of ribavirin is weight-based and is administered orally in two divided doses with food.

Preparation and Administration of Oral Pellets

See the HARVONI oral pellets full Instructions for Use for details on the preparation and administration of HARVONI pellets.

Do not chew HARVONI pellets. If HARVONI pellets are administered with food, sprinkle the pellets on one or more spoonfuls of non-acidic soft food at or below room temperature. Examples of non-acidic foods include pudding, chocolate syrup, mashed potato, and ice cream. Take HARVONI pellets within 30 minutes of gently mixing with food and swallow the entire contents without chewing to avoid a bitter aftertaste.

Renal Impairment

No dosage adjustment of HARVONI is recommended in patients with any degree of renal impairment, including end stage renal disease (ESRD) on dialysis DOSAGE FORMS AND STRENGTHS ADVERSE REACTIONS

Adverse Reactions Reported in ≥5% of Subjects Receiving 8, 12, or

24 Weeks of Treatment with HARVONI 11

Psychiatric disorders: Depression (particularly in subjects with pre-existing history of psychiatric illness) occurred in subjects receiving sofosbuvir containing regimens. Suicidal ideation and suicide have occurred in less than 1% of subjects treated with sofosbuvir in combination with ribavirin or pegylated interferon/ribavirin in other clinical trials.

Laboratory Abnormalities

Bilirubin Elevations: Bilirubin elevations of greater than 1.5xULN were observed in 3%, less than 1%, and 2% of subjects treated with HARVONI for 8, 12, and 24 weeks, respectively.

Lipase Elevations: Transient, asymptomatic lipase elevations of greater than 3xULN were observed in less than 1%, 2%, and 3% of subjects treated with HARVONI for 8, 12, and 24 weeks, respectively.

Postmarketing Experience

Skin rashes, sometimes with blisters or angioedema-like swelling

DRUG INTERACTIONS

Potential for Drug Interaction

After oral administration of HARVONI, sofosbuvir is rapidly absorbed and subject to extensive first-pass hepatic extraction. In clinical pharmacology studies, both sofosbuvir and the inactive metabolite GS-331007 were monitored for purposes of pharmacokinetic analyses.

Ledipasvir is an inhibitor of the drug transporters P-gp and breast cancer resistance protein (BCRP) and may increase intestinal absorption of coadministered substrates for these transporters.

Ledipasvir and sofosbuvir are substrates of drug transporters P-gp and BCRP while GS-331007 is not. P-gp inducers (e.g., rifampin, St. John’s wort) may decrease ledipasvir and sofosbuvir plasma concentrations, leading to reduced therapeutic effect of HARVONI, and the use with P-gp inducers is not recommended with HARVONI.

Established and Potentially Significant Drug Interactions

Clearance of HCV infection with direct acting antivirals may lead to changes in hepatic function, which may impact the safe and effective use of concomitant medications. For example, altered blood glucose control resulting in serious symptomatic hypoglycemia has been reported in diabetic patients. Management of hypoglycemia in these cases required either discontinuation or dose modification of concomitant medications used for diabetes treatment.

Dose adjustments of concomitant medications may be necessary.

Potentially Significant Drug Interactions: Alteration in Dose or Regimen May Be Recommended Based on Drug Interaction Studies or Predicted Interaction USE IN SPECIFIC POPULATIONS

Pregnancy Risk Summary

If HARVONI is administered with ribavirin, the combination regimen is contraindicated in pregnant women and in men whose female partners are pregnant. Refer to the ribavirin prescribing information for more information on ribavirin-associated risks of use during pregnancy .No adequate human data are available to establish whether or not HARVONI poses a risk to pregnancy outcomes.

Lactation

It is not known whether ledipasvir or sofosbuvir, the components of HARVONI, or their metabolites are present in human breast milk, affect human milk production or have effects on the breastfed infant.

DISCLOSURE OF INTERVENTION

Aim of the work

To evaluate the therapeutic efficacy of Sofosbuvir plus ledipasvir , in comparison to the MOHP management guidelines with Oseltamivir (Taminil) combined with Hydroxychloroquine (Plaquinil) plus Azithromycin , on treatment of naïve COVID-19 Egyptian patients.

Objectives:

• To evaluate the therapeutic efficacy of Sofosbuvir plus ledipasvir , in comparison to Oseltamivir (Taminil) combined with Hydroxychloroquine (Plaquinil) plus Azithromycin , on treatment of COVID-19 Egyptian patients.

• To assess side effects of treatments .

• To identify 28-day mortality in both groups.

Ethical Considerations

This study will be assessed bylnstitutional Review Board of Armed Forces Collage of Medicine.

Methodology I. Study design

Prospective randomized controlled study II. Study setting and location

The study will be conducted at (Almaza Military Fever Hospital)

IP. Study population

The study will be designed to recruit 120 pneumonia patients with SARS-COV-2 infection confirmed to be positive by RT-PCR; and demonstrated moderate cases criteria (fever, respiratory symptoms and imaging-confirmed pneumonia) IV. Eligibility Criteria

1. Inclusion criteria

• Age more than 18 and less than 75 years old

• Pneumonia patients with SARS-COV-2 infection confirmed to be positive by RT-PCR; and demonstrated moderate cases criteria (fever, respiratory symptoms and imaging-confirmed pneumonia).

• Women and their partners with no planned pregnancy for 6 months after the study and willing to take effective contraceptive measures within 30 days from the first administration of the study drugs.

• Patients agreed to sign an informed consent to participate in the current study and that they would not participate in other clinical trials within 30 days from the last administration of the study drugs.

2. Exclusion criteria

• Severe COVID-19 patients met one of the following conditions: (1) Respiratory rate (RR) ≥ 30 times / min; (2) Sa02 / Sp02 < 93% in resting state; (3) arterial partial pressure of oxygen (Pa02) / concentration of oxygen (Fi02) < 300 mmHg (lmmhg = 0.133 kPa;

• Critical COVID-19 patients met one of the following conditions: (1) respiratory failure and need mechanical ventilation; (2) shock; (3) other organ failure combined with ICU treatment; severe liver disease (such as child Pugh score ≥ C, AST > 5 times upper limit);

• Patients with contraindications specified specified for sofosbuvir/ledipasvir ; the pregnancy test of female subjects during the screening period was positive; the researchers judged that the patient was not suitable to participate in this clinical study (for example, patients with multiple comorbid diseases, etc.).

• Patients with chloroquine contra-indications: QTc> 500 msec , Myasthenia gravis , Porphyria , Retinal pathology , Epilepsy, G6PD deficiency, Allergy to 4-aminoquinolone, Chronic Heart, Kidney or Liver disease, and Arrythmias.

• All patient who will represent a clinical or radiological deterioration with virologically persistence within at least 5 days of the therapeutic evaluation period of the study will beconsidered as a clinical therapeutic failure of the treatment and will be shifted to the other management protocol.

V. Study Procedures

1. Randomization (in RCT only)

• Once enrolled in the study, patients will be randomly assigned into two groups:

Group I : patients will receive they will receive sofosbuvir plus ledipasvir (n=60). Group II : patient will receive Oseltamivir (Taminil) Plus, Hydroxychloroquine (Plaquinil) plus Azithromycin.

• Randomization will be applied through computer-generated number and concealed using sequentially numbered, sealed opaque envelope.

2. Study procedure

One hundred and twenty patients will be randomized to be assigning equally to either group of the following:

Group I: total number of subjects will be 60 patients.

All patients (60 patients), they will receive sofosbuvir plus ledipasvir (FDA approved Anti-HCY drug since 2014 , with Reference ID: 4081324 ) “ Geneduovir “ , once daily for 14 to 21days as minimum and maximum duration of therapy, respectively . Patients will be evaluated as scheduled on day 0,5,10 & 15 clinically lab. Investigations & C.T chest , also , RT-PCR will be done every 3 days .

Medication will be stopped any time if there is any clinical, radiological or laboratory deterioration.

Group 11: total number of subjects will be 60 naive patients.

All patients (60 patients), will receive ( standard of care treatment according to MOHP management guide) ; Oseltamivir ( Taminil) 150 mg q 12 hours for 10 days Plus, Hydroxychloroquine (Plaquinil) 400 q 12 hours for one day followed by 200mg q 12 hours for 9 days plus azithromycin 500mg once daily followed by 250mg once daily for 10 days.

3. Measurement tools

Basic investigations will be done for all patients on day 0 , will be repeated on days 5 ,10 & 15 or earlier regarding physician order , including :

- CBC , NLR “ neutrophil lymphocyte ratio “.

- AST ,ALT .

- S. Creatinine .

- ESR .

- Fbs .

- ECG.

- C.T chest .

Other investigations will be asked and repeated when needed including :

- LDH .

- TG .

- D. dimer .

- S.Ferritin .

- Abdominal U.S .

RT-PCR will be done on day 0 , and repeated every 3 days .

- Blood samples will be collected during the treatment period at 5:00 am.

VI. Study outcomes 1. Primary outcome

• The 14-21 days therapeutic activity • 28 days in hospital mortality 2. Secondary outcome(s)

• Incidence of clinical failure of treatment.

• Hospital length of stay.

• Time and rate of two consecutive RT-PCR negative tests of SARS-CoV-2 nucleotide acid (respiratory track sampling and sampling interval at least two day apart )

• Lung imaging showing obvious absorption and recovery of acute exudative lesion.

• Significantly improved respiratory symptoms.

• Normal body temperature for at least 3 days.

• Incidence of side effects.

Statistical Analysis

I. Statistical analysis

Data will be summarized and analyzed using SPSS version 17; Data distribution will be assessed for normality using the Kolmogorov-Smimov test. Normally distributed Numerical data will be presented as mean ± SD. Nonparametric data will be presented as median and range. Comparison of the means of the study groups will be done using repeated measure analysis of variance (ANOVA) or Kruskalwallis test according to type of data. Categorical data will be presented as frequency and compared using chi square (X²) test or Fisher’s exact test. Whereas the Gehan-Breslow-Wilcoxon test was used for survival proportion. In all statistical tests, the level of significance will be fixed at the 5% level. A probability (p)-value > 0.05 indicates no significant difference. A p-value < 0.05 indicates significant difference. The smaller the p-value obtained, the more significant will be the difference.

RESULTS

Claims

CLAIMS USE OF sofobovir + ledipasvir COMPOUNDS FOR TREATMENT OF covid 19 CONDITIONS The EPO does not accept responsibility for the accuracy of data originating from authorities other than the EPO, nor does it guarantee that such data is complete, up-to-date or fit for specific purposes.

1. A method for treating covid 19 patients in need of treatment, comprising: administering to said subject an effective amount of sofobovir + ledipasvir.

2. The method of claim 1, wherein said effective amount comprises a fixed dose combination of 400 mg sofobovir+ 90 mg ledipasvir.

3. The method of claim 1, wherein said effective amount comprises a fixed dose combination of 400 mg sofobovir+ 90 mg ledipasvir once daily.

4. The method of claim 1, wherein said administration significantly reduces the complication and manifestation of covid 19 in said subject.

5. The method of claim 1, wherein said administration is oral.

6. The method of claim 1, wherein said administration is once daily.

7. The method of claim 1, wherein said c-reactive protein, procalcitonin, ferritin, D-dimer, total and subpopulations of lymphocytes, IL-6, land other indicators of inflammation and immune status, which can help evaluate clinical progress, alert severe and critical tendencies, and provide a basis for the formulation of treatment strategies.

8. The method of claim 1, wherein said D-dimer levels are significantly elevated in severe cases, which is a potential risk factor for poor prognosis .

9. The method of claim 1, wherein said COVID-19 at the early stage often presents with multifocal patchy shadows or ground glass opacities located in the lung periphery, subpleural area, and both lower lobes on chest CT scans. The long axis of the lesion is mostly parallel to the pleura. Interlobular septa I thickening and intralobular interstitial thickening.

10. The method of claim 1, wherein said performing real-time fluorescence (RT-PCR) to detect the positive nucleic acid of SARS-CoV-2 in sputum, throat swabs, and secretions

11. The method of claim 1, wherein said Cold- or flu-like symptoms usually set in from 2-4 days after a coronavirus infection and are typically mild. However, symptoms vary from person-to-person, and some forms of the virus can be fatal. Symptoms include:

1. Sneezing

2. Runny nose

3. Cough

4. Watery diarrhea

5. Fever in rare cases

6. Sore Throat

7. Exacerbated asthma

12. The method of claim 1, wherein said Sofosbuvir, and ledipasvir can be used against the new strain of coronavirus that emerged with promising results.

13 The method of claim 14, wherein said Severe illness ( Hypoxemia, >50% lung involvement on imaging within 24 to 48 hours) in 14%

14. The method of claim 16, wherein said sofobovir is antiviral used to treat hepatitis C, in vivo activity against SARS-COV-1,

15. The method of claim 1, wherein said said Sofosbuvir, and ledipasvir are very effective owing to their dual inhibitory actions on two viral enzymes.