Patent Application
20230277524
New infectious diseases are constantly emerging, including those caused by viruses. For example, three new coronaviruses have emerged from animal reservoirs over the past two decades to cause serious and widespread illness and death. Currently, the world is experiencing an unprecedented pandemic. Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the United States, more than 1.5 million people have been diagnosed with COVID-19, and over 90,000 cases have been fatal. Accordingly, there is an urgent need for antiviral therapies, such as COVID-19 therapies, particularly those involving drugs which already have been shown to be safe in humans and therefore can be quickly approved.
Rapid development of interventions that may provide clinical efficacy against emerging viral pandemics is relevant, and in the case of SARS-CoV2 critical. To this end, multiple avenues of research have been explored, including extensive machine-based bioinformatics-driven approaches, high-throughput drug screening, re-purposing of existing drugs, and accelerated development of vaccines. While increasingly powered by the rapid growth of data, bioinformatics-based approaches need to be complemented by empirical testing, as hits emerge through prediction-based scoring. High-throughput drug screening is a powerful method to identify novel drugs, especially when a target is identified, but its utility is less effective when the goal is interference with the lifecycle of a virus, such as SARS-CoV2. Singular drugs that have proven highly effective against other viruses are rare. Progress in vaccine development is rapid, and record-breaking, yet it remains unknown if vaccination against SARS-CoV2 may provide lasting immunity. Accordingly, improved anti-viral combinatorial therapies against SARS-CoV2 are needed.
Provided herein are methods of treating a subject infected with a virus, e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or related viruses (e.g., a virus of the family coronaviridae, arenaviridae, and/or filoviridae) by administering an effective amount of a particular combination of compounds which synergistically inhibits the virus and thus treat the infection.
In one embodiment, the combination comprises remdesivir, or analog thereof, and at least one ABCB1 channel transport inhibitor, preferably a dual-specificity ABCB1/ABCG2 inhibitor (Glycoprotein-1). In a particular embodiment, the ABCB1/ABCG2 inhibitor is elacridar, tariquidar, zosuquidar, or analogs thereof. In another embodiment, the combination further comprises an antioxidant. In a particular embodiment, the antioxidant is a Nuclear factor erythroid 2-related factor 2 (NRF2) agonist. In another particular embodiment, the NRF2 agonist is curcumin (also known as diferuloylmethane), or analog thereof,
Accordingly, as described herein, the invention relates to a particular combination of agents (also referred to herein as a “REC combination” or “REC compound”) which synergistically exhibits significantly improved potent anti-viral activity compared to previously known antiviral compounds. As a result, the combinations or compositions of the invention can be administered at significantly lower dosages than previous anti-viral compounds, including oral dosages. In a particular embodiment, the combination comprises remdesivir and elacridar, optionally in combination with curcumin, as well as analogues and functional equivalent variants thereof. In another particular embodiment, the combination comprises remdesivir and tariquidar, optionally in combination with curcumin, as well as analogues and functional equivalent variants thereof. Such combinations can be administered simultaneously (e.g., in a single formulation or concurrently as separate formulations). Alternatively, in another embodiment, the combinations are administered sequentially (e.g., as separate formulations).
Also provided are kits that include the combinations or compositions of the invention in a therapeutically effective amount adapted for use in the methods described herein.
The present disclosure relates to methods and compositions for treating viral infections (e.g., infections by a virus of the family coronaviridae, arenaviridae, and/or filoviridae) in a subject (e.g., a subject diagnosed with a viral infection or a subject at risk of infection by the virus). The disclosure is based, at least in part, upon the discovery that potent antiviral activity (e.g., viral inhibition) is achieved by administering an effective amount of a particular combination of compounds which synergistically inhibits the virus.
In one embodiment, the combination comprises (a) a nucleoside analogue acting as an inhibitor of the virally-encoded polymerase, e.g., remdesivir, an adenosine nucleoside triphosphate analog, (b) a drug efflux inhibitor (e.g., which prevents cytoplasmatic clearance of the nucleoside analogue by way of the ABC-family drug efflux transport system, e.g., a dual-specificity ABCB1/ABCG2 inhibitor, such as elacridar, tariquidar, zosuquidar (as well as other functionally equivalent ABCB1/ABCG2 dual inhibitors); and (c) an effective inducer of Heme Oxygenase 1 (the inducible form of heme oxygenases), such as an NRF2 agonist, e.g., curcumin, to lower the viral replication in the cell by means of removal of cytosolic heme. As demonstrated herein, administration of a combination of compounds representing the above-described three axes (i.e., hereinafter referred to as a “REC combination” or “REC compound”) achieves potent antiviral therapy against RNA-type viral pathogens.
Accordingly, the present disclosure provides antiviral therapies using the combined effects of agents which inhibit one or more of the above-described three aspects of viral replication. The present disclosure also provides beneficial routes of administration (e.g., oral and intravenous, and subcutaneous), as well as preferred administration regimens (e.g., administered simultaneously or sequentially). These and other embodiments are described below.
In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
When trade names are used herein, such trade names independently include the trade name product and the active pharmaceutical ingredient(s) of the trade name product.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
As used herein, “about” will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, “about” will mean up to plus or minus 10% of the particular value.
As used herein, the term “EC50” refers to the concentration of a compound, or combination of compounds, which induces a response, either in an in vitro or an in vivo assay, which is 50% of the maximal response, i.e., halfway between the maximal response and the baseline.
As used herein, the term “effective amount” of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that inhibits a viral infection, an effective amount of an agent is, for example, an amount sufficient to achieve treatment or delay progression of the infection, as compared to the response obtained without administration of the agent. In some embodiments, a therapeutically effective amount is an amount of an agent to be delivered that is sufficient, when administered to a subject with a viral infection, to treat, improve symptoms of, prevent, and/or delay progression of the infection and/or condition.
As used herein, the terms “inhibits,” “blocks,” or “reduces” are used interchangeably and encompass both partial and complete inhibition/blocking. For example, the inhibition/blocking of a virus reduces or eliminates viral cell growth. As used herein, “inhibition”, “blocking”, or “reduces” are also intended to include any measurable decrease in biological function and/or activity of the virus, for example, when a combination or composition of the present invention is in contact with the virus, as compared to the virus not in contact with a combination or composition of the present invention. In some embodiments, a combination or composition inhibits or reduces viral growth and/or activity in a given system by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In other embodiments, a combination or composition inhibits or reduces viral growth and/or activity in a given system by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.
As used herein, the term “inhibits growth” (e.g., referring to cells) is intended to include any measurable decrease in the growth of a cell, e.g., the inhibition of growth of a viral cell by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
As used herein, a subject “in need of prevention,” “in need of treatment,” or “in need thereof,” refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as treatment with a combination or composition of the invention).
The term “in vivo” refers to processes that occur in a living organism.
As used herein, the term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
As generally used herein, “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
As used herein, a “pharmaceutically acceptable carrier” refers to, and includes, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The compositions can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see, e.g., Berge et al. (1977) J Pharm Sci 66:1-19).
As used herein, the term “preventing” when used in relation to a condition, refers to administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.
As used herein, the term “subject” includes any human or non-human animal. For example, the methods and combinations and compositions of the present invention can be used to treat a subject with a viral infection. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
As used herein, the terms “synergy” and “synergistic” refer to the effect achieved when the active ingredients (e.g., compounds) used together is greater than the sum of the effects that results from using the compounds separately. In some aspects, synergy results when a treatment outcome of the active ingredients used together is enhanced, augmented or improved over a treatment outcome of either compound individually. For example, in some aspects, synergy results in vivo when the effect of the active ingredients administered together (e.g., to a subject in need thereof as disclosed herein) provides an enhanced, augmented or improved treatment outcome in the subject, as compared to a treatment outcome when either compound is administered individually. For example, in some aspects synergy results in vivo when the effect of the active ingredients administered together inhibits, reduces or delays viral cell growth or extends or prolongs survival of a subject to a greater extent than the effect of either compound individually on viral cell growth or survival. For example, in some aspects, synergy results in vivo when a combined treatment with the active ingredients inhibits, reduces, or delays viral cell growth to a greater extent than the effect of either compound individually on viral cell growth. For example, in some aspects synergy results in vivo when a combined treatment with the active ingredients inhibits, reduces, or delays viral cell growth by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% or more than the effect of either compound individually on viral cell growth. In some aspects, synergy results in vivo when a combined treatment with the active ingredients extends or prolongs survival in a subject to a greater extent than the survival which results from treatment with either compound individually. For example, in some aspects, synergy results in vivo when a combined treatment with the active ingredients extends, or prolongs the survival in a subject by at least 5 days, at least 10 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days or more than the survival which results from treatment with either compound individually.
In some embodiments, a synergistic effect is attained when the active ingredients (e.g., compounds) are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect is attained when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.
As used herein, the terms “therapeutically effective amount” or “therapeutically effective dose,” or similar terms used herein are intended to mean an amount of an agent (e.g., a combination of compounds of composition that inhibit viral infections) that will elicit the desired biological or medical response (e.g., viral cell death).
The terms “treat,” “treating,” and “treatment,” as used herein, refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration to a subject, in need of such treatment, a combination or composition of the present disclosure, for example, a subject diagnosed with a viral infection or a subject who is at risk of a viral infection, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the infection, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
Viruses
Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea. More than 6,000 virus species have been described in detail. When infected, a host cell is forced to rapidly produce thousands of identical copies of the original virus. When not inside an infected cell or in the process of infecting a cell, viruses exist in the form of independent particles, or virions, consisting of: (i) the genetic material, i.e. long molecules of DNA or RNA that encode the structure of the proteins by which the virus acts; (ii) a protein coat, the capsid, which surrounds and protects the genetic material; and in some cases (iii) an outside envelope of lipids. The shapes of these virus particles range from simple helical and icosahedral forms to more complex structures. Most virus species have virions too small to be seen with an optical microscope as they are one hundredth the size of most bacteria.
According to the ICTV classification system (in conjunction with the Baltimore classification system), classification of viruses is based on the mechanism of mRNA production. Viruses must generate mRNAs from their genomes to produce proteins and replicate themselves, but different mechanisms are used to achieve this in each virus family. Viral genomes may be single-stranded (ss) or double-stranded (ds), RNA or DNA, and may or may not use reverse transcriptase (RT). In addition, ssRNA viruses may be either sense (+) or antisense (−). This classification places viruses into the following seven groups:
Coronaviruses are a group of related RNA viruses that cause diseases in mammals and birds. In humans, these viruses cause respiratory tract infections that can range from mild to lethal. Mild illnesses include some cases of the common cold (which is also caused by other viruses, predominantly rhinoviruses), while more lethal varieties can cause SARS, MERS, and COVID-19. Symptoms in other species vary: in chickens, they cause an upper respiratory tract disease, while in cows and pigs they cause diarrhea. There are as yet no vaccines or antiviral drugs to prevent or treat human coronavirus infections.
Coronaviruses constitute the subfamily Orthocoronavirinae, in the family Coronaviridae, order Nidovirales, and realm Riboviria. They are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry. The genome size of coronaviruses ranges from approximately 26 to 32 kilobases, one of the largest among RNA viruses. They have characteristic club-shaped spikes that project from their surface, which in electron micrographs create an image reminiscent of the solar corona, from which their name derives.
The scientific name for coronavirus is Orthocoronavirinae or Coronavirinae. Coronaviruses belong to the family of Coronaviridae, order Nidovirales, and realm Riboviria. They are divided into alphacoronaviruses and betacoronaviruses which infect mammals—and gammacoronaviruses and deltacoronaviruses, which primarily infect birds. A classification of coronavinis is shown in Table 2.
An arenavirus is a bisegmented ambisense RNA virus that is a member of the family Arenaviridae. Arenaviruses have a segmented RNA genome that consists of two single-stranded ambisense RNAs. As with all negative-sense RNA viruses, the genomic RNA alone is not infectious and the viral replication machinery is required to initiate infection within a host cell. Genomic sense RNA packaged into the arenavirus virion is designated negative-sense RNA, and must first be copied into a positive-sense mRNA in order to produce viral protein. The two RNA segments are denoted Small (S) and Large (L), and code for four viral proteins in a unique ambisense coding strategy. Each RNA segment codes for two viral proteins in opposite orientation such that the negative-sense RNA genome serves as the template for transcription of a single mRNA and the positive-sense copy of the RNA genome templates a second mRNA. The separate coding sequences of the two viral proteins are divided by an intergenic region RNA sequence that is predicted to fold into a stable hairpin structure.
These viruses infect rodents and occasionally humans. A class of novel, highly divergent arenaviruses, properly known as reptarenaviruses, have also been discovered which infect snakes to produce inclusion body disease. At least eight arenaviruses are known to cause human disease. The diseases derived from arenaviruses range in severity. Aseptic meningitis, a severe human disease that causes inflammation covering the brain and spinal cord, can arise from the lymphocytic choriomeningitis virus. Hemorrhagic fever syndromes, including Lassa fever, are derived from infections such as Guanarito virus, Junin virus, Lassa virus, Lujo virus,[2] Machupo virus, Sabia virus, or Whitewater Arroyo virus. Because of the epidemiological association with rodents, some arenaviruses and bunyaviruses are designated as roboviruses.
Within the family Arenaviridae, arenaviruses were formerly all placed in the genus Arenavirus, but in 2014 were divided into the genera Mammarenavirus for those with mammalian hosts and Reptarenavirus for those infecting snakes. Reptarenaviruses and mammarenavirus are separated by an impenetrable species barrier. Infected rodents cannot pass disease onto snakes, and IBD in captive snakes is not transmissible to humans. A third genus, Hartmanivirus, has also been established, including other species that infect snakes. The organisation of the genome of this genus is typical of arenaviruses but their glycoproteins resemble those of filoviruses. Species in this genus lack the matrix protein. A fourth genus, Antennavirus has also been established to accommodate two arenaviruses found in striated frogfish (Antennarius striatus).
Mammarenaviruses can be divided into two serogroups, which differ genetically and by geographical distribution: When the virus is classified “Old World” this means it was found in the Eastern Hemisphere in places such as Europe, Asia, and Africa. When it is found in the Western Hemisphere, in places such as Argentina, Bolivia, Venezuela, Brazil, and the United States, it is classified “New World”. Lymphocytic choriomeningitis (LCM) virus is the only arenavirus to exist in both areas but is classified as an Old World virus.
The family Filoviridae, a member of the order Mononegavirales, is the taxonomic home of several related viruses (filoviruses or filovirids) that form filamentous infectious viral particles (virions) and encode their genome in the form of single-stranded negative-sense RNA. Two members of the family that are commonly known are Ebola virus and Marburg virus. Both viruses, and some of their lesser known relatives, cause severe disease in humans and nonhuman primates in the form of viral hemorrhagic fevers. Table 3 provides the genus, species, and virus names for the family Filoviridae.
Viral Polymerase Inhibitors
Viral polymerases play a central role in viral genome replication and transcription. Based on the genome type and the specific needs of particular virus, RNA-dependent RNA polymerase, RNA-dependent DNA polymerase, DNA-dependent RNA polymerase, and DNA-dependent RNA polymerases are found in various viruses. Viral polymerases are generally active as a single protein capable of carrying out multiple functions related to viral genome synthesis. Specifically, viral polymerases use variety of mechanisms to recognize initial binding sites, ensure processive elongation, terminate replication at the end of the genome, and also coordinate the chemical steps of nucleic acid synthesis with other enzymatic activities.
The present invention provides methods for treating a subject infected with a virus by administering a combination of compounds which comprises a viral polymerase inhibitor, as well as compositions, e.g., a pharmaceutical composition, which comprise a viral polymerase inhibitor. Examples of such inhibitors include remdesivir (Veklury®), and other functionally similar nucleoside analogs, i.e., nucleoside analogs capable of interfering with the action of viral RNA-dependent RNA polymerase and evading proofreading by viral exoribonuclease (ExoN), thus causing a decrease in viral RNA production (Li et al., Drug Discov. Ther. (2020); 14(2):73; Ferner and Aronson, BMJ(2020) 369:m1610). For example, remdesivir, a compound of Formula II
diffuses into cells where it is converted to GS-441524 mono-phosphate via the actions of esterases (CES1 and CTSA) and a phosphoamidase (HINT1); this in turn is further phosphorylated to its active metabolite triphosphate by nucleoside-phosphate kinases. Further information regarding remdesivir and its functionally similar analogs is known in the art, e.g., US 20190255085, U.S. Pat. Nos. 10,251,904, 9,724,360, US 20160122374, and WO 2017049060, which are incorporated herein by reference.
As demonstrated in the present invention, the specificity of remdesivir in the REC combination is remarkable. Various other ribonucleoside analogues (EIDD1931, EIDD2801, Galidesivir, Favipiravir, Rimonavir) were tested, but none were able to substitute for remdesivir. The functional metabolite of remdesivir, GS441524, also failed to substitute for remdesivir. Accordingly, the cellular uptake of remdesivir is more effective than GS441524 and only the pro-drug is subjected to ABC-family export. Considering that remdesivir is currently administered clinically through i.v. infusion mainly due to first-pass hepatic clearing, the enhanced potency of remdesivir attainable by use of ABC-dual inhibition advantageously makes it capable of being administered orally which, in turn, allows it to attain greater potency, and thus extend the use of the drug.
In addition to the data provided herein regarding the REC combination, additional empirical evidence is shown for multiple drugs currently being explored for efficacy against SARS-CoV2. The initial HD-DoE screen included two focus compounds—Favipiravir, and Hydroxychloroquine. Both drugs failed to emerge as efficacious, individually, and in combination with others, against SARS-CoV2 in the VERO6 EGFP assay. Irbesartan, Camostat, Ritonavir, Lopinavir, Rimantadine, EIDD1931, Mefloquine, Arbidol, are all candidate drugs against SARS-CoV-2 and COVID-19. (McKee et al. (2020); Pharmacol. Res.), however, this study did not provide evidence for efficacy. The antihelminth drug Ivermectin, which is currently undergoing clinical testing in COVID-19 (e.g. NCT04351347, NCT04392713, NCT04360356), displayed individual, but limited potency against SARS-CoV2, but was unable to enhance the REC combination.
Efflux Inhibitors
Most microorganisms have highly conserved DNA sequences in their genome that are transcribed and translated to efflux pumps. Efflux pumps are capable of moving a variety of different toxic compounds out of cells, such as antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals, bacterial metabolites and neurotransmitters via active efflux, which is vital part for xenobiotic metabolism. This active efflux mechanism is responsible for various types of resistance to bacterial pathogens within bacterial species—the most concerning being antibiotic resistance because microorganisms can have adapted efflux pumps to divert toxins out of the cytoplasm and into extracellular media. Multidrug efflux pumps represent one of the major mechanisms of drug resistance in bacteria
Mammalian cells also have highly conserved DNA sequences in their genome that are transcribed and translated into efflux pumps and these proteins are evolutionary descendants of the prokaryotic transporters and are thus ancient proteins. All of the efflux transporters belong to the family of ATP-powered pumps. ATP-binding cassette (ABC) transporters are an example of ATP-dependent pumps. ABC transporters are ubiquitous membrane-bound proteins, present in all prokaryotes, as well as plants, fungi, yeast and animals. These pumps can move substrates in (influx) or out (efflux) of cells. ABC-family transporters are responsible for molecular efflux of multiple drug classes. An overview of the mammalian ABC-family is provided in Hum Genomics. 2009 April; 3(3):281-90. doi: 10.1186/1479-7364-3-3-281. The human genome contains 49 ABC genes, arranged in eight subfamilies and named via divergent evolution (as described in ncbi.nlm.nih.gov/pmc/articles/instance/3523235/bin/1479-7364-3-3-281-1.jpg which is shown in
The combination therapy of the present invention includes administration of at least inhibitor of Subfamily B of the ABC family (ABCB) of efflux transporters, preferably dual inhibitors of Subfamily B and Subfamily G (i.e., an inhibitor with dual specificity). This subfamily of 11 genes is unique to mammals and includes four full-transporters and seven half-transporters. Several of the B family members are known to confer multidrug resistance in cancer cells. Hence, subfamily B has also been called the “MDR family of ABC transporters.” The MDR name reflects the fact that ABCB-family efflux transporters are involved in export of known pharmaceutical compounds, which to the body is deemed foreign, and thus exported. In certain cases, the export through the ABC-family leads to an effective lowering of the intracellular concentration of the pharmaceutical agent, such as chemotherapeutic substances (nucleoside analogs), which would be exported by the cancer cells, effectively lowering their potency. Similarly, according to the present invention, the ABC-inhibitory drug (e.g., elacridar) increases the potency of remdesivir.
Elacridar is a third-generation P-glycoprotein non-competitor inhibitor (ABCB1) which also inhibits the ABCG2 protein. As demonstrated herein, the specificity of elacridar to prevent remdesivir export was determined through additional testing. Inhibition of ABCG2 using the ABCG2-specific inhibitor Ko143 failed to complement for elacridar (
Accordingly, the present invention provides methods for treating a subject infected with a virus by administering a combination of compounds which comprises remdesivir in combination with one or more efflux inhibitors, preferably a dual-specificity ABCB1/ABCG2 channel transport inhibitor, which reduces or prevents cytoplasmatic clearance of remdesivir by way of the ABC-family drug efflux transport system (i.e., ABCB1/ABCG2). In one embodiment, the efflux inhibitor is elacridar which is a P-Glycoprotein/ABC-family channel transport inhibitor, i.e., a compound of Formula I;
Accordingly, other members of this class of inhibitors also can be used in the invention. This includes, e.g., specific inhibitors against the ABCB1 drug efflux transporter, in particular, inhibitors with dual specificity against ABCB1 and ABCG2, such as tariquidar or zosuquidar, or P-Glycoprotein Inhibitor, C-4 (CTK8G2344), or HM30181AK. The P-glycoprotein family is known as drug-efflux transporters and serve to eliminate foreign drugs in a cell by an energy dependent (ATP-consuming) transport. The class of proteins is encoded by ABC-family genes, and consists of multiple members, displaying distinct specificities against compounds.
Other examples of such inhibitors include the ABCB1 inhibitor class (P-glycoprotein inhibitors), such as tariquidar, zosuquidar, and other functionally equivalent inhibitors as shown in Table 5. Not all of the listed compounds operate at low nM efficiency and many of the listed compounds are substrates for the ABCB1 transporter and operate as independent pharmaceutical agents on their own. Consequently, for many of the drugs capable of interfering with ABCB1 function, such may be undesirable in the REC combination. Accordingly, a preferred inhibitor is an ABCB1/ABCG2 inhibitor having limited (or no) pharmaceutical effects on the organism, and thus displaying a minimal pharmacodynamic profile, yet able to elicit high ABCB1/ABCG2 inhibition at low concentration, thus displaying a low and sustained pharmacokinetic profile attained by low frequency administration (such as daily and oral administration).
Candida species and Aspergillus species in severely immunocompromised patients.
Streptomyces fulvissimus and related to the enniatins. It is composed of 3 moles each of L-
P. malariae, P. ovale, and P. falciparum. It is also used for
Pneumocystis jirovecii pneumonia (PCP) and for the prevention and treatment of
Plasmodium falciparum malaria.
Heme Oxygenase Agonists/NRF2 Agonists
Heme oxygenase-1 (HO-1), a 32 kDa enzyme containing 288 amino acid residues which is encoded by the HMOX1 gene, is a known anti-oxidant factor. HO-1 expression is induced by oxidative stress, and in animal models increasing this expression has been shown to be protective. HO-1 is found throughout the body with highest concentrations in the spleen, liver, and kidneys.
NRF2, a latent protein within each cell in the human body, is capable of turning on the production of antioxidant enzymes such as Catalase, Glutathione and Superoxide Dismutase (SOD). Once released (i.e., activated) NRF2 migrates into the cell nucleus and bonds to the DNA at the location of the Antioxidant Response Element (ARE) or also called hARE (Human Antioxidant Response Element) which is the master regulator of the total antioxidant system that is available in human cells. These antioxidant enzymes are potent neutralizers of free radicals.
Accordingly, in one embodiment, the anti-viral combination therapy of the present disclosure further comprises an HO-1 inducer (i.e., agonist), such as an NRF2 agonist. Examples of such agonists include curcumin, and other functionally similar inducers, which lower the viral replication in the cell by means of removal of cytosolic heme. In particular, curcumin, a compound of Formula III;
is capable of removing free radicals (ROS, reactive oxygen species) in a cell (Tomeh et al., Int. J. Mol. Sci. (2019); 20(5):1033; Xu et al., Nutrients (2018) October; 10(10):1553). Curcumin impacts cellular iron metabolism, and the expression of the anti-inflammatory molecule Heme Oxygenase (HO-1), as described by Hooper (Cell Stress Chaperones. 2020 Jun. 4; 1-4. doi: 10.1007/s12192-020-01126-9). Wagener et al. described the Heme Oxygenase system as a potential target in COVID-19 patients (Antioxidants (Basel). 2020 Jun. 19; 9(6):540. doi: 10.3390/antiox9060540.). The production of HO-1 exerts multiple effects related to anti-apoptosis, anti-inflammatory, and anti-edema effects in tissues.
Curcumin is a pleiotropic molecule, with multiple cytological effects (Aggarwal and Sung, 2009). As an antioxidant, could improve cellular viability during SARS-CoV2 infection by removal of free radicals. However, N-acetyl cysteine (NAC, not shown), as well as Ascorbate (Vit C, Supp.), both failed to complement the REC combination, or substitute for curcumin. Curcumin is also a functional iron-chelator (Jiao et al. (2009). Curcumin, a cancer chemopreventive and chemotherapeutic agent, is a biologically active iron chelator. Blood), and iron metabolism is important for viral replication (Drakesmith and Prentice (2008) Viral infection and iron metabolism. Nat. Rev. Microbiol.). Clinically approved iron chelators (Deferoxamine (not shown), Deferiprone (
Additional examples of NRF2 agonists include, e.g., Bardoxolone Methyl, Dimethyl Fumarate, Omaveloxolone (RTA-408), Oltipraz, Bardoxolone, 4-Hydroxyphenylacetic acid, Sulforaphane, Obacunone, Mangiferin, Tert-butylhydroquinone (TBHQ), 4-Octyl Itaconate, Diethylmaleate, and functional equivalent compounds.
Examples of HO-1 agonists include, e.g. heavy metals, statins, paclitaxel, rapamycin, probucol, nitric oxide, sildenafil, carbon monoxide, carbon monoxide-releasing molecules, and porphyrins. Phytochemical inducers of HO include: curcumin, resveratrol, piceatannol, caffeic acid phenethyl ester, dimethyl fumarate, fumaric acid esters, flavonoids, chalcones, Ginkgo biloba, anthrocyanins, phlorotannins, camosol, rosolic acid, and numerous other natural products. Endogenous inducers include i) lipids such as lipoxin and epoxyeicosatrienoic acid; and ii) peptides such as adrenomedullin and apolipoprotein; and iii) hemin. NRF2 inducers with downstream HO-1 induction include: genistein, 3-hydroxycoumarin, oleanolic acid, isoliquiritigenin, PEITC, diallyl trisulfide, oltipraz, benfotiamine, auranofin, acetaminophen, nimesulide, paraquat, ethoxyquin, diesel exhaust particles, silica, nanotubes, 15-deoxy-Δ12,14 prostaglandin J2, nitro-oleic acid, hydrogen peroxide, and succinylacetone.
In another aspect, the present invention provides a composition or combination, e.g., a pharmaceutical composition or separately administered combination, of active agents, formulated with a pharmaceutically acceptable carrier, e.g., each component of the combination can be formulated separately or together with the carrier.
Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include a combination or composition of the present invention combined with at least one other therapeutic agent, e.g., a corticosteroid, an anti-inflammatory signal transduction modulator, a β2-adrenoreceptor agonist bronchodilator, an anticholinergic, amucolytic agent, hypertonic saline, and other drugs for treating a virus infection. For example, in one embodiment the other active therapeutic agent is active against Corona virus infections. In another embodiment the other active therapeutic agent is active against Arenaviridae virus infections, particularly Lassa virus and Juninvirus infections. Non-limiting examples of these other active therapeutic agents are ribavirin, favipiravir (also known as T-705 or Avigan), T-705 monophosphate, T-705 diphosphate, T-705 triphosphate, ST-193, and mixtures thereof. The combinations and compositions of the present invention are also intended for use with general care provided patients with viral infections, including parenteral fluids (including dextrose saline and Ringer's lactate) and nutrition, antibiotic (including metronidazole and cephalosporin antibiotics, such as ceftriaxone and cefuroxime) and/or antifungal prophylaxis, fever and pain medication, antiemetic (such as metoclopramide) and/or anti-diarrheal agents, vitamin and mineral supplements (including Vitamin K and zinc sulfate), anti-inflammatory agents (such as ibuprofen), pain medications, and medications for other common diseases in the patient population, such anti-malarial agents (including artemether and artesunate-lumefantrine combination therapy), typhoid (including quinolone antibiotics, such as ciprofloxacin, macrolide antibiotics, such as azithromycin, cephalosporin antibiotics, such as ceftriaxone, or aminopenicillins, such as ampicillin), or shigellosis.
As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for oral, inhalation, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the combination or composition, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
The pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
A pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
For clinical administration of the combinations and compositions, the dosage can range from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months. Preferred dosage regimens of the invention include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the combination or composition being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly.
In a particular embodiment, the ABCB1/ABCG2 inhibitor is administered at about 100 mg to 200 mg, remdesivir is administered at about 20 mg to 300 mg (e.g., 50 mg to 300 mg), and the optional NRF2 agonist is administered at about 100 mg to 1,000 mg, e.g., once daily at about 500 to 25 mg/kg.
In another embodiment:
In a particular embodiment, the ABCB1/ABCG2 inhibitor (e.g., elacridar, tariquidar, or zosuquidar) is administered before administration of remdesivir. For example, in one aspect, administration of tariquidar prior to administration of remdesivir results in ABC-inhibition prior to administration of remdesivir, thus maximizing the effective interference of cytoplasmic export of remdesivir (i.e., reducing the loss of remdesivir).
Alternatively, the combination or composition can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the active ingredient. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A “therapeutically effective dosage” of a combination or composition of the invention preferably results in a decrease in severity of the viral infection symptoms, an increase in frequency and duration of infection symptom-free periods, or a prevention of impairment or disability due to the infection. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
A composition of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. A preferred route of administration for combinations or compositions of the invention includes oral administration.
Other routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
Alternatively, a combination or composition of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally (i.e., by inhalation), vaginally, rectally, sublingually or topically. For example, a combination or composition of the present invention can be administered to a subject by way of the lung. Pulmonary drug delivery may be achieved by inhalation, and administration by inhalation herein may be oral and/or nasal. Examples of pharmaceutical devices for pulmonary delivery include metered dose inhalers, dry powder inhalers (DPIs), and nebulizers. For example, a composition described herein can be administered to the lungs of a subject by way of a dry powder inhaler. These inhalers are propellant-free devices that deliver dispersible and stable dry powder formulations to the lungs. Dry powder inhalers are well known in the art of medicine and include, without limitation: the TurboHaler® (AstraZeneca; London, England); the AIR® inhaler (Alkermes®; Cambridge, Mass.); Rotahaler® (GlaxoSmithKline; London, England); and Eclipse™ (Sanofi-Aventis; Paris, France). See also, e.g., PCT Publication Nos. WO 04/026380, WO 04/024156, and WO 01/78693. DPI devices have been used for pulmonary administration of polypeptides such as insulin and growth hormone. A composition described herein can be intrapulmonarily administered by way of a metered dose inhaler. These inhalers rely on a propellant to deliver a discrete dose of a compound to the lungs.
A composition described herein can be administered to the lungs of a subject by way of a nebulizer. Nebulizers use compressed air to deliver a compound as a liquefied aerosol or mist. A nebulizer can be, e.g., a jet nebulizer (e.g., air or liquid-jet nebulizers) or an ultrasonic nebulizer. Additional devices and intrapulmonary administration methods are set forth in, e.g., U.S. Patent Application Publication Nos. 20050271660 and 20090110679.
The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.
In certain embodiments, combinations and compositions of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 57:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 9:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p 120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.
Uses of the Invention
The combinations and compositions of the present invention have multiple anti-viral in vitro and in vivo diagnostic and therapeutic utilities. For example, these molecules can be administered to cells in culture, e.g. in vitro or ex vivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose a viral infection. The term “subject” as used herein in intended to includes human and non-human animals. Non-human animals includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. The methods are particularly suitable for treating human patients having a virus infection. When combinations of the active ingredients are administered, they can be administered in any order or simultaneously.
Also within the scope of the invention are kits comprising the combinations or compositions of the invention and instructions for use. The kit can further contain a least one additional reagent. Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, Genbank sequences, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.
Experimental Design
Rapid development of interventions that may provide clinical efficacy against emerging viral pandemics is relevant, and in the case of SARS-CoV2 critical. To this end, multiple avenues of research have been explored, including extensive machine-based bioinformatics-driven approaches, high-throughput drug screening, re-purposing of existing drugs, and accelerated development of vaccines. While increasingly powered by the rapid growth of data, bioinformatics-based approaches need to be complemented by empirical testing, as hits emerge through prediction-based scoring. High-throughput drug screening is a powerful method to identify novel drugs, especially when a target is identified, but its utility is less effective when the goal is interference with the lifecycle of a virus, such as SARS-CoV2. Singular drugs that have proven highly effective against other viruses are rare. Progress in vaccine development is rapid, and record-breaking, yet it remains unknown if vaccination against SARS-CoV2 may provide lasting immunity.
Accordingly, in this Example, an HD DoE assay to detect the combinatorial effects of pharmacological compounds was performed as described in Esakov et. al, Endocrine Related Cancer, https://pubmed.ncbi.nlm.nih.gov/31167163/. A composite design was generated in which compounds (i.e., “input drugs”) were administered in combinations through an experimental design constructed based on mathematical algorithms. A master plate of stock compounds (obtained from ApexBio®) was created in which drugs were provided a mM-concentration. A dilution, and reconfiguring, process was executed robotically to create a series of secondary master plates, consisting of drugs in which individual wells contained specific combinations of drugs. This secondary master plate was further diluted to final assay concentrations, which was provided to cells, either in absence (for toxicity screening), or presence (for anti-viral effects) of the SARS-CoV-2 virus. The effect of the combinations of the drugs was measured in the SARS-CoV-2 assay, and the data (relating to survival of cells) was treated as separate response variables. The SARS-CoV-2 assay included the following steps: (a) the cells were seeded (e.g., 25.000 cells are seeded in 100 μl of assay medium and incubated overnight to allow the cells to settle on the bottom and adhere); (b) the next day, the cells are treated and infected, e.g., with 50 μl of input compounds; (c) after 2-3 hours, 50 μl of virus is added to infect the cells (MOI of ˜0.004); and (d) assays were read on days 1-4.
The outcome data based on the response variables (measured as integrated fluorescence related to the EGFP-fluorescence) was mathematically fitted against the effector (i.e., the input drugs) using the mathematical fit method of MLR (multiple linear regression fit). The results are shown in
Imaging scans of VERO-EGPF cells across a high-dimensional design of experiments plate/Toxicology testing were obtained (data not shown) to illustrate the underlying process of obtaining the response data. Each cell of an initial plate was seeded with an equal amount of EGFP-fluorescing VERO-EGFP cells. Twelve independent drugs were administered in combination across the entire plate, and the plate was scanned for fluorescence intensity 4 days later. Specifically, high-content imaging of cellular fluorescence was converted into response data and subsequently fitted onto the design of experiments-based administration of the individual drugs. The response data were converted into specific models based on the toxicology data related to a loss of cells (i.e., death of cells) caused by addition of the drug Brefeldin A. The coefficient plots were generated for all responses and qualitatively inspected by the R2 and Q2 parameters.
A coefficient plot of these data (
In the present invention, >250 distinct antiviral drugs were tested, first in groups of 12 to identify plausible drug/drug interactions which favored interference with the viral cytopathic response. In the first screen, 20 combinatorial plates were analyzed. Subsequent screens focusing on the emerging hits were subsequently performed to validate and, if possible, improve upon any identified combinations. The REC combination was determined from one of the initial plates, wherein 12 separate and distinct compounds where tested for synergistic effects against SARS-CoV-2 as follows: Irbesartan, clemizole, clemizoleHCL, curcumin, elacridar, flavopiridol, GS-9620, camostat, letermovir, remdesivir, trigonelline, and favipiravir, and is described in more detail here.
Using the SARS-CoV-2 assay read out, VERO-EGFP cells were seeded and inoculated with virus on Day 1 after seeding. The input compounds were added to the secondary master plate at designated concentrations (i.e., 250 nM and 500 nM). The EGFP fluorescence (an indicator of cell survival) was measured for four days. In parallel, data for cell survival (Tox) of cells without virus, but with input compounds, was similarly obtained.
Based on the data collected at day four, the mathematical fit of both Tox and Antiviral responses were strong. Logarithmic transformation of the antiviral response data was performed to attain the strongest model. The observed R2 value for virus response at 250 nM was 0.93, Q2=0.84, model validity 0.78, and reproducibility 0.94. These values indicated a very strong approximation of the response space, explaining 93% of the observed variance, and returned an exceptionally strong goodness of fit at 0.78. The values for the corresponding Tox and Viral response at 500 nM is shown in
In conclusion, the data provided herein and below show that the HD-DoE method, when segmented, can be rapidly employed to explore the combination space among known drugs for attaining effective control of viral replication in a relevant mammalian cell. The REC combination provides significant clinical intervention in COVID-19 for both recently diagnosed patients as well patients progressing and experiencing high viral burdens.
The mathematical modeling of the response data described in Example 1 was tuned to attain maximal Q2 (predictive power) as shown in
Synergism among three compounds (remdesivir, elacridar, and curcumin) also was demonstrated. Specifically, inclusion of the triple-interaction term of “Curc*Elac*Rem” in the model signified the existence of triple synergism among these compounds and improved the Q2 value. The triple interaction term also was detected within a 95% confidence interval and kept for further analysis of the model.
The corresponding data for toxicology (as shown in
When performing the same analysis above, but increasing the total amount of input drug to 500 nM, the antiviral response changed to reflect that the relevance of curcumin is less important, i.e., strong synergism still exists between remdesivir and elacridar, and the data supports antiviral activity based on the triple interaction term of Curc*Elac*Rem. Therefore, curcumin is still shown to be potently enhancing the other two, yet with less significance than if the drugs were applied at lower concentration. Corresponding toxicology analysis at the higher concentration of 500 nM (as shown in
Based on the mathematical model of the response space, it was possible to perform in-silico analysis of the factor effects (attributed to the triple combination therapy of remdesivir, elacridar, and curcumin), and their relative criticality. This process is entirely data driven and based on statistical analysis. Creating the conditions, in which all irrelevant drugs in the screen are set to zero, the system response is shown in
In
In
At higher concentrations of elacridar and remdesivir, however, curcumin was not needed to inhibit the viral effect. In
In
Altogether, the data show strong triple synergism and independent criticality of each of the compounds in this combination at the specified concentrations. In
Three-dimensional plots were created which visualize the synergistic effects of the compounds according to the two-way interactions (data not shown). These three-dimensional plots were created with all other drugs set to zero and the components in the combination not plotted set at maximal. For example, one plot showed the response to elacridar and curcumin, with 250 nM of remdesivir added and Vero-EGFP cells/4 days exposure to SARS-CoV-2 virus. This plot exhibited a sloped response plane which correlates to synergism. Maximum was attainable at max concentration of either compound. A second plot showed the response to elacridar and remdesivir, with 250 nM of curcumin added and Vero-EGFP cells/4 days exposure to SARS-CoV-2 virus. This plot also exhibited a sloped response plane which correlates to synergism. Maximum was attainable at max concentration of either compound. A third plot showed the response to curcumin and remdesivir, with 250 nM of elacridar added and Vero-EGFP cells/4 days exposure to SARS-CoV-2 virus. Like the other two plots, this plot exhibited a sloped response plane is shown which correlates to synergism. Maximum was attainable at max concentration of either compound.
Accordingly, as discussed above, the particular combination of the ribonucleoside analogue remdesivir, the ABCB1/ABCG2 dual inhibitor elacridar, and optionally curcumin, was highly effective in blocking the viral lethality of the SARS-CoV2 host cell. As shown, the potency of the combination was remarkable and able to completely abolish viral cytopathy below 70 nM of administration of the compounds. Moreover, as further discussed below, more specific inhibitors of either ABCB1 or ABCG2 alone were unable to substitute for elacridar in the combination. However, the known dual-specificity inhibitor tariquidar was able to substitute for elacridar, indicating that remdesivir is exported by both ABCB1 and ABCG2, and therefore that effective interference with cytoplasmic export requires a dual-specificity inhibitor of the elacridar/tariquidar type.
Effective interference of SARS-CoV2 cytopathy was also achieved using remdesivir and elacridar alone. A gradual reduction in the relevance of curcumin at higher concentrations of remdesivir/elacridar was observed. However, at lower concentrations, the importance of curcumin was noted.
The specificity of the identified combination of remdesivir, elacridar, and curcumin (R+E+C) is further supported by the observed lack of interaction the combination exhibits with another compound, favipiravir, which has been reported to have efficacy against SARS-CoV2. As shown in a 4-dimensional plot (data not shown), favipiravir had no effect on the efficacy of the remdesivir, elacridar, and curcumin combination. Specifically, the four-dimensional plot included remdesivir, elacridar, curcumin and farvipiravir in Vero-EGFP cells/4 days exposure to SARS-CoV-2 virus. This plot showed the inhibitory effect of the triple combination effect and that the farvipiravir compound did not impact the effect of the R+E+C combination.
Focus testing of the REC combination was performed using known ABC-family inhibitors. As shown in
The specificity of elacridar to remdesivir export was determined through additional testing. Inhibition of ABCG2 using the ABCG2-specific inhibitor Ko143 failed to complement for elacridar (
Quercetin is a known ABCB1 inhibitor but operates at a higher concentration than elacridar/tariquidar/zosuquidar. It failed to complement for elacridar (
Focus testing of the REC combination was performed to identify substitutes for remdesivir. Remdesivir is cleaved inside cells to an active metabolite, referred to as GS441524. Therefore, GS441524 was tested as a possible substitute for remdesivir. As shown, while GS441524 exerted a smaller, yet significant, antiviral effect, it did not replace remdesivir in the REC combination (
The specificity of remdesivir in the REC combination was shown to be remarkable. Various other ribonucleoside analogues (EIDD1931, EIDD2801, galidesivir, favipiravir, rimonavir) were tested, but none were able to substitute for remdesivir. The functional metabolite of remdesivir, GS441524, also failed to substitute for remdesivir. Therefore, cellular uptake of remdesivir was more effective than GS441524, and only the pro-drug was subjected to ABC-family export. Considering that remdesivir is currently administered clinically through intravenous (i.v.) infusion (mainly due to first-pass hepatic clearing) the enhanced potency of remdesivir attained by use of ABC-dual inhibition offers an opportunity for oral administration of the drug, as well as greater potency.
In our high-dimensional testing of compounds against SARS-CoV2 we explored approximately 250 unique, and known, anti-viral type drugs. In the initial screen, which encompassed 20 HD-DoE experiments, several emerging hits were identified. These hits were distributed across the library and, therefore, had not been evaluated against the REC combination. Accordingly, these hits were tested to identify additional compounds that could enhance the REC combination through possible quadruple synergism. None were found. The best emerging hits included JQ-1 (a BRD2/4 bromodomain inhibitor), ponatinib (a tyrosine kinase inhibitor), Z-VAD (an apoptosis inhibitor, targeting the Caspase apoptotic protein class), Q-VD-Oph (also an apoptosis inhibitor, targeting the Caspase apoptotic protein class). As shown in
As demonstrated, the specificity of remdesivir in the REC combination is remarkable. Various other ribonucleoside analogues (EIDD1931, EIDD2801, galidesivir, favipiravir, rimonavir) were tested, but none were able to substitute for remdesivir. The functional metabolite of remdesivir, GS441524, also failed to substitute for remdesivir. Accordingly, the cellular uptake of remdesivir is more effective than GS441524 and only the pro-drug is subjected to ABC-family export. Considering that remdesivir is currently administered clinically through i.v. infusion mainly due to first-pass hepatic clearing, the enhanced potency of remdesivir attainable by use of ABC-dual inhibition makes it capable of being administered orally which, in turn, allows it to attain greater potency, and thus extend the use of the drug.
In addition to the data provided herein regarding the REC combination, additional empirical evidence is shown for multiple drugs currently being explored for efficacy against SARS-CoV2. The initial HD-DoE screen included two focus compounds—favipiravir, and hydroxychloroquine. Both drugs failed to emerge as efficacious, individually, and in combination with others, against SARS-CoV2 in the VERO6 EGFP assay, irbesartan, camostat, ritonavir, lopinavir, rimantadine, EIDD1931, mefloquine, arbidol, reviewed in (McKee et al. (2020) Candidate drugs against SARS-CoV-2 and COVID-19. Pharmacol. Res.), are all strong candidates for SARS-CoV2; however, this study did not provide evidence for efficacy. The antihelminth drug ivermectin, which is currently undergoing clinical testing in COVID-19 (e.g. NCT04351347, NCT04392713, NCT04360356), displayed individual, but limited potency against SARS-CoV2, but was unable to enhance the REC combination.
A serial dilution assay was performed to determine the IC50 of the REC combination. Starting at 10 μM equimolar mixtures of the components of the REC combination, the viability of VERO-EGFP cells was determined. For all dilutions, extending to the lowest tested (at 50 nM) the REC combination exerted complete protection from the SARS-CoV2 virus (
Cell Culture and Preparation of Virus Stock for Assays in Huh7 Cells
The human hepatoma cell line, Huh7, was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2% HEPES 1M, 5 mL sodium bicarbonate 7.5%, 1% non-essential amino acids, and 1% Penicillin-Streptomycin 10,000 U/mL in a humidified 5% CO2 incubator at 37° C. Assay medium for producing virus stocks and antiviral testing was prepared by supplementing DMEM with 4% FBS, 2% HEPES 1M, 5 mL sodium bicarbonate 7.5% and 1% NEAA.
To quantify antiviral activity of remdesivir, combinations on Huh7 cells (a SARS-CoV-2 virus strain that produces sufficient cytopathic effect (CPE) on this cell line) was prepared as follows. Starting from passage 6 of the SARS-CoV-2 strain BetaCov/Belgium/GHB-03021/2020 (EPI ISL 40797612020-02-03; which was isolated from a Belgian patient returning from Wuhan in February 2020) three additional passages were completed on Huh7 cells. Cultures which showed the greatest CPE were selected. This resulted in a virus stock (passage 9) that confers full CPE on Huh7 (5.6×10{circumflex over ( )}4 TCID50/mL), as well as on Vero E6 cells (1.8×10{circumflex over ( )}7 TCID50/mL). The genotype of this virus stock shows four nucleotide changes as compared with the mother virus stock (P6).
Assays in Huh7 Cells
Remdesivir alone, and in combination with elacridar, was tested in antiviral assays with Huh7 cells to determine whether a synergistic benefit could be observed in the combination. The assay measures the extent to which these drugs interfere with the viral cytopathy of SARS-CoV-2 in host cells. Considering that viral load drives the clinical presentation and progression of viral-induced disease, interference with viral production in host cells provides significant clinical benefits. Huh7 is an immortalized cell line composed of epithelial-like cells derived originally from a human liver tumor. This line is susceptible to viral infection and supports viral replication, and thus provides a useful model of viral infection in human tissue.
The anti-viral effects of drug combinations were assayed in untreated, infected control cells, i.e., under conditions where the virus-induced cytopathic effect (CPE) was fully in effect. Huh7 cells were seeded in 96-well plates at a density of 6000 cells per well in assay medium. After overnight growth, cells were preincubated with candidate antiviral combinations for 2 hours prior to infection with multiplicity of infection (MOI) of 0.01 TCID50/cell. Cytopathic effect (CPE) was determined using MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) as an indicator of residual metabolic activity in cells 4 days post-infection. For this assay, an MTS/phenazine methosulphate (PMS) stock solution (2 mg/mL MTS and 46 μg/mL PMS in PBS at pH 6-6.5) was diluted 1/20 in MEM without phenol red. Medium was aspirated from wells of the test plates and 70 μL of MTS/PMS solution was added. After 0.5-1 hour incubation at 37° C. absorbance was measured at 498 nm. Cytotoxic effects caused by compound treatment alone were monitored in parallel plates containing mock-infected cells.
Remdesivir alone, and remdesivir in combination with elacridar, were tested for antiviral activity at a range of concentrations. Percent inhibition of virus-induced cytopathic effect was calculated as 100%×[A498(treated, infected cells)−A498(untreated, infected cells)]/[A498(treated, mock-infected cells)−A498(untreated, infected cells)], where A498 was the absorbance measurement in the MTS assay. Compounds were tested at a range of concentrations using an 8-point dilution series with 3-fold serial dilutions starting at 0.5 μM.
Cytoxicity of remdesivir alone was determined for Huh7 cells using the same method at a higher range of remdesivir concentrations, but without introducing virus. The 50% cytotoxic concentration (CC50) for remdesivir was 3±0.07 μM. Thus, the antiviral effect of remdesivir was measured in these studies at concentrations significantly below cytotoxic concentration.
A significant enhancement in the reduction of CPE was observed for the combination of remdesivir and elacridar in comparison with remdesivir alone. The ratios of EC50 for remdesivir alone to EC50 for remdesivir combinations is tabulated as ‘X-fold’ in
Cell Culture and Preparation of Virus Stock for Assays in Vero E6 Cells
Vero E6 WT cells and Vero E6 GFP cells were maintained in DMEM supplemented with heat-inactivated 10% v/v fetal calf serum (FCS) and 500 μg/mL Geneticin and kept under 5% CO2 at 37° C. Assay medium for Vero E6 cells was DMEM supplemented with 2% v/v FCS. Virus stock was prepared as described above for assays in Huh7 cells.
Assays in Vero E6 Cells
Combinations of remdesivir, elacridar, and curcumin were also tested in cell-based antiviral assays with Vero E6 cells as the host cell. Both wild type cells (Vero E6 WT) and cells transformed to express green fluorescent protein (Vero E6-GFP) were used. Vero E6 is a cell line derived from kidney epithelial cells from African green monkey.
Cells were preincubated with candidate antiviral combinations for 2 hours prior to infection with SARS-CoV-2 virus (MOI=0.01). Three days post-infection, cytopathic effect (CPE) was determined using either MTS as described above. Compounds were assayed in the following combinations: (1) remdesivir alone, (2) curcumin alone, (3) elacridar alone, (4) remdesivir with elacridar, and (5) remdesivir with curcumin (Vero E6-GFP cells only). Percent inhibition of virus-induced cytopathic effect was calculated as for Huh7 cells. Compounds were tested at concentrations ranging from 0.0046 μM to 10 μM.
In all the Vero E6 assays, a significant enhancement in the reduction of CPE was observed for the combination of remdesivir and elacridar in comparison with remdesivir alone. The addition of elacridar to remdesivir improves EC50 for the antiviral effect by a factor of 9.2 (Vero E6 WT cells) to 19.2 (Vero E6 GFP cells). In addition, in Vero E6-GFP cells, the combination of remdesivir and curcumin exhibited a greater antiviral effect than remdesivir alone. The beneficial effect of adding curcumin is seen most clearly at the 1.11 μM treatment concentration. At this concentration, the cytopathic effect was eliminated in cells treated with remdesivir and curcumin. For cells treated with remdesivir only at the same concentration (i.e., 1.11 μM) the virus-induced cytopathic effect was 50-60% inhibited. Curcumin alone did not result in any significant antiviral effect in these assays (data not shown). Elacridar alone had a modest antiviral effect: EC50 for % inhibition of virus-induced CPE was 1.07±0.5 μM in Vero E6-WT cells, and 2.16±0.5 μM in Vero E6-GFP cells. Therefore, the antiviral effect of the combinations was greater than one would expect if the drugs were acting together additively.
Preclinical virus challenge assays in Syrian golden hamster were performed to test the synergistic effect of combining the antiviral drug remdesivir with tariquidar (a dual-specificity ABC-family inhibitor). The Syrian golden hamster model of SARS-CoV-2 infection is a valuable model system for evaluating therapeutic interventions for COVID-19 as the animals exhibit clinical signs of viral-induced morbidity and viral replication in relevant tissues, including lung, blood, and nasal washes.
Hamsters were infected intranasally with SARS-CoV-2 virus (2×106 TCID50 inoculum). Following infection, animals were treated twice daily on days 0-3 with vehicle, remdesivir alone (20 mg/kg, subcutaneous route (SC)), or remdesivir (20 mg/kg, SC) combined with other treatments, including tariquidar (5 mg/kg, SC). On day 4, animals were sacrificed, and lung tissue was harvested and analyzed to determine both the viral genome levels and the level of infectious virus in the lungs.
Viral genome levels and infectious virus levels were determined in the different experimental groups. Statistical comparisons between groups were made using a two-tailed Mann-Whitney test. Notably, the group treated with remdesivir and tariquidar showed improved anti-viral effect when compared to the group treated with remdesivir alone and the group treated with vehicle control. That is, a pronounced reduction of viral genome level was observed in individual animals in the remdesivir plus tariquidar group (data not shown).
To further examine the combinatorial effect of remdesivir and tariquidar, the data were re-analyzed using a group pooling strategy.
Accordingly, administration of tariquidar (which alone would be expected to have no antiviral effect) enhanced the antiviral potency of remdesivir in lung tissue, and further demonstrated that the combination of tariquidar with remdesivir will lower the dose of remdesivir required for antiviral efficacy.
Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents of the specific embodiments described herein described herein. Such equivalents are intended to be encompassed by the following claims.
This application claims priority to U.S. Provisional Patent Application Nos. 63/028,844 (filed May 22, 2020), 63/057,829 (filed Jul. 28, 2020), and 63/071,848 (filed Aug. 28, 2020) the contents of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/033689 | 5/21/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63071848 | Aug 2020 | US | |
| 63057829 | Jul 2020 | US | |
| 63028844 | May 2020 | US |
