Patent Application
20240009213
The present disclosure relates to a leukocyte tyrosine kinase (LTK) inhibitor for use in the treatment of a viral infection in a subject, wherein the viral infection is caused by an RNA virus. The LTK inhibitor may be an acyclic ALK (anaplastic lymphoma kinase) inhibitor such as ceritinib, brigatinib, ensartinib, alectinib, crizotinib, entrectinib, belizatinib, CEP-28122 or CEP-37440. Similarly, the disclosure provides a method of treating a viral infection in a subject, wherein the viral infection is caused by an RNA virus, the method comprising administering to the subject an LTK inhibitor, and the use of an LTK inhibitor in the manufacture of a medicament for the treatment of a viral infection, wherein the viral infection is caused by an RNA virus. In particular, the disclosure relates to the treatment of influenza or coronavirus infections in human subjects.
Viral diseases pose a substantial and increasing disease burden on the human population with high morbidity and mortality. Globally, seasonal influenza (or flu) causes hundreds of thousands of deaths every year (despite the existence of vaccines) while many other potentially lethal viruses, for which no vaccines or specific treatments exist, are now endemic across much of the world, such as Dengue virus and West Nile virus.
In addition to the general viral disease burden, certain viruses have pandemic potential. This has been demonstrated to devastating effect by the 2020 COVID-19 pandemic (caused by the coronavirus SARS-CoV-2), while recent years have also witnessed outbreaks of Ebola virus in West Africa, Middle Eastern Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). Both MERS and SARS are also caused by coronaviruses (MERS-CoV and SARS-CoV-1, respectively). The enduring threat of an influenza pandemic is also a public health concern, particularly in respect of Highly Pathogenic Avian Influenza (HPAI), primarily of the subtypes H5N1 and H7N9 which have emerged in East Asia in recent decades.
Newly emerging coronaviruses (such as those mentioned above) and HPAI viruses have particular pandemic potential and can cause fatal acute lung injury. The mechanisms, cells and inflammatory mediators are only partly characterised for SARS-CoV-2, but it is known to cause diffuse alveolar damage (DAD) and cytokine storm (Guo et al., Military Medical Research 7: 11, 2020). This is similar to SARS-CoV-1, where mortality was generally a result of acute lung injuries in association with cytokine storms (IFN-γ, IL-1, IL-6, IL-12, CCL2, CXCL10, CXCL9, and IL-8) and low levels of IL-10 (Zhang et al., Infect Immun 72: 4410-4415, 2004). Alveoli were described to contain massive mixed inflammatory infiltrates (activated Th1 and cytotoxic T cells, macrophages and NK-cells), epithelial necrosis and erosions, vascular leakage, fibrin and hyaline deposition, squamous metaplasia, and multinucleated giant cells. HPAI and MERS have similarly been associated with cytokine storms and acute lung injury.
While the ultimate aim in any pandemic situation is a vaccine that can be used to eliminate the spread of infection, vaccine development is a lengthy process. The development of treatments for viral infections is therefore essential in order to reduce mortality. To date, however, research into treatments for viral infections, such as anti-virals, has been limited and severely underfunded (with the exception of treatments for HIV).
Currently, treatments for respiratory viruses with pandemic potential (e.g. coronaviruses and influenza) are limited: corticosteroids such as dexamethasone are the only drugs to have been shown to significantly reduce mortality in COVID-19 patients (The RECOVERY Collaborative Group, N Engl J Med NEJMoa2021436, 2020), presumably by dampening the aberrant immune response that causes cytokine storms. However, corticosteroid therapy has been found to be ineffective in MERS treatment. The viral RNA-dependent RNA polymerase inhibitor remdesivir has activity against coronavirus polymerases and has been shown to reduce recovery time in COVID-19 patients (Beige) et al., N Engl J Med NEJMoa2007764, 2020), though trial results in respect of its impact on mortality are so far inconclusive.
A handful of influenza drugs have historically been globally available: the adamantane derivatives rimantadine and amantadine and the neuraminidase inhibitors (NAIs) oseltamivir and zanamivir. However, from 2004 the adamantane derivatives were no longer recommended due to the emergence of resistance in most circulating influenza viruses. Resistance to NAIs has also been reported, e.g. 90% of circulating strains exhibited resistance to oseltamivir during the 2007-2009 influenza seasons (Hurt et al., N Engl J Med 365: 2541-2542, 2011).
More recently, two further NAIs (laninamivir and peramivir) have been introduced in Japan, China, South Korea, and the USA. These have been licensed for influenza prevention and treatment. In 2018 the RNA-dependent RNA polymerase inhibitors baloxavir marboxil (BAM), and favipiravir reached the market (the first in Japan and the USA, and the latter in Japan), these drugs inhibit viral replication. However, use of these drugs was associated with a rapid emergence of resistant strains and mutated viruses could be identified in 2.2-9.7% of cases (Hayden et al., N Engl J Med 379: 913-923, 2018).
The development of antiviral drugs for use in pandemic scenarios is challenging, since many drugs are effective against only a small subset of viruses and so may not be effective against an emerging virus. Moreover, many viruses, particularly RNA viruses such as influenza, have a high rate of mutation meaning that drug-resistant strains can rapidly emerge. To address both these challenges, there is a need for new drugs for treatment of viral infections that target host mechanisms that are hijacked by viruses, rather than viruses themselves. Wide ranges of virus types hijack the same host mechanisms during infection, meaning drugs which target these mechanisms can be expected to have a broad spectrum of activity, including against new or emerging viruses. Moreover, targeting a host pathway will negate development of viral escape mutations and serve as a durable and efficient druggable target in severe infections.
In COVID-19, fatal lung injury occurs only in a small subset of the population and, without being bound by theory, one hypothesis is that lung injury caused by novel coronavirus infection is caused by insufficient immunity and unchecked secretion of the virus that over-activates macrophages, resulting in cytokine storms (Liu et al., JCI Insight 4(4): e123158, 2019). If so, reducing the viral load is a key step in preventing mortality in COVID-19 and other such infections.
The receptor tyrosine kinase LTK has recently been shown by the present inventors to be an endoplasmic reticulum (ER)-resident kinase that regulates trafficking from the ER to the Golgi (Centonze et al., J Cell Biol 218(8): 2470-2480, 2019). Inhibition or knockdown of LTK was found to result in retardation of ER-to-Golgi trafficking. The present inventors have found that by blocking this pathway, inhibition of LTK is effective in treating cancer associated with protein hypersecretion, in particular multiple myeloma (co-pending application PCT/EP2020/056698).
RNA viruses are known to hijack the secretory pathway: viral proteins are synthesised and glycosylated in the ER, and virions are assembled in compartments distal to the ER such as the ERGIC (ER-Golgi Intermediate Compartment) or the Golgi. Such pathways are known to be used by the influenza virus and coronaviruses (Fung & Liu, Front Microbiol 5: 296, 2014).
The present disclosure demonstrates that inhibition of LTK blocks assembly and secretion of HPAI and SARS-CoV-2 in vitro. It has been found that LTK, but not ALK, is well expressed in lung and GI tissue (see
In a first aspect, provided herein is a leukocyte tyrosine kinase (LTK) inhibitor for use in the treatment of a viral infection in a subject, wherein the viral infection is caused by an RNA virus.
In a related aspect, provided herein is a method of treating a viral infection in a subject, wherein the viral infection is caused by an RNA virus, the method comprising administering to the subject a leukocyte tyrosine kinase (LTK) inhibitor.
In particular, the LTK inhibitor is administered to a subject in need thereof in an amount effective to treat the viral infection.
In another related aspect, provided herein is the use of a leukocyte tyrosine kinase (LTK) inhibitor in the manufacture of a medicament for the treatment of a viral infection, wherein the viral infection is caused by an RNA virus.
In another related aspect, provided herein is a pharmaceutical composition comprising a leukocyte tyrosine kinase (LTK) inhibitor for use in the treatment of a viral infection in a subject, wherein the viral infection is caused by an RNA virus.
In more particular aspects, the disclosure concern the treatment of influenza and coronavirus infections in human subjects.
Particularly in relation to influenza, the LTK inhibitor is selected from ceritinib, brigatinib, ensartinib, alectinib, crizotinib, entrectinib, belizatinib, CEP-28122, CEP-37440 or ALK-IN-1, and the influenza is caused by a Type A influenza virus of subtype H1N1, H3N2, H5Nx, H7N1 or H7N9, or a Type B influenza virus.
Particularly in relation to coronavirus infections, for example by SARS-CoV-2, the LTK inhibitor is selected from ceritinib, brigatinib, ensartinib, alectinib, crizotinib, belizatinib, CEP-28122, CEP-37440 or ALK-IN-1.
Also provided herein is a method of inhibiting assembly and/or secretion of influenza virus, comprising contacting cells infected with influenza virus or at risk of infection by influenza virus with a leukocyte tyrosine kinase (LTK) inhibitor, wherein the LTK inhibitor is selected from ceritinib, brigatinib, ensartinib, alectinib, crizotinib, entrectinib, belizatinib, CEP-28122, CEP-37440 or ALK-IN-1.
The method may be performed in vitro, ex vivo or in vivo. Thus, the contacting may be by contacting isolated cells or tissues in vitro or ex vivo, or by administering the inhibitor to a subject (i.e. a subject comprising the cells).
Further provided herein is a leukocyte tyrosine kinase (LTK) inhibitor for use in inhibiting the assembly and/or secretion of influenza virus in a human subject thereby to treat or prevent infection of said subject by the virus,
wherein the LTK inhibitor is selected from ceritinib, brigatinib, ensartinib, alectinib, crizotinib, entrectinib, belizatinib, CEP-28122, CEP-37440 or ALK-IN-1,
and wherein the influenza is caused by a Type A influenza virus of subtype H1N1, H3N2, H5Nx, H7N1 or H7N9, or a Type B influenza virus.
Research into treatments for viral infections has traditionally focused on antiviral therapies that directly target viruses. For instance, as detailed above, common targets for antivirals include viral RNA-dependent RNA polymerases, and in the case of influenza, neuraminidase. In the case of COVID-19 monoclonal neutralising antibodies have also been developed to block viral entry into host cells (Abbasi, JAMA 324(2):128, 2020). The present inventors have adopted the rarer approach of treating viral infections by targeting host cell mechanisms that play a role in viral replication, specifically LTK. Surprisingly, inhibition of LTK has been found to be highly effective in inhibiting assembly/secretion of influenza and SARS-CoV-2, as demonstrated in the examples below.
Provided herein is thus an LTK inhibitor for use in treating a viral infection in a subject, wherein the viral infection is caused by an RNA virus. The term “RNA virus”, in line with the standard definition in the art, is used to refer to any virus with an RNA genome (that is to say, a virus with genetic material composed of RNA).
A viral infection is, in line with the standard meaning of the term, an infection caused by a virus. That is to say, the present invention provides a new therapy for infections caused by viruses (specifically infections caused by RNA viruses). As discussed further below, RNA viruses include retroviruses. In an embodiment, the RNA virus treated according to the invention is a ribovirus (i.e. an RNA virus that is not a retrovirus).
In a particular embodiment, the RNA virus that causes the viral infection treated according to the uses and methods herein is an enveloped RNA virus. That is to say, an LTK inhibitor may be used in the treatment of a viral infection in a subject, wherein the infection is caused by an enveloped RNA virus. In a more particular embodiment, the RNA virus that causes the infection treated according to the uses and methods herein is an enveloped ribovirus.
As is well known in the art, all virus particles (or virions) comprise a protein capsid encasing the viral genetic material. The capsid consists of capsid proteins, also known as viral coat proteins. An enveloped virus comprises an additional external layer known as the viral envelope. The viral envelope is a membranous layer derived from the membrane of the host cell in which the virion was formed. The envelope is formed when the virus particle buds off from the host cell, and so comprises host cell membrane and membrane proteins, in addition to viral glycoproteins which recognise host cell surface receptors. Upon binding of the enveloped virus to a new host cell, the envelope fuses with the host cell membrane, causing the centre of the virion (i.e. the capsid and genetic material) to enter the cell.
The majority of RNA viruses are enveloped. Medically-significant enveloped RNA virus families include Coronaviridae, Orthomyxoviridae, Flaviviridae, Togaviridae, Paramyxoviridae, Rhabdoviridae and Filoviridae. An LTK inhibitor may be used to treat a viral infection caused by a virus of any one of these families, i.e. an LTK inhibitor may be used to treat a viral infection caused by a known or future emergent virus of the family Coronaviridae, Orthomyxoviridae, Flaviviridae, Togaviridae, Paramyxoviridae, Rhabdoviridae or Filoviridae.
In an embodiment, the RNA virus that causes the viral infection treated according to the invention is a double-stranded RNA virus (double-stranded RNA viruses constitute Baltimore classification Group III). Double stranded RNA viruses have a genome consisting of double-stranded RNA (i.e. complementary positive and negative RNA strands). Rotavirus is a medically-significant genus of double-stranded RNA virus, that is the most common cause of gastroenteritis in infants and young children. An LTK inhibitor may be used to treat a rotavirus infection, e.g. rotaviral enteritis.
In a particular embodiment, the RNA virus that causes the viral infection to be treated is a single-stranded RNA virus (ssRNA virus, i.e. having a genome consisting of a single RNA strand). That is to say, an LTK inhibitor may be used to treat a viral infection in a subject, wherein the viral infection is caused by a single-stranded RNA virus. In particular, the RNA virus that causes the viral infection to be treated may be an enveloped ssRNA virus. In a specific embodiment, the RNA virus that causes the viral infection to be treated is an enveloped single-stranded ribovirus.
In a particular embodiment, the single-stranded RNA virus that causes the viral infection is a positive-sense single-stranded RNA virus (+ssRNA virus). That is to say, an LTK inhibitor may be used to treat a viral infection in a subject, wherein the viral infection is caused by a positive-sense single-stranded RNA virus, in particular an enveloped positive-sense single-stranded RNA virus. A+ssRNA virus has a genome consisting of a single strand of positive-sense RNA. The virus's RNA genome can thus act as messenger RNA (mRNA) in a host cell, and be directly translated into viral proteins.
In a particular embodiment, the positive-sense single-stranded RNA virus is a ribovirus, particularly an enveloped ribovirus. Positive-sense single-stranded RNA riboviruses constitute Baltimore classification Group IV. An LTK inhibitor may thus be used to treat a viral infection caused by a positive-sense single-stranded ribovirus.
In certain embodiments, the +ssRNA virus that causes the viral infection is of the family Coronaviridae or the family Flaviviridae. That is to say, an LTK inhibitor may be used to treat a viral infection caused by a positive-sense single-stranded RNA virus of the family Coronaviridae or the family Flaviviridae.
The family Coronaviridae includes coronaviruses. Coronaviruses are respiratory viruses, and are so-called because of their characteristic spike (S) proteins which project from the viral surface and resemble solar corona in electron micrographs. Seven different coronavirus strains which infect humans are currently known: while four of these are associated with only mild common cold infections (HCoV-0043, HCoV-HKU1, HCoV-229E and HCoV-NL63) the other three are recently emerged zoonotic pathogens which are associated with severe infections and have significant mortality rates. SARS-CoV-1 and SARS-CoV-2 are the causative agents of SARS and COVID-19, respectively. It is believed that both viruses originated in horseshoe bats. MERS-CoV is the causative agent of MERS, and is also believed to have originated in bats, spreading first to camels and then from camels to humans.
In principle, any viral infection caused by a coronavirus may be treated with an LTK inhibitor according to the uses and methods herein. Thus, the viral infection to be treated may be caused by any one of the above-listed seven coronaviruses that infect humans, or may be caused by any other coronavirus not currently known to science, e.g. a novel strain of a known coronaviruses or a new zoonotic coronavirus. However, mild, common cold infections are generally self-limiting and do not necessarily require medical intervention. The present uses and methods are therefore particularly relevant to the treatment of severe coronavirus infections. In particular, an LTK inhibitor may be used to treat SARS, MERS or COVID-19. These diseases may be diagnosed using standard diagnostic methods, e.g. reverse transcriptase PCR (RT-PCR) performed on nose or throat swabs.
In the specific context of COVID-19, it is well known that the large majority of COVID-19 infections are either asymptomatic or mild (not requiring medical intervention). The present uses and methods may thus particularly be used for treatment of severe COVID-19, though in some instances (e.g. in individuals with risk factors for suffering severe disease) a clinician may consider it appropriate to treat even mild COVID-19 with an LTK inhibitor. Alternatively, an LTK inhibitor may be used to treat mild or asymptomatic COVID-19, in order to prevent the development of severe disease.
SARS, MERS and severe COVID-19 infections are characterised by symptoms including dyspnea (shortness of breath), pneumonia and/or acute respiratory distress syndrome (ARDS). Thus, more broadly, an LTK inhibitor may be used to treat a coronavirus infection associated with dyspnea, pneumonia and/or ARDS, or systemic symptoms of viral infection such as damage to the heart, brain, kidneys or other vital organs. That is to say, an LTK inhibitor may be used to treat a coronavirus infection in a subject, wherein the subject has (or is suffering from) dyspnea, pneumonia and/or ARDS or systemic symptoms from infection of the heart, brain, kidneys or other vital organs. These conditions can easily be diagnosed by a trained physician, based on e.g. physical and neurological examination; standard blood tests including for acute phase proteins, elevated liver enzymes, LDH, troponins, creatine kinase-MB, D-dimer, urea, creatinine, procalcitonin or IL-6; cardiac function tests, kidney function tests; reduced blood oxygen level; or an X-ray or CT scan of the chest.
SARS-CoV-1 and SARS-CoV-2 are related coronavirus strains. Many other similar SARS-like coronavirus strains have previously been isolated from bats, all of which may have human pandemic potential (Menachery et al., Nature Medicine 21: 1508-1513, 2015). It is likely that many of these viruses would cause infections with the same symptoms as SARS and COVID-19, and which can be treated using an LTK inhibitor according to the present invention. The present invention thus provides a therapeutic that can not only be used in the treatment of currently-known coronavirus infections, but can also be used in the event of any future coronavirus pandemic, or indeed smaller outbreak.
Viruses of the family Flaviviridae are insect-borne (primarily by ticks or mosquitos). The family contains several medically important viruses. In particular, the family includes the genus Flavivirus, which contains several viruses capable of causing potentially-fatal encephalitis, and in accordance with the present uses and methods, an LTK inhibitor may be used to treat a viral infection caused by a virus of the genus Flavivirus. Flaviviruses of particular relevance include Dengue virus (causative agent of Dengue fever), West Nile virus (causative agent of West Nile fever), tick-borne encephalitis virus (causative agent of tick-borne encephalitis), Japanese encephalitis virus (causative agent of Japanese encephalitis) and yellow fever virus (causative agent of yellow fever). Thus, an LTK inhibitor may be used in the treatment of an infection caused by Dengue virus, West Nile virus, tick-borne encephalitis virus, Japanese encephalitis virus or yellow fever virus. That is to say, an LTK inhibitor may be used in the treatment of Dengue fever, West Nile fever, tick-borne encephalitis, Japanese encephalitis or yellow fever. LTK inhibitors may also be used to treat other diseases caused by flaviviruses.
Diagnosis of these diseases can be made by a trained physician, using suitable diagnostic procedures, e.g. physical examination, RT-PCR performed on blood samples, etc. As in the context of coronaviruses, any infection caused by a flavivirus can in principle be treated with an LTK inhibitor. In practice, clinicians may prefer not to intervene in mild cases of disease, and administer treatment only in the case of more serious illness or to at-risk individuals. Such treatment decisions lie within the routine duties of the skilled clinician.
In another embodiment, the positive-sense single-stranded RNA virus that causes the viral infection to be treated is a retrovirus. LTK inhibitors can thus be used to treat retroviral infections (i.e. viral infections caused by a retrovirus). As is well known to the skilled person, retroviruses are characterised by their use of the enzyme reverse transcriptase to reverse transcribe their RNA genomes into DNA, which is then integrated into the host genome using the integrase enzyme. RNA retroviruses constitute Baltimore classification Group VI.
Retroviruses that cause human disease include in particular the human immunodeficiency viruses HIV-1 and HIV-2, and the oncoretrovirus human T-lymphotropic virus (HTLV), which causes adult T-cell leukaemia/lymphoma (ATL). Thus an LTK inhibitor can be used to treat an infection caused by HIV or HTLV, and accordingly can be used in treatment of HIV/AIDS. As a cancer caused by a virus, ATL can also be treated with an LTK inhibitor. ATL tends only to occur in a subset of individuals infected with HTLV, generally many years after infection. Development of ATL following HTLV infection is associated with high viral load (i.e. HTLV-infected individuals with a high viral load are at risk of developing ATL, whereas those with a low viral load are generally considered not to be at risk of developing ATL). An LTK inhibitor may therefore be used to prevent ATL. This may be achieved by administration of an LTK inhibitor to an individual who has been infected with HTLV, but has not (or has not yet) developed ATL. Herein, an individual who does not have detectable ATL is defined as having not developed ATL; equivalently, only an individual who has detectable ATL is defined as having developed ATL.
This prophylactic therapy may be particularly targeted at individuals who have been infected with HTLV, and who have not yet developed ATL, but have a high HTLV viral load (alternatively referred to as a high proviral load). A high HTLV viral load may be defined as at least 5% of peripheral blood mononuclear cells (PBMCs) being infected with HTLV. Alternatively, a high HTLV viral load may be defined as at least 10% of PBMCs being infected with HTLV. Viral load may be measured by qPCR, or any other appropriate standard laboratory technique.
In another embodiment, the single-stranded RNA virus that causes the viral infection to be treated is a negative-sense single-stranded RNA virus (−ssRNA virus), in particular an enveloped negative-sense single-stranded RNA virus. That is to say, an LTK inhibitor may be used to treat a viral infection in a subject, wherein the viral infection is caused by a negative-sense single-stranded RNA virus. A −ssRNA virus has a genome consisting of a single strand of negative-sense RNA. During infection by a −ssRNA virus, the negative-sense RNA strand acts as template for transcription of mRNA by the viral RNA-dependent RNA polymerase, and during viral replication the same enzyme generates a positive-sense RNA antigenome that is used as template for the synthesis of the negative-sense RNA genome. Negative-sense single-stranded RNA viruses constitute Baltimore classification Group V.
In a particular embodiment, the negative-sense single-stranded RNA virus that causes the viral infection to be treated is an influenza virus. That is to say, an LTK inhibitor may be used to treat influenza in a subject. Any influenza infection may be treated.
As is known to the skilled person, there are four genera of influenza virus: influenza A, influenza B, influenza C and influenza D (also known as Types A-D). Types A-C are known to infect humans, and thus an LTK inhibitor may be used to treat influenza caused by a Type A, Type B or Type C influenza virus. It is unclear whether Type D influenza is capable of infecting humans. If this proves to be possible, an LTK inhibitor may be used to treat influenza caused by a Type D influenza virus.
Seasonal flu epidemics are caused by Type A and B influenza viruses. Accordingly, in a particular embodiment an LTK inhibitor is used to treat influenza caused by a Type A influenza virus or a Type B influenza virus.
Type B influenza is capable of infecting only humans and seals. Since influenza is a segmented virus (all its genes are encoded on separate viral RNA segments), influenza strains are capable of reassortment, in which two strains mix and exchange vRNA segments, giving rise to a new viral strain. Due to its limited host range, Type B influenza is not capable of antigenic shift (in which two strains of influenza from different host organisms undergo reassortment, yielding a new subtype with a significantly different phenotype). Influenza B is not therefore capable of causing human pandemics (or is not believed to be so capable).
Type A influenza, on the other hand, has a wide host range, being capable of infecting not only humans but also many bird species (both wild and domestic) and several other mammalian species including dogs, horses and pigs. Type A influenza therefore has high potential for antigenic shift, and all known influenza pandemics have been caused by Type A influenza, including the 1918 Spanish flu. A particularly important embodiment is therefore the use of an LTK inhibitor in the treatment of influenza caused by a Type A influenza virus.
Influenza A viruses are classified into subtypes based on their haemagglutinin (H) and neuraminidase (N) serotypes. An LTK inhibitor may be used to treat influenza caused by any Type A influenza virus of any subtype, i.e. having any neuraminidase serotype and any haemagglutinin serotype. Examples of influenza A subtypes include e.g. H1N1 (i.e. having haemagglutinin serotype 1 and neuraminidase serotype 1), the subtype responsible for both the 1918 Spanish flu pandemic and the mild 2009 swine flu pandemic, and H3N2 (i.e. having haemagglutinin serotype 1 and neuraminidase serotype 2), the subtype responsible for the 1968 Hong Kong flu pandemic. As mentioned above, an LTK inhibitor may be used to treat influenza caused by any influenza A subtype, including these.
In recent years and decades certain highly pathogenic strains of avian influenza have developed, i.e. that show high pathogenicity in birds. These strains are referred to as highly pathogenic avian influenza (HPAI). Though no HPAI strain has to-date gained the ability to transmit efficiently between humans, in rare cases of bird-to-human transmission HPAI strains have also shown high pathogenicity in humans. HPAI strains are therefore considered a major risk for future influenza pandemics, should they gain the ability to transmit efficiently between humans. Certain strains of low pathogenic avian influenza (LPAI), i.e. strains that show low pathogenicity in birds, have also been found to show high pathogenicity in humans, and these strains are therefore also considered a major risk for future pandemics.
Accordingly, an LTK inhibitor may be used to treat influenza caused by an avian influenza strain. In a particular embodiment, an LTK inhibitor may be used to treat influenza caused by highly pathogenic avian influenza (HPAI). In another embodiment, an LTK inhibitor may be used to treat influenza caused by low pathogenic avian influenza (LPAI).
In recent years, particular strains of avian influenza have caused significant concern as to their potential to cause a pandemic, due to their high pathogenicity in humans. These include H5Nx strains, particularly H5N1, H5N2, H5N6 and H5N8, and H7N9. An LTK inhibitor may be used to treat influenza caused by an H7N1 or H7N9 influenza strain or an H5Nx influenza strain, such as influenza caused by an H5N1, H5N2, H5N6 or H5N8 influenza strain.
In a particular embodiment the influenza which is treated is caused by a Type A influenza virus of subtype H1N1, H3N2, H5Nx, H7N1 or H7N9, or a Type B influenza virus.
The methods and used disclosed herein may have particular utility in treating subjects suffering from seasonal flu. The seasonal flu may be caused by a Type A influenza virus of subtype H1N1, H3N2, H5Nx, H7N1 or H7N9, or a Type B influenza virus. In another embodiment the influenza may be pandemic flu. The pandemic flu may be caused by a Type A influenza virus of subtype H1N1, H3N2, H5Nx, H7N1 or H7N9. In the context of these particular influenza infections the subject may be a human subject.
In certain embodiments, the subject may be a subject who has been hospitalised for the influenza infection. Thus, the subject may be a human subject who is presenting with severe symptoms of influenza, and in particular symptoms which require in-patient hospital care.
An LTK inhibitor, as referred to herein, is a compound which is capable of interacting with LTK (specifically human LTK) and reducing or abrogating its activity. Human LTK has the UniProt accession number P29376. Methods for testing compounds for activity in LTK inhibition are known, and described in Centonze et al. (supra). In particular, LTK activation requires autophosphorylation on Tyr672. Inhibition of LTK therefore inhibits Tyr672 autophosphorylation, which may be detected by immunoblot using an anti-phospho-LTK(Y672) antibody. Accordingly, to determine whether a compound has activity in LTK inhibition (i.e. is an LTK inhibitor), the compound of interest may be applied to cells expressing human LTK, the cells lysed and LTK autophosphorylation on Tyr672 analysed.
Many commercial antibodies exist that are suitable for detecting LTK phosphorylated on Tyr672. Human LTK has high homology to human ALK (UniProt accession number Q9UM73). Similarly to LTK, ALK activation requires autophosphorylation on Tyr1278 (Tyr1278 of human ALK is equivalent to Tyr672 of human LTK) and several antibodies that recognise phospho-ALK(Y1278) are commercially available. Many of the commercially available antibodies which recognise phospho-ALK(Y1278) also recognise phospho-LTK(Y672), and can thus be used to determine whether a compound is an LTK inhibitor. An example of such an antibody, which can be used to detect phospho-LTK(Y672), is antibody D59G10, available from Cell Signaling Technology (USA).
Thus the LTK inhibitor for use according to the uses and methods herein may be defined as having activity in reducing LTK autophosphorylation of Tyr672, as determined using antibody D59G10 to detect phosphor-LTK(Y672). This determination may be made by any technique known in the art, e.g. immunoblotting (particularly Western blotting).
In one embodiment, the LTK inhibitor completely abrogates LTK autophosphorylation of Tyr672, such that following treatment with the inhibitor phospho-LTK(Y672) is undetectable. In another embodiment, the LTK inhibitor reduces LTK autophosphorylation of Tyr672, but does not completely abrogate it. In this embodiment, some phospho-LTK(Y672) is detectable following treatment with the LTK inhibitor, but less phospho-LTK(Y672) is detectable than in a control sample not contacted with an LTK inhibitor. Levels of phospho-LTK(Y672) in a sample may be quantified using any method known in the art. For instance, if phospho-LTK(Y672) detection is performed by immunoblotting, levels of phospho-LTK(Y672) in each sample may be quantified by densitometry. An LTK inhibitor may reduce LTK autophosphorylation of Tyr672 (and thus the level of phospho-LTK(Y672) in a sample) by e.g. at least 50%, at least 60%, at least 70%, at least 80% or at least 90%.
As detailed above, many existing ALK inhibitors have been discovered also to have activity in inhibiting LTK. Specifically, acyclic ALK inhibitors (which may alternatively be referred to as linear ALK inhibitors) have been found to have activity in inhibiting LTK, while macrocyclic ALK inhibitors have been found to have no or minimal activity against LTK. In line with standard nomenclature in the art, a macrocyclic molecule is defined herein as a molecule that contains a cyclic framework of at least twelve atoms. As described in Basit et al. (European Journal of Medicinal Chemistry 134: 348-356, 2017), an example of a macrocyclic ALK inhibitor is lorlatinib (Formula I).
Macrocyclic ALK inhibitors may be referred to as 3rd generation ALK inhibitors. Examples of other macrocyclic ALK inhibitors include repotrectinib and TPX-0131. As noted above, macrocyclic ALK inhibitors have been found to be ineffective in LTK inhibition, and thus are not suitable for use according to the present methods and uses.
In an embodiment, the LTK inhibitor for use herein is not a macrocyclic ALK inhibitor, or more generally is not macrocyclic. In another embodiment the term “LTK inhibitor” does not include lorlatinib.
As noted above, acyclic ALK inhibitors (which may alternatively be referred to as non-macrocyclic ALK inhibitors) are effective in inhibition of LTK. Thus, the LTK inhibitor for use according to the present uses and methods may be an acyclic ALK inhibitor. 1st and 2nd generation ALK inhibitors are acyclic ALK inhibitors. Acyclic ALK inhibitors can in particular be defined as lacking a cyclic framework, as is seen in macrocyclic inhibitors such as lorlatinib. Although an acyclic ALK inhibitor lacks a cyclic framework, as is known to the skilled person, an acyclic ALK inhibitor may nonetheless comprise one or more cyclic functional groups, which may be connected in series.
Examples of acyclic ALK inhibitors which may be used as an LTK inhibitor according to the current invention include ceritinib, brigatinib, ensartinib, alectinib, crizotinib, entrectinib, belizatinib, CEP-28122, CEP-37440 and ALK-IN-1.
In an embodiment, the LTK inhibitors for use herein do not include entrectinib (in other words, in an embodiment the LTK inhibitor is not entrectinib).
In another embodiment, the LTK inhibitor is ceritinib, brigatinib, ensartinib, alectinib, crizotinib, entrectini or belizatinib.
In another embodiment, where the viral infection is a coronavirus infection, the LTK inhibitor is not entrectinib.
In another embodiment, where the viral infection is selected from COVID-10, SARS or MERS, the LTK inhibitor is not entrectinib.
In another embodiment, where the viral infection is caused by SARS-CoV-2, the LTK inhibitor is not entrectinib.
Ceritinib (5-chloro-2-N-(5-methyl-4-piperidin-4-yl-2-propan-2-yloxyphenyl)-4-N-(2-propan-2-ylsulfonylphenyl)pyrimidine-2,4-diamine) has the trade name Zykadia and the structure shown in Formula II:
Brigatinib (5-chloro-4-N-(2-dimethylphosphorylphenyl)-2-N-[2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl]pyrimidine-2,4-diamine) has the trade name Alunbrig and the structure shown in Formula III:
Ensartinib (6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-N-[4-[(3R,5S)-3,5-dimethylpiperazine-1-carbonyl]phenyl]pyridazine-3-carboxamide) has the structure shown in Formula IV:
Alectinib (9-ethyl-6,6-dimethyl-8-(4-morpholin-4-ylpiperidin-1-yl)-11-oxo-5H-benzo[b]carbazole-3-carbonitrile) has the trade name Alecensa and the structure shown in Formula V:
Crizotinib (3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine) has the trade name Xalkori and the structure shown in Formula VI:
Entrectinib (N-[5-[(3,5-difluorophenyl)methyl]-1H-indazol-3-yl]-4-(4-methylpiperazin-1-yl)-2-(oxan-4-ylamino)benzamide) has the trade name Rozlytrek and the structure shown in Formula VII:
Belizatinib (4-fluoro-N-[6-[[4-(2-hydroxypropan-2-yl)piperidin-1-yl]methyl]-1-[4-(propan-2-ylcarbamoyl)cyclohexyl]benzimidazol-2-yl]benzamide) has the structure shown in Formula VIII:
CEP-28122 ((1S,2S,3R,4R)-3-[[5-chloro-2-[[(7S)-6,7,8,9-tetrahydro-1-methoxy-7-(4-morpholinyl)-5H-benzocyclohepten-2-yl]amino]-4-pyrimidinyl]amino]-bicyclo[2.2.1]hept-5-ene-2-carboxamide) has the structure shown in Formula IX:
CEP-37440 (2-[[5-chloro-2-[[(6S)-6,7,8,9-tetrahydro-6-[4-(2-hydroxyethyl)-1-piperazinyl]-1-methoxy-5H-benzocyclohepten-2-yl]amino]-4-pyrimidinyl]amino]-N-methyl-benzamide) has the structure shown in Formula X:
ALK-IN-1 5-chloro-N2-[4-[4-(dimethylamino)-1-piperidinyl]-2-methoxyphenyl]-N4-[2-(dimethylphosphinyl)phenyl]-2,4-pyrimidinediamine) has the structure shown in Formula XI:
In another embodiment, the LTK inhibitor has the general structure shown in Formula XII:
Pharmaceutically acceptable salts or esters, or other pro-drugs of the LTK inhibitors described herein may be used, in accordance with principles well known in the pharmaceutical arts. The identified LTK inhibitors referred to herein by name thus include salts and esters, and pro-drugs more generally, particularly salts and esters thereof. Such pharmaceutically acceptable salts include acid and base addition salts, e.g. with organic or inorganic acids or bases.
In a particular embodiment the LTK inhibitor is ceritinib, brigatinib, ensartinib, alectinib, crizotinib or entrectinib. For treatment of influenza, ceritinib may in particular be used. Alternatively, for treatment of influenza, entrectinib may be used. For treatment of an infection caused by a coronavirus, particularly COVID-19, entrectinib may in particular be used. However, in other embodiments, as discussed above, for treatment of an infection caused by a coronavirus, particularly COVID-19, entrectinib is not used.
The LTK inhibitor is administered to the subject at a pharmaceutically-effective dosage (i.e. a dosage that is sufficient to have an effect on the viral infection being treated). The existing FDA-approved dosing regimen for ceritinib is for treatment of non-small cell lung cancer (NSCLC), and is a once daily 750 mg oral dose. Ceritinib may be used (i.e. administered to a subject to treat a viral infection caused by an RNA virus) at a dosage of up to 750 mg/day. The maximum ceritinib dose may therefore be e.g. 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150 or 100 mg/day. A minimum pharmaceutically-effective dosage of ceritinib to be used may be e.g. 50, 100, 150, 200, 250 or 300 mg/day.
The existing FDA-approved dosing regimen for brigatinib is for treatment of NSCLC, and is a once daily 90 mg oral dose for the first 7 days. If the 90 mg/day dose is tolerated, the dosage is increased to 180 mg/day. Brigatinib may be used (i.e. administered to a subject to treat a viral infection caused by an RNA virus) at an initial dosage of up to 90 mg/day for 7 days, and if tolerated the dosage may be increased to up to 180 mg/day. The maximum initial brigatinib dosage may be 90, 80, 70, 60, 50, 40 or 30 mg/day. The maximum subsequent brigatinib dosage (if the initial dosage is tolerated) may be 180, 160, 140, 120, 100, 80, 60 or 40 mg/day. A minimum pharmaceutically-effective initial dosage of brigatinib to be used may be e.g. 20, 30, 40, 50 or 60 mg/day. A minimum pharmaceutically-effective subsequent dosage of brigatinib (to be used of the initial dosage is tolerated) may be 30, 40, 50, 60, 70, 80 or 90 mg/day.
Ensartinib is currently in phase III clinical trials for treatment of NSCLC. It is being trialed at a dosage of 225 mg/day, orally. Ensartinib may thus be used (i.e. administered to a subject to treat a viral infection caused by an RNA virus) at a dosage of up to 225 mg/day. The maximum ensartinib dose may therefore be 225, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110 or 100 mg/day. A minimum pharmaceutically-effective dosage of ensartinib to be used may be e.g. 80, 90, 100, 110, 120, 130, 140 or 150 mg/day.
The existing FDA-approved dosing regimen for alectinib is for treatment of NSCLC, and is a twice daily oral dose of 600 mg. Alectinib may thus be used (i.e. administered to a subject to treat a viral infection caused by an RNA virus) at a dosage of up to 600 mg twice daily. The maximum alectinib dose may thus be 600, 550, 500, 450, 400, 350 or 300 mg twice daily. A minimum pharmaceutically-effective dosage of alectinib to be used may be e.g. 100, 150, 200, 250, 300, 350 or 400 mg twice daily. Alternatively, dosing may be once daily, or different doses may be administered at different times of the day, as determined by a physician.
The existing FDA-approved dosing regimen for crizotinib is for treatment of NSCLC and is a twice daily oral dose of 250 mg. Crizotinib may thus be used (i.e. administered to a subject to treat a viral infection caused by an RNA virus) at a dosage of up to 250 mg twice daily. The maximum crizotinib dose may thus be 250, 225, 200, 175, 150, 125 or 100 mg twice daily. A minimum pharmaceutically-effective dosage of crizotinib to be used may be e.g. 80, 100, 120, 140 or 150 mg twice daily. Alternatively, dosing may be once daily, or different doses may be administered at different times of the day, as determined by a physician.
The existing FDA-approved dosing regimen for entrectinib is for treatment of NSCLC and other solid tumours, and is a once daily oral dose of 600 mg. Entrectinib may thus be used (i.e. administered to a subject to treat a viral infection caused by an RNA virus) at a dosage of up to 600 mg/day. The maximum entrectinib dose may thus be 600, 550, 500, 450, 400, 350 or 300 mg/day. A minimum pharmaceutically-effective dosage of entrectinib to be used may be e.g. 100, 150, 200, 250, 300, 350 or 400 mg/day.
For each drug, suitable dosing schedules may be selected by the skilled physician, e.g. based on clinical studies or patient condition or response to the drug.
The subject treated is generally and most particularly a human. However, other animals, particularly other mammals, may also be treated, including livestock, domestic or sports animals. For instance, the subject may be a farm animal such as a cow, horse, sheep, pig or goat, or a pet animal such as a cat or dog, or a racing animal such as a horse. For example the subject may be a cat infected with feline leukemia virus (FeLV) the most common fatal pathogen affecting cats worldwide. FeLV is an enveloped, positive-sense, single-stranded RNA retrovirus. In another example, the subject may be a dog infected with canine coronavirus (CCoV), an enveloped, positive-sense, single-stranded RNA virus that can cause severe diarrhoea, vomiting, and anorexia, or canine respiratory coronavirus (CRCoV) that may cause respiratory disease. In another example the subject may be a cow or calf infected with bovine coronavirus (BCV-2 or BCoV-3) that causes calf enteritis/pneumonia entailing profuse diarrhoea, dehydration, depression, reduced weight gain and anorexia, pneumonia and a serous to purulent nasal discharge. In another example the subject may be a horse infected with equine coronavirus (ECoV) an emerging virus that can cause fever, depression, anorexia, colic or diarrhoea, disruption of the gastrointestinal barrier and severe systemic infections in adult horses.
The subject is an individual who has been diagnosed with a viral infection caused by an RNA virus, who may therefore be treated with an LTK inhibitor according to the present uses and methods. The diagnosis may be made by any suitable method known to the skilled person, e.g. RT-PCR performed on a swab, an alternative nucleic acid amplification technique (e.g. LAMP), antigen detection by e.g. ELISA, lateral flow assay, or based on clinical presentation.
The LTK inhibitor may be administered to the subject by any suitable route. Existing ALK inhibitors are currently formulated for oral administration, but alternative administration routes may be utilised as desired. In particular the LTK inhibitor may be formulated for administration to the subject intravenously or as an aerosol. In particular, existing acyclic ALK inhibitors may be reformulated for administration to the subject intravenously or as an aerosol.
The LTK inhibitor is particularly administered in the form of a pharmaceutical composition. Suitable pharmaceutical compositions may include liquid solutions, suspensions or syrups, and solid compositions such as powders, granules, tablets or capsules. In an embodiment, the LTK inhibitor is provided as a liquid solution or suspension for intravenous or aerosol administration. In another embodiment the LTK inhibitor is provided for oral administration.
Pharmaceutically-acceptable diluents, carriers and excipients for use in such pharmaceutical compositions are well known in the art. For instance, suitable excipients include lactose, sodium starch glycolate, maize starch or derivatives thereof, stearic acid or salts thereof, vegetable oils, waxes, fats and polyols. Suitable carriers or diluents include carboxymethylcellulose (CMC), methylcellulose, microcrystalline cellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose (HPMC), dextrose, trehalose, liposomes, polyvinyl alcohol, pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose (and other sugars), magnesium carbonate, silica, gelatin, oil, alcohol, detergents and emulsifiers such as polysorbates. Stabilising agents, wetting agents, emulsifiers, sweeteners etc. may also be used.
Liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following: sterile diluents such as water for injection, saline solution (preferably physiological), Ringer's solution, fixed oils such as synthetic mono- or diglycerides which may serve as a solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral preparation (e.g. for intravenous administration) can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. A solution for aerosol administration may be provided in a nebuliser or inhaler. A pharmaceutical composition is typically sterile.
The existing acyclic ALK inhibitors which may be used according to the present uses and methods may be formulated with the excipients and suchlike for which their use has previously been approved. Such information can be found on the regulatory approvals for these drugs issued by the FDA or EMA.
The LTK inhibitor may be used as a monotherapeutic agent (i.e. it may be the only drug used to treat the viral infection). More commonly, the LTK inhibitor may be used in combination with one or more additional active agents to treat the viral infection. The additional active agent(s) used to treat the viral infection in combination with the LTK inhibitor may be any agent useful in (or that may be useful in) treatment of the viral infection.
For instance, the LTK inhibitor may be used in combination with an antiviral drug. For instance, in the treatment of influenza, the LTK inhibitor may be used in combination with one or more anti-flu drugs, such as a neuraminidase inhibitor (e.g. oseltamivir, zanamivir or peramivir) or viral RNA polymerase inhibitor (e.g. favipiravir or baloxavir marboxil). In the treatment of a coronavirus infection, the LTK inhibitor may be used with one or more drugs useful in the treatment of such infections, such a viral RNA polymerase inhibitor (e.g. remdesivir). Any other suitable antiviral drug may also be used in combination with an LTK inhibitor.
Alternatively, the LTK inhibitor may be used in combination with another drug that targets host mechanisms associated with the viral infection. For instance, in the case of a viral infection associated with a cytokine storm (e.g. COVID-19), the LTK inhibitor may be used in combination with an immunosuppressant, such as a corticosteroid (e.g. dexamethasone, hydrocortisone or methylprednisolone).
As noted above, the methods and uses herein are predicated at least in part on the effect of LTK inhibitors of inhibiting viral assembly and/or secretion. Thus, in this manner the replication, or more particularly the multiplication or reproduction, of the RNA virus may be inhibited, e.g. prevented, blocked or reduced. In other words the production of virus (or virions or virus particles) in the infected subject may be inhibited. This may be of benefit in the treatment of infected subjects. Thus, as noted above, viewed from another aspect the disclosures herein can be seen to provide methods and uses for inhibiting assembly and/or secretion of an RNA virus in a subject, particularly a human subject. In particular, such methods and uses may be for inhibiting assembly and/or secretion of an RNA virus in a subject, particularly a human subject, thereby to treat or prevent infection of said subject by the virus. Alternatively viewed, the methods and uses may be defined as being for inhibiting production or multiplication of the virus in the subject. In a certain embodiment of said aspect, the RNA virus is an influenza virus. More particularly, the influenza virus may be a Type A influenza virus of subtype H1N1, H3N2, H5Nx, H7N1 or H7N9, or a Type B influenza virus.
The invention may be further understood by reference to the non-limiting examples below, and the figures.
Madin-Darby canine kidney (MDCK) cells or Vero E6 cells were plated out into 96-well cell culture plates at 1×104 cells/well. The next day 100×TCID (tissue culture infective dose) of virus (influenza A/California/07/2009 (H1N1), influenza A/PR/8/1934 (H1N1), influenza A/Vietnam/1194/2004 (H5N1), influenza A/turkey/Italy/3889/1999 (H7N1), or SARS-CoV-2) were mixed with 5-fold dilutions of titrated drugs. Following a 1 h incubation 37° C. in a 5% CO2 humidified atmosphere, the mixture was added to the plated cells.
Plates were incubated for 50 h at 37° C. in a 5% CO2 humidified atmosphere. Next, monolayers were washed with PBS, and fixed in cold 80% acetone for 10 min. Influenza virus was detected by ELISA using biotinylated mAb against influenza nucleoprotein (HB75, ATCC) and streptavidin-alkaline phosphatase (GE Healthcare). Plates were developed using phosphatase substrate (P4744-10G, Sigma-Aldrich) dissolved in substrate buffer, and read with a Tecan reader using the Magellan v5.03 program.
The SARS-CoV-2 virus Human 2019-nCoV strain 2019-nCoV/Italy-InMI1 (008V-03893) from the European Virus Archive (EVA) was detected in Vero E6 cell cultures by ELISA using a mAb against SARS-CoV-2 nucleocapsid (40143-R004, SinoBiological) and HRP-conjugated goat anti-rabbit IgG-Fc mAb (SSA003, SinoBiological). Plates were developed using TMB substrate buffer (se-286967, Santa Cruz), and read with the Tecan reader.
The expression of LTK and ALK in relevant tissues was tested by analysing data depositories. Both influenza and SARS-CoV-2 target respiratory epithelium and alveolar cells in the lungs. The SARS-CoV-2 virus also infects gastrointestinal tissue via the viral entry receptor ACE2. LTK but not ALK was abundantly expressed in lung tissue in 423/427 donors. 2 donors were double positive, and 2 donors expressed neither LTK nor ALK. A similar dominance was found in the small intestine where 126/137 samples were LTK+ALK− (
The effect of LTK inhibitors on virus assembly/secretion was tested by adding influenza virus to MDCK (
Inhibition of SARS-CoV-2 was tested in cultures of VERO E6 cells. The LTK inhibitors, but not lorlatinib, inhibited SARS-CoV-2 within the 50 h incubation time (
The protective capacity of LTK inhibitors was tested by infecting BALB/c mice with 5×LD50 of influenza A/PuertoRico/1934 (H1N1) (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2015584.2 | Oct 2020 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/077037 | 9/30/2021 | WO |
