Patent Application
20230295236
The present invention relates to the treatment of inflammation, and in particular, inflammation associated with or as a result of activation of Toll-like receptors. The invention also relates to the treatment of pathogen-induced inflammation.
Inflammation is one of the main responses of the immune system to tissue damage and infections, and involves the major immune cells and extracellular mediators and regulators, such as cytokines.
In the case of infection, inflammation is triggered when the innate immune system is activated. Innate immune cells, such as macrophages, dendritic cells and neutrophils are all important players of innate immunity and infection. These cells express PRRs (pattern recognition receptors) that detect microbial components, the so-called pathogen-associated molecular patterns (PAMPS). One class of PRRs are the Toll-like receptors (TLRs, of which there are 10 sub-types TLR1-10), which can detect various pathogens from viruses, bacteria, protozoa and fungi. For example, TLRs can detect bacterial lipopolysaccharide, lipoproteins, flagellin, bacterial CpG-DNA and viral single and double-stranded RNA. After recognition of PAMPS, PRRs are activated and trigger an intracellular signalling cascade ultimately resulting in the expression of pro-inflammatory cytokine molecules. One signalling cascade results in the activation of the canonical NF-κB pathway, which is the pivotal regulator of inflammation and the central mediator of pro-inflammatory gene induction. Activation of NF-κB transduction is responsible for the transcriptional induction of pro-inflammatory cytokines, chemokines and additional inflammatory mediators in different types of immune cells. These inflammatory mediators can both directly engage in the induction of inflammation and act indirectly through promoting the differentiation of inflammatory T cells.
However, although the generation of a potent immune response is a crucial part of a host’s response to a pathogen infection, excessive or inappropriate inflammation – e.g. hyperinflammation can be harmful. Indeed, excessive inflammation plays an important role in the pathogenesis of many infectious diseases. In one example, excessive inflammation following pathogen infection leads to hypercytokinemia, commonly known as a cytokine storm. A cytokine storm results from an uncontrolled and excessive release of pro-inflammatory cytokines. This sudden and excessive release of cytokines is particularly harmful to the host, and can lead to multi-system organ failure and death. In particular, acute lung injury (ALI) is a common consequence of a cytokine storm in the lung alveolar environment and systemic circulation. Pathogen induced lung injury can progress into ALI or the more severe form - called acute respiratory distress syndrome (ARDS), which is seen with SAR-CoV-2 infections. Hypercytokinemia can also result from a number of infectious agents, as well as non-infectious hosts.
In addition, pathogen infection also leads to the activation of macrophages. Activated macrophages are twice the size of resting macrophages and are more “aggressive”, having increased levels of lysosomal proteins and a greater ability to phagocytose. Classically activated macrophages also release proteases, neutrophil chemotactic factors, reactive oxygen species and pro-inflammatory cytokines (such as IL-1 beta/IL-1F2, IL-6, and TNF-alpha/TNFSF1A), leading to inflammation and tissue destruction. As such, macrophage effector function significantly affects the quality, duration and magnitude of an inflammatory response, and while this response is important for host defence, when uncontrolled, activated macrophages can cause significant tissue damage. As such, over-activation of macrophages are a key contributor to disorders characterised by excessive inflammation. Most notably, in Wang et al., 2020 it was concluded that activated alveolar macrophages are central to the cytokine storm caused by SARS-CoV-2 infection. For this reason alone, methods to reduce levels of macrophage activation and/or recruitment are also key to preventing and treating inflammation arising from pathogen infection, and in particular, hypercytokinemia.
As such, inflammation and cytokine storms are a common clinical feature in serious and lethal infections by a number of viral infections. These include respiratory viruses such as Orthomyxoviridae. They also include infections by single stranded (+) RNA viruses, including members of the families Flaviviridae, Picornaviridae, Togaviridae, Caliciviridae, Roniviridae, Retroviridae and Coronaviridae. In the majority of deadly infections, the agent responsible for the lethality is not the cytolytic activity of the virus, but the immunopathological response of the host. Among the ss (+) RNA viruses that cause serious illnesses in human and animals of economic importance: Flaviviridae, Picornaviridae, Togaviridae, Caliciviridae, Retroviridae, Coronaviridae, Orthomyxoviridae, Phlebovirus, Arenaviridae and Herpesviridae are particularly associated with inflammation and cytokine storms.
As such, there is a need to provide new treatments for inflammation, and in particular, excessive inflammation or hyperinflammation such as that which arises as a result of pathogen infection. The present invention address this need.
In one aspect, the present invention is directed to a compound of general formula I, or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein X is selected from O and NH;
In one aspect, the present invention is directed to a compound as defined above (and herein), for use in the treatment of inflammation associated with activation of Toll-like receptors.
In another aspect, the present invention is directed to a compound as defined above (and herein), for use in the treatment of a disorder selected from acute respiratory syndrome (ARDS), pneumonia and immunopathology, and in particular hypercytokinemia (cytokine storm syndrome), sepsis and graft-versus-host disease.
In one aspect, the present invention is directed to a compound as defined above (and herein), for use in the treatment of pathogen-induced inflammation. In one embodiment, the pathogen is a bacteria or virus.
In one aspect, the present invention is directed to a compound as defined above (and herein), for use in the combined treatment of inflammation associated with activation of Toll-like receptors or inflammation associated with pathogen-induced inflammation and in the treatment of a viral infection.
In one aspect, the present invention is directed to a compound as defined above (and herein), for use as an anti-inflammatory and an antiviral.
In a particular aspect, the compound of general formula I is PLD, or a pharmaceutically acceptable salt or stereoisomer thereof.
In a particular aspect, the compound of general formula I is DidemninB, or a pharmaceutically acceptable salt or stereoisomer thereof.
In another aspect, the present invention is also directed to a pharmaceutical composition comprising a compound as defined herein, and a pharmaceutically acceptable carrier, for use according to the present invention.
In another aspect, the present invention is directed to the use of a compound as defined herein, in the manufacture of a medicament for the treatment of inflammation.
In another aspect, the present invention is directed to the use of a compound as defined herein, in the manufacture of a medicament for the treatment of inflammation associated with activation of Toll-like receptors.
In another aspect, the present invention is directed to the use of a compound as defined herein, in the manufacture of a medicament for the treatment of a disorder selected from pneumonia, acute respiratory syndrome (ARDS), hypercytokinemia (cytokine storm syndrome), sepsis and graft-versus-host disease.
In another aspect, the present invention is directed to the use of a compound as defined herein, in the manufacture of a medicament for the treatment of pathogen-induced inflammation.
In another aspect, the present invention is directed to the use of a compound as defined herein, in the manufacture of a medicament for the combined treatment of inflammation associated with activation of Toll-like receptors or inflammation associated with pathogen-induced inflammation and in the treatment of a viral infection.
In another aspect, the present invention is directed to the use of a compound as defined herein, in the manufacture of a medicament for use as an anti-inflammatory and an antiviral.
In another aspect, the present invention is directed to a method for treating inflammation in any mammal, preferably a human, wherein the method comprises administering to an individual in need thereof a therapeutically effective amount of a compound as defined herein.
In another aspect, the present invention is directed to a method for treating any mammal, preferably a human, for inflammation associated with activation of Toll-like receptors, wherein the method comprises administering to an individual in need thereof a therapeutically effective amount of a compound as defined herein.
In another aspect, the present invention is directed to a method for treating any mammal, preferably a human, for a disorder selected from pneumonia, acute respiratory syndrome (ARDS), hypercytokinemia (cytokine storm syndrome), sepsis and graft-versus-host disease, wherein the method comprises administering to an individual in need thereof a therapeutically effective amount of a compound as defined herein.
In another aspect, the present invention is directed to a method for treating any mammal, preferably a human, for pathogen-induced inflammation, wherein the method comprises administering to an individual in need thereof a therapeutically effective amount of a compound as defined herein.
In another aspect, the present invention is directed to a method of combined treatment of any mammal, preferably a human, for inflammation associated with activation of Toll-like receptors or inflammation associated with pathogen-induced inflammation and a viral infection, wherein the method comprises administering to a individual in need thereof a therapeutically effective amount of a compound as defined herein.
In another aspect, the present invention is directed to a method of anti-inflammatory and antiviral treatment, wherein the method comprises administering to an individual in need thereof a therapeutically effective amount of a compound as defined herein.
In a further aspect of the invention, there is provided a kit comprising the compound as defined herein, together with instructions for treating inflammation; for treating inflammation associated with activation of Toll-like receptors; for treating a disorder selected from pneumonia, hypercytokinemia (cytokine storm syndrome), sepsis and graft-versus-host disease; for treating pathogen-induced inflammation; for the combined treatment of inflammation associated with activation of Toll-like receptors or inflammation associated with pathogen-induced inflammation and in the treatment of a viral infection; or for use as an anti-inflammatory and an antiviral. In a further embodiment, the kit may also comprise instructions for treating a viral infection, preferably a SARS-CoV or SARS-CoV-2 infection.
The following embodiments apply to all aspects of the present invention.
The pathogen may be a virus. The Toll-like receptors may be activated by the virus. Preferably, the virus is a RNA virus. More preferably, the virus is selected from. Flaviviridae, Picornaviridae, Togaviridae, Caliciviridae, Retroviridae, Coronaviridae, Orthomyxoviridae, Phlebovirus and Arenaviridae. Alternatively, the virus is a dsDNA virus, preferably selected from Herpesviridae.
Alternatively or additionally, the virus is a respiratory virus.
R3 and R4 may be independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl. R3 may be isopropyl and R4 may be hydrogen. R3 and R4 may be methyl (this compound is also designated a compound of general formula II).
R11 may be selected from hydrogen and substituted or unsubstituted C1-C6 alkyl. R11 may be methyl or isobutyl. R11 may be methyl and n=1 (this compound is also designated a compound of general formula III).
R1, R5, R9, and R15 may be independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl. R1 may be selected from sec-butyl and isopropyl, R5 may be isobutyl, R9 may be p-methoxybenzyl, and R15 may be selected from methyl and benzyl.
R8, R10, R12, and R16 may be independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl. R8, R10 and R12 may be methyl, and R16 may be hydrogen.
R6 and R14 may be independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl. R6 may be selected from hydrogen and methyl, and R14 may be hydrogen.
R7 and R13 may be independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl. R7 may be methyl and R13 may be selected from hydrogen, methyl, isopropyl, isobutyl, and 3-amino-3-oxopropyl.
R6 and R7 and/or R13 and R14 together with the corresponding N atom and C atom to which they are attached may form a substituted or unsubstituted pyrrolidine group.
R2 may be selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, and CORa, and wherein Ra may be a substituted or unsubstituted C1-C6 alkyl. R2 may be hydrogen.
R17 may be selected from hydrogen, CORa, COORa, CONHRb, (C═S)NHRb, and SO2Rc, and wherein each Ra, Rb, and Rc may be independently selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. R17 may be selected from hydrogen, COObenzyl, CObenzo[b]thiophen-2-yl, SO2(p-methylphenyl), COCOCH3 and COOC(CH3)3.
X may be NH. X may be O. Y may be CO. Y may be —COCH(CH3)CO—.
The compound may be PLD, or pharmaceutically acceptable salts or stereoisomers thereof. The compound may be PLD.
The compound may be didemninB, or pharmaceutically acceptable salts or stereoisomers thereof. The compound may be didemninB.
The inflammation may be due to COVID-19.
The CoV infection may be mild infection; and/or moderate infection; and/or severe infection.
The CoV infection may be acute CoV infection, preferably wherein the CoV infection is acute COVID-19 infection; and/or may be ongoing symptomatic CoV infection, preferably wherein the CoV infection is ongoing symptomatic COVID-19 infection; and/or may be post-CoV syndrome, CoV persistent or long CoV; preferably wherein the CoV infection is post-COVID-19 syndrome, COVID persistent or long COVID. The post-CoV syndrome, CoV persistent or long CoV may include one or more symptoms arising from the cardiovascular, respiratory, gastrointestinal, neurological, musculoskeletal, metabolic, renal, dermatological, otolaryngological, haematological and autonomic systems; psychiatric problems, generalised pain, fatigue and/or persisting fever.
The use may include use in the treatment of a patient with signs and symptoms of CoV infection (preferably COVID-19) for up to 4 weeks; and/or from 4 weeks to 12 weeks; and/or for more than 12 weeks.
The use may include use in the prophylaxis, reduction or treatment of COVID persistent, long COVID or post-COVID syndrome; preferably wherein the prophylaxis, reduction or treatment minimises the likelihood that a patient suffers from COVID persistent, long COVID or post-COVID syndrome symptoms; and/or reduces the severity of such symptoms; further preferably wherein the treatment minimising the symptoms of CoV infection.
The use may reduce the infectivity of CoV patients; including wherein the patient is asymptomatic or not very symptomatic yet has a high viral load. The use may reduce the occurrence of supercontagators (asymptomatic or not very symptomatic patients with high viral loads (e.g. TC <25)).
The use may reduce complications associated with pathogen infection, including hospitalization, ICU and death. The pathogen is preferably a CoV infection.
The use may be in the prophylaxis, reduction or treatment of COVID persistent (also known as long COVID or post-COVID syndrome).
The compound may be administered in combination with a corticosteroid, preferably dexamethasone. The compound and corticosteroid may be administered concurrently, separately or sequentially.
The compound may be administered according to a regimen of a once daily dose for 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day; preferably 2-5 days, 3-5 days, or 3, 4 or 5 days; most preferably 3 days or 5 days; most preferably 3 days.
The compound may be administered at a dose of 5 mg a day or less, 4.5 mg a day or less, 4 mg a day or less, 3.5 mg a day or less, 3 mg a day or less, 2.5 mg a day or less or 2 mg a day or less; 0.5 mg/day, 1 mg/day, 1.5 mg/day, 2 mg/day, 2.5 mg/day, 3 mg/day, 3.5 mg/day, 4 mg/day, 4.5 mg/day, or 5 mg/day; preferably 1 mg/day, 1.5 mg/day, 2 mg/day or 2.5 mg/day; preferably 1.5-2.5 mg/day; further preferably 1.5 mg/day, 2 mg/day or 2.5 mg/day.
The compound may be administered at a total dose of 1-50 mg, 1-40 mg, 1-30 mg, 1-20 mg, 1-15 mg, 3-15 mg, 3-12 mg, 4-12 mg, 4-10 mg, or 4.5-10 mg; 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg or 10 mg; preferably 4.5 mg, 5 mg, 6 mg, 7.5 mg, 8 mg, 9 mg or 10 mg; more preferably 4.5-7.5 mg/day. The total dose may be split over 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, preferably 3 days or 5 days; most preferably 3 days.
The compound may be administered at a once daily dose for 3 days at a dose of 1.5-2.5 mg/day. The dose may be 1.5 mg/day. The dose may be 2.5 mg/day.
The compound may be PLD administered as a 1.5-hour infusion, once a day for 3 consecutive days. 1.5 mg of PLD may be administered as a 1.5-hour infusion, once a day for 3 consecutive days. 2 mg of PLD may be administered as a 1.5-hour infusion, once a day for 3 consecutive days. 2.5 mg of PLD may be administered as a 1.5-hour infusion, once a day for 3 consecutive days. 1 mg of PLD may be administered as a 1.5-hour infusion, once a day for 5 consecutive days. 2 mg of PLD may be administered as a 1.5-hour infusion, once a day for 5 consecutive days.
The regimen may be a single dose (1 day). The compound may be administered as a single dose of 1-10 mg, 4-10 mg, 4.5-10 mg; 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg or 10 mg; preferably 4.5 mg, 5 mg, 6 mg, 7.5 mg, 8 mg, 9 mg or 10 mg; more preferably 5-9 mg, 6.5-8.5 mg, 7-8 mg or 7.5 mg. The compound may be PLD administered as a single dose 1.5-hour infusion.
The single dose regimen may be utilised with all therapies set out in the present invention. The combination use with corticosteroids (including subsequent corticosteroid administration) may in embodiments be used with the single dose regimen. The multi-day regimen may be utilised with all therapies set out in the present invention.
The corticosteroid may be administered daily on the same day(s) as administering a compound according to the present invention. The corticosteroid may be administered on one or more subsequent days. The corticosteroid may be administered on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more subsequent days. The corticosteroid may be administered at a higher dose when administered on the same day(s) as a compound according to the present invention and at a lower dose on subsequent days. The corticosteroid may be dexamethasone.
The compound according to the present invention may be administered at a dose according to the present invention on days 1-3 of the dosage regimen. The corticosteroid may be administered intravenously on days 1-3 of the dosage regimen. The corticosteroid may thereafter be administered by oral administration or IV from Day 4 and up to Day 10 (as per physician judgement according to patient clinical condition and evolution). The corticosteroid may be dexamethasone. The dose may be 6.6 mg/day IV on Days 1 to 3 (for example 8 mg dexamethasone phosphate), followed by dexamethasone 6 mg/day (for example 7.2 mg dexamethasone phosphate or 6 mg dexamethasone base) oral administration or IV from Day 4 and up to Day 10.
In embodiments, dexamethasone is dexamethasone phosphate and is administered at a dose of 8 mg/day IV on Days 1 to 3, followed by dexamethasone 7.2 mg/day oral administration or IV from Day 4 and up to Day 10.
The compound according to the present invention may be administered as an infusion, preferably a 1 hour infusion, a 1.5 hour infusion, a 2 hour infusion, a 3 hour infusion or longer; particularly preferably a 1.5 hour infusion.
The regimen may be 1.5 mg of plitidepsin administered as a 1.5-hour infusion, once a day for 3 consecutive days; or 2 mg of plitidepsin administered as a 1.5-hour infusion, once a day for 3 consecutive days; or 2.5 mg of plitidepsin administered as a 1.5-hour infusion, once a day for 3 consecutive days; or 1 mg of plitidepsin administered as a 1.5-hour infusion, once a day for 5 consecutive days; or 2 mg of plitidepsin administered as a 1.5-hour infusion, once a day for 5 consecutive days.
The regimen may be 7.5 mg of plitidepsin administered as a 1.5-hour infusion, as a single dose on day 1.
The compound according to the present invention may be administered using a loading dose and a maintenance dose.
The regimen according to the present invention may be:
The compound according to the present invention may be administered in combination with a corticosteroid. The corticosteroid may be administered on the same day(s) as administration of the compound.
The corticosteroid may also be administered on one or more subsequent days. For example, the corticosteroid is administered with the compound on days 1-3 and the corticosteroid is further administered on one or more of days 4-10.
The corticosteroid may be administered intravenously on days when the compound is administered but administered by oral administration or IV on subsequent days.
The corticosteroid may be dexamethasone. Dexamethasone may be administered at a dose of 6.6 mg/day IV on days when the compound is administered.
Dexamethasone may be administered at a dose of 6 mg/day oral administration or IV on subsequent days, preferably one or more of days 4, 5, 6, 7, 8, 9 and 10.
The dexamethasone dose as defined herein refers to the base weight. The dose can therefore be adjusted if used in salt form. For example, the dexamethasone may be dexamethasone phospate such that 8 mg/day is equivalent to 6.6 mg of dexamethasone base, and 7.2 mg/day is equivalent to 6 mg of dexamethasone base.
The compound according to the present invention, particularly PLD, may be administered 1.5 mg/day intravenous (IV) combined with dexamethasone 6.6 mg/day IV on Days 1 to 3, followed by dexamethasone 6 mg/day oral administration (PO)/IV from Day 4 and up to Day 10 (as per physician judgement according to patient clinical condition and evolution).
The compound according to the present invention, particularly PLD, may be administered 2.0 mg/day intravenous (IV) combined with dexamethasone 6.6 mg/day IV on Days 1 to 3, followed by dexamethasone 6 mg/day oral administration (PO)/IV from Day 4 and up to Day 10 (as per physician judgement according to patient clinical condition and evolution).
The compound according to the present invention, particularly PLD, may be administered 2.5 mg/day intravenous (IV) combined with dexamethasone 6.6 mg/day IV on Days 1 to 3, followed by dexamethasone 6 mg/day oral administration (PO)/IV from Day 4 and up to Day 10 (as per physician judgement according to patient clinical condition and evolution).
The corticosteroid may be administered 20 to 30 minutes prior to starting treatment with the compound as defined herein.
In regimens according to the present invention, the patient may additionally receive the following medications, preferably 20 to 30 minutes prior to starting treatment with the compound according to the present invention:
In regimens according to the present invention, on Days 4 and 5, patients may receive ondansetron (or equivalent) 4 mg twice a day PO.
When administered as a single dose, patients may receive the following prophylactic medications 20-30 minutes prior to plitidepsin infusion:
Ondansetron 4 mg orally may be given every 12 hours for 3 days after plitidepsin administration to relieve drug-induced nausea and vomiting. If plitidepsin is administered in the morning the patient may receive the first dose of ondansetron in the afternoon.
The invention is further described in the following non-limiting figures:
The following embodiments apply to all aspects of the present invention.
The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects or embodiment or embodiments unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
In the present application, a number of general terms and phrases are used, which should be interpreted as follows.
“Treat”, “treating”, and “treatment” in the context of a viral infection may refer to one or more of the following: 1) reduction in the number of infected cells; 2) reduction in the number of virions present in the serum, including reduction in viral titre (which can be measured by qPCR); 3) inhibition (i.e., slowing to some extent, preferably stopping) the rate of viral replication; 4) reduction in the viral RNA load; 5) reduction in the viral infectivity titre (the number of virus particles capable of invading a host cell); and 6) relieving or reducing to some extent one or more of the symptoms associated with the viral infection. This may include inflammation associated with viral infection.
“Patient” includes humans, non-human mammals (e.g., dogs, cats, rabbits, cattle, horses, sheep, goats, swine, deer, and the like) and non-mammals (e.g., birds, and the like). The patient my require hospitalisation for management of inflammation and/or infection.
Plitidepsin (PLD) is a cyclic depsipeptide originally isolated from the marine tunicate Aplidium albicans. PLD is also known as Aplidin or Aplidine. Such terms are used interchangeably herein. PLD analogues are those analogues as defined herein as compounds of Formula I, II or III. In a preferred embodiment, the present invention relates to the use of PLD.
We have found that PLD
These properties means that PLD has particular efficacy in the treatment of inflammation. In particular, we have found that PLD has particular efficacy in treating inflammation as a result of pathogen infection. As shown in the Examples, we have found that PLD can inhibit the secretion of a number of key pro-inflammatory cytokines. In particular, we have found that PLD can inhibit the transactivation of NF-kB through the Toll-like receptors (TLR) and subsequent secretion of pro-inflammatory cytokines. As explained herein, the Toll-like receptors are activated in response to a number of infectious stimuli. Binding of a TLR ligand (i.e. a stimuli) to a Toll-like receptor (TLR) triggers a downstream signalling cascade that ultimately leads to the activation of the transcription factor nuclear factor-kappa B (NF-kB), which controls induction of pro-inflammatory cytokines and chemokines. We have found that PLD significantly blocks this cascade, consequently leading to a reduction in the release or secretion of pro-inflammatory cytokines. As a result, in one example, PLD can be used to prevent inflammation following activation of the Toll-like receptors. In another example, PLD can be used to inhibit NF- kB transactivation or NF- kB-induced expression or secretion of pro-inflammatory cytokines. In another example, PLD can be used to inhibit expression and/or secretion of pro-inflammatory cytokines.
We have also found that PLD significantly reduces levels of macrophage activation and/or recruitment, particularly in response to a pathogen stimulus. Activated macrophages are a key mediator of inflammation and inhibition of activation is central to treating inflammation. Accordingly, in a further example, PLD can also be used to treat pathogen-induced inflammation, particularly by reducing macrophage activation and/or recruitment.
PLD has also been shown to bind to the human translation elongation factor eEF1A (eukaryotic translation elongation factor 1 alpha 1) with a high-affinity and a low rate of dissociation. FLIM-phasor FRET experiments on tumor cells demonstrate that PLD localises in tumor cells sufficiently close to eEF1A to suggest the formation of a drug-protein complex in living cells. PLD-resistant cell lines also show reduced levels of eEF1A protein and ectopic expression of eEF1A in these resistant cells restores the sensitivity to PLD, demonstrating that eEF1A is directly involved in the mechanism of action of PLD.
The N protein of a number of viruses, such as SARS-CoV and SARS-CoV-2, also bind to eEF1A, and this binding is essential for viral replication. However, administration and subsequent binding of PLD to eEF1A prohibits or reduces the binding of the viral N-protein to eEF1A. This in turn prevents virus replication. The interaction between PLD and EF1A could therefore reduce the efficiency of de novo viral capsid synthesis and consequently cause a decrease in viral load.
In addition to the above, PLD binding to eEF1A prevents eEF1A from interacting with its usual binding partners. One such binding-partner is the dsRNA-activated protein kinase (PKR or eIF2AK2). Binding of PLD to eEF1A releases PKR from a complex with eEF1A leading to the activation of PKR. PKR is a key player in anti-viral immune responses. Specifically,
Of note, protein 4a of viruses such as CoVs potently suppresses the activation of PKR through the sequestration of dsRNA. PLD bypasses this viral response, leading to activation of PKR by releasing PKR from the eEF1A complex, as can be seen from the activation of PKR in the absence of viral infection.
Finally - and in addition to the above, binding of PLD to eEF1A also activates the ER-stress induced unfolded protein response (UPR), which in turn leads to a number of anti-viral responses, including again the phosphorylation of eIF2α.
Through a combination of these mechanisms – (i) inhibition or reduction of the N-protein/ eEF1A interaction; (ii) activation of PKR and (iii) activation of the UPR; PLD prevents viral replication and causes the activation of host responses that lead to the elimination of the virus. Both of which contribute to an effective viral therapy. An additional advantage of targeting eEF1A is that it is a human target and as such will not mutate to evade PLD the way viral proteins do.
Accordingly, compounds of the present invention (including PLD) can be used to treat both a viral infection and any inflammation arising from the viral infection. In other words, compounds of the present invention (including PLD) as a single therapy can be used to treat two indications that arise from a viral infection – the infection itself (inhibition of viral synthesis and elimination of virus) and any (subsequently) excessive host inflammatory response – i.e. hypercytokinemia.
Accordingly, compounds of the present invention (including PLD and DidemninB), particularly PLD, can be used in the treatment inflammation, and in particular inflammation associated with either the activation of Toll-like receptors and/or inflammation as a result of pathogen infection.
The present invention may be useful in relation to the following viruses:
Flaviviridae viruses are responsible for many important diseases that affect public health worldwide. The Flaviviridae viruses include yellow fever virus (YFV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), West Nile Virus (WNV), hepatitis C virus (HCV) and dengue virus (DENV). Flaviviridae pathogens usurp cellular pathways in infected cells, generating an environment that is permissive for viral replication. Through its interaction with the 3′(+) stem loop of the viral RNA and with several replication complex proteins, eEF1A has an important role in minus-strand RNA synthesis and viral replication. Elongation factor eEF1A interacts with the 3′-terminal stem loop of the RNAs of several divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus replication complexes in infected cells. These results suggest that eEF1 A plays a similar role in RNA replication for all flaviviruses. Also characteristic of all flavivirus-caused diseases is that a fraction of the patients suffer from the exacerbation of the inflammatory response to the viral infection (cytokine storm), which eventually generates tissue damage and worsen the pathological process caused by the virus.
Dengue is caused by any of the four dengue viruses (DENV-1–4), belonging to the Flaviviridae family. DENV are small enveloped viruses containing a single-stranded (+) RNA approximately 10 kilobases in length that encodes a single polyprotein that is cleaved to produce 10 viral proteins. DENV infection is a major health problem in the tropical and subtropical regions of the world. Initial DENV infection can be asymptomatic or produce dengue fever (DF), an acute febrile disease accompanied by headache, arthralgia and rash, from which patients usually recover without complications. A second infection with a heterologous dengue virus serotype can produce the Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS) characterized by plasma leakage and abnormal bleeding that can lead to shock and death. The hallmark of severe dengue is a transient perturbation in blood vessel integrity and coagulation. Recovery is usually rapid and complete, suggesting that the key mechanisms are functional rather than structural changes in the vasculature, most likely due to effects of locally produced cytokines. Studies demonstrating exaggerated immune activation in severe dengue strongly suggest a critical role of the immune response in the pathogenesis of dengue. Both innate and adaptive immunity (and their interactions) are involved in this process. Early gene expression in peripheral blood cells of dengue patients is dominated by type I IFN-mediated response genes, in addition to type IIFN, dendritic cells and monocyte/macrophages also produce proinflammatory cytokines that can increase vascular permeability. This tsunami of cytokines over a short period induces the disruption of vascular endothelial cells and deregulation of the coagulation system, leading to plasma leakage, hemorrhage and shock.
Eukaryotic elongation factor 1A (eEF1A) is a direct activator of SphK1. DENV-2 RNA co-localizes and co-precipitates with eEF1A from infected cells. The reduction in SpbK1 activity late in DENV-2-infected cells could be a consequence of DENV-2 out-competing SpbK1 for eEF1A binding and hijacking cellular eEF1A for its own replication strategy.
Zika virus (ZIKV), the causative pathogen of Zika fever, belongs to the Flaviviridae family. ZIKV is a small enveloped ss (+) RNA virus with a genome of around 11 kilobases in length that encodes a single polyprotein that is cleaved to produce 10 viral proteins. ZIKV infections in humans were sporadic before emerging in the Pacific and the Americas in the last decade. Indeed, ZIKV infection was associated with only mild illness prior to the large French Polynesian outbreak in 2013 and 2014, when severe neurological complications were reported, and the emergence in Brazil of a dramatic increase in severe congenital malformations (microcephaly) associated with ZIKV infection during pregnancy. The majority of cases (around 80%) are asymptomatic. When symptoms occur, they are typically mild, self-limiting, and similar to other arbovirus infections (e.g., DENV and CHIKV). Commonly reported symptoms include rash, fever, arthralgia and headache. Rare deaths have been described in patients infected with Zika virus. ZIKV is now known to cause fetal infection and congenital Zika syndrome, which includes microcephaly, cerebral malformations, ophthalmological and hearing defects, and arthrogryposis.
Mild elevations in inflammatory markers have been described during ZIKV. Similarly to what happens in DENV infection, in ZIKV infection the interferon system is the central mediator of host defense and target of viral counterattack. A polyfunctional immune activation was seen during the acute phase of ZIKV infection, with elevated cytokine profiles associated with Th1 (IL-2), Th2 (IL-4, IL-13), Th17 (IL-17), and also Th9 (IL-9) responses (16). Increased IL-4, IL-6, IL-8, IL-10, and IP-10 levels are also observed in ZIKV infected patients. Although no “cytokine storm” is observed in ZIKV patients, in ZIKV-infected placentas a massive inflammation has been observed, that may hamper the success of pregnancy (17). ZIKV perturbs the pro-/anti-inflammatory equilibrium of the placenta leading to tissue damage and massive infiltration of the villous core by inflammatory Hofbauer cells.
Yellow fever (YF) is a lethal viral hemorrhagic fever (VHF) caused by the Yellow Fever Virus (YFV) belonging to the Flaviviridae family. YFV is a small enveloped ss (+) RNA virus with a genome of around 11 kilobases in length that encodes a single polyprotein that is cleaved to produce 10 viral proteins. While most YFV infections are asymptomatic or have very mild symptoms, severe YF occurs in around 12% of patients, manifesting with jaundice, hemorrhage and multi-organ failure. YFV infection is characterized by severe hepatitis, renal failure, hemorrhage, and rapid terminal events with shock and multi-organ failure.
There are three phases in the course of a Yellow fever (YF): the “infection” phase, a flu-like illness characterized by fever, headache, nausea and myalgia, starts after an incubation period of three to six days. The peak of viremia occurs during this infection phase. Then follows a “remission” period after which most patients recover. Finally, in the severe form of the disease, after this remission patients progress to the intoxication phase, in which the hemorrhagic and hepatic signs of illness occur, along with multi-organ dysfunction.
The early signs of infection are probably due to the innate immune response to infection, including the production of acute-phase reactants like interferon-α and TNF-α. In the severe form of the disease, a sudden systemic inflammatory response syndrome (‘cytokine storm’) contributes to terminal events and death. In monkeys, YF virus predominantly replicates in lymphoid tissues and this replication extends beyond the appearance of neutralizing antibodies. Lymphoid tissues undergo profound changes in YF infection and the activation of cells in these tissues contributes to the systemic terminal features of YF, characterized by release of pro-inflammatory cytokines. The terminal events occur swiftly and are characterized clinically by cardiovascular shock and multi-organ failure. The features of this phase strongly suggest that they are mediated by an inflammatory cascade, although few patients have been directly studied. In naturally acquired disease, pro- and anti-inflammatory cytokines (IL-6, IL-8, TNF-α, monocyte chemoattractant protein-1, IL-1-receptor antagonist, IL-10), resembling bacterial sepsis were significantly elevated in fatal cases. Immune clearance by antibodies and T cells result in the release of pro-inflammatory cytokines, GPCR signaling, NFκB activation, and production of oxygen free radicals, contributing to pathogenesis and cytokine storm.
West Nile virus (WNV) is an important emerging neurotropic virus, responsible for increasingly severe encephalitis outbreaks in humans and horses worldwide. WNV is a member of the Flaviviridae family and is encoded by a ~11 kb positive-sense single-stranded RNA (ssRNA) genome. The genome is translated as a single polyprotein, and subsequent cleavage of this polyprotein by viral and host proteases generates 10 viral proteins. WNV pathogenesis follow three phases, the early phase initial infection and spread (the early phase), peripheral viral amplification (the visceral-organ dissemination phase) and neuroinvasion (the central nervous system (CNS) phase).The innate immune response, including type I interferon (IFN) and innate cell-mediated responses is responsible for the early control of WNV, whereas the adaptive immune response, including humoral and adaptive immune cell mediated responses (CD4+, CD8+ and regulatory T cells), is essential for WNV clearance and limiting possible immune response-mediated damage in the later stages of infection.
The early phase after subcutaneous infection is defined by WNV replication in keratinocytes and skin-resident DCs, followed by viral amplification within the draining lymph node, which results in viremia and spread to visceral organs. The specific target cells for WNV infection are not well defined, but are thought to be subsets of DCs, macrophages and possibly neutrophils. WNV invasion of the CNS tissues (for example, the brain and spinal cord) constitutes the third phase of the infection. WNV may enter the brain though a combination of mechanisms that facilitates viral neuroinvasion, such as direct infection with or without a breakdown of the blood-brain barrier (BBB) and/or virus transport along peripheral neurons.
Innate antiviral defenses are essential for the control of WNV infection, including the production of type I IFNs and pro-inflammatory cytokines, the expression of antiviral genes and the subsequent activation of the adaptive immune response. In WNV infection the innate immune activity is mainly triggered by RIG-1 like receptor (RLR) signaling, although Toll-like receptors (TLRs) could also contribute to NF-κB activation and the production of type I interferons and pro-inflammatory cytokines. DCs and macrophages, both of which are innate immune sentinel cells and target cells of WNV infection, are pivotal in linking innate and adaptive immune responses. Macrophages and DCs are readily activated by WNV, releasing pro-inflammatory cytokines and chemokines such as type I IFN, TNF, IL-1β, CCL2, CCL3, CCL5 and IL-8. These cytokines are important in regulating innate cell-mediated responses (involving NK cells, neutrophils and γδ T cells) as well as in developing adaptive immune responses. A major hallmark of WNV pathogenesis is neuroinflammation, which is caused by exaggerated innate and acquired immune response. Accumulation of inflammatory monocytes into the brain and their differentiation to macrophages and microglia can also worsen neuroinflammation and CNS injury, as demonstrated in a murine model of nonlethal WNV infection. Recognition of WNV nucleic acid in monocytes/microglia by TLRs may lead to the production of TNF-α, which results in a loss of tight junctions, allowing the entry of WNV and immune cells into the perivascular space of the brain in mice. Thus, activation of cells of the monocyte/macrophage system by WNV appears to result in important neuropathological consequences, and exaggerated innate responses may cause inflammation, altering the blood brain barrier permeability and allowing the virus to enter the CNS. Indeed, treatment of infected neuronal cells with antibodies blocking TNF-α and other pro-inflammatory mediators results in a significant reduction of WNV-mediated neuronal death, suggesting that such mediators play a major role in the pathogenesis of WNV infection in the CNS.
Hepatitis C virus (HCV) infection causes a progressive liver disease that deteriorates from chronic inflammation to fibrosis, cirrhosis and even to hepatocellular carcinoma. HCV-caused hepatitis has an estimated prevalence of 71 million infected people and causes around 400000 deaths per year. Belonging to the Flaviviridae family, Hepatitis C Virus (HCV) is a small enveloped virus containing a single-stranded (+) RNA approximately 10 kilobases in length, which encodes a single polyprotein that is cleaved by cellular and viral proteases to generate 10 viral proteins. Upon HCV infection, hepatocytes and immune cells (macrophages, mast cells, dendritic cells and natural killer cells) recruited to the liver initiate the innate immune response, resulting in the spontaneous elimination of acute HCV infection. However, in 70%-80% of cases, the immune responses fail to eliminate the virus during the acute phase, leading to chronic infection. Persistent HCV replication in hepatocytes leads to uncontrolled inflammation and chemokine production. The excessive cytokines, as inflammatory agents, further cause inflammation in the liver, which eventually exacerbates tissue damage and liver disease progression.
Direct antiviral treatment is the first choice for HCV treatment, but antivirals alone are insufficient to block the severe inflammation and liver injury in HCV-infected individuals. HCV is just the trigger for pathophysiological processes, but persistent inflammatory cytokine storms and HCV-induced hepatocyte damage exacerbate the progression of severe liver diseases. HCV RNA elicits TLR-mediated NF-κB activation and inflammatory cytokine release, while HCV proteins activate NLRP3. The released inflammatory factors bind to their corresponding receptors and then induce NF-κB activation, leading to downstream inflammatory response. Therefore, a long-term, persistent and uncontrolled inflammatory response is a hallmark of these diseases and further leads to hepatic injury and more severe disease progression. Some patients who achieve a sustained antiviral response after direct antiviral therapy are still at a long-term risk for progression to liver cirrhosis and hepatocellular carcinoma. Therefore, coupling direct antiviral therapies with anti-inflammatory/hepatoprotective agents is a promising therapeutic regimen for HCV patients.
Tick-borne encephalitis virus (TBEV) causes a severe disease that can lead to permanent neurological conditions or even death. The severity of TBE varies depending on the viral subtype, the European (TBEV-Eu), the Siberian (TBEV-Sib), and the Far-Eastern (TBEV-FE). TBEV-Eu is the mildest variant (< 2% lethality), while TBEV-FE is the worst variant with important rates of neurological sequelae and up to 40% fatalities. TBEV is a small enveloped virus containing a single-stranded (+) RNA of approximately 11 kilobases, encoding a single polyprotein that is cleaved by cellular and viral proteases to generate 10 viral proteins. Tick-borne encephalitis (TBE) is a syndrome with a triphasic course, beginning with a flu-like illness (characterized by fever, fatigue and body pain) after which 65-70% of infected individuals clear the virus. Around one third of the patients follow with the asymptomatic disease phase, and finally the neuroinvasive phase with neurological symptoms of variable severity, ranging from meningitis to severe meningoencephalitis with or without myelitis.
Immune and none-immune mechanisms have been proposed to contribute to TBEV crossing through the blood-brain barrier (BBB) and invasion of the CNS. Cytokines may facilitate this process. Cytokines such as TNF-α and IL-6 have an impact on endothelial cell permeability that may induce a BBB disruption, leading to crossover of the virus into the CNS. TBEV-infected immune cells such as dendritic cells, neutrophils, monocytes, macrophages, and T cells could also migrate into the parenchymal compartment causing infection of neurons or other cells in the brain and the spinal cord.
TBEV infection activates type 1 IFN production through TLR3 recognition of viral dsRNA in the extracellular environment or in the cytoplasm. The IFN-α system appears to play a key role in activation of the innate immunity. It affects activation of immunocompetent cells and induction of other pro-inflammatory cytokines. In mice models, TBEV induces a cytokine storm during the terminal stage of infection, which could be considered as a cytokine storm.
Interestingly, during the phase of acute infection in TBEV-infected patients, the levels of S1P in blood and cerebrospinal fluid are highly elevated. This increase might promote a proinflammatory response. An increased production of extracellular S1P can be regulated by modulators of the S1P pathway. Indeed, elongation factor eEF1A binds to and activates SPHK to phosphorylate sphingosine and produce S1P. Plitidepsin targets eEF1A, inhibiting the production of S1P in treated cells.
Classical swine fever (CSF) remains one of the most important transboundary viral diseases of swine worldwide. It has tremendous impact on animal health and pig industry and is therefore notifiable to the World Organization for Animal Health. Classical swine fever virus (CSFV) is an small enveloped virus belongs to the Flaviviridae family. It has a single-stranded (+) RNA genome of approximately 12.3 kb which is translated into one polyprotein. Co- and post-translational processing of the precursor protein by viral and cellular proteases results in 13 mature proteins. A putative receptor for the virus in porcine cells is CD46. Classical swine fever can be divided into the following forms of the disease: an acute (transient or lethal), a chronic and a persistent course, which usually requires infection during pregnancy (34). During the first two weeks upon infection, the acute phase is characterized by unspecific (often referred to as “atypical”) clinical signs like high fever, anorexia, gastrointestinal symptoms, general weakness, and conjunctivitis. Around two to four weeks after infection neurological signs can occur including incoordination, paresis, paralysis and convulsions. At the same time, skin hemorrhages or cyanosis can appear in different locations of the body such as the ears, limbs, and ventral abdomen. These late signs are the textbook cases and are therefore referred to as “typical” CSF signs. In acute-lethal progressions, death usually occurs 2-4 weeks after CSFV infection and mortality can reach up to 100%. Chronic course occurs when an infected pig is not able to mount an adequate immune response. In general, only non-specific clinical signs are observed in infected animals like remittent fever, depression, wasting and diffuse dermatitis.
A hallmark of acute classical swine fever is the high interferon (IFN)-α levels found in the serum early after infection, followed by an inflammatory cytokine storm. Activated macrophages seem to play a crucial role in the immuno-pathogenesis of CSF. Indeed, direct damage by the virus could be almost excluded for many lesions occurring in the course of CSFV infection.
Retroviridae are a family of positive-sense single stranded RNA viruses characterised by their expression of reverse transcriptase, an RNA-dependent DNA polymerase that generates DNA from their RNA genome, which is then subsequently integrated into the host genome of infected plants. Retroviridae viruses include HIV-1 and HIV-2 which cause AIDS and HTLV (human T-lymphotropic virus). HIV infection in particular triggers an immune response that manifests as acute retroviral syndrome (ARS) and in some individuals or advanced cases of infection, an inflammatory syndrome consistent with cytokine storm syndrome.
Picornaviruses are non-enveloped, small, single-stranded (+) RNA viruses. Their small genome spans over 8 kilobases, containing an open reading frame that is translated into a singlepolypeptide which is subsequently processed by viral proteases into 11-12 individual viral proteins. The family comprises enteroviruses (EVs), hepatoviruses, parechoviruses, rhinoviruses, aphthoviruses, and cardioviruses. One of the major features of severe pathogenic diseases as a result of several picornavirus infections (type 1 diabetes, myocarditis, or paralysis) is a strong association with autoimmunity. The simplest explanation may be that infection with cytopathic infectious agents results in cell death or injury, thus releasing either sequestered or cellular autoantigens which are present at low concentrations prior to infection, thus preventing autosensitization. Since several picornaviruses, including poliomyelitis virus (PV), coxsackievirus (CVB), and foot-and-mouth disease virus, can induce persistent viral infections, chronic activation of MDA5 and upregulation of IFN-I could produce the adjuvant effect attributed by some investigators as the major contributing factor in picornavirus infections to induce autoimmunity.
Foot-and-mouth disease virus (FMDV) is the causative agent of an acute vesicular disease affecting pigs, cattle and other domestic and wild animals worldwide. FMDV, a small non-enveloped virus from the Picornaviridae family, has a single stranded (+) RNA genome of about 8.5 Kb in length encoding a single polyprotein which undergoes proteolytic processing by viral proteases to generate 14 viral proteins.
Viral infections can stimulate multiple pathways to induce type-I and type-III IFNs which have antiviral, antiproliferative, and immunomodulatory functions. Maturation of dendritic cells (DC) is promoted by IFN-I, influencing the efficacy of the adaptive immune responses induced. Some viral proteases can inhibit type-I IFN production and NF-κB signaling through the cleavage of receptors, adaptors, and regulators participating in these pathways. The IFN response is then amplified and spread to surrounding uninfected cells by the expression of hundreds of IFN-stimulated genes. MDA5 is involved in recognizing picornaviruses. The relevance of the type 1 IFN induced double-stranded RNA-dependent protein kinase R (PKR) in the inhibition of FMDV replication has been well-documented in swine and bovine cells. Treatment with an inhibitor of PKR increased the virus yield for several folds compared with that of the untreated infected cells and PKR down-regulation by RNA interference also resulted in higher viral titers, further confirming a direct function of PKR in controlling FMDV replication. Elongation factor eEF1A2 binds PKR and maintains the kinase on hold. Plitidepsin binds to eEF1A2, releasing PKR from the complex with the elongation factor and activating the kinase (48). This way plitidepsin could enhance the response against FMDV infection.
Hand, foot, and mouth disease (HFMD) is a contagious viral disease and mainly affects infants and young children. The main manifestations are fever, vesicular rashes on hand, feet and buttocks and ulcers in the oral mucosa. Usually, HFMD is self-limiting, with patients presenting fever, a maculopapular or papulovesicular rash on the hands and soles of the feet, and painful oral ulcerations that usually resolve in less than ten days. Nevertheless, a small proportion of children may experience severe complications including meningitis, encephalitis, acute flaccid paralysis and neurorespiratory syndrome, and even death. HFMD is caused by two pathogens, the enterovirus 71 (EV-A71) and the coxsackievirus A16 (CV-A16). EV-A71 is a small, icosahedral, non-enveloped, single-stranded (+) RNA virus from the Picornaviridae family. The approximately 7.4-kb genome of EV71 encodes a single polyprotein that is proteolytically cleaved to various structural and nonstructural viral proteins.
Lethal EV-A71 infections course with extensive neuronal degeneration, severe CNS inflammation and necrosis, and pulmonary congestion and hemorrhaging. A systemic inflammatory response coupled with CNS inflammation and cytokine storm may play an important role in the development of EV71-related fulminant pulmonary edema. Brain-derived proinflammatory cytokines may enter the systemic circulation after the occurrence of postencephalitis blood-brain barrier disruption to systemically activate an inflammatory cascade, thereby contributing to the development of postencephalitis systemic inflammatory response syndrome (SIRS) and subsequent cardiopulmonary failure and pulmonary edema.
Coxsackieviruses (CVs) are relatively common enteroviruses associated with a number of serious human diseases, including myocarditis and meningoencephalitis. Coxsackieviruses are small, icosahedral, non-enveloped, single-stranded (+) RNA viruses from the Picornaviridae family, with a genome of 7-8 kilobases covalently linked to the viral protein VPg, which acts as a primer for RNA synthesis. Transmission is through the fecal-oral route. Coxackieviruses can be divided into two groups, A and B. Group A viruses infect preferentially the skin and mucous membranes, causing diseases as hand, foot and mouth disease (HFMD). Group coxsackieviruses (CVB) tend to infect inner organs instead, causing pancreatitis, hepatitis, myocarditis, etc. In particular, serotype B3 viruses (CVB3) are associated with the development of myocarditis in humans.
CV is a relatively common pediatric virus, typically causing mild infections ranging from subclinical to flu-like symptoms and mild gastroenteritis. CV has been shown to infect the heart, pancreas, and CNS. In some cases CVs cause severe systemic inflammatory diseases such meningoencephalitis, pancreatitis, and myocarditis, all of which can be fatal or result in lasting organ dysfunction, including dilated cardiomyopathy and encephalomyelitis, with a mortality rate as high as 10%.
Following uptake through the host gastrointestinal tract, CVB3 infects and replicates in lymphocytes and macrophages of Peyer’s patches and the spleen. Subsequently, infectious virions are released into the bloodstream and disseminate into organs such as the heart and pancreas. The development of viral myocarditis is generally divided into three distinct phases. The first 3-4 days are the ‘acute’ phase, which is characterized by virus replication. Cell lysis produced by the virus and the recognition of PAMPs by TLRs induce the expression of proinflammatory cytokines including IL-1b, IL-6, IL-18, TNF-α, and type I and type II interferons (IFNs). These cytokines are produced by cardiac resident cells, including myocytes, fibroblasts, endothelial cells, and dendritic cells (DCs). These cytokine signals activate local macrophages and upregulate endothelial adhesion molecules as well as chemokines and chemokine receptors to collectively trigger the recruitment of innate immune cells. The subacute phase, from day 4 or 5 to approximately day 14, commences with infiltration of the heart by cells of the innate immune system. Monocytes engage in phagocytosis of dead cells and strongly augment the expression of proinflammatory cytokines. During the subacute phase, the immune response not only eliminates infected and dead cells but also significantly contributes to irreversible cardiac damage. The third stage of viral myocarditis starts after complete elimination of the virus and is characterized by cardiac repair and replacement of dead tissue by a fibrotic scar, affecting cardiac function in the long term.
Human rhinoviruses (HRVs) are responsible for more than one-half of cold-like illnesses and cost billions of dollars annually in medical visits and missed days of work. HRVs are transmitted from person to person via contact (either direct or through a fomite) or aerosol (small or large particle). HRVs belong to the family Picornaviridae and are single stranded (+) RNA viruses with a genome of around 7,200 bp encompassing a single ORF which encodes a poly-protein that is cleaved by virally encoded proteases to produce the 11 viral proteins. There are three genetically distinct HRV groups, designated groups A, B, and C.
The majority of HRV-A and -B serotypes (the major receptor group) enter airway epithelial cells via ICAM-1, a member of the immunoglobulin superfamily. HRV is infrequently associated with cytopathology of the upper respiratory tract. In addition to a direct effect on respiratory epithelial cells, the innate and adaptive host responses also have a role in the pathogenesis of HRV infection. The type I interferon (IFN) response is mediated by MDA-5 and RIG-1. These receptors maximize the induction of type I and II IFNs and proinflammatory cytokines, including RANTES, IP-10, IL-6, IL-8, and ENA-78. HRV stimulation of IL-8 production is mediated in part by an NF-κβ-dependent transcriptional activation pathway. Levels of IL-8 correlated with symptom severity (rhinorrhea and nasal obstruction) and peaked at 48 to 72 h after virus inoculation.
Hepatitis A virus (HAV) causes around 1.4 million cases of enterically transmitted hepatitis per year (WHO Fact sheet N°328, Hepatitis A, 2013), being still a source of mortality despite the existence of a successful vaccine. Unlike the rest of picornaviruses, HAV has an envelope that makes it tremendously stable and cannot shut down host protein synthesis. The cell surface molecule TIM-1 acts as a receptor for HAV. Disease severity is age-dependent, being mild or asymptomatic in young children and presenting with acute hepatitis (jaundice, fatigue, general malaise, etc) and a higher incidence of fulminant hepatitis at older ages. Fulminant hepatitis affects those aged over 50, with mortality rates up to 5.4%.
HAV is a single stranded (+) RNA pseudo-enveloped virus belonging to the Picornaviridae family. Its genome spans 7.5 kb, containing a single open reading frame that encodes for a polyprotein that is cut by viral proteases into 10 viral proteins. The RNA genome lacks a 5′ cap structure but is bound to a VPg protein.
Infection with HAV courses with initial symptoms, as fever or jaundice, in about 70% of cases, with about one third of patients remaining asymptomatic. HAV infection has no chronic carrier state and does not lead to chronic hepatitis or cirrhosis. The cytosolic RNA of picornaviruses such as HAV is sensed by MDA5 but, in contrast to HCV, HAV minimally stimulates IFN responses in the infected liver. HAV 3C protease cleaves NF-κB essential modulator (NEMO), thereby attenuating nuclear factor-κB (NF-κB) activation downstream of both MAVS and TLR3. Liver injury could not be directly caused by HAV but caused by immune-mediated mechanisms instead. Treg cells from AHA patients undergo qualitative changes resulting in the production of TNF-α. Moreover, TNF-α-producing Treg cells from AHA patients exhibit Th17-like features in terms of their phenotype and have an attenuated suppression function. Inflammatory Treg cells are associated with the immunopathological liver injury in AHA.
Togaviridae family contains only one genus, Alphaviridae, with 31 species. Alphavirus are small enveloped icosahedral viruses with a single stranded (+) RNA genome of around 11-12 kilobases. This capped RNA genome is divided into a nonstructural domain encoding the nonstructural proteins (5′-terminal two-thirds of the genome) and a structural domain encoding the three structural proteins of the virus (3′-terminal one-third of the genome). The nonstructural proteins are translated as one or two polyproteins from the genomic RNA itself. These polyproteins are cleaved to produce nsP1, nsP2, nsP3, and nsP4, as well as a number of cleavage intermediates that possess important functions distinct from the final products. The structural domain is translated as a polyprotein from a capped subgenomic mRNA, the 26S mRNA.
The alphaviruses are a serious or potential threat to human health in many areas. Eastern Equine Encephalitis Virus (EEEV) and Western Equine Encephalitis Virus (WEEV) regularly cause fatal encephalitis in humans in both North America and South America, although the number of cases is small. Venezuelan Equine Encephalitis Virus (VEEV) also causes human illness. Chikungunya Virus (CHIKV) and its close relative O’Nyong-Nyong Virus (ONNV) have caused millions of cases of serious, but not deathly, disease characterized by fever, rash, and a painful arthralgia. Ross River Virus (RRV), Barmah Forest Virus (BFV) and Sindbis Virus (SINV) cause epidemic polyarthritis in humans, with symptoms that last for years. Semliki Forest Virus (SFV) cause a disease characterized by exceptionally severe headache, fever, myalgia, and arthralgia.
Chikungunya virus (CHIKV) is the etiological agent of the mosquito-borne disease chikungunya fever, a debilitating arthritic disease that causes immeasurable morbidity and some mortality in humans, including newborn babies. CHIKV has a single-stranded (+) RNA genome of approximately 12 kilobases. This genome is organized in two open reading frames, the 5′ ORF which is translated from genomic RNA and encodes four non-structural proteins, and the 3′ ORF which is translated from a subgenomic 26S RNA and encodes for a polyprotein that is cleaved into 5 structural proteins.
Chikungunya fever (CF) has an incubation period of less than 10 days. Asymptomatic patients are less than 30%, fewer than in other alphaviruses diseases. Symptomatic patients suffer from a sudden onset of high fever, polyarthralgia, headache and fatigue. Polyarthralgia represents the most characteristic symptom, especially peripheral joints pain. Myalgia is also frequent. Cutaneous manifestation happens in half of the patients, including macular rash. A variety of other clinical symptoms have been reported during the acute stage of chikungunya fever, such as conjunctivitis, neuroretinitis, iridocyclitis, myocarditis, pericarditis, pneumonia, dry cough, lymphadenopathy, nephritis, hepatitis and pancreatitis. The reported causes of death were heart failure, multiple organ failure syndrome, toxic hepatitis, encephalitis, bullous dermatosis, respiratory failure, renal failure, pneumonia, acute myocardial infarction, cerebrovascular disease, hypothyroidism or septicemia.
The CHIKV RNA genome can trigger host pattern recognition receptors (PRRs) including endosomal Toll-like (TLR3 and TLR7) and cytoplasmic RIG-I-like (RIG-I and MDA5) receptors, which activate downstream adaptor molecules (TRIF, MyD88, and MAVS) to induce nuclear translocation of interferon-regulatory (IRF1, IRF3, IRF5, and IRF7) and NF-κB transcription factors, which induce expression of type I IFNs, IFN-stimulated genes (ISGs), and pro-inflammatory cytokines and chemokines. Inflammatory cytokines and chemokines are thought to be involved in the pathogenesis of CHIKV and are associated with severe clinical presentations as well as with development of abrupt and persistent arthralgia. A significant increase of IL-1β and IL-6 levels in severe compared with non-severe CF cases has been reported. In a deadly case described in Italy, the analysis of inflammatory cytokines revealed a remarkable and strong increase of circulating type-I IFN, as well as of the IL-6 pro-inflammatory cytokine, consistent with a possible role of type-I IFN in the CHIKV-driven cytokine storm that, in turn, may have contributed to enhanced tissue injury and death.
Sindbis virus (SinV) is the most widely distributed of all known arthropod-transmitted viruses. The Pogosta, Ockelbo and Karelian fever viruses belong to the Sindbis group. Ockelbo disease, characterized by arthritis, rash, and sometimes fever, was first recognized in Ockelbo village in central Sweden. Later on, the same disease was described in Finland as Pogosta disease. In Russia the disease was named Karelian fever. There is no evidence of any SinV-induced diseases in other vertebrates than humans. In general, the incubation time varies from 2 to 10 days, the symptomatic illness starts suddenly and the duration of the disease is short, less than 20 days. There are, however, a good proportion of SinV infections that may course with chronic illness. As with other alphaviruses, children are often infected but develop only subclinical diseases. Main symptoms are fever, muscle pain, headache, sore throat, maculopapular rash and joint pain. In human macrophages the infection promotes activation, leading to the release of macrophage migration inhibitor factor (MIF) from intracellular stores and inducing the secretion of TNF-α, IL-1β, and IL-6. In mice, SinV replication resulted in induction of high levels of TNF-α, IFN-γ, IFN-a/β, IL-6, and corticosterone in serum, two causes of Systemic Inflammatory Response Syndrome (SIRS, cytokine storm) (76). Therefore, there is a strong circumstantial case for involvement of SIRS in disease induced by virulent Sindbis virus.
Semliki Forest virus (SFV) is a single stranded (+) RNA virus of the genus Alphavirus of the family Togaviridae. The genome of SFV is 13 kilobases in length, encoding for only 9 proteins in two open reading frames (ORF). Four non-structural proteins are encoded as a polyprotein in the first ORF, located in 5′ region of the genome and directly translated from the genomic DNA. The remaining 5 structural proteins are encoded as a polyprotein in a second ORF which is translated from a subgenomic RNA species called 26S and located in the 3′ region of the genome. Polyproteins are then cut by viral proteases into individual proteins.
The original L10 isolate of SFV shows complete virulence in inbred mouse strains, causing lethal encephalitis by infection of the central nervous system (CNS). A critical difference between virulent and nonlethal strains could be the swift induction of neuronal damage in the CNS, causing the death of infected mice before the immune system can intervene. Infection with nonlethal SFV strains induces inflammatory spongiform lesions in mouse brain. Those lesions are delayed in mice treated with the immunosuppressive drug cyclophosphamide (Berger ML, 1980). Therefore, direct viral cytolysis is not essential in the production of pathology. The fact that nude mice suffer the same inflammatory spongiform lesions although lacking functional T cells indicates that the immunopathology does not depend on them. Instead, the lesions seem to be due to the presence of mononuclear cells from the monocyte-macrophage lineage, which can be recruited into the infected tissue and produce spongiform necrosis.
Three viruses in the genus Alphavirus (Togaviridae family), Eastern, Western and Venezuelan equine encephalitisviruses (EEEV, WEEV and VEEV, respectively), can cause severe outbreaks of encephalitis in equines and humans. EEEV was first identified in humans in 1938 during an important outbreak affecting Massachusetts. Most EEEV infections in adult humans are asymptomatic or produce a mild illness characterized by fever, chills, headache, nausea, vomiting and myalgia. The fatality rate in symptomatic patients approaches 80%, many survivors exhibiting important sequelae as mental retardation, convulsions, and paralysis. Lethality of WEEV ranges between 3 and 7% with 15-50% of the encephalitis survivors, especially young children, suffering permanent neurological damage. VEEV-associated encephalitis has caused serious morbidity and mortality in outbreaks in Columbia and Venezuela, with more than 75000 affected people, over 3000 patients suffering neurologic conditions and over 300 deaths.
As the rest of alphaviruses, EEEV, WEEV and VEEV are single stranded (+) RNA viruses with a short, capped genome of around 11.7 kilobases. The genome has two ORFs, the 5′ ORF encoding four non-structural proteins which are translated as a single polypeptide from genomic RNA, and the 3′ ORF which is translated from a subgenomic 26S RNA as a single polypeptide containing three structural proteins. Polypeptides are cut into individual proteins by viral proteases.
In experimentally infected laboratory mice, EEEV produces neurologic disease that resembles that following human and equine infections. Virus is detected in the brain on day 1 postinfection (PI), and signs of disease are evident on days 3 to 4 PI. Clinical signs of murine disease include ruffled hair, anorexia, vomiting, lethargy, posterior limb paralysis, convulsions, and coma. Pathological changes in infected mice include neuronal degeneration, cellular infiltration, and perivascular cuffing, similar to the CNS pathological changes described in naturally infected humans. VEEV infects dendritic cells (DCs) and macrophages in lymphoid tissues, fueling a serum viremia and facilitating neuroinvasion. In contrast, EEEV replicates poorly in lymphoid tissues.
Rubella virus (RV) infection of children and adults is usually asymptomatic (up to 50% of cases) or characterized by a mild fever, sore throat, lymphadenopathy and a maculopapular rush. However, when infecting pregnant women, RV causes birth defects and fetal death. RV is a major public health concern since more than 100,000 cases of congenital rubella syndrome (CRS) occur every year. Rubella virus (RV), the sole member of the rubivirus genus within the Togaviridae family, has a single stranded (+) RNA genome of around 10 kilobases that encodes for five proteins. The genome contains two ORFs, 5′ ORF which encodes two nonstructural proteins directly translated from the genomic RNA and a second 3′ ORF which encodes three structural proteins translated from a subgenomic mRNA.
RV infection during critical stages of organ development in fetus (in the first trimester of pregnancy) can result in CRS, a syndrome characterized by severe defects in sensory organs (eyes and ears), central nervous system and cardiovascular system. In CRS, rubella virus is able to infect the placenta, spread to the fetus, and alter the function of multiple fetal systems by interfering with organ formation and causing systemic inflammation. Rubella virus-associated uveitis (RVU) is one of the inflammatory syndromes caused by RV. This inflammatory process is characterized by high intraocular levels of inflammatory cytokines, including IL-6, IL-6ra and MCP-1.
Caliciviridae family is composed by 11 genera, with seven genera (Lagovirus, Norovirus, Nebovirus, Recovirus, Sapovirus, Valovirus and Vesivirus) infecting mammals, two genera (Bavovirus and Nacovirus) infecting birds and two genera (Minovirus and Salovirus) infecting fish. Caliciviruses cause species-specific infections, with most noroviruses, sapoviruses and neboviruses restricted to the gastrointestinal tract, while lagoviruses, saloviruses and vesiviruses can cause severe systemic infections in their natural hosts. The family includes non-enveloped viruses with single stranded (+) RNA genomes of around 7-8 kilobases, organized in two, three or four ORFs. Caliciviruses are similar to picornaviruses in the presence of VPg protein attached to the 5′ terminus of their genome and the in sequence of several proteins.
Norovirus (NoV) is a genus of the Caliciviridae family composed by a single species, Norwalk virus. Human norovirus is a major cause of epidemic and sporadic acute gastroenteritis worldwide, and can chronically infect immunocompromised patients. NoV infection generates typical gastroenteritis symptoms, including diarrhea, vomiting, and stomach pain, and may also provoke fever, headache and body aches. Novoviruses are non-enveloped viruses with a single stranded (+) RNA genome of around 7.5 kilobases. The genome is organized into three or four ORFs. The 5′ ORF1 encodes a large polyprotein that is cleaved by viral proteases into 6 nonstructural proteins. ORF2 encodes a single structural capsid protein. ORF3 encodes for a minor structural protein. A subgenomic RNA encodes for two additional structural proteins. Finally, ORF4 (which overlaps with ORF2) in murine novovirus encodes for an additional protein, the virulence factor 1. The 5′ ends of NoV genomic and subgenomic RNAs are covalently linked to a small virus-encoded protein known as VPg and the 3′ ends are polyadenlyated.
Compelling evidence has accumulated in recent years demonstrating that a major target of NoVs is antigen presenting cells (APCs). MuNoVs efficiently replicate in primary dendritic cells and macrophages, as well as a number of macrophage-like cell lines, in vitro. The ability of NoVs to infect intestinal immune cells undoubtedly has a significant impact on NoV pathogenesis and the host immune response to NoV infection. Consistent with the short duration of NoV symptoms, innate immunity - and in particular type I IFNs - is critical for controlling acute NoV infections.
Coronaviruses are pathogens that can infect animal and human hosts, mostly causing mild to severe enteric or respiratory diseases that can be life threatening in some cases, as with MERS-CoV, SARS-CoV or the new SARS-CoV-2, this latter causing the COVID-19 disease which recently originated in Wuhan, Hubei province, China. Both SARS-CoV-2 and the closely related SARS-CoV belong to the subgenus Sarbecoviridae, within the genus Betacoronaviridae (to which also MERS-CoV belongs), one of the four genus of Coronaviridae. Coronaviruses are enveloped, single stranded (+) RNA viruses with genomes of around 26-32 kilobases in size, the largest known genome for an RNA virus. They were identified for the first time in 1964, by June Almeida. Their name comes from the similarity their electron microscope images share with the solar corona. Their genome encodes for 14 open reading frames, i.e., the replicase ORFs 1a and 1b (which encode for 15 or 16 mature nonstructural proteins also known as replicase proteins), the four common CoV structural protein genes (S, E, M, and N) and the ORFs encoding so-called accessory proteins. The replicase proteins seem to have multiple functions, being essential for the synthesis or processing of viral RNA, but also to stablish virus-host interactions to facilitate viral entry, gene expression, RNA synthesis or virus release. The CoV N proteins are structural proteins of 350 to 450 amino acids, broadly phosphorylated in serine residues, highly basic, and its main function is to associate with viral RNA to form a long helical ribonucleoprotein. N proteins are also involved in viral RNA transcription, translation, and virus exit. The N protein of SARS-CoV shares a high homology with its SARS-CoV-2.
MERS-CoV, SARS-CoV and SARS-CoV-2 can cause acute respiratory distress syndrome (ARDS), the most acute and fatal stage of the disease, characterized by wide-spread inflammation in the lungs resulting from the aberrant immune response to the viral infection.
Severe Acute Respiratory Syndrome (SARS), caused by SARS-CoV, started in Guangdong province of China in 2002 and affected 8,096 people worldwide, resulting in 774 deaths (10% mortality rate). The genome of SARS-CoV comprises 29.7 kilobases, containing 11 ORFs that encode for 23 proteins.
The most common symptoms of SARS are high fever, myalgia, dry cough, and lymphopenia. Around 30% of patients also developed an atypical form of pneumonia, with acute respiratory distress resulting from acute lung damage. Several factors including advanced age, male sex, a high peak lactate dehydrogenase level, a high peak creatine kinase level, and a high initial absolute neutrophil count were significant predictive factors for intense care unit admission and death. An excessive inflammatory response in the lungs explains the development of acute lung injury, moreover when patients still manifest lung injury at a time when the viral load is falling.
Acute viral infection might produce damage to host cells by direct cytopathy or by indirect immunopathological damage. In the early stage, cytopathy is accompanied by viral amplification, time at which the treatment with antiviral drugs may be especially effective. In the later stage, when an adaptive immune response is mounted, viral clearance can be accompanied by severe inflammatory damage. An IFN-γ-related cytokine storm may happen in this latter phase of SARS coronavirus infection, and this cytokine storm might be involved in the immunopathological damage in SARS patients.
Middle East respiratory syndrome (MERS), caused by MERS-CoV, started in Saudi Arabia in 2012 and affected 2506 people, causing 862 deaths worldwide with a 35% mortality rate. The genome of MERS-CoV is 30.1 kilobases in length, encompassing 10 ORFs that encode for 16 nontstructural proteins and 5 structural proteins.
The most common symptoms of MERS are fever, cough, chills, sore throat, myalgia, and arthralgia, followed by dyspnoea and rapid progression to pneumonia within the first week, often requiring ventilatory and other organ support. While younger individuals experience mild-moderate clinical illness, elderly individuals or those with comorbid conditions such as diabetes, obesity, heart failure, and renal failure among others experience severe disease after infection with MERS-CoV. In its most severe form, MERS causes an acute highly lethal pneumonia. Individuals with severe MERS show elevated levels of serum pro-inflammatory cytokines (IL-6 and IFN-α) and chemokines (IL-8, CXCL-10, and CCL5) compared to those with mild to moderate disease. High serum cytokine and chemokine levels in MERS patients correlated with increased neutrophil and monocyte numbers in lungs and in the peripheral blood, being those cells the predominant source of cytokines and chemokines associated with the lethal acute respiratory distress syndrome that kills MERS patients.
COVID-19, caused by SARS-CoV-2, started in Wuhan city in China in December 2019, quickly spreading throughout the world and growing into a global pandemic. Mortality rate is still uncertain, although it seems smaller than in SARS and MERS diseases. SARS-CoV-2 genome encodes for 14 open reading frames, including the replicase ORFs 1a and 1b (which encode for 15 or 16 mature nonstructural proteins also known as replicase proteins), the four common CoV structural protein genes (S, E, M, and N) and the ORFs encoding so-called accessory proteins. The replicase proteins seem to have multiple functions, being essential for the synthesis or processing of viral RNA, but also for other steps of the virus life cycle. The N protein is a broadly phosphorylated, highly basic structural protein, whose main function is to associate with viral RNA to form a long helical ribonucleoprotein. N protein is also involved in viral RNA transcription, translation, and virus exit. The N protein of SARS-CoV and SARS-CoV-2 share a high homology. Several cellular proteins collaborate with viral proteins to support viral proliferation, among them eEF1A, which interacts with N protein to support viral replication and spreading.
Common mild symptoms of the early stage of infection are fever, dry cough, myalgia and fatigue. Less common are headache, hemoptysis and diarrhea. In the transition to the severe stage of the disease, symptoms such as dyspnea and hypoxia develop. Finally, the severe form of the disease manifests with Acute Respiratory Distress Syndrome (ARDS), together with severe lung inflammation and damage. Complications at the severe stage of disease associate with virus-induced hyperinflammation, similar to what has been previously seen in SARS-CoV and MERS-CoV infections.
In SARS-CoV-2, macrophages present CoV antigens to Th17 cells, leading to a massive release of pro-inflammatory cytokines such as IL-1, IL-6, IL-8, IL-21, TNF-b and MCP-1. This is responsible for immune amplification and is partly responsible for the tissue damage in the respiratory alveoli, bronchioles and pulmonary interstitial walls. In addition, the increased expression of the inflammatory mediators has a down regulatory effect on NK cells and CD8 cells, which are also important to clear the virus (Kaul. D (2020)). As such, in SARS-CoV-2 infections, hypercytokinemia has emerged as a main factor driving a more severe clinical outcome.
In relation to SARS-CoV-2, the present invention may be treating SARS-CoV-2 infection. The treatment may be treating COVID-19 infection. The treatment may be the treatment of COVID-19. The treatment may be the treatment of a disease that results from infection by CoV. The treatment may be the treatment of a disease that results from infection by SARS-CoV-2. The treatment may be the treatment of pneumonia caused by infection by CoV. The treatment may be the treatment of pneumonia caused by infection by SARS-CoV-2. The treatment may be the treatment of pneumonia caused by infection by COVID-19. The treatment may be the treatment of pneumonia caused by COVID-19. The treatment may be the treatment of acute respiratory syndrome (ARDS) caused by infection by SARS-CoV-2.
The infection may be moderate infection. The infection may be severe infection. The infection may be mild infection.
The treatment may be reducing complications associated with CoV infection, including hospitalization, ICU and death.
The present invention may be useful to treat acute COVID-19 infection (signs and symptoms of COVID-19 for up to 4 weeks); treat (or miniminse) ongoing symptomatic COVID-19 (signs and symptoms of COVID-19 from 4 weeks up to 12 weeks); or treat or minimise post-COVID-19 syndrome (signs and symptoms that develop during or following an infection consistent with COVID-19, continue for more than 12 weeks and are not explained by an alternative diagnosis. It usually presents with clusters of symptoms, often overlapping, which can fluctuate and change over time and can affect any system in the body. Post-COVID-19 syndrome may be considered before 12 weeks while the possibility of an alternative underlying disease is also being assessed). The compounds of the present invention may treat a patient with signs and symptoms of COVID-19 for up to 4 weeks. The compounds of the present invention may treat a patient with signs and symptoms of COVID-19 from 4 weeks to 12 weeks. The compounds of the present invention may treat a patient with signs and symptoms of COVID-19 for more than 12 weeks.
The treatment may be prophylaxis, reduction or treatment of COVID persistent (also known as long COVID or post-COVID syndrome). The compounds according to the present invention can minimise the likelihood of a patient suffering from COVID persistent symptoms. The compounds according to the present invention may alternatively reduce the severity of such symptoms, preferably may minimise the symptoms of CoV infection.
Post-COVID syndrome may be considered as signs and symptoms that develop during or following an infection consistent with COVID-19 which continue for more than 12 weeks and are not explained by an alternative diagnosis. The condition usually presents with clusters of symptoms, often overlapping, which may change over time and can affect any system within the body. Many people with post-COVID syndrome can also experience generalised pain, fatigue, persisting high temperature and psychiatric problems. Symptoms include (but are not limited to) symptoms arising in the cardiovascular, respiratory, gastrointestinal, neurological, musculoskeletal, metabolic, renal, dermatological, otolaryngological, haematological and autonomic systems, in addition to psychiatric problems, generalised pain, fatigue and persisting fever.
The treatment may be reducing the infectivity of CoV patients. The present invention achieves a rapid and significant reduction in the viral burden. Reducing the viral burden may reduce the infectiveness of patients. This is particular beneficial with patients who are asymptomatic or not very symptomatic yet have a high viral loads (e.g. TC <25). Such patients may be supercontagators or superspreaders. Administration of compounds according to the present invention upon detection of infection can reduce the viral burden and therefore reduce the infectiveness of the patient.
The treatment may result in a reduction of viral load. This may be expressed as a replication cycle threshold (Ct) value greater than 30 (Ct> 30), on day 6 after the administration. The treatment may reduce viral load from baseline. This may be expressed as a reduction in the percentage of patients requiring hospitalisation following administration. This may be expressed as a reduction in the percentage of patients requiring invasive mechanical ventilation and / or admission to the ICU following administration. This may be expressed as a reduction of patients who develop sequelae related to persistent disease. This may be expressed as an increase in the percentage of patients with normalization of analytical parameters chosen as poor prognosis criteria (including, for example, lymphopenia, LDH, D-dimer or PCR). This may be expressed as an increase in the percentage of patients with normalization of clinical criteria (disappearance of symptoms), including, for example: headache, fever, cough, fatigue, dyspnea (shortness of breath), arthromyalgia or diarrhoea.
Phleboviruses are negative or ambisense single-stranded, enveloped RNA viruses from the Bunyavirus family with genomes in the range of approximately 11-12 kb. Phleboviruses can be broadly divided into two groups, the sandfly fever virus group (SFV) and the Uukuniemi-like virus (ULV) group. SFVs are transmitted by dipterans, including phlebotomines and mosquitoes, and are endemic in the Mediterranean and other regions. Symptoms of SFV infection include fever, headache, muscle and joint pain, fatigue, and abdominal pain. Patients typically recover within 7-14 days. In some cases, infection of the CNS can lead to encephalitis and meningitis. The ULV group is transmitted by ticks but they are non-pathogenic in humans.
The best-studied phlebovirus is Rift Valley fever (RVF) virus, which predominantly infects domesticated animals but it can also cause moderate-to-severe infection in humans. Human outbreaks of RVF have occurred in sub-Saharan Africa and Arabia via infected animals or mosquitoes. Symptoms are usually mild, flu-like and may include a biphasic fever. A small proportion of patients develop serious complications, such as hemorrhagic fever, encephalitis or retinitis. Neurological disease is a delayed-onset, often fatal, complication, which may occur 5-30 days after the initial illness. During the 2000 Saudi outbreak, there was a 50% mortality rate for patients with CNS complications.
The interaction of viral proteins with the host immune response may play a role in pathogenicity and may explain why the clinical presentation of RVF varies between individuals. Chemokines, pro-inflammatory cytokines and anti-inflammatory cytokines were significantly increased (IL-8, CXCL9, MCP-1, IP-10, IL-10) or decreased (RANTES) in fatal cases during the 2010/2011 South African outbreak. In three Ugandan patients with severe hemorrhagic infection, levels of IL-8, IL-10, IP-10, MCP-2, MCP-3, fractalkine and GRO-α were increased, with MCP-2 and IP-10 levels correlating with viral load. The elevated levels of pro-inflammatory markers in the three Ugandan patients normalised as patients recovered. Furthermore, genetic polymorphisms in innate immune pathways, including IL-6, may be associated with the varying severity of RVFV symptoms. The host inflammatory response may affect the outcome of RVFV infection in humans and an uncontrolled inflammatory response may contribute to fatal outcomes.
Arenaviruses (AVs) are a family of enveloped, pleomorphic, single-stranded RNA viruses, with an approximate genome size of 11 kb spanning a small and large segment, with each segment encoding for two proteins in an ambisense manner. Transmission to humans occurs by exposure to rodents. Some arenaviruses can cause mild-to-severe illness in humans, including haemorrhagic illness. After a long incubation period of 6-21 days, AV infection usually starts with fever, general weakness and malaise, followed by headache, sore throat, nausea, vomiting, diarrhea, and abdominal pain. In more severe cases, symptoms may include hemorrhaging, shock, seizures, coma and even death.
AVs can be divided into two serogroups, ‘Old World’ and ‘New World’. ‘Old World’ viruses include Lassa fever and lymphocytic choriomeningitis virus. Lassa fever is endemic in Western African regions, causing approximately 5,000 deaths per year. Lassa fever causes systemic disease but the majority of infections are asymptomatic. Severe disease caused by viral haemorrhagic fever occurs in 20% of individuals. The overall fatality is about 1%. For patients admitted to hospital, the mortality rate is 15%- 25%. Fatal cases in humans are characterized by necrosis in the liver, spleen and adrenal glands.
‘New World’ viruses include Pichinde virus and the Argentine, Bolivian and Venezuelan haemorrhagic fevers. Pichinde virus is non-pathogenic in humans. South American hemorrhagic fever has a 6–14-day prodromic phase. After 8 –12 days, approximately 20–30% of patients progress to the neurologic and hemorrhagic phase. Symptoms include confusion, convulsions, coma and bleeding from body orifices. Case fatality rates within endemic regions can be 10–30%.
Arenaviruses initially target macrophages and dendritic cells in humans, which produce high levels of interferons and cytokines. Infection of these antigen-presenting cells is likely responsible for spreading the virus, establishing systemic infections and allowing for significant viral product and release. Infection with Lassa virus suppresses the type I interferon response, which is critical for controlling the infection, leading to uncontrolled virus production. In contrast, infection with ‘New World’ arenaviruses is associated with high levels of interferons and cytokines in severe and fatal cases. Indeed, patients infected with Junin virus, the causative agent of Argentine haemorrhagic fever, can develop a cytokine storm and show increased levels of TNF-α, IFN-α, IL-6 and IL-10. Patients with severe illness and fatal cases consistently show elevated levels of TNF-α and IFN-α.
Herpesviruses (HSVs) are large, enveloped, double-stranded DNA viruses with complex genomes. HSV genomes range in size from 125-240 kbp and encode for dozens of viral genes. Transmission of HSVs is via skin or oral contact between infected humans, with the main route being sexual transmission. Symptoms of herpes include painful blisters or ulcers at the site of infection, but most infections are asymptomatic. As such, HSVs are very prevalent in humans and animals, with an estimated 90% of the world’s population is infected with either one or both of herpes simplex virus type-1 (HSV-1) and herpes simplex virus type-2 (HSV-2). HSV-1 is associated with orofacial infections, while HSV-2 can cause genital infections. Infants can also be infected with HSV-2 during childbirth, causing serious disease of the central nervous system or disseminated infection.
Infection with HSVs is incurable due to their ability to establish latent infection in host cells. Following uptake at the primary infection site, HSVs can establish latency in neuronal cells. For example, HSV-1 can establish latency in trigeminal ganglia, while HSV-2 can establish latency in sacral ganglia. During latency, the viral genome may become integrated into host cell DNA or it may remain extra-chromosomal. Latency-associated RNA transcripts are expressed which regulate the host cell genome and suppress host cell death mechanisms, preserving the viral infection of host cells and allowing subsequent recurrences of non-latent periods. The non-latent periods are usually symptomatic and allow the virus to infect a new host.
In some cases, HSV infection can lead to encephalitis. HSV encephalitis begins with flu-like symptoms and progresses into neurological deterioration that can be fatal if left untreated. Furthermore, in neonates with severe disseminated infection, the systemic inflammatory response to viral HSV infection can lead to organ damage and dysfunction, which may lead to death.
The family Orthomyxoviridae is a family of negative-sense RNA viruses and contains significant pathogens of both humans and animals. There are seven genera in the family, including Influenzavirus A, Influenzavirus B, Influenzavirus C, Thogotovirus, Quaranjavirus, and Isavirus
There are four classes of influenza viruses: A, B, C and D, with influenza virus A and B in particular causing winter epidemics of flu, causing around 300 to 650 thousand deaths a year. Influenza A viruses are of particular clinical significance as they have been the cause of a number of flu pandemics where many countries have been affected by large outbreaks. Influenza A viruses are are negative-sense, single-stranded, segmented RNA viruses that are divided into subtypes based on two proteins on the surface of the virus: hemagglutinin (H) and neuraminidase (N). There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes (H1 through H18 and N1 through N11, respectively), therefore there are many subtypes of influenza virus that may be circulating in the population at any given time, causing the disease known as flu.
Flu is characterized by a mild to severe disease which symptoms include high fever, runny nose, sore throat, muscle and joint pain, headache, coughing and tiredness, although vomiting and diarrhea can also occur in children infected with the virus. These symptoms typically appear one to four days after exposure and are generally self-limiting, however in some cases, particularly in those with weaker immune systems, complications such as pneumonia and sepsis can occur.
Severe influenza infection has been associated with significant pathological changes in pulmonary tissues associated with heightened levels of inflammatory cytokines and chemokines. This exuberant immune response, known as the cytokine storm, is associated with high levels of pro-inflammatory cytokines and widespread tissue damage. In fact, the term “cytokine storm” was first coined when describing the immune response to influenza-associated encephalopathy. It is thought that influenza infection of epithelial cells in the respiratory tract leads to a wave of inflammatory cytokine production from these cells, driving various interferon-regulated genes that go on to cause further downstream cytokine production through activation of innate immune cells such as macrophages, neutrophils and dendritic cells which, in severe cases, can proceed to cause tissue damage and chronic inflammation. It is this positive feedback loop of inflammation that can result in complications relating to influenza infection, and can ultimately result in death for patients most severely affected.
In compounds of the present invention; the groups can be selected in accordance with the following guidance:
Alkyl groups may be branched or unbranched, and preferably have from 1 to about 12 carbon atoms. One more preferred class of alkyl groups has from 1 to about 6 carbon atoms. Even more preferred are alkyl groups having 1, 2, 3 or 4 carbon atoms. Methyl, ethyl, n-propyl, isopropyl and butyl, including n-butyl, tert-butyl, sec-butyl and isobutyl are particularly preferred alkyl groups in the compounds of the present invention. As used herein, the term alkyl, unless otherwise stated, refers to both cyclic and noncyclic groups, although cyclic groups will comprise at least three carbon ring members.
Preferred alkenyl and alkynyl groups in the compounds of the present invention may be branched or unbranched, have one or more unsaturated linkages and from 2 to about 12 carbon atoms. One more preferred class of alkenyl and alkynyl groups has from 2 to about 6 carbon atoms. Even more preferred are alkenyl and alkynyl groups having 2, 3 or 4 carbon atoms. The terms alkenyl and alkynyl as used herein, unless otherwise stated, refer to both cyclic and noncyclic groups, although cyclic groups will comprise at least three carbon ring members.
Suitable aryl groups in the compounds of the present invention include single and multiple ring compounds, including multiple ring compounds that contain separate and/or fused aryl groups. Typical aryl groups contain from 1 to 3 separated or fused rings and from 6 to about 18 carbon ring atoms. Preferably aryl groups contain from 6 to about 10 carbon ring atoms. Specially preferred aryl groups include substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl, substituted or unsubstituted phenanthryl, and substituted or unsubstituted anthryl.
Suitable heterocyclic groups include heteroaromatic and heteroalicyclic groups containing from 1 to 3 separated or fused rings and from 5 to about 18 ring atoms. Preferably heteroaromatic and heteroalicyclic groups contain from 5 to about 10 ring atoms, most preferably 5, 6 or 7 ring atoms. Suitable heteroaromatic groups in the compounds of the present invention contain one, two or three heteroatoms selected from N, O or S atoms and include, e.g., coumarinyl including 8-coumarinyl, quinolyl including 8-quinolyl, isoquinolyl, pyridyl, pyrazinyl, pyrazolyl including pyrazol-3-yl, pyrazol-4-yl and pyrazol-5-yl, pyrimidinyl, furanyl including furan-2-yl, furan-3-yl, furan-4-yl and furan-5-yl, pyrrolyl, thienyl, thiazolyl including thiazol-2-yl, thiazol-4-yl and thiazol-5-yl, isothiazolyl, thiadiazolyl including thiadiazol-4-yl and thiadiazol-5-yl, triazolyl, tetrazolyl, isoxazolyl including isoxazol-3-yl, isoxazol-4-yl and isoxazol-5-yl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indolizinyl, phthalazinyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, pyridazinyl, triazinyl, cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzothiophenyl including benzo[b]thiophen-2-yl and benzo[b]thiophen-3-yl, benzothiazolyl, benzoxazolyl, imidazo[1,2-a]pyridinyl including imidazo[1,2-a]pyridine-2-yl and imidazo[1,2-a]pyridine-3-yl, quinazolinyl, quinoxalinyl, naphthyridinyl and furopyridyl. Suitable heteroalicyclic groups in the compounds of the present invention contain one, two or three heteroatoms selected from N, O or S atoms and include, e.g., pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydrothiopyranyl, piperidinyl including piperidin-3-yl, piperidin-4-yl and piperidin-5-yl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydropyrrolyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, 3H-indolyl, and quinolizinyl.
In the above mentioned groups one or more hydrogen atoms may be substituted by one or more suitable groups such as OR′, =O, SR′, SOR′, SO2R′, NO2, NHR′, NR′R′, ═N—R′, NHCOR′, N(COR′)2, NHSO2R′, NR′C(=NR′)NR′R′, CN, halogen, COR′, COOR′, OCOR′, OCONHR′, OCONR′R′, CONHR′, CONR′R′, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group, wherein each of the R′ groups is independently selected from the group consisting of hydrogen, OH, NO2, NH2, SH, CN, halogen, COH, COalkyl, CO2H, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list. When a substituent group terminates with a double bound (such as =O and ═N—R′) it replaces 2 hydrogen atoms in the same carbon atom.
Suitable halogen substituents in the compounds of the present invention include F, Cl, Br and I.
The term “pharmaceutically acceptable salts” refers to any salt which, upon administration to the patient is capable of providing (directly or indirectly) a compound as described herein. It will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the invention since those may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts can be carried out by methods known in the art. For instance, pharmaceutically acceptable salts of compounds provided herein are synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two. Generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of the acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate. Examples of the alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, N,N-dialkylenethanolamine, triethanolamine and basic amino acids salts.
The compounds of the invention may be in crystalline form either as free compounds or as solvates (e.g. hydrates, alcoholates, particularly methanolates) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art. The compounds of the invention may present different polymorphic forms, and it is intended that the invention encompasses all such forms
Any compound referred to herein is intended to represent such specific compound as well as certain variations or forms. In particular, compounds referred to herein may have asymmetric centres and therefore exist in different enantiomeric or diastereomeric forms. Thus any given compound referred to herein is intended to represent any one of a racemate, one or more enantiomeric forms, one or more diastereomeric forms, and mixtures thereof. Likewise, stereoisomerism or geometric isomerism about the double bond is also possible, therefore in some cases the molecule could exist as (E)-isomer or (Z)-isomer (trans and cis isomers). If the molecule contains several double bonds, each double bond will have its own stereoisomerism, that could be the same or different than the stereoisomerism of the other double bonds of the molecule. Furthermore, compounds referred to herein may exist as atropisomers. All the stereoisomers including enantiomers, diastereoisomers, geometric isomers and atropisomers of the compounds referred to herein, and mixtures thereof, are considered within the scope of the present invention.
In compounds of general formula I and II, particularly preferred R1, R5, R9, R11, and R15 are independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl. More preferred R1, R5, R9, R11, and R15 are independently selected from hydrogen, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted n-propyl, substituted or unsubstituted isopropyl and substituted or unsubstituted butyl, including substituted or unsubstituted n-butyl, substituted or unsubstituted tert-butyl, substituted or unsubstituted isobutyl, and substituted or unsubstituted sec-butyl. Preferred substituents of said groups are OR′, =O, SR′, SOR′, SO2R′, NO2, NHR′, NR′R′, ═N—R′, NHCOR′, N(COR′)2, NHSO2R′, NR′C(=NR′)NR′R′, CN, halogen, COR′, COOR′, OCOR′, OCONHR′, OCONR′R′, CONHR′, CONR′R′, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group, wherein each of the R′ groups is independently selected from the group consisting of hydrogen, OH, NO2, NH2, SH, CN, halogen, COH, COalkyl, CO2H, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list. Hydrogen, methyl, n-propyl, isopropyl, isobutyl, sec-butyl, 4-aminobutyl, 3-amino-3-oxopropyl, benzyl, p-methoxybenzyl, p-hydroxybenzyl, and cyclohexylmethyl are the most preferred R1, R5, R9, R11, and R15 groups. Specifically, particularly preferred R1 is selected from sec-butyl and isopropyl, being sec-butyl the most preferred. Particularly preferred R5 is selected from isobutyl and 4-aminobutyl, being isobutyl the most preferred. Particularly preferred R11 is methyl and isobutyl. Particularly preferred R9 is selected from p-methoxybenzyl, p-hydroxybenzyl, and cyclohexylmethyl, being p-methoxybenzyl the most preferred. Particularly preferred R15 is selected from methyl, n-propyl, and benzyl, being methyl and benzyl the most preferred.
In compounds of general formula III, particularly preferred R1, R5, R9, and R15 are independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl. More preferred R1, R5, R9, and R15 are independently selected from hydrogen, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted n-propyl, substituted or unsubstituted isopropyl and substituted or unsubstituted butyl, including substituted or unsubstituted n-butyl, substituted or unsubstituted tert-butyl, substituted or unsubstituted isobutyl, and substituted or unsubstituted sec-butyl. Preferred substituents of said groups are OR′, =O, SR′, SOR′, SO2R′, NO2, NHR′, NR′R′, =N-R′, NHCOR′, N(COR′)2, NHSO2R′, NR′C(=NR′)NR′R′, CN, halogen, COR′, COOR′, OCOR′, OCONHR′, OCONR′R′, CONHR′, CONR′R′, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group, wherein each of the R′ groups is independently selected from the group consisting of hydrogen, OH, NO2, NH2, SH, CN, halogen, COH, COalkyl, CO2H, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list. Hydrogen, methyl, n-propyl, isopropyl, isobutyl, sec-butyl, 4-aminobutyl, 3-amino-3-oxopropyl, benzyl, p-methoxybenzyl, p-hydroxybenzyl, and cyclohexylmethyl are the most preferred R1, R5, R9, and R15 groups. Specifically, particularly preferred R1 is selected from sec-butyl and isopropyl, being sec-butyl the most preferred. Particularly preferred R5 is selected from isobutyl and 4-aminobutyl, being isobutyl the most preferred. Particularly preferred R9 is selected from p-methoxybenzyl, p-hydroxybenzyl, and cyclohexylmethyl, being p-methoxybenzyl the most preferred. Particularly preferred R15 is selected from methyl, n-propyl, and benzyl, being methyl and benzyl the most preferred.
In compounds of general formula I, II and III, particularly preferred R8, R10, R12, and R16 are independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl. More preferred R8, R10, R12, and R16 are independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl and butyl, including n-butyl, tert-butyl, isobutyl and sec-butyl, and even more preferred they are independently selected from hydrogen and methyl. Specifically, particularly preferred R8, R10 and R12 are methyl, and particularly preferred R16 is hydrogen.
In compounds of general formula I and III, particularly preferred R3 and R4 are independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl. More preferred R3 and R4 are independently selected from hydrogen, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted n-propyl, substituted or unsubstituted isopropyl, and substituted or unsubstituted butyl, including substituted or unsubstituted n-butyl, substituted or unsubstituted tert-butyl, substituted or unsubstituted isobutyl and substituted or unsubstituted sec-butyl. Preferred substituents of said groups are OR′, =O, SR′, SOR′, SO2R′, NO2, NHR′, NR′R′, ═N—R′, NHCOR′, N(COR′)2, NHSO2R′, NR′C(═NR′)NR′R′, CN, halogen, COR′, COOR′, OCOR′, OCONHR′, OCONR′R′, CONHR′, CONR′R′, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group, wherein each of the R′ groups is independently selected from the group consisting of hydrogen, OH, NO2, NH2, SH, CN, halogen, COH, COalkyl, CO2H, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list. Hydrogen, methyl, isopropyl, and sec-butyl are the most preferred R3 and R4 groups. Specifically, particularly preferred R3 is selected from methyl and isopropyl and particularly preferred R4 is methyl or hydrogen.
In one embodiment of compounds of general formula I, II and III, particularly preferred R6 and R7 are independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl. More preferred R7 is selected from hydrogen, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted n-propyl, substituted or unsubstituted isopropyl and substituted or unsubstituted butyl, including substituted or unsubstituted n-butyl, substituted or unsubstituted tert-butyl, substituted or unsubstituted isobutyl, and substituted or unsubstituted sec-butyl. Preferred substituents of said groups are OR′, =O, SR′, SOR′, SO2R′, NO2, NHR′, NR′R′, ═N—R′, NHCOR′, N(COR′)2, NHSO2R′, NR′C(═NR′)NR′R′, CN, halogen, COR′, COOR′, OCOR′, OCONHR′, OCONR′R′, CONHR′, CONR′R′, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group, wherein each of the R′ groups is independently selected from the group consisting of hydrogen, OH, NO2, NH2, SH, CN, halogen, COH, COalkyl, CO2H, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list. More preferred R6 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl and butyl, including n-butyl, tert-butyl, isobutyl and sec-butyl. Most preferred R6 is selected from hydrogen and methyl and most preferred R7 is methyl.
In another embodiment of compounds of general formula I, II and III, it is particularly preferred that R6 and R7 together with the corresponding N atom and C atom to which they are attached form a substituted or unsubstituted heterocyclic group. In this regard, preferred heterocyclic group is a heteroalicyclic group containing one, two or three heteroatoms selected from N, O or S atoms, most preferably one N atom, and having from 5 to about 10 ring atoms, most preferably 5, 6 or 7 ring atoms. A pyrrolidine group is the most preferred.
In one embodiment of compounds of general formula I, II and III, particularly preferred R13 and R14 are independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl. More preferred R13 is selected from hydrogen, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted n-propyl, substituted or unsubstituted isopropyl and substituted or unsubstituted butyl, including substituted or unsubstituted n-butyl, substituted or unsubstituted tert-butyl, substituted or unsubstituted isobutyl, and substituted or unsubstituted sec-butyl. Preferred substituents of said groups are OR′, =O, SR′, SOR′, SO2R′, NO2, NHR′, NR′R′, ═N—R′, NHCOR′, N(COR′)2, NHSO2R′, NR′C(═NR′)NR′R′, CN, halogen, COR′, COOR′, OCOR′, OCONHR′, OCONR′R′, CONHR′, CONR′R′, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group, wherein each of the R′ groups is independently selected from the group consisting of hydrogen, OH, NO2, NH2, SH, CN, halogen, COH, COalkyl, CO2H, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list. More preferred R14 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl and butyl, including n-butyl, tert-butyl, isobutyl and sec-butyl. Most preferred R13 is selected from hydrogen, methyl, isopropyl, isobutyl, and 3-amino-3-oxopropyl and most preferred R14 is hydrogen.
In another embodiment of compounds of general formula I, II and III, it is particularly preferred that R13 and R14 together with the corresponding N atom and C atom to which they are attached form a substituted or unsubstituted heterocyclic group. In this regard, preferred heterocyclic group is a heteroalicyclic group containing one, two or three heteroatoms selected from N, O or S atoms, most preferably one N atom, and having from 5 to about 10 ring atoms, most preferably 5, 6 or 7 ring atoms. A pyrrolidine group is the most preferred.
In compounds of general formula I, II and III, particularly preferred R2 is selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, and CORa, wherein Ra is a substituted or unsubstituted C1-C6 alkyl, and even more preferred Ra is methyl, ethyl, n-propyl, isopropyl and butyl, including n-butyl, tert-butyl, sec-butyl and isobutyl. More preferably R2 is hydrogen.
In compounds of general formula I, II and III, particularly preferred R17 is selected from hydrogen, CORa, COORa, CONHRb, (C=S)NHRb, and SO2Rc, wherein each Ra, Rb, and Rc is preferably and independently selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. Preferred substituents of said groups are OR′, =O, SR′, SOR′, SO2R′, NO2, NHR′, NR′R′, ═N—R′, NHCOR′, N(COR′)2, NHSO2R′, NR′C(=NR′)NR′R′, CN, halogen, COR′, COOR′, OCOR′, OCONHR′, OCONR′R′, CONHR′, CONR′R′, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group, wherein each of the R′ groups is independently selected from the group consisting of hydrogen, OH, NO2, NH2, SH, CN, halogen, COH, COalkyl, CO2H, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list. Hydrogen, CORa, COORa, and SO2Rc are the most preferred R17 groups, and hydrogen, COObenzyl, CObenzo[b]thiophen-2-yl, SO2(p-methylphenyl), COCOCH3 and COOC(CH3)3 are even most preferred.
In another embodiment of compounds of general formula I, II and III, it is particularly preferred that Y is CO. In another embodiment, it is particularly preferred that Y is –COCH(CH3)CO-.
In another embodiment of compounds of general formula I, II and III, it is particularly preferred that X is O. In another embodiment, it is particularly preferred that X is NH.
In another embodiment of compounds of general formula I and II, it is particularly preferred that n, p and q are 0. In another embodiment, it is particularly preferred that n is 1 and p and q are 0. In another embodiment, it is particularly preferred that n and p are 1 and q is 0. In another embodiment, it is particularly preferred that n, p, and q are 1. In another embodiment, it is particularly preferred that n and p are 1 and q is 2.
In another embodiment of compounds of general formula III, it is particularly preferred that p and q are 0. In another embodiment, it is particularly preferred that p is 1 and q is 0. In another embodiment, it is particularly preferred that p and q are 1. In another embodiment, it is particularly preferred that p is 1 and q is 2.
In additional preferred embodiments, the preferences described above for the different substituents are combined. The present invention is also directed to such combinations of preferred substitutions of formula I, II and III above.
In the present description and definitions, when there are several groups Ra, Rb, and Rc present in the compounds of the invention, and unless it is stated explicitly so, it should be understood that they can be each independently different within the given definition, i.e. Ra does not represent necessarily the same group simultaneously in a given compound of the invention.
In compounds of general formula I, II and III when q takes a value of 2 there are two groups R15 and two groups R16 in the compound. It is hereby clarified that each R15 and each R16 group in a given compound may be independently selected among the different possibilities described above for such groups.
A particularly preferred stereochemistry for compounds of general formula I is
wherein X, Y, n, p, q, and R1-R17 are as defined above, and when Y is —COCH(CH3)CO— it has the following stereochemistry:
A particularly preferred stereochemistry for compounds of general formula II is
wherein X, Y, n, p, q, R1, R2, and R5-R17 are as defined above, and when Y is —COCH(CH3)CO—it has the following stereochemistry:
A particularly preferred stereochemistry for compounds of general formula III is
wherein X, Y, p, q, R1-R10, and R12-R17 are as defined above, and when Y is —COCH(CH3)CO— it has the following stereochemistry:
Particularly preferred compounds of the invention are the following:
or pharmaceutically acceptable salts or stereoisomers thereof.
The compounds of general formula I, II and III may be prepared following any of the synthetic processes disclosed in Vera et al. Med. Res. Rev. 2002, 22(2), 102-145, WO 2011/020913 (see in particular Examples 1-5), WO 02/02596, WO 01/76616, and WO 2004/084812, which are incorporated herein by reference.
The preferred compound is PLD or pharmaceutically acceptable salts or stereoisomers thereof. Most preferred is PLD.
The chemical name of plitidepsin is (-)-(3S,6R,7S,10R,11S,15S,17S,20S,25aS)-11-hydroxy-3-(4-methoxybenzyl)-2,6,17-trimethyl-15-(1-methylethyl)-7-[[(2R)-4-methyl-2-[methyl[[(2S)-1-(2-oxopropanoyl)pyrrolidin-2-yl]carbonyl] amino]pentanoyl]amino]-10- [(1S)-1-methylpropyl]-20-(2-methylpropyl)tetradecahydro-15H-pyrrolo[2,1-f]-[1,15,4,7,10,20]dioxatetrazacyclotricosine-1,4,8,13,16,18,21(17H)-heptone corresponding to the molecular formula C57H87N7O15. It has a relative molecular mass of 1110.34 g/mol and the following structure:
Plitidepsin is a cyclic depsipeptide originally isolated from a Mediterranean marine tunicate (Aplidium albicans) and currently manufactured by full chemical synthesis. It is licensed and marketed in Australia under the brand name plitidepsin for the treatment of multiple myeloma.
In eukaryotic cells, plitidepsin has been shown to target the eukaryotic elongation factor (eEF1A), which has a key role in modulating interaction with other proteins, some of which are believed to be essential in viral replication. It is noteworthy that one of the aforementioned proteins is the coronavirus N protein, which is produced abundantly within infected cells and is known to interact with elongation factor EF1A. As said above, the interaction between plitidepsin and EF1A could therefore reduce the efficacy of de novo viral capsid synthesis and consequently lead to a decrease in viral load.
The present invention provides the use of a compound of the present invention in the treatment of inflammation. In particular, the invention provides the use of a compound of the present invention in the treatment of inflammation, and in particular inflammation associated with either the activation of Toll-like receptors and/or inflammation as a result of pathogen infection.
In one aspect of the invention, there is provided a compound of the present invention, for use in the treatment of inflammation. In another aspect of the invention, there is provided the use of a compound of the present invention, in the manufacture of a medicament for the treatment of inflammation. In another aspect of the invention, there is provided a method for the treatment of inflammation, the method comprising administering to an individual in need thereof a therapeutically effective amount of a compound of the present invention.
In one embodiment, the inflammation is characterised by excessive or increased production or secretion of pro-inflammatory cytokines, and preferably at least one of IL-12, IL-10, IL-1, IL-6, IL-8, CCL-2 and TNF-α and more preferably at least one of IL-1, IL-6, IL-8 and TNF-α. In another embodiment, the inflammation is hyperinflammation. Hyperinflammation is characterised by excessive cytokine production or secretion (also known as a cytokine storm). In one embodiment, hyperinflammation is defined by high levels (higher than normal levels) of at least one of the following cytokines: interleukin (IL)-1, IL-2, IL-6, GM-CSF, IFN-γ, and TNF, as well as the chemokines, C-C motif chemokine ligand (CCL)-2, CCL-3, and CCL-5. In another embodiment, the inflammation is chronic inflammation.
In particular embodiments, the virus is SARS-CoV-2 and the associated COVID-19 disease. Mortality associated with COVID-19 disease appears to be associated with a) severe respiratory failure secondary to respiratory distress and b) an inflammatory status caused by a cytokine storm. Thus, the proportion of patients with severe disease requiring hospitalisation with or without high-flow oxygen supplements and patients requiring mechanical ventilatory support was estimated to be close to 15% and 5%, respectively, in the initial series from China. However, in Europe, the figures reported by the health authorities are higher, reaching 30% of serious cases requiring hospitalisation (in the city of Madrid) without the need for mechanical ventilation and close to 10% of patients requiring mechanical ventilation. Likewise, the duration of the need for mechanical ventilation in the Chinese series is much shorter than that reported in cities such as Madrid, so the usual flow of patients to intensive care units is being altered by the prolonged stay of patients. This is putting an enormous burden on hospital services, which has made it necessary to take extraordinary, unprecedented measures. It is believed that the magnitude of the complications initially described can be avoided or reduced through the use of the present invention in patients with early-stage COVID-19 pneumonia, since once the cytokine storm and respiratory distress take place, it is typically harder for an antiviral drug to have a beneficial therapeutic effect. However, in embodiments, the compounds of the present invention are also useful at a later stage of the viral infection, for example in patients where cytokine storm and respiratory distress have taken place.
As mentioned above, in eukaryotic cells, FLIM-FRET experiments demonstrated that plitidepsin localises sufficiently close to eEF1A to suggest the formation of drug-protein complexes. A separate set of experiments carried out with 14C-plitidepsin and eEF1A purified from rabbit muscle showed that plitidepsin binds eEF1A with high affinity and a low rate of dissociation.
Several in vitro experiments aimed at determining the effect of plitidepsin on SARS-CoV-2 were carried out and are disclosed herein. Two studies, each using Vero E6 cells infected with SARS-CoV-2 and direct quantitation of SARS-CoV-2 nucleocapsid (N) protein, which is clearly involved in the mechanism of plitidepsin-induced antiviral activity, showed that plitidepsin is a potent inhibitor of SARS-CoV-2 growth in vitro, with IC50 of 0.7 to 3.0 nM. In another study, human stem cell-derived pneumocyte like cells were prophylactically exposed to 10 nm plitidepsin for 1 hour and then infected with SARS-CoV-2 (4 × 104 plaque forming units). After a 48 hour incubation period, both antiviral and cytotoxic plitidepsin effects were determined. Results showed that plitidepsin completely eliminated replication of SARS-CoV-2 with no observable cytotoxicity against the pneumocyte like cells.
Plitidepsin demonstrated potent antiviral effects in vivo, using a previously described mouse model of adenovirus-mediated hACE2 infected with SARS-CoV-2. Plitidepsin also demonstrated potent antiviral effects in vivo using a previously described model of transgenic mice expressing hACE2 driven by the cytokeratin-18 gene promoter (K18-hACE2) infected with SARS-CoV-2.
Similar to SARS CoV, infection with SARS-CoV-2 also produces hypersecretion of several cytokines, with increasing plasma levels as the disease progresses, suggesting a possible relation between cytokine release and disease severity.
Innate immunity is the first line of defence against invading pathogens. In the case of SARS-CoV-2, the entry of the virus into host epithelial cells is mediated by the interaction between the viral envelope spike (S) protein and the cell surface receptor ACE2. Viral RNAs, as pathogen associated molecular patterns, are then detected by the host pattern recognition receptors, which include the family of toll like receptors. Toll like receptors then upregulate antiviral and proinflammatory mediators, such as interleukin (IL) 6, IL 8, and interferon (IFN)-y, through activation of the transcription factor nuclear factor kappa B (NF-κB). The importance of NF-κB in pro-inflammatory gene expression, particularly in the lungs, has been highlighted by studies exploring SARS CoV infection in nonclinical species as well as in patients. In mice infected with SARS CoV, the pharmacologic inhibition of NF-κB resulted in higher survival rates and reduced expression of tumour necrosis factor alpha (TNFα), CCL2, and CXCL2 in lungs.
As shown herein, plitidepsin induces down-regulation of NFκB in tumour cells. Subsequently, both in vitro and ex vivo studies were also performed to assess the effects of plitidepsin on immune cells.
In vitro studies were performed using THP-1 cells, a spontaneously immortalised monocyte-like cell line derived from the peripheral blood of a childhood case of acute monocytic leukaemia, that is widely used for investigating monocyte structure and function. As shown in
An ex vivo study assessed the effect of plitidepsin on expression of the cytokines IL 6, IL 10, and TNFα in the lungs of mice. As shown in
In one aspect of the invention, there is provided a compound as defined herein, for use in the treatment of inflammation associated with (or caused by) activation of Toll-like receptors. In another aspect of the invention, there is provided the use of a compound as defined herein, in the manufacture of a medicament for the treatment of inflammation associated with activation of Toll-like receptors. In another aspect of the invention, there is provided a method for the treatment of inflammation associated with activation of Toll-like receptors, the method comprising administering to an individual in need thereof a therapeutically effective amount of a compound as defined herein.
By “inflammation associated with activation of Toll-like receptors” is meant inflammation that arises from agonism (i.e. stimulation) of one or more Toll-like receptor (TLR) and activation of the toll-like receptor signalling cascade or increased signalling through at least one TLR (that may be caused by increased expression of at least one TLR). Methods for measuring activation of TLR signalling in response to a known or possible TLR agonist would be well-known to the skilled person, but in one example, levels of NF-κB transactivation may be used as an indicator of TLR activation. As described herein NF-κB transactivation may be measured using luciferase-tagged NF-κB transactivation as described in the Examples. In another example, TLR activation can be determined by measuring any one of IRAK1 (IL-receptor-associated kinase), IRAK4 phosphorylation and TAK1 activation (transforming growth factor-β-activated kinase-1). Other indicators of TLR activation would be known in the art (see for example, Kawai & Akira, 2007, which describes the TLR pathway). In one embodiment, the TLR is TLR-3, TLR4, TLR7 or TLR8.
Toll-like receptors or TLRs can be activated by microbial pathogens. In one embodiment, the microbial pathogen may be a bacteria, a virus, a parasite, fungus or other microorganism.
In a preferred embodiment, the Toll-like receptor is activated by a virus. In one embodiment, the virus is an RNA virus.
In one embodiment, the virus is a single stranded (+) RNA virus. In a further preferred embodiment, the virus is selected from the family: Flaviviridae, Picornaviridae, Togaviridae, Caliciviridae, Retroviridae and Coronaviridae. In an alternative embodiment, the virus is a respiratory virus.
In one embodiment, where the virus is a Flaviviridae virus, the virus is selected from Hepatitis C, Yellow fever virus (YFV), West Nile Virus (WNV), Dengue virus (DENV), Japanese encephalitis virus (JEV), Tick-borne encephalitis virus (TBEV), classical swine fever virus and Zika virus (ZIKV). In one embodiment, where the virus is a Picornaviridae virus, the virus is selected from foot and mouth disease virus, enterovirus A71, Coxsackieviruses, Rhinovirus and Hepatitis A. In another embodiment, where the virus is a Togaviridae virus, the virus is selected from Alphaviridae, and in particular, Chikungunya Virus, Sindbis Virus, Semliki Forest Virus, Eastern Equine Encephalitis Virus / Western Equine Encephalitis Virus / Venezuelan Equine Encephalitis Virus and Rubella virus. In another embodiment, where the virus is a Caliciviridae virus, the virus is selected from Lagovirus, Norovirus, Nebovirus, Recovirus, Sapovirus, Valovirus and Vesivirus, and in particular, Norwalk virus.
In one embodiment, the viral infection is a CoV infection. The term “CoV” infection means any infection from a virus in the family Coronaviridae and the sub-family Orthocoronavirinae. In one embodiment, the infection is from a virus in the genus Betacoronavirus, which includes Betacoronavirus 1, Human coronavirus HKU1, Murine coronavirus, Pipistrellus bat coronavirus HKU5, Rousettus bat coronavirus HKU9, Severe acute respiratory syndrome-related coronavirus (SARS-CoV), Tylonycteris bat coronavirus HKU4, Middle East respiratory syndrome-related coronavirus, Human coronavirus OC43 and Hedgehog coronavirus 1 (EriCoV). Preferably, the virus is selected from the α-type HCoV-229E, HCoV-NL63; the β-type HCoV-HKU1, SARS-CoV, MERS-CoV, and HCoV-OC43; and SARS-CoV-2. SARS-CoV-2 was previously called 2019-nCoV and such terms may be used interchangeably herein.
In another embodiment, where the virus is Retroviridae the virus is HIV (Human Immunodeficiency Virus).
In another embodiment, the virus is a negative sense single-stranded RNA virus. In one embodiment the virus is selected from the family Orthomyxoviridae, Phlebovirus and Arenaviridae.
In one embodiment, the Orthomyxoviridae virus is selected from Influenzavirus A, Influenzavirus B, Influenzavirus C, Thogotovirus, Quaranjavirus, and Isavirus. In another embodiment, the Orthomyxoviridae virus is Influenza A, preferably selected from H1N1, H1N2 and H3N2. In another embodiment, the Orthomyxoviridae virus is influenza B, preferably selected from the Yamagata or Victoria lineages.
In one embodiment, where the virus is from the family Phlebovirus, the virus is Sandfly fever virus or Rift valley fever virus. In one embodiment, where the virus is selected from the family Arenaviridae, the virus is Pichinde virus.
In another embodiment, the virus is a dsDNA virus. In one embodiment the virus is selected from the family Herpesviridae, preferably, Herpes simplex virus 1 or 2.
In another embodiment, the virus is a respiratory virus. Respiratory viruses include rhinoviruses and enteroviruses (Picornaviridae), orthomyxoviridae, (influenza) parainfluenza, metapneumoviruses and respiratory syncytial viruses (Paramyxoviridae), coronaviruses (Coronaviridae), and several adenoviruses. With the exception of adenoviruses, all possess an RNA genome.
In a further embodiment, the compound of the invention may be further administered in combination with an anti-viral agent. The anti-viral agent may be administered concurrently, sequentially or separately to administration of a compound of the invention. In this example, the compound of the invention may be used as an anti-inflammatory to treat inflammation or hyperinflammation associated or as a consequence of the viral infection.
In another aspect of the invention, there is provided a compound as defined herein, for use in the treatment of a disorder selected from pneumonia and immunopathology, in particular hypercytokinemia (cytokine storm syndrome), sepsis and, graft-versus-host disease In another aspect of the invention, there is provided the use of a compound as defined herein, in the manufacture of a medicament for the treatment of a disorder selected from pneumonia and immunopathology, in particular hypercytokinemia (cytokine storm syndrome), sepsis and graft-versus-host disease. In another aspect of the invention, there is provided a method for the treatment of a disorder selected from pneumonia and immunopathology, in particular hypercytokinemia (cytokine storm syndrome) sepsis and, graft-versus-host disease,, the method comprising administering to an individual in need thereof a therapeutically effective amount of a compound as defined herein. In a particularly preferred embodiment, the disorder is immunopathology and in particular, hypercytokinemia.
In a further aspect of the invention, there is provided a compound as defined herein, for use in the treatment of pathogen-induced inflammation. In another aspect of the invention, there is provided the use of a compound as defined herein, in the manufacture of a medicament for the treatment of pathogen-induced inflammation. In another aspect of the invention, there is provided a method for the treatment of pathogen-induced inflammation, the method comprising administering to an individual in need thereof a therapeutically effective amount of a compound as defined herein.
In a further aspect of the invention, there is provided a compound as defined herein, for use in the combined treatment of a viral infection and inflammation associated with activation of Toll-like receptors or inflammation associated with pathogen-infection. In another aspect of the invention, there is provided the use of a compound as defined herein, in the manufacture of a medicament for the combined treatment of a viral infection and inflammation associated with activation of Toll-like receptors or inflammation associated with pathogen-infection. In another aspect of the invention, there is provided a method for the combined treatment of a viral infection and inflammation associated with activation of Toll-like receptors or inflammation associated with pathogen-infection, the method comprising administering to an individual in need thereof a therapeutically effective amount of a as defined herein.
In one embodiment, the inflammation may be hypercytokinemia.
By “pathogen-induced inflammation” is meant any inflammation or immunopathology caused by infection of a pathogen. Accordingly, in one aspect of the invention there is provided compounds and methods as described herein for the treatment of immunopathology. The pathogen may be a microbial pathogen. More preferably, the microbial pathogen may be a bacteria, a virus, a parasite, fungus or other microorganism. In one embodiment, the pathogen causes activation of the TLRs. In an alternative embodiment, the pathogen does not cause activation of the TLRs. In a further embodiment, the pathogen causes macrophage activation. The inflammation may be chronic inflammation. In a preferred embodiment, the pathogen is a virus.
In a preferred embodiment, the Toll-like receptor is activated by a virus.
In one embodiment, the virus is a positive sense single stranded (+) RNA virus. In a further preferred embodiment, the virus is selected from the family: Flaviviridae, Picornaviridae, Togaviridae, Caliciviridae, Retroviridae and Coronaviridae. In one embodiment, where the virus is a Flaviviridae virus, the virus is selected from Hepatitis C, Yellow fever virus (YFV), West Nile Virus (WNV), Dengue virus (DENV), Japanese encephalitis virus (JEV), Tick-borne encephalitis virus (TBEV), classical swine fever virus and Zika virus (ZIKV). In one embodiment, where the virus is a Picornaviridae virus, the virus is selected from foot and mouth disease virus, enterovirus A71, Coxsackieviruses, Rhinovirus and Hepatitis A. In another embodiment, where the virus is a Togaviridae virus, the virus is selected from Alphaviridae, and in particular, Chikungunya Virus, Sindbis Virus, Semliki Forest Virus, Eastern Equine Encephalitis Virus / Western Equine Encephalitis Virus / Venezuelan Equine Encephalitis Virus and Rubella virus. In another embodiment, where the virus is a Caliciviridae virus, the virus is selected from Lagovirus, Norovirus, Nebovirus, Recovirus, Sapovirus, Valovirus and Vesivirus, and in particular, Norwalk virus.
In one embodiment, the viral infection is a CoV infection. The term “CoV” infection means any infection from a virus in the family Coronaviridae and the sub-family Orthocoronavirinae. In one embodiment, the infection is from a virus in the genus Betacoronavirus, which includes Betacoronavirus 1, Human coronavirus HKU1, Murine coronavirus, Pipistrellus bat coronavirus HKU5, Rousettus bat coronavirus HKU9, Severe acute respiratory syndrome-related coronavirus (SARS-CoV), Tylonycteris bat coronavirus HKU4, Middle East respiratory syndrome-related coronavirus, Human coronavirus OC43 and Hedgehog coronavirus 1 (EriCoV). Preferably, the virus is selected from the α-type HCoV-229E, HCoV-NL63; the β-type HCoV-HKU1, SARS-CoV, MERS-CoV, and HCoV-OC43; and SARS-CoV-2. SARS-CoV-2 was previously called 2019-nCoV and such terms may be used interchangeably herein.
In another embodiment, where the virus is Retroviridae the virus is HIV (Human Immunodeficiency Virus).
In another embodiment, the virus is a negative sense single-stranded RNA virus. In one embodiment the virus is selected from the family Orthomyxoviridae, Phlebovirus and Arenaviridae.
In one embodiment, the Orthomyxoviridae virus is selected from Influenzavirus A, Influenzavirus B, Influenzavirus C, Thogotovirus, Quaranjavirus, and Isavirus. In another embodiment, the Orthomyxoviridae virus is Influenza A, preferably selected from H1N1, H1N2 and H3N2. In another embodiment, the Orthomyxoviridae virus is influenza B, preferably selected from the Yamagata or Victoria lineages.
In one embodiment, where the virus is from the family Phlebovirus, the virus is Sandfly fever virus or Rift valley fever virus. In one embodiment, where the virus is selected from the family Arenaviridae, the virus is Pichinde virus.
In another embodiment, the virus is a dsDNA virus. In one embodiment the virus is selected from the family Herpesviridae, preferably, Herpes simplex virus 1 or 2.
In another embodiment, the virus is a respiratory virus. Respiratory viruses include rhinoviruses and enteroviruses (Picornaviridae), parainfluenza, metapneumoviruses and respiratory syncytial viruses (Paramyxoviridae), coronaviruses (Coronaviridae), and several adenoviruses. With the exception of adenoviruses, all possess an RNA genome.
Accordingly, in a specific embodiment, there is provided a compound as defined herein for use in the combined treatment of a SARS-CoV-2 infection and hypercytokinemia. The host inflammatory response to infection may also cause pneumonia or ARDS. Accordingly, in a further embodiment, there is provided a compound as defined herein for use in the combined treatment of a SARS-CoV-2 infection and (associated) pneumonia (also referred to as COVID-19 pneumonia) or associated ARDS. In another aspect of the invention, there is provided the use of a compound of as defined herein in the manufacture of a medicament for the combined treatment of a SARS-CoV-2 infection and hypercytokinemia. In another aspect, there is provided the use of a compound as defined herein in the manufacture of a medicament for the combined treatment of a SARS-CoV-2 infection and COVID-19 pneumonia. In another aspect of the invention, there is provided the use of a compound of as defined herein in the manufacture of a medicament for the combined treatment of a SARS-CoV-2 infection and ARDS. In another aspect of the invention, there is provided a method for the combined treatment of a SARS-CoV-2 infection and hypercytokinemia, the method comprising administering to an individual in need thereof a therapeutically effective amount of a compound as defined herein. In another embodiment, there is provided a method for the combined treatment of a SARS-CoV-2 infection and COVID-19 pneumonia, the method comprising administering to an individual in need thereof a therapeutically effective amount of a compound as defined herein. In another embodiment, there is provided a method for the combined treatment of a SARS-CoV-2 infection and ARDS, the method comprising administering to an individual in need thereof a therapeutically effective amount of a compound as defined herein. Current strategies to treat both the viral infection and the host inflammatory response to the infection (i.e. hypercytokinemia) (which may result in pneumonia or ARDS) require the presence of multiple agents to target each indication (i.e. viral replication and elimination) and inflammation separately. As shown in Example 12 and
Alternatively, the viral infection is a HIV (human immunodeficiency virus) infection. Accordingly, in a specific embodiment, there is provided a compound as defined herein use in the combined treatment of a HIV infection and hypercytokinemia. In another aspect of the invention, there is provided the use of a compound as defined herein in the manufacture of a medicament for the combined treatment of a HIV infection and hypercytokinemia. In another aspect of the invention, there is provided a method for the combined treatment of a HIV infection and hypercytokinemia, the method comprising administering to an individual in need thereof a therapeutically effective amount of a compound as defined herein.
Compounds of the invention may be used in pharmaceutical compositions having biological/pharmacological activity for the treatment of the above mentioned conditions. These pharmaceutical compositions comprise a compound of the invention together with a pharmaceutically acceptable carrier. The term “carrier” refers to a diluent, adjuvant, excipient or vehicle with which the active ingredient is administered. Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E. W. Martin, 1995. Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions, emulsions, etc.) compositions for oral, topical or parenteral administration. Pharmaceutical compositions containing compounds of the invention may be delivered by liposome or nanosphere encapsulation, in sustained release formulations or by other standard delivery means.
An exemplary composition is in the form of powder for solution for infusion. For example, compositions as described in WO9942125. For example, a lyophilised preparation of a compound of the invention including water-soluble material and secondly a reconstitution solution of mixed solvents. A particular example is a lyophilised preparation of PLD and mannitol and a reconstitution solution of mixed solvents, for example PEG-35 castor oil, ethanol and water for injections. Each vial, for example may contain 2 mg of PLD. After reconstitution, each mL of reconstituted solution may contain: 0.5 mg of PLD, 158 mg of PEG-35 castor oil, and ethanol 0.15 mL/mL.
The specific dosage and treatment regimen for any particular patient may be varied and will depend upon a variety of factors, including the activity of the specific compound employed, the particular formulation being used, the mode of application, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, reaction sensitivities, and the severity of the particular disease or condition being treated.
In embodiments of the invention, the compounds of the present invention may be administered according to a dosing regimen of a daily dose.
In embodiments of the invention, the compounds of the present invention may be administered according to a dosing regimen of a once daily dose.
In further embodiments, the compounds of the present invention may be administered according to a dosing regimen of a daily dose for 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day. Preferred regimen is 2-5 days, or 3-5 days, or 3, 4 or 5 days, most preferably 3 days or 5 days.
The dose may be a dose of 5 mg a day or less, 4.5 mg a day or less, 4 mg a day or less, 3.5 mg a day or less, 3 mg a day or less, 2.5 mg a day or less or 2 mg a day or less.
Particular doses include 0.5 mg/day, 1 mg/day, 1.5 mg/day, 2 mg/day, 2.5 mg/day, 3 mg/day, 3.5 mg/day, 4 mg/day, 4.5 mg/day, or 5 mg/day. Preferred doses are 1 mg/day, 1.5 mg/day, 2 mg/day and 2.5 mg/day.
In further embodiments, the compounds of the present invention may be administered according to a total dose of 1-50 mg, 1-40 mg, 1-30 mg, 1-20 mg, 1-15 mg, 3-15 mg, 3-12 mg, 4-12 mg, 4-10 mg, or 4.5-10 mg. Total doses may be 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg or 10 mg. Preferred total doses are 4.5 mg, 5 mg, 6 mg, 7.5 mg, 8 mg, 9 mg or 10 mg. The total dose may be split over 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, preferably 3 days or 5 days.
In a particular embodiment, the compounds of the present invention may be administered according to a dosing regimen of a once daily dose for 5 days, at a dose of 2.5 mg a day or less.
In a further embodiment, the compounds of the present invention may be administered according to a dosing regimen of a once daily dose for 5 days, at a dose of 2 mg a day or less.
In a further embodiment, the compounds of the present invention may be administered according to a dosing regimen of a once daily dose for 3 days, at a dose of 1.5 mg a day or less.
In a further embodiment, the compounds of the present invention may be administered according to a dosing regimen of a once daily dose for 3 days, at a dose of 2 mg a day or less.
In a further embodiment, the compounds of the present invention may be administered according to a dosing regimen of a once daily dose for 3 days, at a dose of 2.5 mg a day or less.
In a further embodiment, the compounds of the present invention may be administered according to a dosing regimen of a once daily dose for 3 days, at a dose of 1.5 mg a day.
In a further embodiment, the compounds of the present invention may be administered according to a dosing regimen of a once daily dose for 3 days, at a dose of 2.0 mg a day.
In a further embodiment, the compounds of the present invention may be administered according to a dosing regimen of a once daily dose for 3 days, at a dose of 2.5 mg a day.
In a further embodiment, the compounds of the present invention may be administered according to a dosing regimen of a once daily dose for 3 days, at a dose of 1.5 to 2.5 mg a day.
An alternative regimen is a single dose on day 1. The single dose may be 1-10 mg, 4-10 mg, 4.5-10 mg; 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg or 10 mg; preferably 4.5 mg, 5 mg, 6 mg, 7.5 mg, 8 mg, 9 mg or 10 mg; more preferably 5-9 mg, 6.5-8.5 mg, 7-8 mg or 7.5 mg.
In a further embodiment, the compounds of the present invention may be administered according to the present invention, wherein the compounds of the present invention are administered with a corticosteroid. Preferably the corticosteroid is dexamethasone.
The corticosteroid may be administered daily with the compounds of the present invention. Administration may be sequential, concurrent or consecutive. The corticosteroid may be further administered on the days following administration of compounds according to the present invention. By way of example, with a 3 day dosing regimen, the corticosteroid may be administered on days 1-3 and then further administered daily for 3, 4, 5, 6, 7, 8, 9 or 10 or more further days.
In a particular embodiment, the corticosteroid may be administered is administered on days 1-3 as an intravenous administration and then on days 6-10 as an oral administration. In a further embodiment, the dosage of corticosteroid may be higher during the co-administration phase with the compounds of the present invention, and is lowered during the subsequent days.
Particular dosing schedules include:
In an embodiment, to avoid administration-related infusion reactions, patients may receive the following medications 20 to 30 minutes prior to starting the infusion with a compound according to the present invention:
Additionally, on Days 4 and 5 patients treated with compounds according to the present invention may receive ondansetron 4 mg twice a day PO.
Doses of dexamethasone, ondansetron and ranitidine are herein defined on the basis of the base form. The dose of diphenhydramine hydrochloride is given on the basis of the hydrochloride salt. Doses of compounds of the invention are given on the basis of the base form.
The daily doses may be administered as an infusion. The infusion may be a 1 hour infusion, a 1.5 hour infusion, a 2 hour infusion, a 3 hour infusion or longer. Preferably, the infusion is 1.5 hours.
In certain embodiments, the dose may be administered according to a regimen which uses a loading dose and a maintenance dose. Loading/maintenance doses according to the present invention includes:
According to a further embodiment, the daily dose may be reduced in the final day or days of the regimen.
According to a further embodiment, if the daily dose is 2 mg, the dose may be reduced to 1 mg on days 4 and 5.
Particular regimens include:
A single dose regimen includes:
According to further embodiments, patients may be selected for treatment with compounds of the present invention based on clinical parameters and/or patient characteristics. Suitable parameters may be measurements disclosed in the present application.
The regimens and doses outlined above apply to both methods of treatment according to the present invention, use, and use of a compound as defined herein in the manufacture of medicaments as defined herein. In a further aspect of the invention, there is provided a method of decreasing the expression and/or secretion of one or more pro-inflammatory cytokine in a subject or in a sample obtained from a subject or in a cell culture, the method comprising administering a compound as defined herein.
In another aspect of the invention, there is provided a method of increasing macrophage activation in a subject or in a sample obtained from a subject or in a cell culture, the method comprising administering a compound as defined herein.
In embodiments, the present invention is directed to a compound for use according to the present invention, wherein the compound is administered in combination with one or more of the following prophylactic medications: diphenhydramine hydrochloride, ranitidine, dexamethasone, ondansetron. In particular, one or more of diphenhydramine hydrochloride 25 mg iv or equivalent; Ranitidine 50 mg iv or equivalent; Dexamethasone 8 mg intravenous; Ondansetron 8 mg i.v. in slow infusion of 15 minutes or equivalent. Patients may receive said prophylactic medications 20-30 minutes before the infusion of plitidepsin. Dexamethasone 8 mg intravenous may be dexamethasone phosphate leading to 6.6 mg dexamethasone base.
To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term “about”. It is understood that, whether the term “about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value.
While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.
Compounds of the present invention can be obtained according to the processes set out in the literature, for example: Vera et al. Med. Res. Rev. 2002, 22(2), 102-145, WO 2011/020913 (see in particular Examples 1-5), WO 02/02596, WO 01/76616, and WO 2004/084812, the contents of which are incorporated herein by reference.
Particular compounds used in experiments of the present invention are:
Following the procedures described in WO 02/02596 and in the specification, and further disclosed in the previous examples, the following compounds are obtainable:
Following the procedures described in WO 02/02596 and in the specification, and further disclosed in the previous examples, the following compounds are obtainable:
A further compound is Compound 240, known as DidemninB and shown by the structure below:
As shown in
We checked whether the transcriptional activity of NFκB was regulated by plitidepsin. To that end, we took advantage of THP-1 cells stably transfected with an NFκB luciferase reporter plasmid. We treated the cells with 100 ng/mL TNFα (an activator of NF-κB), 500 µg/mL poly I:C (TLR3 ligand), 10 µg/mL LPS-B5 (TLR4 ligand) or 10 µg/mL Resiquimod (TLR-⅞ ligand). The compounds were used either alone (
As shown in
To investigate whether plitidepsin inhibits the TLR-trigged cytokine secretion, we treated THP-1 cells with 100 ng/mL TNFα (an activator of NF-κB), 500 µg/mL poly I:C (TLR3 ligand), 10 µg/mL LPS-B5 (TLR4 ligand) or 10 µg/mL Resiquimod (TLR-⅞ ligand). The compounds were used either alone (grey bars) or combined with 100 nM of plitidepsin (red bars) for 6 hours. We compared the variations in cytokine secretion in the cell culture supernatants between the different treatments by ELISA assays. As can be seen in
In the presence of each one of the TLR ligands, plitidepsin clearly inhibited the secretion of pro-inflammatory cytokines IL-1, IL-6, IL-8.
In a further in vitro experiment, the effect of plitidepsin (APL) pre-treatment on THP-1 cells was studied. Using a THP-1 NFκB luc line, 1, 10 or 50 nM of APL or DMSO (0.2%) was added 8 hours before stimulus with Resiquimod (RQ) at 2.5 or 5 µg/mL. RQ is a TLR⅞ agonist and mimics ssRNA. At 24 hours the level of cytokines or cell viability was measured. As shown in
As shown in
We checked whether plitidepsin inhibits the LPS-trigged cytokine secretion in alveolar macrophages. To that end mice were injected i.v. with plitidepsin (1 mg/kg) or vehicle and 12 hours after administration bronchoalveolar lavage fluid (BAL) was collected. Cells were plated and treated ex-vivo or not with 15 µg/mL of LPS-B5 for 3 or 6 hours and secreted cytokines were measured. As can be seen LPS induce the secretion of IL-6, IL-10 and TNF-α (grey bars). Furthermore, in the animals treated with plitidepsin, the drug clearly inhibited the production of pro-inflammatory cytokines IL-6, and TNF-α induced by LPS (red bars) and led to an overall anti-inflammatory effect.
This is further shown again in
We further checked whether plitidepsin inhibits resiquimod (RQ)-trigged cytokine secretion in BALF. Mice were injected i.v. with plitidepsin (1 mg/kg) or vehicle 1 hour before a 50 µg/mouse intranasal inoculation with resiquimod. At 1 or 3 hours after intranasal administration of RQ bronchoalveolar lavage fluid (BALF) was collected. Cells were plated and secreted cytokines were measured. As can be seen, RQ induces the secretion of TNFα at both 1 and 3 hours following administration. In vivo administration of PLD prevented the increased production of TNFα. As can be seen in
Also we checked the effect of plitidepsin on alveolar macrophage recruitment. Activated monocyte-derived macrophages contribute to the COVID-19 cytokine storm by releasing massive amounts of pro- inflammatory cytokines. Bronchoalveolar lavage cells were stained and analyced by flow cytometry. Plitidepsin decrease the percentage of macrophages presents on bronchoalveolar lavage without cytotoxic effects.
As shown in
To investigate whether plitidepsin decreases the percentage of alveolar macrophage in animals with acute inflammation, we treated mice with plitidepsin (1 mg/kg) i.v., with LPS (20 µg/kg) i.p. in sterile saline or with plitidepsin (1 mg/kg; i.v.) in combination with LPS (20 µg/kg, i.p.). Three hours later, bronchoalveolar lavages were collected. Bronchoalveolar lavage cells were obtained by centrifugation and analyzed by flow cytometry (
As shown in
The concentration of plitidepsin in lungs was consistently higher than that in plasma at any sampling time, with a lung-to-plasma ratio (calculated as lungAUC0-∞/plasma AUC0-∞) in mice, rats and hamsters of 133, 460 and 909, respectively, thus confirming the distribution of plitidepsin into the lung.
†Schedule:
a Maximum Tolerated Dose.
b Calculated from the Recommended Dose of 5 mg/m2, 3-h infusion or 9.5 mg/patient.
c Equivalent to 1.5 mg/patient, this being a dose used in APLICOV (1-h infusion on days 1, 2 and 3).
d Estimated from plitidepsin’s population PK model (CPR/2016/01), following 3 daily doses of 1.5 mg/patient.
NF-κB transactivation was assayed using the Bright-Glo™ Luciferase Assay System following the manufacturer’s instructions. The NF-κB reporter (Luc)-THP-1 human monocytic, cells stably transfected with NF-κB-Luc plasmid (containing four NF-κB binding sites, a minimal promoter and a luciferase gene), were exposed to 100 ng/mL TNFα (positive control), 500 µg/mL poly (I:C) (Polyinosinic-polycytidylic), 10 µg/mL LPS-B5 (Lipopolysaccharide from Escherichia coli 055:B5) or 10 µg/mL Resiquimod. The compounds were used either alone or combined with 100 nM plitidepsin for 6 hours. Luminescence was measured in a Perkin-Elmer EnVision reader. A MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay was simultaneously performed to control the cytotoxicity of the compounds. Cell survival was expressed as percentage of control cell growth. The data presented are the average of three independent experiments performed in triplicate.
THP1-NFκB-LUC cell cultures were treated as described above, and the culture medium was sampled at 6 hours post-treatment to assay for secreted cytokines by ELISA. Media samples were stored at 4° C. IL-8, IL-1β, IL-6 and TNF-α protein secretion into culture medium was quantitated using highly specific and sensitive ELISA kits. Human IL-1b, human IL-6, human IL-8 and human TNF OptEIA™ ELISA kits were obtained from BD Biosciences and performed as described by the manufacturer. The data presented are the average of three independent experiments performed in triplicate.
Cells were seeded in 96 well microtiter plates and allowed to stand for 24 hours at 37° C. and 5% CO2 before treatment described above. After 6 hours of continuous treatment, cellular viability was estimated from conversion of MTT to its coloured reaction product, MTT formazan, which was dissolved to measure its absorbance at 540 nm. Data presented here are representative from a series of at three independent experiments performed in triplicate.
Mice were randomized into groups of five animals to receive the treatments. Mice were injected intra venous (i.v.) with plitidepsin (1 mg/kg) and 12 hours after administration were euthanised. Control group received plitidepsin vehicle diluted with saline (Cremophor/Ethanol/Water). Bronchoalveolar lavage fluid (BAL) of each group was collected and centrifugated to obtain bronchoalveolar lavage cells. Cells underwent red blood cell lysis (Roche) and were plated and treated ex-vivo or not with 15 µg/mL of LPS-B5 for 3 or 6 hours. Secreted cytokines were measured using highly specific and sensitive ELISA kits. Mouse IL-6, mouse IL-10 and mouse TNF DuoSet ELISA kits were obtained from R&D Systems and performed as described by the manufacturer. Data presented here are representative from a series of at three independent experiments.
Mice were randomiced into groups of two animals to receive the treatments. Mice were challenged with plitidepsin (1 mg/kg) intra venous (i.v.), with LPS (20 µg/kg) intra peritoneal (i.p.) in sterile saline or with plitidepsin (1 mg/kg; i.v.) in combination with LPS (20 µg/kg, i.p.). The control group received plitidepsin vehicle (Cremophor/Ethanol/Water) diluted with saline. Three hours later, animals were euthanised and bronchoalveolar lavage collected (a total of 1.2 ml, PBS). Bronchoalveolar lavage cells were obtained by centrifugation and analyzed by flow cytometry. Data presented here are representative from a series of at three independent experiments.
In another inflammation model, mice were randomized into groups of two animals to receive the treatments. Mice were challenged with plitidepsin (1 mg/kg) intra venous (i.v.) followed by Resiquimod (50 µg/mouse, intranasal;) 1 hour later. The control group received plitidepsin vehicle (Cremophor/Ethanol/Water) diluted with saline. One and 3 hours later, animals were euthanized, bronchoalveolar lavage collected (a total of 1.2 ml, PBS) and then, TNFα quantified by ELISA kits. Data presented here are representative from a series of at three independent experiments.
Bronchoalveolar lavage cells were stained with anti- F4/80-BV510, CD45-APC700, CD11b-BV650, CD11c-APC-Fire, CD24-PC7 and Ly6C-BV605 monoclonal antibodies (Biolegend) and a LIVE/DEAD™ Fixable Green Dead Cell Stain Kit, for 488 nm excitation (Thermofisher). Macrophages (F4/80+) were gated on alive immune cells (CD45+ LIVE/DEAD dye-), while alveolar macrophages (F4/80+ CD24-) were specifically gated on CD11c+ CD11b- population from alive immune cells. Isotype controls and compensation beads were used to set compensations and gating strategies.
The recombinant virus assay was performed in both, MT-2 cells and PBMCs previously activated with PHA + IL-2. Cells were infected with supernatants obtained from 293t cells transfected with full-length infectious HIV-1 plasmids: pNL4.3-Luc (X4 tropic virus), pNL4.3-Renilla (X4 tropic virus able to develop more than one round of replication), pNL4.3-Δenv-Luc plus pVSV-env (HIV pseudotyped with the G protein of VSV) or pJR-Renilla (R5 tropic virus able to develop more than one round of replication). Resistant viruses were obtained cloning in NL4.3-Renilla the pol gene of viruses from different infected donors. Virus 9D carry the following mutations: 41L, 67N, 70R, 98G, 118I, 184V, 215F, 219Q, 74I and virus 4D: K65R, K70R, V75I, F77L, F116Y, Q151M, M184I, L10I. The assay was then performed in 96 well microplates seeded with 100 µl containing 250.000 (PBMCs) or 100.000 (MT-2) cells/well. The compounds to be tested were added to the culture in concentrations ranging from 50 to 0.0016 µg/ml (100 µl/well). Finally, cell culture was infected with supernatants obtained form transfection of the different plasmids described above. After 48 hours, cell culture supernatant was removed and cells were lysed with Luciferase assay system or Renilla assay system (both from Promega) following the specifications of the manufacturer, and the luciferase-renilla activity was measured in a luminometre (Berthold Detection systems). All the experiments were controlled with cells treated with the vehicle (DMSO) and non-treated cells.
HIV-1 replication inhibition was evaluated by measuring the reduction of luciferase-renilla activity or RLUs (Relative light units) in a luminometre, being the 100% the infection of non-treated cells.
Compound 3 (
Compound 8 (
Compounds 9 (
Antiviral activity of compounds of the invention were tested in Huh-7 cells (human hepatoma cell line) infected with HCoV-229E. HCoV-229E has a multiplication and propagation mechanism very similar to SARS-COV-2. Indeed, the N protein of HCoV-229E has a protein homology greater than 90% with the homologous N protein in SARS-CoV-2. It is believed that all coronaviruses need their N (nucleocapsid) protein to bind to EF1A in order to replicate effectively and synthesise viral proteins. Reducing or abolishing the binding of N to EF1A reduces the viability for the spread of the virus.
Compounds of the invention as set out in Table 2 below were reconstituted in DMSO and stored at -20° C.
Huh-7 cells (human hepatoma cell line) grown to confluence in a M96 well plate, were infected with HCoV-229E-GFP virus at a moi (multiplicity of infection) of 0.01 pfu. Virus stock was HCoV-229E-GFP (from 31 Jan. 2013) at 3×107 pfu/ml.
At 8 hpi (hours post infection), media is replaced by media with the appropriated compound dilutions (DMSO final concentration 2%), following the scheme:
Fluorescent cells were observed 24 hpi. Photos were obtained using an automated system. Cells were fixed for 30 min with PFA 4%, washed with PBS, and cell nuclei were stained with DAPI 1:200 in PBS 20 min RT. Images in green show GFP tagged vial particles. Images in blue show DAPI stained nuclei.
For a short while, confluent cultures of Huh-7 were infected at a multiplicity of infection (MOI) of 0.01 pfu/cell, with a viral inoculum of 3×107 pfu/ml and after 8 hours, plitidepsin was added at concentrations ranging from 0.5 nM to 50 µM. The cultures with plitidepsin were incubated for 48 hours and then viral viability was measured by fluorescence. The results obtained showed an antiviral effect induced by plitidepsin at concentrations as low as 0.5 nM (0.555 µg/l), much lower than those reported with other antivirals.
It is shown that compounds of the invention are effective antiviral agents across a range of tested concentrations, whilst retaining cell viability.
A multicenter, randomized, parallel and proof of concept study was undertaken to evaluate the safety profile of three doses of Plitidepsin in patients with COVID-19 requiring hospitalization. Study details are available through ClinicalTrials.gov Identifier: NCT04382066.
The primary objective of the study was to determine the safety and toxicological profile of plitidepsin at each dose level administered according to the proposed administration scheme in patients admitted for COVID-19.
The secondary objectives were to assess the efficacy of plitidepsin in patients with COVID-19 at the proposed dose levels by reference to: change in SARS-CoV-2 viral load from baseline; time until negative detection of SARS-CoV-2 by PCR; cumulative incidence of disease severity (evaluation based on: mortality; need for invasive mechanical ventilation and/or ICU admission; need for non-invasive mechanical ventilation; need for oxygen therapy) and selection of the recommended dose levels of plitidepsin for a phase II/III efficacy study.
Patients included in the study were randomised in a 1:1:1 ratio to receive:
All patients received the following prophylactic medications 20-30 minutes before the infusion of plitidepsin:
Patients included in the study received treatment for 3 days.
Plitidepsin is supplied as a powder for concentrate for solution for infusion at a concentration of 2 mg/vial. Before use, the vials are reconstituted with 4 ml of reconstitution solution to obtain a colourless to slightly yellowish solution containing 0.5 mg/ml of plitidepsin, 25 mg/ml of mannitol, 0.15 ml/ml of macrogolglycerol ricinoleate oil, 0.15 ml/ml of ethanol and 0.70 ml/ml of water for injection. An additional dilution should be made in any suitable intravenous solution prior to infusion.
Plitidepsin 2 mg is supplied in a Type I clear glass vial with a bromobutyl rubber stopper covered with an aluminium seal. Each vial contains 2 mg of plitidepsin.
The solvent for the reconstitution of macrogolglycerol ricinoleate (polyoxyl 35 castor oil)/absolute ethanol/water for injection, 15%/15%/70% (v/v/v) is supplied in a Type I colourless glass vial. The ampoules have a volume of 4 ml.
Plitidepsin will be labelled with the study protocol code, the batch number, the content, the expiry date, the storage conditions, the name of the investigator and the sponsor. The study drug will be labelled in accordance with Annex 13 of the European Good Manufacturing Practices. Plitidepsin should be stored between 2° C. and 8° C. and the vials should be kept in the outer carton to protect them from light. The drug in these conditions is stable for 60 months.
After reconstitution of the 2 mg plitidepsin vial with 4 ml of the solution for reconstitution of macrogolglycerol ricinoleate/ethanol/water for injection, the reconstituted solution should be diluted and used immediately after preparation. If not used immediately, storage times and conditions until use are the responsibility of the user. The reconstituted concentrated solution of the drug product has been shown to be physically, chemically and microbiologically stable for 24 hours under refrigerated conditions (5° C.±3° C.) and for 6 hours when stored in the original vial under indoor light at room temperature. If storage is required before administration, solutions should be stored refrigerated and protected from light and should be used within 24 hours after reconstitution.
A further dosage regimen is 1.5 mg daily for 5 days. A further regimen is illustrated in
To date, data is available for nine patients. PLD was administered as a 90 minute IV infusion daily for 3 consecutive days (day 1-3) with viral load assessed by PCR at baseline, day 4, day 7 and day 15 and day 31.
Patient 1-50 year old male, bilateral pneumonia. Received PLD 1.5 mg × 3. PCR COVID 19 test: POSITIVE at baseline, converted to NEGATIVE (no viral load) by day 4. Acute clinical improvement. Hospital discharge by day 7. As such, PLD 1.5 mg × 3 removed viral load by day 4. PLD achieved an acute clinical improvement, including removing all viral burden and treating bilateral pneumonia to enable hospital discharge by day 7.
Patient 2: 40 year old male, bilateral pneumonia. Received PLD 1.5 mg × 3. By day six, lack of improvement and cross over to Remdesivir + TOL + Corticoids + Opiates. PCR converted to negative by day 15, Hospital discharge by Day 19.
Patient 3: 53 year old male, bilateral pneumonia. Received PLD 1.5 mg × 3. PLD prevented clinical deterioration. Hospital discharge by day 10, PCR converted to negative by day 31.
Patient 4: 42 year old male, bilateral pneumonia. Received PLD 2.0 mg × 3. Corticoid therapy required. PCR COVID 19 test: POSITIVE at baseline, and still positive at day 7. By day 15 the patient was PCR negative, as shown in
Patient 5: 33 year old female, bilateral pneumonia at entry. Received PLD 1.5 mg × 3. PCR COVID 19 test: POSITIVE at baseline, converted to NEGATIVE (no viral load) by day 4 as shown in
Patient 6: 69 year old female, highly symptomatic COPD. Unilateral pneumonia on entry. Received PLD 1.5 mg × 3. PCR COVID 19 test: POSITIVE at baseline, converted to NEGATIVE (no viral load) by day 7 as shown in
Patient 7: 39 year old female, pulmonary infiltrates. Received PLD 2.0 mg × 3. PCR COVID 19 test: POSITIVE at baseline, converted to NEGATIVE (no viral load) by day 7 as shown in
Patient 8: 32 year old male. Received PLD 1.5 mg × 3. Not evaluable for efficacy, hospital discharge by day 4.
Patient 9: 34 year old male. Received PLD 2.0 mg × 3. PCR COVID 19 test: POSITIVE at baseline and still positive at day 7. However, major clinical improvement and hospital discharge by day 8.
The effect of PLD on inflammatory cytokines was also measured for patients 5, 7 and 9 and the results of C-reactive protein tests are shown in
The preliminary results of the PLD clinical trial on COVID-19 patients further demonstrate the remarkable properties of PLD in the treatment of SARS-CoV-2 infection and COVID-19.
6 out of 8 evaluable patients demonstrated SARS-CoV- PCR conversions to negative, with a median time for PCR conversion of 7 days (4-31). The antiviral properties of PLD on CoV infection, namely SARS-CoV-2, is clinically demonstrated with total removal of viral burden.
Remarkably, of the 9 patients currently tested, due to administration of PLD, none of the patients required mechanical ventilation or ICU admission, and there were no deaths on study. PDL induced disease control and major clinical improvements in 6 out of 8 evaluable patients with only 2 out of 8 requiring post PLD specific anti COVID 19 therapy. The median time to hospital discharge was 8 days (7 -19). These results demonstrate that PLD is an effective therapy for SARS-CoV-2 infection and COVID-19, and pneumonia caused by SARS-CoV- infection.
Ad hoc analysis of the first cohort at 1.5 mg and 2 mg confirmed that PLD at the doses administered was active, achieved acute improvement and complete disease control. The complete removal of viral burden was noted in the vast majority of patients.
Upon completion of the study, 45 patients hospitalised for COVID 19 were randomised to treatment with plitidepsin at doses of 1.5, 2.0, and 2.5 mg daily for 3 days. Treatment was well tolerated in all 3 dose cohorts. Treatment outcomes, assessed by hospital discharge rate, were driven by disease severity and viral load at baseline. Across dose cohorts, 100% (9/9) patients with mild disease, 82% (23/28) with moderate disease, and 57% (4/7) with severe disease were discharged by Day 15. Across dose cohorts, median viral load at baseline was 6.2 (0 to 10.6) log10 copies/mL and a mean reduction in viral load of -3.1 log10 copies/mL was achieved by Day 7 and -4.5 log10 copies/mL by Day 15.
The study was a Phase 1, multicentre, open-label study in which 45 patients hospitalised for management of COVID-19 were randomised into 3 dose groups, comprising 1.5, 2.0, and 2.5 mg plitidepsin administered as a 1.5-hour IV infusion once a day for 3 consecutive days. The primary objective of this study was to determine the safety and toxicological profile at each dose level, based on (1) frequency of Grade ≥3 treatment-emergent adverse events (TEAE) at Days 3, 7, 15, and 31 using National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 criteria; (2) percentage of patients unable to complete treatment and reasons; (3) percentage of patients with TEAEs and SAEs at Days 3, 7, 15, and 31; (4) change from baseline haematologic and non-haematologic parameters on Days 3, 7, 15, and 31; and (5) percentage of patients with ECG abnormalities on Days 2, 3, 4, 5, 6, 7, 15, and 31. A secondary objective was to select a recommended dose for a pivotal study.
Findings for protocol-specified safety endpoints are summarised as follows:
Based on these findings, it is concluded that plitidepsin treatment was well tolerated and it was not possible to detect a difference in safety between the 3 doses studied.
A total of 44 patients were evaluable for efficacy; 1 patient in the 1.5-mg cohort who experienced an anaphylactic reaction during the first infusion of plitidepsin had treatment discontinued and was not considered evaluable for efficacy. Results for protocol-specified efficacy endpoints showed equivalent results across the 3 dose cohorts (Table 3). Treatment outcomes were driven by baseline disease severity and viral load. Consistent with these findings, a recent study showed that SARS-CoV-2 viral load is associated with increased disease severity and mortality. Across dose cohorts, 100% (9/9) patients with mild disease were discharged by Day 15, compared to 82% (23/28) with moderate disease and 57% (4/7) with severe disease.
A: Patient who experienced an anaphylactic reaction during the first plitidepsin infusion had treatment discontinued and was not considered evaluable for efficacy
B: Results based on 39 patients: 1 patient missed baseline viral load assessment and 4 patients had baseline viral load below limit of quantitation despite having positive polymerase chain reaction test within 48 hours prior to enrolment
In the APLICOV-PC study, most patients (84%) had mild to moderate disease and 82% of patients were discharged by Day 15. As progressive deterioration of respiratory function and development of cytokine release syndrome typically occur at a mean of 10 days from onset of symptoms, the endpoint of hospital discharge rate at Day 15 is considered to reflect successful amelioration of life-threatening complications.
Post-hoc analyses showed that treatment response, assessed by hospital discharge rate, was correlated with baseline disease severity and viral load. Across dose cohorts, 100% (9/9) of patients with mild disease were discharged by Day 15, compared to 82% (23/28) with moderate disease and 57% (4/7) with severe disease.
In the APLICOV-PC study, median baseline viral load was 6.1 log10 copies/mL and by Day 15 a mean -4.2 log10 reduction in viral load was observed. These results support a conclusion that plitidepsin reduces viral replication.
Considering the low rate of drug-related Grade ≥3 AEs and the high discharge rate at Day 15, along with an average 4.2-log10 reduction in baseline viral load by Day 15 (-3.0-log reduction reported for those patients with moderate disease), demonstrates a positive benefit risk for plitidepsin for treatment of patients hospitalised for COVID 19 infection.
The activity of plitidepsin against SARS-CoV-2 was further confirmed in an in vitro assay using vero cells.
SARS-CoV-2 was obtained from Korea Centers for Disease Control and Prevention (KCDC). Vero cells were acquired from the American Type Culture Collection (ATCC CCL-81).
The compound was prepared a two-fold serial dilutions at 20-point concentrations with DMSO and Ampolla (Cremophor:Ethanol:Water (15:15:70)) respectively. 24 hours after cell seeding, the compound was treated in the cells with the top concentration at 5uM. After an hour, plates were transferred into the BSL-3 containment facility for viral infection and SARS-CoV-2 was added at a multiplicity of infection (MOI) of 0.0125. The plates were incubated at 37° C. for 24 hours. The cells were fixed at 24 hpi with 4% paraformaldehyde (PFA) for permeabilization. Anti-SARS-CoV-2 Nucleocapsid (N) 1st antibody and 488-conjugated goat anti-rabbit IgG 2nd antibody were treated to the cells and Hoechst 33342 were treated to dye the cells for the analysis by immunofluorescence. The acquired images with Operetta (Perkin Elmer) were analyzed using in-house software to quantify cell numbers and infection ratios, and antiviral activity was normalized to positive (mock) and negative (0.5% DMSO) controls in each assay plate.
DRCs were fitted by sigmoidal dose-response models, with the following equation: Y = Bottom + (Top Bottom)/(1 + (IC50/X)Hillslope), using XLfit 4 Software or Prism7. IC50 values were calculated from the normalized activity dataset-fitted curves. All IC50 and CC50 values were measured in duplicate, and the quality of each assay was controlled by Z′-factor and the coefficient of variation in percent (%CV).
Dose-response curves are shown in
The activity of plitidepsin against SARS-CoV-2 was further confirmed in a separate in vitro assay using vero cells.
Vero E6 cells (ATCC CRL-1586) were cultured in Dulbecco’s modified Eagle medium, (DMEM; Lonza) supplemented with 5% fetal calf serum (FCS; EuroClone), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM glutamine (all ThermoFisher Scientific).
SARS-CoV-2 virus was isolated from a nasopharyngeal swab collected from an 89-year-old male patient giving informed consent and treated with Betaferon and hydroxychloroquine for 2 days before sample collection. The swab was collected in 3 mL medium (Deltaswab VICUM) to reduce viscosity and stored at -80° C. until use. Vero E6 cells were cultured on a cell culture flask (25 cm2) at 1,5 × 106 cells overnight prior to inoculation with 1 mL of the processed sample, for 1 h at 37° C. and 5% CO2. Afterwards, 4 mL of 2% FCS-supplemented DMEM were supplied and cells were incubated for 48 h. Supernatant was harvested, centrifuged at 200 × g for 10 min to remove cell debris and stored at -80° C. Cells were assessed daily for cytopathic effect and the supernatant was subjected to viral RNA extraction and specific RT-qPCR using the SARS-CoV-2 UpE, RdRp and N assays (Corman et al., 2020). The virus was propagated for two passages and a virus stock was prepared collecting the supernatant from Vero E6.
Viral RNA was extracted directly from the virus stock using the Indimag Pathogen kit (Indical Biosciences) and transcribed to cDNA using the PrimeScript™ RT reagent Kit (Takara) using oligo-dT and random hexamers, according to the manufacturer’s protocol. DNA library preparation was performed using SWIFT amplicon SARS-CoV-2 panel (Swift Biosciences). Sequencing ready libraries where then loaded onto Illumina MiSeq platform and a 300 bp paired-end sequencing kit. Sequence reads were quality filtered and adapter primer sequences were trimmed using trimmomatic. Amplification primer sequences were removed using cutadapt (Martin, 2011). Sequencing reads were then mapped against coronavirus reference (NC_045512.2) using bowtie2 tool (Langmead, B. and Salzberg, S, 2012). Consensus genomic sequence was called from the resulting alignment at a 18×1800×879 average coverage using samtools (Li et al., 2009). Genomic sequence was deposited at GISAID repository (http://gisaid.org) with accession ID EPI_ISL_510689.
Plitidepsin was used at a concentration ranging from 100 µM to 0.0512 nM at ⅕ serial dilutions, and also assayed from 10 µM to 0.5 nM at ⅓ dilutions.
Increasing concentrations of Plitidepsin was added to Vero E6 cells together with 101.8 TCID50/mL of SARS-CoV-2, a concentration that achieves a 50% of cytopathic effect. Non-exposed cells were used as negative controls of infection. In order to detect any drug-associated cytotoxic effect, Vero E6 cells were equally cultured in the presence of increasing drug concentrations, but in the absence of virus. Cytopathic or cytotoxic effects of the virus or drugs were measured at 3 days post infection, using the CellTiter-Glo luminescent cell viability assay (Promega). Luminescence was measured in a Fluoroskan Ascent FL luminometer (ThermoFisher Scientific).
Response curves were adjusted to a non-linear fit regression model, calculated with a four-parameter logistic curve with variable slope. Cells not exposed to the virus were used as negative controls of infection and set as 100% of viability, and used to normalize data and calculate the percentage of cytopathic effect. Statistical differences from 100% were assessed with a one sample t test. All analyses and figures were generated with the GraphPad Prism v8.0b Software.
The cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of plitidepsin is shown in
A constant concentration of SARS-CoV-2 was mixed with increasing concentrations of plitidepsin and added to Vero E6 cells. To control for drug-induced cytotoxicity, Vero E6 cells were also cultures with increasing concentrations of plitidepsin in the absence of SARS-CoV-2. Plitidepsin was able to inhibit viral-induced cytophatic effects (red squares) at concentrations where no cytotoxic effects of the drug were observed (grey circles). The mean IC50 value and standard deviation of plitidepsin in two experiments with two replicates each was 0.06 ± 0.02 µM.
The aim here was to evaluate in vivo the effects of plitidepsin in the treatment of severe pneumonia caused by the mouse-adapted A/H1N1 influenza virus infection (A/Puerto Rico/8/34), and also on viral titre levels.
Experimental set-up: To achieve this objective we employed an in vivo model of viral pathogenesis based on the administration of high-dose of PR8 influenza virus (2×105 pfu), which generated a severe infection in the lungs. We then evaluated the therapeutic effect of plitidepsin on severe influenza virus infection in mice. Female mice at the age of 9 weeks were anesthetized by intraperitoneal injection of ketamine-xylazine solution and infection was performed by intranasal administration of virus solution PBS into 20 ul per nares.
Mice that were receiving the treatment were injected subcutaneously with 0.3 mg/kg or 0.15 mg/kg of plitidepsin. Subsequently, survival and body weight loss was monitored until day 3 p.i.. No death mice or mice with a weight loss of more than 30% of the starting body weight was recorded during the time of the treatment.
The control of influenza infection in the airways is mediated by enhanced inflammation in the bronchoalveolar lavage fluid (BALF).
The viral titer in the lungs was also assessed. As shown in
The BALF cellular composition is defined as a marker of lung immune response viral infection. Quantitative measurement of infiltrating cells in correlation to inflammatory cytokine levels was assessed in influenza infected mice. Treatment with plitidepsin did not reduce the total cellular composition of the BALF (CD45+ × 106). As shown in
All together, these results confirmed that three subsequent administrations of (total dose of 0.9 mg/kg) of plitidepsin in influenza infected mice can positively reduce inflammation, as shown by the reduction of the early pro-inflammatory cytokines by the treatment. In addition, we detected an increase in alveolar macrophage absolute numbers, which could suggest that AMs play a critical role in viral spread and protection. We also saw a diminished viral titer in the lung of plitidepsin high-dose treated mice.
In this example, the antiviral activity of plitidepsin on the spread of West Nile virus in cell culture was studied.
(a) Recombinant virus WNV-GFP (lineage 2; molecular clone WN956) in human hepatoma cells (Huh7) and African green monkey kidney cells (Vero-E6).
In order to determine the antiviral potential of plitidepsin against WNV-GFP, target cell lines Vero-E6 and Huh7 were inoculated with an infectious virus stock dilution in the presence of increasing concentrations of plitidepsin, starting at 2.3 pM or 2.5 pg/ml and using 4.5 µM or 5 µg/ml as the highest concentration. Infection efficiency was evaluated at 48 hours, after the virus has spread to a substantial fraction of the target cells in vehicle-treated cultures.
Relative infection efficiency was assessed by automated fluorescence microscopy in the green channel and overall cell biomass/well was estimated by nuclear staining with DAPI in the blue channel, as a preliminary assessment of the compound effective doses as well as overall compound cytotoxicity.
As shown in
In Huh7 cells, as shown in
The above data shows that plitidepsin interferes with WNV-GFP propagation in cell culture infection models in both Vero-E6 and Huh-7 cells. In one example, antiviral activity in the absence of measurable interference with cell viability may be observed at 1.5 nM (p<0.05) in Huh-7 cells and 7.2 nM (p<0.05) in Vero-E6 cells.
(b) Recombinant virus with wild-type WNV-NY99 genome (lineage 1; molecular clone NY99) in Huh7 human hepatoma cells and African green monkey kidney cells (Vero-E6).
In this example, the impact of plitidepsin on WNV-NY99 propagation using viral RNA load as readout of the infection efficiency was assessed. Vero-E6 or Huh-7 cells were inoculated (MOI 0.01) with a recombinant virus based on the NY99 WNV strain. Infection was performed in the presence of the vehicle or a dose range of plitidepsin
displaying bioactivity in the WNV-GFP model (45, 15, 5, 1.5 nM; see above). Inoculated cells were incubated for 48 hours, time at which samples of the supernatants were processed to determine infection efficiency by extracellular infectivity titration. In addition, total RNA was extracted from control and plitidepsin-treated cells to determine viral RNA load and independently assess overall viral infection efficiency.
Extracellular infectivity titers show that the presence of plitidepsin strongly interfered with WNV propagation as shown in
Intracellular WNV RNA load was determined by RT-qPCR in control and plitidepsin- treated cells. Remarkably, as shown in
The WNV infection propagation efficiency data suggest that plitidepsin interferes with viral replication at doses above 5 nM in both Vero-E6 and Huh-7 cells, although some degree of interference could be observed at lower concentrations in Huh-7 cells. The results measuring overall propagation efficiency using functional (infectivity) as well as molecular (RT-qPCR) approaches confirm that plitidepsin reduces virus propagation efficiency in the expected range of concentrations based on the data obtained with WNV-GFP.
In summary, supplementation of the cell culture medium with increasing doses of plitidepsin resulted in strong reduction in WNV propagation efficiency in the two infection (WNV-GFP and WNV/NY99) models and in both Vero-E6 and Huh-7 cells.
Compound preparation: Pre-weighted solid was diluted to a final 1 mg/ml solution in dimethylsulfoxyde (DMSO) and aliquoted at -20° C. until further use. Cell culture: Subconfluent Vero-E6 cells and Huh-7cell cultures were maintained in complete media [(DMEM supplemented with 10 mM HEPES, 1x non-essential amino acids (Gibco), 100 U/ mL penicillin-streptomycin (Gibco) and 10% Fetal Bovine Serum (heat-inactivated at 56° C. for 30 min)].
Viruses: WNV (NY99) and WNV-GFP recombinant viruses were rescued from cloned cDNA as previously described. Stock infectivity titers were determined by plaque assay on Vero- E6 cells as previously described.
Infection experiments: Cells were seeded onto 96-well plates (2 ×104 cells/well). The day after, serial 5-fold dilutions of plitidepsin were prepared in 2% FCS-containing complete media to achieve the indicated final concentrations. WNV-GFP stock was diluted in complete media containing 2% FCS to achieve the required multiplicity of infection (MOI 0.01). Compound and virus dilutions were mixed 1:1 and added onto the target cells. Cells were incubated for 48 hours at 37° C.; 5% CO2 and 95% humidity.
Cells were fixed by addition of a 5X formaldehyde solution to achieve a 4% final concentration for 30 mins at room temperature. Cells were washed with PBS and stained with DAPI (4′,6-diamidino-2-phenylindole) following manufacturer’s recommendations. Relative infection efficiency was estimated by image analysis in an automated microscopy device (Tecan Spark Cyto). Uninfected cells and vehicle-treated controls were included in each plate.
Cells were seeded onto a 24-well plate using 1.2 ×105 cells /well. The day after, cells were inoculated with a WNV/NY99 stock to achieve a multiplicity of infection of 0.01 (MOI 0.01) and the indicated plitidepsin concentration in a final volume of 1 ml. Cultures were maintained at 37° C. for 48 hours, time at which supernatants were collected and preserved at -80° C. Total RNA was collected from cells by adding TrizolTM reagent directly to the cells and following the manufacturer’s instructions.
Infectivity titration: infectivity titers were determined using endpoint dilution and immunofluorescence microscopy using a monoclonal antibody against Flavivirus E protein (4G2; ATCC® HB-112™). Briefly, Huh-7 cells were inoculated with supernatant dilutions in a 96-well format. Forty-eight hours post infection, cells were fixed for 30 minutes at room temperature with a 4% formaldehyde solution in PBS, washed twice with PBS and incubated with binding buffer (0.3% Triton X100, 3%BSA in PBS) for 1 hour. Primary antibody was diluted in binding buffer and incubated with the cells for 1-hour, time after which the cells were washed with PBS and subsequently incubated with a 1:500 dilution of a goat anti-mouse conjugated to Alexa 488 (ThermoFisher). DAPI (4′,6-diamidino-2-phenylindole; ThermoFisher) was used as nucleus staining reagent to evaluate cell number. Cells were washed with PBS and infection foci number was determined under a fluorescence microscope.
Reverse-transcription and qPCR: 60 ng of total cellular RNA were subjected to RT-qPCR using NZYSpeedy One-Step qPCR probe master mix, using manufacturer’s recommendations and the primers.
Statistical Analysis: Means and SEM were calculated using Excel. Means were compared using one-way ANOVA and a Dunnet’s post-hoc analysis (2-tails; alpha=0.05) using IBM- SPSS
Indication: Treatment of patients hospitalised for management of moderate COVID 19 infection
Primary objective:
Key secondary objectives of this study are to compare plitidepsin 1.5 and 2.5 mg versus control on the following:
Other secondary objectives of this study are:
Change in proinflammatory biomarkers (C-reactive protein [CRP], lactate dehydrogenase [LDH], ferritin, interleukin [IL]-6, IL-1β, IL-10, and tumour necrosis factor alpha [TNFα]) in each study arm from baseline to Days 2, 3, 4, 8, 15, and 31
Methodology/study design:
Randomisation will be stratified for 2 factors:
From treatment initiation on Day 1, patients will be followed in the hospital for at least 4 days and then through Day 31 or resolution/stabilisation of TEAEs that occurred through Day 31. Patients discharged from the hospital prior to Day 8 will return to an out-patient clinic for assessments on Days 8 and 31.
An Independent Data Monitoring Committee (IDMC) will oversee study conduct (safety and primary endpoint), including analysis of summary safety data as per the trial requirements.
Diagnosis and main criteria for inclusion and exclusion:
The following are the exclusion criteria:
est products, dose, and mode of administration:
For prevention of plitidepsin related infusion reactions, all patients must receive the following medications 20 to 30 minutes prior to starting the plitidepsin infusion:
Additionally, on Days 4 and 5 patients treated with plitidepsin must receive ondansetron 4 mg twice a day PO.
Reference therapy, dose, dose form, and mode of administration:
Indication: Treatment of patients with mild type COVID 19 infection.
Patients will be included in the study if presenting with acute clinical infection (onset of symptoms in the previous 5 days), in which the diagnosis of COVID-19 infection is reached through a diagnostic method that could be a positive antigen test or a positive PCR test.
The study comprises two arms:
All patients receive the following prophylactic medications 20-30 minutes prior to plitidepsin infusion:
Ondansetron 4 mg orally is given every 12 hours for 3 days after plitidepsin administration to relieve drug-induced nausea and vomiting. If plitidepsin is administered in the morning the patient receives the first dose of ondansetron in the afternoon.
The study will show that a single dose of plitidepsin administered to patients results in a reduction of viral load. This may be expressed as a replication cycle threshold (Ct) value greater than 30 (Ct> 30), on day 6 after the administration. For example, the study will show that patients with COVID-19 infection who are to be discharged from the Emergency Department show a reduction in viral load on day 6 after discharge of emergencies expressed as a replication cycle threshold (Ct) value greater than 30 (Ct> 30), when administered with a single dose of plitidepsin. This may be expressed as a reduction in SARS-CoV-2 viral load from baseline. This may be expressed as a reduction in the percentage of patients requiring hospitalisation following administration. This may be expressed as a reduction in the percentage of patients requiring invasive mechanical ventilation and / or admission to the ICU following administration. This may be expressed as a reduction of patients who develop sequelae related to persistent disease. This may be expressed as an increase in the percentage of patients with normalization of analytical parameters chosen as poor prognosis criteria (including, for example, lymphopenia, LDH, D-dimer or PCR). This may be expressed as an increase in the percentage of patients with normalization of clinical criteria (disappearance of symptoms), including, for example: headache, fever, cough, fatigue, dyspnea (shortness of breath), arthromyalgia or diarrhea.
The study will show that single-dose treatment with plitidepsin can eliminate the SARS-CoV-2 viral load in the patient on day 6, which, according to several studies, leads to clinical improvement, and therefore a decrease in complications, understood as hospitalization, ICU and death. In addition to improving the prognosis of patients in the short term, the decrease in viral load is believed to be key to two other objectives. Firstly, reducing the infectivity of asymptomatic or not very symptomatic patients with high viral loads (TC <25), known as supercontagators. Secondly, decreasing viral load can be decisive to avoid long-term complications known as COVID persistent or long COVID.
A validated plitidepsin population pharmacokinetic model (Nalda-Molina R, et al. Population pharmacokinetics meta-analysis of plitidepsin in cancer subjects. Cancer Chemother Pharmacol. 2009 Jun;64(1):97-108. doi: 10.1007/s00280-008-0841-4) was used to confirm total plasma concentration will reach the estimated lung target concentrations.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20382152.5 | Mar 2020 | EP | regional |
| 20382192.1 | Mar 2020 | EP | regional |
| 20382266.3 | Apr 2020 | EP | regional |
| 20382339.8 | Apr 2020 | EP | regional |
| 20382815.7 | Sep 2020 | EP | regional |
| 20382816.5 | Sep 2020 | EP | regional |
| 21382059.0 | Jan 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/055187 | 3/2/2021 | WO |
