Patent Application
20230338310
The present invention relates to the use of compounds, and compositions comprising such compounds, for treating viral infection. More specifically, the compounds for use in the present invention are comprised of structural formula (1), or a pharmaceutically acceptable salt thereof. As an example, the compounds may be used for treating infection with a coronavirus, e.g. acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, known as COVID-19.
Viral infection in the human and animal populations is not a new problem. However, with the emergence of a more global society, the ability for particularly virulent infections to spread to the point of epidemic or pandemic proportions is becoming more common. In 2002-2003 the world experienced infection with Severe Respiratory Syndrome (SARS) caused by SARS-associated coronavirus (SARS-CoV), in 2012 it was the turn for infection with the Middle East Respiratory Syndrome (MERS) caused by Middle East Respiratory Syndrome Coronavirus (MERS-CoV). In 2019-2020, we have seen the emergence of an even more transmissible viral infection in the form of Coronavirus disease 2019 (COVID-19), caused by SARS-Associated Coronavirus 2 (SARS-CoV-2).
As the identification of sufficiently effective vaccines and therapeutic treatments of coronavirus infections, such as SARS-CoV-2, has thus far eluded science, the principle strategy to halt such pandemics has been to “lock-down” society, thereby using social distancing to prevent transmission of the virus. Such strategies are clearly incredibly detrimental to world economies, our way of life and the population's mental health in general. There is therefore a need for new therapeutics that can target the treatment, including prophylactically, of viral infections in general, and currently those relating to coronavirus infection. This is particularly an urgent requirement for emerging viral infection, such as COVID-19. Approximately 5% of patients infected with COVID-19 require admission to Intensive Therapy Units (ITUs) and require mechanical ventilatory support because of respiratory failure, the mortality rate for these patients is 50-60%, in addition many patients will require prolonged stay in ITU (>14 days) further increasing pressure on ITU resources. The pathological processes resulting in respiratory failure are not entirely clear but are likely to include direct damage to bronchial epithelial/alveolar cells by COVID-19, superadded bacterial infection and associated inflammatory reaction. There is currently no approved therapeutic composition in Europe or the US for treating coronavirus infection.
Clinical interventions designed to directly address viral infection essentially fall into three categories; (1) antiviral (2) viricidal therapeutics or (3) vaccines. Viricidal therapeutics deactivate virions and so are aimed at destroying the viral infection. Antiviral therapeutics are aimed at interrupting viral replication (for example, Acyclovir). A vaccine is designed to activate the acquired immune responses of the body to the viral invasion by stimulating B lymphocytes, thereby invoking an antibody response (e.g. IgA, IgM, or IgG). Each of these strategies can present difficulties. Designing an effective vaccine is a process fraught with technical hurdles and often takes a long time to arrive at an effective preparation. Viricidals often have off-target effects that can result in damage to the host and so are seldom developed for therapeutic purposes. Perhaps the most successful antiviral drug strategies are to administer non-functional “analogues” of the nucleotides that are used by the virus during replication, which block viral polymerase and so replication itself. Acyclovir, ribavirin and azidothymidine (AZT) are examples of these drugs and have been demonstrated to be effective at treating herpes simplex virus, hepatitis C and HIV/AIDS. Unfortunately, coronavirus can resist such strategies as it “proofreads” and edits out non-authentic nucleotides during replication. A further problem associated with both antiviral and viricidal therapeutics is that for optimum effect they often have to be administered directly to the site of infection. For example, in the case of COVID-19 this would normally be the lungs. Administering therapeutics to the lung is difficult and often painful.
A number of compounds according to formula 1 have been considered to have therapeutic potential. For example, cystamine (2,2′-Dithiobis(ethylamine)) has been suggested to have radioprotective properties and to have potential for treating Huntington's disease. Cysteamine (2-Aminoethanethiol) is an aminothiol that possesses a number of characteristics that are currently used for therapeutic benefit. Cysteamine has been licensed for over 30 years for the treatment of the inherited metabolic disorder cystinosis, and in development for more than 16 years for the bacterial respiratory infections associated with of cystic fibrosis. Cysteamine has been shown to have powerful antibacterial (including antivirulence and antibiofilm), antibiotic-potentiating, mucolytic and anti-inflammatory properties.
Some limited work has been carried out on the potential use of cysteamine for treating HIV/AIDS and specific influenzas. In these studies, it was concluded that the cysteamine has antiviral effects on HIV by its cleavage of the envelope glycoprotein GP120 that is specific to HIV. Similar effects were found in connection with the treatment of specified influenza viruses. Cysteamine is therefore considered to act as a viricidal agent that targets envelope glycoproteins that are specific for these two classes of virus (ie HIV, H1N1 and H7N9). As the envelope glycoprotein populations are specific to each class of virus, it is not possible to have any reasonable level of expectation that cysteamine may be able to act on any other given virus.
To-date, there has been no empirical evidence that demonstrates that any compound of formula (1), for example cysteamine, has any antiviral effect on a coronavirus infection.
The applicants have been investigating the therapeutic utility of a number of compounds according to formula 1 in respiratory and bacterial and fungal infectious disease for over 16 years and have a unique insight into the therapeutic potential that such compounds have to offer. Much of the work has been involved in elucidating the mechanisms of action of the compounds when directed to bacterial or fungal infections; mechanisms that have no relevance to antiviral activity. As discussed above, cysteamine has been proposed as having antiviral activity, but the results to-date, and explanations for mode of action, only point to the therapeutic effect being restricted to specific viruses. It has therefore been surprisingly found by the applicants that compounds according to formula 1 are able to treat a coronavirus infection and indeed viral infection in general.
Accordingly, in a first aspect of the present invention, there is provided a compound comprising a structure of formula (1), or a pharmaceutically acceptable salt thereof, for use in the treatment of viral infection, wherein the viral infection is not HIV, H1N1, or H7N9, and wherein formula (1) is:
The viral infection can be caused by any virus, excepting HIV, H1N1, or H7N9. The virus may therefore be influenza virus, except H1N1, or H7N9. Therefore, the virus can be an influenza C virus. The virus can be an influenza virus with a haemagluttinin content that does not include H1 and is not H7N9, unless optionally the neuraminidase is not N1. Consequently, the virus can have a haemagluttinin content of any of H2-17, or H1 when the virus does not including N1, and is not H7N9. Consequently, the virus can have a neuraminidase content of any of N2-9, or N1 when the virus does not include H1 and is not H7N9. The virus can be an influenza virus with a haemagluttinin content that does not include H7 and is not H1N1, unless optionally the neuraminidase is not N9. Consequently, the virus can have a haemagluttinin content of any of 1-6 or 8-17, or H7 when the virus does not including N9, and is not H1N1. Consequently, the virus can have a neuraminidase content of any of N1-8, or N9 when the virus does not include H7 and is not H1N1. The viral infection can be a Ribovirus (but not H1N1, H7N9). The viral infection can be any of ebola, coronavirus, hepatitis C, hepatitis E, West Nile Fever, Norovirus, Rotavirus, Polio, rabies, measles, rhinovirus. The virus may be a coronavirus. For example, any virus of the family Coronaviridae. Such viruses are characterised as enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry. The genome size of coronaviruses may be rather large, ranging from about 26 to 32 kilobases. The virus can be SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-299E, HCoV—OC43, HCoV-NL63, CoV-HKU1, Human Coronavirus 229E (ATCC No. VR/740) or any combination thereof. The virus is preferably SARS-CoV-2. SARS-CoV-2 is a betacoronavirus with 79% genetic homology with SARS-CoV, and 98% homology to the bat coronavirus RaTG13 (Zhou et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin, Nature, 2020;579 (7798):270-3). It is spread in respiratory droplets and infects nasal, bronchial and alveolar epithelial cells by binding of the viral spike protein to its cellular receptor, ACE2 (Walls et al. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell. 2020; 181(2):281-92 e6). Owing to the error prone nature of the viral replication process, RNA viruses such as SARS-CoV-2 accumulate mutations resulting in some sequence diversity. Nevertheless, different strains of SARS-CoV-2 can be recognised by sequencing and phylogenetic sequence trees. Exemplification of such phylogenetic tree analysis with rapid sequencing of isolates is reported for example by Meredith et al. Rapid Implementation of SARS-CoV- 2 sequencing to investigate cases of health-care associated COVID-19: a prospective genomic surveillance study in The Lancet, published on-line Jul. 14, 2020. Sequences of amplified SARS virus genome can be compared with the NCBI Reference sequence NC_045512.2 or equivalent GenBank reference MN908947.3 for SARS-CoV-2 corresponding to the SARS-CoV-2 isolate Wuhan-Hu-1 complete genome (Wu et al. Nature 579, 265-269). SARS2-CoV-2 variant strains can thus be recognised and can be expected to have high homology to the reference genome, e.g. at least 90%, at least 95%, at least 98% or at least 99%. Such sequencing surveillance can equally enable any new SARS virus infecting humans to be identified. The use of compounds of the present invention may therefore be used for treating humans infected with any SARS-CoV virus, especially any SARS-CoV-2 viral strain (which can include variants thereof).
The applicants have surprisingly discovered that the antiviral mode of action for the compounds of the present invention (eg when addressed to a coronavirus infection) is different to the general known antiviral (eg anti-HIV/AIDS) mechanisms of action of some of those compounds. The applicant has determined that it is the action of the compound to inhibit the activity of glycine decarboxylase in the host that results in the anti-coronavirus effect. Based on their further investigations, this has enabled the applicants to identify a number of further inventive concepts associated with the use of the compounds of the present invention for treating viral infections.
Because glycine decarboxylase is ubiquitous in viruses, the realisation that, for example, coronavirus infection can be treated with compounds of the present invention in this manner supports the understanding that all viruses are treatable in this manner.
Consequently, in a further aspect of the present invention, there is a provided a compound comprising a structure of formula (1), or a pharmaceutically acceptable salt thereof, for use in treating viral infection by inhibiting the activity of glycine decarboxylase.
The viral infection may be that defined in connection with other aspects of the present invention.
Those subjects with an elevated activity level of glycine decarboxylase are as a result of the above understanding particularly susceptible to treatment with cysteamine for viral infection. Consequently, in a further aspect of the present invention, there is provided a compound comprising a structure of formula (1), or a pharmaceutically acceptable salt thereof, for use in treating viral infection in a subject, wherein subject has a glycine decarboxylase activity that is greater than normal. Determining such a subject may be achieved by a quantitative analysis of any biomarker for glycine decarboxylase activity, for example that found in the urine or blood. A lower level for that biomarker for the subject than normal would be indicative of an individual that would respond well to treatment for viral infection by the compounds of the present invention. Determining when the level is lower than normal may be possible by comparing the level for the biomarker with that in a sample provided by the subject to be treated prior to infection. If such samples are not available, it is well within the skill of a clinician to determine if the level for any individual biomarker is lower than normal. As an example, the biomarker can be glycine. The viral infection may be that defined in connection with other aspects of the present invention.
Consequently, in a further aspect of the present invention, there is provided a compound comprising a structure of formula (1), or a pharmaceutically acceptable salt thereof, for use in treating viral infection in a subject, wherein subject has a plasma concentration of glycine that is lower than or equal to 300, 290, 280, 250, 200, 150, 125 μmol/L. A preferred range is lower than or equal to 300 μmol/L. A further preferred range is lower than or equal to 125 μmol/L. The viral infection may be that defined in connection with other aspects of the present invention.
Therefore, before administering any of the compounds described, a subject may be tested for glycine levels in any bodily fluid, for example, blood (e.g. plasma) or urine. Glycine levels vary according to age and health. The skilled person, for example, a physician, would be aware of the appropriate levels of glycine for a subject. Example ranges are given below for guidance:
There is therefore also disclosed a method of: a) determining the level of glycine in a sample obtained from a subject; and b) comparing the level of glycine to a control range, wherein if the level of glycine is lower than the control or at a low level compared to the control the subject is treated with cysteamine or any of the compounds described below.
By low level is meant, for example, the glycine may be in the lower 10% of the control range.
Alternatively, the glycine may be in the lower 15% of the control range.
Alternatively, the glycine may be in the lower 20% of the control range.
Alternatively, the glycine may be in the lower 25% of the control range.
Alternatively, the glycine may be in the lower 30% of the control range.
Alternatively, the glycine may be in the lower 35% of the control range.
Alternatively, the glycine may be in the lower 40% of the control range.
Alternatively, the glycine may be in the lower 45% of the control range.
Alternatively, the glycine may be in the lower 50% of the control range.
The control range in plasma for healthy adults is 120-554 with average levels of between 200-300 μmol/L. Therefore, the glycine may be below 120 or from 120-200 μmol/L. Alternatively, the glycine may be below 120 or from 120-150 μmol/L.
Alternatively, the glycine may be below 200-300 μmol/L. For example, below 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150 μmol/L.
For obese or type II diabetic subjects, the glycine may be below 190, 180, 170, 160, 150, 140, 130 or 120 μmol/L.
Other biomarkers which would identify patients eligible for treatment include: glycine decarboxylase or ammonia (which is a by-product of the glycine cleavage pathway). Therefore, also disclosed is a method of: a) determining the activity of glycine decarboxylase in a sample obtained from a subject; and b) comparing the activity to a control range, wherein if the activity is higher than the control, the subject is treated with cysteamine of any of the compounds described below. The skilled person would be aware of normal activity levels of glycine decarboxylase in a subject. Also disclosed is a method of: a) determining the level of ammonia in a sample obtained from a subject; and b) comparing the level to a control range, wherein if the level is higher than the control, the subject is treated with cysteamine or any of the compounds described below. The normal range for ammonia in the blood is 15 to 45 p/dL (11 to 32 μmol/L).
Other markers for increased flux through the glycine cleavage pathway may also be tested. Therefore, there is also disclosed a general method of a) determining the activity or level of a biomarker which indicates increased flux through the glycine cleavage pathway; and administering a compound described below. For example, any of the compounds below, for use in a method of treating a viral infection, wherein the method comprises: a) determining the activity or level of a biomarker which indicates increased flux through the glycine cleavage pathway; and b) administering the compound.
As well as testing for glycine or ammonia levels, or glycine decarboxylase activity, a physician may instruct the tests to be carried out.
A number of conditions are associated with an elevated level of activity of glycine decarboxylase, for example metabolic syndrome, diabetes (particularly type II) and obesity. The elderly (eg over 75) also tend to have elevated levels of glycine decarboxylase, particularly when suffering from dementia. Consequently, the compositions of the present invention may have particular utility when directed for use in treating viral infection in a subject, wherein subject is a diabetic (particularly type II), obese, suffers from dementia or elderly (particularly with dementia), or a combination thereof. The viral infection may be that defined in connection with other aspects of the present invention.
The applicant has furthermore identified that compounds capable of inhibiting the reaction that is catalysed by glycine decarboxylase in vivo (ie glycine cleavage) are able to optimise the anti-viral activity of cysteamine, particularly those that achieve such inhibition through means that does not involve inhibit glycine decarboxylase activity. For example, a suitable compound is aminooxyacetic acid or bicarbonate. Preferably the compound is bicarbonate.
Consequently, in a further aspect of the present invention there is a provided a composition comprising a compound comprising a structure of formula (1), or a pharmaceutically acceptable salt thereof, and an inhibitor of glycine cleavage, eg bicarbonate. It is preferred that such in inhibitors carry out this activity via a mechanism other than the inhibition of glycine decarboxylase. As such a composition has therapeutic value, the present invention may relate to a pharmaceutical composition comprising a compound comprising a structure of formula (1), or a pharmaceutically acceptable salt thereof, and an inhibitor of glycine cleavage (eg bicarbonate) and one or more pharmaceutically acceptable excipient. In a further aspect of the present invention, there is provided a composition comprising a compound comprising a structure of formula (1), or a pharmaceutically acceptable salt thereof, and bicarbonate for use in therapy. The compound and inhibitor of glycine cleavage (eg bicarbonate) may be prepared for separate, sequential or simultaneous administration. That therapeutic use may be the treatment of viral infection. The viral infection may be that defined in connection with other aspects of the present invention.
As the compounds of all aspects of the present invention comprise a structure of formula (1), or a pharmaceutically acceptable salt thereof, they may therefore be cysteamine, a prodrug thereof (ie cysteamine produced in vivo by a subject being treated), or a pharmaceutically acceptable salt thereof. The prodrug may be an amine prodrug compound, a nitrosothiol prodrug compound, a thiophosphate prodrug compound or a disulfide prodrug compound as described herein, or any combination thereof. The prodrug can therefore be cystamine.
The compound of any of the aspects of the present invention may be provided as a composition with antiviral therapeutic activity. Consequently, it maybe that the composition comprises the compound as the only compound with antiviral activity in the composition. The composition may consist of the compound, optionally with additional pharmaceutically acceptable excipients.
Opportunistic bacterial infection often follow infection from viruses, not least infections associated with coronavirus, which can, on top of the viral infection, exacerbate adverse immunological responses further; pushing the subject closer to very serious conditions such as cytokine shock syndrome. Only treating viral infection will leave the bacterial infection to flourish, and vice versa if one were to treat the bacterial infection alone. Combinations therapies can be unpredictable. Consequently, a surprising advantage of the work carried out by the applicant is that one can treat a bacterial and viral infection with a single therapeutic that is effective for both, eg one of the compounds of formula (1), such as cysteamine, a prodrug thereof, or a pharmaceutically acceptable salt thereof. Immuno-suppressive activity of compounds of the present invention aids further in the reduction in progression of the deleterious effects of both such infections. Consequently, the compounds or compositions of the present invention show surprising utility for treating a viral infection (such as coronavirus) and bacterial infection simultaneously. Treating a subject with only viral (eg coronavirus) infection will also have the benefit of providing prophylactic treatment of the bacterial-co-infection. The subjects to be treated in the present invention may therefore be those with a viral and bacterial infection, or those with a viral infection and that may be vulnerable to a bacterial infection (for example, due to weekend state from a severe viral infection, or their presence in a hospital). The viral infection may be that defined in connection with other aspects of the present invention.
Because conventional antiviral therapeutics work in a different way to that of the present invention, co-administration of compounds or compositions of the present invention and a further antiviral therapeutic can provide an improved level of efficacy compared to the use of either therapeutic alone, for example, a synergistic level of improvement. This advantage can be found when combined with any conventional anti-viral therapeutic, eg ones that does not act via inhibition of glycine decarboxylase. As an example an additional antiviral agent may be Idoxuridine, Trifluridine, Brivudine, Vidarabinea, Entecavir, Telbivudine, Foscarnet, Zidovudine, Didanosine, Zalcitabinea, Stavudine, Lamivudine, Abacavir, Emtricitabine, Nevirapine, Delavirdinea, Efavirenz, Etravirine, Rilpivirine, Saquinavir, Ritonavir, Indinavir, Nelfinavir, Amprenavira, Lopinavir-ritonavir, Atazanavir, Fosamprenavir, Tipranavir, Darunavir, Telaprevira, Boceprevira, Simeprevir, Asunaprevirb, Paritaprevirb, Grazoprevirb, Raltegravir, Elvitegravir, Dolutegravir, RSV-IGIVa, Palivizumab, Docosanol, Enfuvirtide, Maraviroc, VZIGa, VariZIG, Acyclovir, Ganciclovir, Famciclovir, Valacyclovir, Penciclovir, Valganciclovir, Cidofovir, Tenofovir disoproxil fumarate, Adefovir dipivoxil, Amantadinea, Ribavirin, Rimantadine, Zanamivir, Oseltamivir, Laninamivir octanoate, Peramivir, Favipiravir, Pegylated interferon alfa 2b, Interferon alfacon 1a, Fomivirsena, Podofilox, Imiquimod, Sinecatechins, Remdesivir, Flavipiravir, or any combination thereof. A preference may be for Remdesivir, Flavipiravir, Lopinavir-ritonavir or any combination thereof.
Consequently, compositions for use in any aspect of the present invention may include one or more further antiviral therapeutic. Alternatively, the compounds or compositions of the present invention may be administered separately, but as part of the same regimen, for treating viral infection (optionally coronavirus). The compositions or compounds of the present invention may therefore be provided for separate, simultaneous or sequential use with one or more further antiviral therapeutic for use in treating viral infection (optionally coronavirus).
The compositions of the present invention may be prepared for administration intravenously, orally, rectally, parentally, topically. Optionally, the compositions are prepared for administration to the site of infection, for example the lungs. In a further aspect of the present invention, there is provided a method of treating viral infection in a subject, the method comprising the step of administering any of the preceding compounds or compositions in a therapeutically effective amount to the subject. The viral infection maybe a coronavirus infection, as defined above. The method may be prophylactic treatment.
The inventors have also seen beneficial reduction in the damaging IL-6 induced inflammatory response as a result of cysteamine treatment (see the discussion of
The hyperinflammation may be in Acute respiratory distress syndrome (ARDS). The hyperinflammation may be in sepsis or toxic shock syndrome. The hyperinflammation may be as a result of other causes, i.e. not infection. For example, the hyperinflammation may be in an autoimmune disease, for example in a flare up of an autoimmune disease. An autoimmune flare-up is a period of worsening and intensification of symptoms. This can be diagnosed by using various biomarkers depending on the autoimmune condition. The autoimmune disease may be rheumatoid arthritis, systemic lupus erythematosus or inflammatory bowel disease (IBD). Hyperinflammation is systemic, acute inflammation and can be diagnosed by elevated levels of proinflammatory cytokines. Current treatments include IL-6 receptor antibodies and steroids. Patients have an increased incidence of shock, multiorgan failure and higher mortality. Hyperinflammation may also be described as hyperinflammatory disease or syndrome. Alternatively it may be referred to as systemic inflammatory response syndrome (SIRS).
The compound comprising the structure of formula (1) includes the therapeutically active agent or a prodrug thereof. Whether the compound is the therapeutically active agent or the prodrug depends on the group(s) present at the X and/or Y position(s) in formula (1).
When X is selected
from —C(O)—, —C(O)O—, —C(O)NR5—, —C(O)OC(O)—, —C(O)NR5C(O)—, —S(O)2—, —S(O)2O— and —S(O)2NR5—, the dotted line to X in formula (1) is a single bond (e.g. as represented in each of the preceding substituents). All of these substituents comprise a carbonyl group or a sulfonyl group and form either an amide group (i.e. carboxamide group) or a sulfonamide group with the nitrogen atom shown in the structure. The amide and sulfonamide groups can be hydrolysed in vivo to produce a primary amine (e.g. as shown in the structure of formula (2)). Thus, when X is a group other than hydrogen, a compound having the structure of formula (1) can be a prodrug.
When X is a group other than hydrogen (i.e. selected
from —C(O)—, —C(O)O—, —C(O)NR—, —C(O)OC(O)—, —C(O)NR5C(O)—, —S(O)2—, —S(O)2O— and —S(O)2NR5—), then the resulting class of compounds may be referred to herein as an “amine prodrug compound”.
When X is hydrogen, then the compound comprises a structure of formula (2):
When Y is a group other than —S—, then the compound comprises a structure of formula (3):
In formula (3), Y1 may be selected from hydrogen, nitroso and phosphate.
When Y or Y1 is hydrogen, then the compound has a thiol group. Thus, the compound may have a structure of formula (4):
The therapeutically active agent is believed to comprise a thiol group.
Y or Y1 can be nitroso. The resulting compound comprises an S-nitrosothiol group (e.g. —S—NO). The S-nitrosothiol group may be converted in vivo to a thiol group. Thus, when the compound has the structure of formula (3) where Y1 is nitroso, then the resulting class of compounds may be referred to herein as a “nitrosothiol prodrug compound”.
Y or Y1 can be a phosphate group. The resulting group can be converted in vivo to a thiol group. Thus, when the compound has the structure of formula (3) where Y1 is phosphate, then the resulting class of compounds may be referred to herein as a “thiophosphate prodrug compound”.
When Y is —S—, the dotted line to Y in formula (1) is a single bond, such as shown in the structure of formula (5). The wavy line represents an undefined group.
As shown above, the resulting compound has a disulfide group. The disulfide group can be cleaved in vivo to produce a thiol group (e.g. as shown in the structure of formula (4)). Thus, when Y is —S—, a compound having the structure of formula (5) can be a prodrug. Thus, the resulting class of compounds may be referred to herein as a “disulfide prodrug compound”.
When Y is —S—, then it may be preferable for X to be hydrogen. Thus, the compound may comprise a structure of formula (6):
It may be preferable that the compound is either a compound having a structure of formula (3) or formula (4) [e.g. an amine prodrug compound] or a compound having the structure of formula (6) [e.g. a disulfide prodrug compound]. This may avoid issues associated with different in vivo mechanisms for the conversion of the prodrug forms of both the amine and the thiol functional groups.
The compound may preferably have a structure of formula (7):
In formulae (3), (3A), (7) or (7A), it is preferred that Y1 is selected from hydrogen and phosphate, more preferably Y1 is hydrogen.
Generally, each of R1 and R2 is independently selected from hydrogen and unsubstituted C, to C6 alkyl. It is preferred that each of R1 and R2 is independently selected from hydrogen and unsubstituted C, to C3 alkyl. More preferably, each of R1 and R2 is independently selected from hydrogen and methyl. Even more preferably, R1 and R2 is each hydrogen.
Typically, each R3 and R4 is independently selected from hydrogen and unsubstituted C, to C6 alkyl. It is preferred that each R3 and R4 is independently selected from hydrogen and unsubstituted C, to C3 alkyl. More preferably, each R3 and R4 is independently selected from hydrogen and methyl. Even more preferably, each R3 is hydrogen and each R4 is hydrogen.
R5 is typically selected from hydrogen and unsubstituted C1 to C6 alkyl. It is preferred that R5 is independently selected from hydrogen and unsubstituted C1 to C3 alkyl.
More preferably, R5 is selected from hydrogen and methyl. Even more preferably, R5 is hydrogen.
When the compound is an amine prodrug compound (e.g. X is not hydrogen), then preferably X is selected from —C(O)O—, —C(O)NR5—, —C(O)OC(O)—, —C(O)NR5C(O)—, —S(O)2O— and —S(O)2NR5—. More preferably, X is selected from —C(O)O—, —C(O)NH—, —C(O)OC(O)—, —C(O)NHC(O)—, —S(O)2O— and —S(O)2NH—.
It may preferred that X is selected from —C(O)O—, —C(O)NR5—, —C(O)OC(O)—, and —C(O)NR5C(O)—, such as —C(O)O—, —C(O)NH—, —C(O)OC(O)—, and —C(O)NHC(O)—. Even more preferably, X is —C(O)O— or —C(O)NR5—, particularly —C(O)O— or —C(O)NH—.
The carbon chain that directly connects the nitrogen atom and the sulfur atom shown in the structure of formula (1) can be two carbon atoms in length (e.g. an ethylene group) or three carbon atoms in length (e.g. a propylene). In the formulae above, this variation in the carbon chain length is represented by the integer n in relation to the carbon atom shown in brackets to which R3 and R4 are attached. When n is 1, then a single carbon atom is present within the brackets and the length of the carbon chain that directly connects the nitrogen atom and the sulfur atom is two carbon atoms in length. When n is 2, then two carbon atoms are present within the brackets and the length of the carbon chain that directly connects the nitrogen atom and the sulfur atom is three carbon atoms in length.
In general, it is preferred that the integer n is 1.
When n is 1 and each of R1, R2, R3 and R4 is hydrogen, then the compound comprises a structure of formula (1A):
In formula (1A), when X is hydrogen, then the compound comprises a structure of formula (2A):
In formula (1A), when Y is a group other than —S—, then the compound comprises a structure of formula (3A):
In formula (1A) or formula (3A), when Y or Y1 is hydrogen, then the compound has a thiol group. Thus, the compound may have a structure of formula (4A):
When the compound has a structure of formula (4A), then preferably the compound is pantethine.
When Y is —S—, the dotted line to Y in formula (1A) is a single bond, such as shown in the structure of formula (5A).
When Y is —S— in formula (1A), then it may be preferable for X to be hydrogen. Thus, the compound may comprise a structure of formula (6A):
It is preferred that the compound having a structure of formula (6A) is cystamine.
The compound may preferably have a structure of formula (7A):
It is preferred that the compound (e.g. having a structure of formula (7A)) is cysteamine, S-nitrosocysteamine or cysteamine S-phosphate. The compound is most preferably cysteamine.
In the formulae (1), (1A), (2), (2A), (5), (5A), (6) and (6A), the disulfide bond may connect the compound to a second amine group or a prodrug form thereof.
The compound may comprise a structure represented by formula (1B):
When the compound has a structure of formula (1B), then it may be preferable that each X is hydrogen. The compound may therefore have a structure of formula (2B):
When each n is 1 and each of R1, R2, R3 and R4 is hydrogen, then the compound having the structure of formula (1B) will have the structure of formula (5B):
Generally, in formulae (1B), (2B) and (5B) it is preferred, where present, that each n is the same, each R1 is the same, each R2 is the same, each R3 is the same, each R4 is the same, each X is the same, and each R5 is the same.
The compound having the structure of formula (1) may be a compound having a structure as represented by formula (1C) or by formula (1AC):
Thus, in the formulae (1), (1A), (2) and (2A) above when Y is —S—, then the dashed line may be a bond to the group Q. Similarly, in formulae (5), (5A), (6) or (6A) above, the wavy bond may represent a bond to the group Q.
The compound having the structure of formula (1) may be a compound having a structure as represented by formula (1D) or by formula (1AD):
Thus, in the formulae (1), (1A), (2) and (2A) above when Y is —S—, then the dashed line may be a bond to the group Q. Similarly, in formulae (5), (5A), (6) or (6A) above, the wavy bond may represent a bond to the group Q.
The compound having the structure of formula (1) may be a compound having a structure as represented by formula (1D) or by formula (1AD):
Thus, in the formulae (1), (1A), (3), (3A), (4), (4A), (5) and (5A) above when X is a group other than hydrogen, then the dashed line may be a bond to the group Z.
The compound having the structure of formula (1) may be a compound having a structure as represented by formula (1E) or by formula (1AE):
In the formulae above, the moiety —S-Q represents a compound selected from glutathione, cysteine, N-acetylcysteine, homocysteine and gamma-L-glutamyl-L-cysteine (e.g. the S atom in the moiety —S-Q represents the S atom in each of these compounds, which forms the disulfide bond). Additionally or alternatively, Q may be selected from optionally substituted C1 to C26 alkyl, optionally substituted C2 to C26 alkenyl, optionally substituted C2 to C26 alkynyl, optionally substituted C6 to C14 aryl, optionally substituted C3 to C14 cycloalkyl, optionally substituted heteroaryl having 5 to 14 ring atoms and at least one heteroatom selected from nitrogen, oxygen and sulfur, and optionally substituted heterocyclyl having 5 to 14 ring atoms and at least one heteroatom selected from nitrogen, oxygen and sulfur.
Also in the formulae above, Z may comprise an antiviral compound or an anti-inflammatory compound and an optional linker group. Z is covalently linked to X optionally by the linker group. The linker group may be a fatty acid side chain, such as described below.
A fatty acid may be used to form an amide group with the nitrogen atom shown in the formulae above. Thus, the carbonyl group in X is derived from the carboxylic acid group of the fatty acid. The fatty acid side chain is the carbon chain that is connected to the carboxylic acid group in the fatty acid.
Thus, Z may be a fatty acid side chain (e.g. C1 to C26 alkyl or C2 to C26 alkenyl), such as from a fatty acid selected from caprylic acid, capric acid, lauric acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid and docosahexaenoic acid.
Z may be selected from hydrogen, optionally substituted C1 to C26 alkyl, optionally substituted C2 to C26 alkenyl, optionally substituted C2 to C26 alkynyl, optionally substituted C6 to C14 aryl, optionally substituted C3 to C14 cycloalkyl, optionally substituted heteroaryl having 5 to 14 ring atoms and at least one heteroatom selected from nitrogen, oxygen and sulfur, optionally substituted heterocyclyl having 5 to 14 ring atoms and at least one heteroatom selected from nitrogen, oxygen and sulfur, —(CH2)2-S—Y1 and —(CH2)3-S—Y1, where Y1 is as defined above.
The compound can be any of 2-methylthio ethylamine (cinnamate), 2-methyl thio ethylurea, N-(2-methylthio ethyl) p-acetamido benzamide, 2-aminoethanethiol, N-(2-methylthio ethyl)p-acetamido benzenesulfonamide,N-(2-propylthioethyl)-p-methoxy benzamide, N-(butylthio ethyl) nicotinamide, N-(2-dodecylthio ethyl) p-butoxybenzamide, N— (2-methylthio ethyl) p-toluenesulfonamide, N-(2-isopropylthio ethyl) propionamide, N-(2-octylthio ethyl) acetamide, N-(2-butylthio ethyl) methanesulfonamide, N-(2-isopentylthio ethyl)butane, bis 1,4-(2-acetamido ethylthio), 2,3-butanediol, 2-hexadecylthio ethylamine hydrochloride, 2-allylthio ethylamine malate,9-octadecene 2-ylthio ethylamine hydrochloride, 2-dodecylthio ethylamine hydrochloride, 2-isopentylthio ethylamine mandelate, 2-octadecylthio ethylamine salicylate, 2-·beta·-hydroxyethyl thio ethylurea, 2-·beta·-hydroxyethylthio ethylamine hydrochloride, 2-(2,3-dihydroxy propylthio)ethylamine p-toluenesulfonate, 2-(2-hydroxypropylthio)ethylamine oxalate, N-(2-methylthio ethyl)phenylacetamide, 2-(2,2-dimethoxy ethylthio) ethylamine hydrochloride, 2-(2,2-dimethoxy ethylthio) ethylamine undecylenate, 2-(2,2-diethoxy ethylthio) ethylamine undecylenate, 2-(2,2-diethoxy ethylthio)ethylamine acetate, 2-undecenylthio ethylamine, 2-·beta·-ureidoethylthio ethylamine hydrochloride, 2-·beta·-acetamidoethylthio ethylamine tropate, 2,2′-thio diethylamine fumarate, 2,2′-thio diethylurea, 3-·beta·-aminoethylthio propylamine hydrochloride, S-·beta·-ureidoethyl thiocarbamate, 2-ethoxycarbonylthio ethylamine hydrochloride, 2-dimethylamino carbonylthio ethylamine sulfate, 2-butoxycarbonyl methylthio ethylurea, 2-ethyloxycarbonylmethylthio ethylamine hydrochloride, 6-·beta·-aminoethylthio hexanoate of methyl hydrochloride, 5-·beta·-aminoethylthio pentanoic acid, 2-phenylthio ethylamine dihydrogen phosphate, 2-p-t-butylphenylthio ethylamine trichloracetate, 2-p-methoxyphenylthio ethylamine ditartrate, 2-tolylthio ethylamine hydrobromide, 2-(I-biphenyl thio) ethylamine hydrochloride, 2-N-pentachlorophenylthio ethyl acetamide, 2-benzylthio ethylamine malate, 2-benzylthio ethylamme nicotinate, 2-benzylthio 2-methyl propylamine hydrochloride, 2-benzylthio propylamine lactate, N-(2-benzylthio ethyl)nicotinamide hydrochloride, N-(2-benzylthio ethyl) 10-undecene amide, N-(2-benzylthio ethyl) hexadecanamide, S-·beta·-aminoethyl mercaptobutyric acid, N-(2-benzylthio ethyl)formamide, N-(2-benzylthio ethyl)phenylacetamide, N-[2-(2,6-dimethyl phenyl)ethyl] hexanamide, 2-o-aminophenylthio ethylamine succinate, N-(2-benzylthio ethyl) glutamine, S— ·beta·-aminoethyl mercapto acetic acid (3-S-·beta·-aminoethyl) mercapto propionic acid, (3-S— ·gamma·-amino propyl) mercapto acetic acid, S(2-p-methoxybenzamido ethyl) mercapto 2-(2-naphtyl methylthio) ethylamine hydrochloride, 2-(2-naphtyl methylthio) ethylamine disuccinate, (2-thenyl) 2-thio ethylamine hydrobromide, 2-N-acetyl (2-thenylthio-ethylamine, 2-o-chlorobenzylthio ethylamine hydrochloride, 2-p-chlorobenzylthio ethylamine glycolate, 2-o-fluorobenzylthio ethylamine hydrochloride, 2-furfurylthio ethylamine hydrochloride, 2-tetrahydrofurfurylthio ethylamine p-amino-benzoate, 2-·beta·-phenylethylthio ethylamine glutamate, 2-diphenylmethylthio ethylamine hydrochloride, 2-triphenyl methylthio ethylamine hydrochloride hemihydrate, 2-(2-pyridyl ethylthio)ethylamine hydrochloride, 2-(2-p-toluene sulfonamido ethyl thio) pyridine N-oxide, 2-·beta·-aminoethylthiomethyl pyridine N-oxide dihydrochloride, 2-·beta·-aminoethylthio pyridine N-oxide hydrochloride, 2,4-dichloro 2-benzylthio ethylamine aspartate, N-[2-(3,4-dichloro benzylthio)ethyl] butyramide, N-[2-(2,6-dichloro benzylthio)ethyl] dodecanamide, N-[2-(3,5-dichloro benzylthio)ethyl]trifluoroacetamide hydrochloride, 2-p-ethoxybenzylthio ethylamine hydrochloride, N-[2-m-fluorobenzylthio ethyl] chloroacetamide, 2-p-bromobenzylthio ethylamine succinate, 2-(3,4-dimethoxy benzylthio)ethylamine malate, 2-(3,4-methylenedioxy benzylthio)ethylamine hydrochloride, 2-(2,4-dichloro cetylthio)ethylamine, 2 (3,4,5-trimethoxy benzylthio)ethylamine hydrocinnamate, 2-p-methoxy benzylthio ethylamine salicylate, 2-o-methylbenzylthio ethylamine phenyl-acetate, N-[2-p-dimethylaminobenzylthio ethyl]methane-sulfonamide,2-p-phenoxybenzylthio ethylamine hydrochloride, 2-·beta·-aminoethylthio pyridine hydrochloride, 2-benzylthio ethylamine citrate, N-[2-benzylthio ethyl] 2,4-dihydroxy 3,3-dimethyl butyramide, N-(2-benzylthio ethyl) 6,8-dihydroxy 7,7-dimethyl 5-oxo 4-aza octanamide, N-[2-(2-pyridyl thio)ethyl] propionamide, 2-(2-pyridyl methylthio)ethylamine dihydrochloride, 2-benzylthio ethylamine pantothenate, S—(·beta·-acetamidoethyl)mercaptoacetate of beta·-morpholinoethyl, S—(·beta·-phenylacetamidoethyl)mercaptoacetate N′-methyl 2-piperazino ethyl, S—(·beta·-ureidoethyl)mercaptoacetate of beta·-pyrrolidino-ethy, S—(·beta·-trifluoroacetamidoethyl)-·beta·mercapto-propionate of ·beta·-dimethylaminoethyl, 2-p-nitrobenzylthio ethylamine crotonate, 2-·beta·-morpholinocarbonyl ethylthio ethylamine hydrochloride, N,N-di(hydroxyethyl)S—(·beta·-benzamido-ethyl) mercapto-acetamido, N[2-N— methyl piperazino carbonylthio ethyl] acetamide, 2-(I-naphthyl thio)ethylamine hydrochloride, N-(3-·beta·-ureidoethylthio propyl) succinamic acid, 3-allylthio propylamine, 3-(2,2′-dimethoxy ethylthio)propylamine, 3-(2,2′-dimethoxy ethylthio)propylamine sulfate, S-·beta·-aminoethylmercapto acetic acid, the hydrochloride of S-·beta·-aminoefhyl mercapto acetic acid, N-(2-benzylthioethyl)acetamide, N-(2-benzylthioethyl)propionamide, N-(2-benzylthioethyl)butyramide, N-(2-benzylthioethyl)methanesulfonamide, N-(2-benzylthioethyl)ethanesulfonamide, N-(2-benzylthioethyl-propanesulfonamide, N-(2-benzylthioethyl)butanesulfonamide, S-(2-p-acetamidobenzenesulfonamido ethyl) mercapto acetic acid, S-(2-p-acetamidobenzamido ethyl) mercapto acetic acid, N-(2-thenylthioethyl)acetamide, 2-benzylthio propylamine, 2-benzylthio 2-methyl propylamine, 2-(2-p-toluenesulfonamido ethylthio) pyridine N-oxide, S-(2-p-butoxybenzamidoethyl)mercapto acetic acid, 2-t-butylthio ethylamine hydrochloride, 2-methoxycarbonyl methylthio ethylamine hydrochloride, 2-ethoxycarbonylmethylthio ethylamine hydrochloride, 2-propoxycarbonylmethyl thio ethylamine hydrochloride, 2-butoxycarbonylmethylthio ethylamine hydrochloride, 2,2′-thio diethylamine dihydrochloride, 3-(2-aminoethylthio)alanine hydrochloride, 2-benzylthio ethylammonium diacid phosphate, 2-methylthio ethylamine, N-(methylthioethyl) p-acetamidobenzamide, N-(2-methylthioethyl)nicotinamide, N-(2-methylthioethyl)benzamide, N-(2-methylthioethyl) p-butoxybenzamide, N-(2-methylthioethyl) butyramide, N-(2-methylthioethyl) propionamide, N-(2-methylthioethyl) acetamide, N-(2-methylthioethyl) butanesulfonamide, N-(2-octylthioethyl) methanesulfonamide, 2-cetylthio ethylamine hydrochloride, 2-(2-hydroxyethylthio) ethylamine hydrochloride, 2-methylthio ethylamine phenylacetatesnd 2-methylthio ethylamine undecylenate.
The compound may also be amifostine. Amifostine is an organic thiophosphate prodrug which is hydrolysed in vivo by alkaline phosphatase to the active cytoprotective thiol metabolite, WR-1065.
The invention also relates to a pharmaceutically acceptable salt of the compound. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, “Pharmaceutically Acceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19.
If the compound is cationic, or has a functional group which may be cationic, then a salt may be formed with a suitable anion or a conjugate acid thereof. Examples of suitable inorganic anions include chloride, bromide, iodide, phosphate, dihydrogenphosphate, sulfate, bisulfate, hemisulfate, persulfate, sulfonate, and nitrate. Examples of suitable organic anions include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, butyrate, camphorate, camphorsulfonate, cinnamate, citrate, crotonate, cyclopentanepropionate, detartrate, digluconate, disuccinate, dodecylsulfate, ethanesulfonate, fumarate, gluconate, glucoheptanoate, glycerophosphate, glycolate, heptanoate, hexanoate, 2-hydroxyethane-sulfonate, lactate, maleate, mandelate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pantothenate, pectinate, picrate, pivalate, propionate, salicylate, succinate, tartrate, thiocyanate, tosylate, tropate, trichloroacetate, undecanoate, and undecylenate.
If the compound is anionic, or has a functional group which may be anionic, then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include alkali metal ions, such as Na* and K*, and the alkaline earth metal cations, such as Ca2+ and Mg2+. Examples of suitable organic cations include ammonium ion (i.e. NH4+) and substituted ammonium ions (e.g. NH3R+, NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, triethylamine, as well as amino acids, such as lysine and arginine.
Generally, it is preferred that the pharmaceutically acceptable salt of the compound is a chloride salt or a bitartrate salt.
The compound may be cysteamine bitartrate.
Any compound comprising the structure of formula (1) according to any aspect of the present invention will preferably inhibit the activity of glycine decarboxylase activity. How to determine such inhibition is described above.
Where the context permits, all optional features of the first aspect of the present invention apply equally to the further aspects of the present invention.
Throughout the specification, unless the context demands otherwise, the terms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or ‘comprising’, ‘includes’ or ‘including’ will be understood to imply the method or kit includes a stated integer or group of integers, but not the exclusion of any other integer or group of integers.
Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in the text is not repeated in this text is merely for reasons of conciseness. Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known in any country.
Aspects of the present invention will now be illustrated by way of example only and with reference to the following experimentation and to the figures.
“Virus” or “viral infection” relates to any virus in the context of the present invention. As the invention provides therapeutic utility principally via inhibition of glycine decarboxylase, and all viruses require their hosts to utilise this enzyme in order to enable viral replication, virus and viral infection can relate to any virus. Consequently, the virus or viral infection can be a member Duplodnaviria, Monodnaviria, Riboviria, Varidnaviria, or any combination thereof. The virus can therefore be any one or combination of the following: a Ribovirus, an influenza, ebola, coronavirus, hepatitis C, hepatitis E, West Nile Fever, Norovirus, Rotavirus, Polio, rabies, measles, rhinovurus. Preferably the virus is a coronavirus. The virus can be SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-299E, HCoV—OC43, HCoV-NL63, CoV-HKU1, Human Coronavirus 229E (ATCC No. VR/740) or any combination thereof. The virus is most preferably SARS-CoV-2.
The expression “optionally substituted”, as used herein, refers to a parent group, which is unsubstituted or substituted. The term “substituted,” as used herein, refers to a parent group which bears one or more substitutents. The term “substituent” is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to the parent group.
The term “alkyl” as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a linear or branched, saturated aliphatic hydrocarbon compound. The prefix (e.g. C1 to C26) denotes the number of carbon atoms. It is preferred that the alkyl group is C1 to C10 alkyl, such as C1 to C6 alkyl. It may be preferable that the alkyl group is an unsubstituted alkyl group.
The term “alkenyl” as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a linear or branched aliphatic hydrocarbon compound, which has at least one carbon-carbon double bond. An alkenyl group may have two or more double bonds, which may or may not be conjugated. An alkenyl group typically does not include a carbon-carbon triple bond. It is preferred that the alkenyl group is C2 to C10 alkenyl, such as C2 to C6 alkenyl. It may be preferable that the alkenyl group is an unsubstituted alkenyl group.
The term “alkynyl” as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a linear or branched aliphatic hydrocarbon compound, which has at least one carbon-carbon triple bond. An alkynyl group may have two or more triple bonds. An alkynyl group may include a carbon-carbon double bond. It is preferred that the alkynyl group is C2 to C10 alkynyl, such as C2 to C6 alkynyl. It may be preferable that the alkynyl group is an unsubstituted alkynyl group.
The term “aryl” as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 6 to 14 ring atoms (as indicated by the prefix C6 to C14). Preferably, the aryl group is a C6 to C14 aryl group, more preferably the aryl group is a phenyl group. It may be preferable that the aryl group is an unsubstituted aryl group.
The term “cycloalkyl” as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring carbon atom of a carbocyclic ring of a carbocyclic compound. The carbocyclic ring may be saturated or unsaturated. Preferably, the carbocyclic ring is saturated. It is preferred that the cycloalkyl group is C3 to C10 cycloalkyl, such as C3 to C6 cycloalkyl. It may be preferable that the cycloalkyl group is an unsubstituted cycloalkyl group.
The term “heteroaryl” as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic heterocyclic compound. The heterocyclic compound has from 5 to 14 ring atoms, preferably 5 to 10 ring atoms, and even more preferably 5 or 6 ring atoms. The heterocyclic compound comprises at least one heteroatom, which is a ring atom. The heteroatom may be selected from nitrogen, oxygen and sulfur. Preferably, the heteroaryl group is a pyridyl group. It may be preferable that the heteroaryl group is an unsubstituted heteroaryl group.
The term “heterocyclyl” as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a non-aromatic heterocyclic compound. The heterocyclic compound has from 5 to 14 ring atoms, preferably 5 to 10 ring atoms, and even more preferably 5 or 6 ring atoms. The heterocyclic compound comprises at least one heteroatom, which is a ring atom. The heteroatom may be selected from nitrogen, oxygen and sulfur. Preferably, the heterocyclyl group is a morphinyl group.
Any reference herein to an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl group or an optionally substituted cycloalkyl group includes a group (e.g. the alkyl, alkenyl or alkynyl group) substituted with one or more substituents selected from halo, hydroxy (—OH), C1 to C6 alkoxy, C6 to C10 aryloxy, nitro (—NO2), nitrile (—CN), oxo (═O), carboxylic acid (—COOH), carboxylic ester (—C(O)OR), carboxamide (—CONR2), carbonyloxy (—O(C(O)R), carbonylamino (—NRC(O)R), -carbonate (—OC(O)OR), ureido (—NRC(O)NR2), carbamate (—OC(O)NR2) and urethane (—NRC(O)OR). Each of the substituents are typically unsubstituted.
Any reference herein to an optionally substituted aryl, an optionally substituted heteroaryl or an optionally substituted heterocyclyl includes a group (e.g. the aryl, heteroaryl or heterocyclyl group) substituted with one or more substituents selected from C1 to C6 alkyl, halo, hydroxy (—OH), C1 to C6 alkoxy, C6 to C10 aryloxy, nitro (—N02), nitrile (—CN), carboxylic acid (—COOH), carboxylic ester (—C(O)OR), carboxamide (—CONR2), carbonyloxy (—O(C(O)R), carbonylamino (—NRC(O)R), -carbonate (—OC(O)OR), ureido (—NRC(O)NR2), carbamate (—OC(O)NR2) and urethane (—NRC(O)OR). Each of the substituents are typically unsubstituted.
In the optional substituents above, each group represented by “R” can be hydrogen or an unsubstituted C1 to C6 alkyl group.
The term “alkoxy” as used here refers to a group of the formula —OR, where R is an alkyl group as defined above.
The term “aryloxy” as used here refers to a group of the formula —OAr, where Ar is an aryl group as defined above.
The term “halo” as used here refers to the halogens, chloro, bromo or fluoro. It is preferred that the halo is chloro or bromo.
The effect of treatment of coronavirus-infected pulmonary cells with cysteamine was studied. Two separate pulmonary cell lines (ie A549 and MRC-5) were used as the basis for the study. A549 (sample obtained from deposit, deposited under ATCC CCL-185) being a human lung epithelial cell line, whilst MRC-5 (sample obtained from deposit, deposited under ATCC CCL-171) being a human lung fibroblast cell line. Prior to viral challenge cells were grown at 37° C. in 5% CO2 atmosphere in growth media (EMEM+10% FBS+pen-strep for MRC-5 cells/F-12 with Kaighn's modification+10% FBS+pen-strep) to 70-80% confluency. Immediately prior to viral challenge, cells were washed in Hanks balanced salt solution and media was replaced with 100 μl challenge medium per well (EMEM for MRC-5 and F-12 for A549) which contained 1% serum. A separate plate of cells was used for each cysteamine treatment concentration as well as one for no treatment. Cells were incubated at 33° C. in a 5% CO2 atmosphere and treated daily with the same concentration of cysteamine until the 5th day when immunoperoxidase assay was conducted according to the protocol described in Lambert et al., 2008. 4 wells at a time were infected with a human coronavirus hCoV 229E (a coronavirus related to SARS-CoV-2, obtained from a deposit, deposited under ATCC VR/740), the concentration of virus diluted with each subsequent 4 well-group. It was determined that the stock inoculum from which each dilution was derived (determined in the untreated cells) was about 1.12×1014 pfu/ml.
All the infected cell cultures were treated with daily doses of cysteamine free-base. Two dosing regimens were tested on the infected cell cultures; an application of 1 mg/L cysteamine once a day for 5 days was applied one set of plates, and an application of 2 mg/L cysteamine once a day for 5 days was applied to another set of plates (5 microlitres of 20×stock into 100 microlitres on the plate, with controls being provided with a mock treatment). The above procedure was carried out in quadruplicate (ie in four parallel wells, as the above was practiced in a 96 well plate).
The effect of the treatment of each infected cell culture with each dosing regimen on viral infection was assayed by microscopic examination of the cultured cells when compared to the same infected cell cultures over the same period but in the absence of cysteamine treatment. Uninfected cell lines (treated and untreated with cysteamine) were provided as controls. Viral titre of the undiluted stock of virus used as the originator stock for the study and as provided by the depository institute was determined as the 50% tissue culture infectivity dose (TCID50: ie the number of viruses required to cause cytopathology in 50% of wells; a value that can be converted into the viral titre-pfu/ml by multiplying by 0.7) following immunoperoxidase assay (Lambert, et al, 2008, Methods in Molecular Biology, 454:93-102). From this undiluted stock, the range of concentrations of virus were prepared; 16 dilutions extending form a 10-1 to a 10-16 dilutions by volume from the original stock was prepared and used in the study.
Results Summary: Results of Treatment of Coronavirus-Infected Pulmonary Cells
Application of the 2 mg/L cysteamine daily for 5 days was shown to confer protection against infection with hCoV 229E in both the A549 and MRC-5 cell lines over all concentrations of virus used in the study.
The high level of pfu/ml of the inoculum demonstrates that the concentrations tested contained a very high number of infectious 229E virions representing a physiologically very heavy viral load of infection.
A lower pfu/ml value, relative to that of the control in which cysteamine was not applied, demonstrates that cysteamine can reduce viral load in both cell types at 2 mg/L (which is physiologically achievable in vivo).
These results clearly demonstrate an ability for cysteamine to both reduce viral load in a coronavirus infection and to provide cell-protective role against viral induced cell death.
To investigate how cysteamine is reducing viral load, the following experiment looked at cytokines which are modulated by the glycine cleavage system.
Results summary: These results show that cysteamine treatment modulates cytokines downstream from the glycine cleavage pathway. The glycine cleavage pathway feeds into the biosynthetic pathways for purines and pyrimidines, the building blocks for nucleotide synthesis. When a cell is infected by a virus, purine and pyrimidine levels are reduced as these are being used for viral replication. These modulated cytokines are induced in response to this reduction in pyrimidines/purines, as a host response. The inventors hypothesize that cysteamine is acting on the enzyme glycine decarboxylase in the glycine cleavage pathway to cause this cytokine modulation. By acting on glycine decarboxylase, the actions of cysteamine are two-fold: 1) it reduces viral load by inhibiting the production of pyrimidines and purines which could otherwise be used for viral replication; and 2) as a result of this reduction in these nucleotide building blocks, boosts the cell's natural response to viral attack by inducing cytokines which help viral clearance.
This multi-pronged action of cysteamine is summarised in
Cell Challenge
MRC-5 airway fibroblast cells were grown to 90% confluence in 2×12-well plates in EMEM+10% FBS. Cell culture media was replaced with:
Plate 1
Plate 2
As above, but with the addition of cysteamine to a final concentration of 10 mg/L Cells were then incubated at 37° C. and 5% C02 for 4 h. After incubation cell supernatants were harvested and transferred to labelled cryotubes and stored at −80° C. prior to assessment of glycine content. Insulin strongly stimulates glycine utilisation via glycine decarboxylase.
Glycine Assessment
The supernatants were thawed prior to determining the glycine concentration using the fluorometric glycine assay kit from Abcam (Ab211100).
Manufacturer's instructions were followed and fluorometric assessment of glycine concentration in supernatants and standards was performed on the Biotek Synergy plate reader using 96-well black-walled plates at 535/587 nm (ex/em).
Results summary: The results are shown in
Glycine Decarboxylase (GLDC) activity depends upon PLP cofactor. Pyridoxal-5-phosphate (PLP) is yellow green in aqueous solution with an absorption maxima at 388 nm.
A reaction with cysteamine will form a thiazolidine compound with a blue-shifted absorbance maxima at 320 nm.
10 ml equimolar solutions of 20 mM pyridoxal 5′-phosphate monohydrate (Sigma: 82870), 20 mM cysteamine free base (CFB), 20 mM cysteamine bitartrate (CBT) and 20 mM cystamine dihydrochloride (CTM) were prepared in carbonate-bicarbonate buffer pH 7.4 In a 96-well microtitre plate 6×replicates of 100 μl total volume containing each of the following reaction mixes were prepared:
Plates were mixed gently by tapping and incubated for 1 h in the dark at room temperature prior to full spectral scan (300-700 nm) performed on Biotek Powerwave and the absorbances recorded. Absorbances at 390 and 320 nm compared for reaction components and products.
Results summary: Cysteamine is shown to react with the GLDC co-factor PLP. The inventors hypothesize cysteamine may inhibit GLDC via access to, and reaction with the PLP co-factor rather than directly with the GLDC enzyme. Cysteamine forms a thiazolidine ring with PLP and as shown in
Following on from Example 1, to further investigate the efficacy of cysteamine to reduce viral titer, cysteamine was applied to infected A549 lung epithelial cells and viral load was measured by PCR.
The effect of treatment of coronavirus-infected pulmonary cells with cysteamine was studied using A549 (sample obtained from deposit, deposited under ATCC CCL-185) being a human lung epithelial cell line. Prior to viral challenge A549 cells were grown at 37° C. in 5% CO2 atmosphere in growth media F-12 with Kaighn's modification+10% FBS +pen-strep [F-12K]) to 70-80% confluency in F12-K in tissue-culture-treated 96-well plates. Immediately prior to viral challenge, cells were washed in Hanks balanced salt solution and media was replaced with 100 μl challenge medium per well (F-12K) which contained 1% serum. A separate plate of cells was used for each cysteamine treatment concentration as well as one for no treatment. Cells were incubated at 33° C. in a 5% CO2 atmosphere and treated daily with the same concentration of cysteamine (0, 1 or 2 mg/L) until the 5th day when supernatants were removed and viral RNA was extracted using the Qiagen viral RNA mini kit and RNA was frozen at −85° C. until required. Viral RNA was reverse transcribed using Superscript IV reverse transcriptase (Thermo-Fisher)). Quantitative (real-time) polymerase chain reaction (qPCR) was carried out using PrecisionPLUS qPCR Master Mix (PrimerDesign) and forward and reverse oligonucleotides (229E Sense & 229E Antisense) specific to the hCoV 229E M protein gene. Amplification was carried out using a MiniOpticon Real-Time PCR system (Bio-Rad) and data was analysed using Opticon Monitor software (Bio-Rad). Analysed samples had been exposed to 10−3 (1.12×1012 pfu/ml), 10−4 (1.12×1011 pfu/ml) and 10−5 (1.12×1010 pfu/ml) of hCoV 229E and conducted from quadruplicate technical replicates. The number of copies of the 229E M protein gene were determined from a standard curve containing known number of copies of the 229E M protein gene.
Results Summary: The results are shown in
Materials:
Method:
Vero E6 MESO cells were seeded in 96-well plates and cultured overnight. The following day, 2-fold serial dilutions of cysteamine starting at 256 mg/L were prepared in (after addition of the virus the final compound concentrations are 128 mg/L, 64 mg/L, 32 mg/L, 16 mg/L, 8 mg/L, 4 mg/L, 2 mg/L, 1 mg/L, 0.5 mg/L, 0.25 mg/L, 0 μM). Serial dilutions of the compounds were added to cells as depicted in the plate layout below, and infected with one dose of SARS-CoV-2.
Plates were incubated at 37° C. Every 24 h one plate was fixed and stained. Prior to fixing supernatant from the cells was harvested into Trizol. A daily dose of cysteamine of 2 mg/L final was added to wells 1 to 11 in rows E to H in the remaining plates. The remaining half of the plate (i.e. rows A to D) was left untouched. Daily dosing regimen for each plate and timepoint is presented in
Results Summary: The results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007768.1 | May 2020 | GB | national |
| 2012298.2 | Aug 2020 | GB | national |
| 2102106.8 | Feb 2021 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2021/051269 | 5/25/2021 | WO |
