Patent Application
20230142547
The present invention relates to the treatment or prevention of a Coronaviridae infection, and conditions related thereto and infections with other viruses that are dependent on Dynamin 2; in particular a Coronaviridae infection in humans.
In particular, the invention relates to compounds, pharmaceutical compositions and medicaments for treating and/or preventing a Coronaviridae infection, and conditions related thereto.
The invention further relates to uses and methods for assessing a Coronaviridae infection or the efficacy of a treatment of such infections, and conditions related thereto.
Viruses are one of the major causes of diseases around the world. Viruses are generally defined as small, non-living, infectious agents that replicate only within living cells, as they do not possess a completely autonomous replication mechanism. Although diverse in shape and size, they typically consist of a virus particle (known as a “virion”), made from a protein coat which comprises at least one nucleic acid molecule and optionally, depending on the type of virus, one or more proteins or nucleoproteins.
Even though their replication cycle varies greatly between species, it is generally recognized that the life cycle of viruses includes six basic steps: attachment, penetration, uncoating, replication, assembly and release.
Depending on the nature of the targeted virus, therapeutic molecules have been designed which may interfere with one or more of those mechanisms.
Among those, the replication step involves not only the multiplication of the viral genome, but also the synthesis of viral messenger RNA, of viral protein synthesis, and the modulation or use of the transcription or translation machinery of the host. However, it is also clear that the type of genome (single-stranded, double-stranded, RNA, DNA . . . ) characterizes dramatically this replication step. For instance, most DNA viruses assemble in the nucleus while most RNA viruses develop solely in the cytoplasm. Also, there is increasing evidence that single-stranded RNA viruses use the host RNA splicing and maturation machinery.
Accordingly, and considering the implications of a given type of genome in the replication step, the Baltimore classification of viruses was developed. This classification clusters viruses into families (or “groups”) depending on their type of genome. The present virus classification, as in 2018, comprises seven different groups:
There are few cures for diseases caused by RNA virus infections, in particular single-stranded RNA viruses, and more specifically RNA virus infections from viruses belonging to group IV of the Baltimore classification.
Strikingly, an acute respiratory disease was recently found to be caused by a novel coronavirus (SARS-CoV-2, previously known as 2019-nCoV), also referred herein as the coronavirus disease 2019 (COVID-19), which belongs to the Coronaviridae family and which part of the group IV of the Baltimore classification.
This coronavirus shows sustained human-to-human transmission, along with many exported cases across the globe. World Health Organization (WHO) has officially declared the COVID-19 pandemic as a public health emergency of international concern. The novel coronavirus uses the same receptor, angiotensin-converting enzyme 2 (ACE2) as that for Severe Acute Respiratory Syndrome (SARS)-CoV, and mainly spreads through the respiratory tract. The elderly and people with underlying diseases are susceptible to infection and prone to serious outcomes, which may be associated with acute respiratory distress syndrome (ARDS) and cytokine storm.
Four strategies for therapy are currently explored:
(i) to limit the spreading of the SARS-CoV-2 infection by blocking the replication of the virus. This can be achieved through inhibition of RNA dependent polymerase (RdRp) of the virus or by preventing the entry of the virus in pulmonary and other tissues target cells;
(ii) to dampen the inflammation of the pulmonary tracts and of other tissues;
(iii) to promote tissue repair of the pulmonary tracts and of other tissues;
(iv) to promote a vaccination strategy.
Antiviral drugs for managing infections with human coronaviruses are not yet approved (except one anti-IL6 product in the US with limited efficacy data), posing a serious challenge to current global efforts aimed at containing the outbreak of COVID-19. Some companies are testing several combinations in clinical trials to find antiviral compounds, mainly nucleoside analogues, in order to block the RNA-dependent RNA polymerase (RdRp) of the virus, and anti-proteases in order to block the entry of the virus. Other strategies involve biologics and the combination of drugs, in order to block the inflammation.
The response to each of these therapies is highly uncertain at the moment and requires other options to be explored.
WO2014111892 teaches the use of miR-124 as a biomarker of an HIV infection.
WO2016135052 and WO2016135055 teach the use of quinoline derivatives and metabolites thereof for treating or preventing viral infections, including HIV infections. Indeed, originally developed as an inhibitor of HIV replication and HIV reservoir reduction, a set of quinoline derivatives including the ABX464 compound has now been found to bind to the Cap Binding Complex (CBC) at the interface between the two “20” and “80” subunits of a large complex that regulates splicing and export of mRNA from the nucleus. The active metabolite of ABX464, a N-glucuronidated form referred herein as “ABX464-N-Glu”, also binds to the CBC complex. The ABX464-CBC interaction has been shown to strengthen the RNA quality control of HIV-RNA biogenesis, thus preventing the production of unspliced HIV RNA and to reduce reservoirs of HIV infected patients.
While capable of altering directly the splicing of a small number of genes in cells, the examination of the effects of ABX464 on the microRNAs profile has shown that the expression of a single miRNA was significantly increased by ABX464: miR-124.
WO2020011810 now further teaches the use of quinoline derivatives for treating or preventing a RNA virus infection, and more particularly RNA viruses belonging to the group IV of the Baltimore classification, to which the Coronaviridae family belongs.
However there remains a need for novel compounds for treating or preventing a RNA virus infection, and especially a Coronaviridae infection.
The invention has for purpose to meet the above-mentioned needs.
The inventors have surprisingly found that dynamin 2 (DNM2), which is a GTPase responsible for vesicle scission, is a target of miR-124, especially in the context of a Coronaviridae infection.
Dynamin-2 is a well-known pleiotropic GTPase which is involved in many membrane-remodeling events, including membrane scission during vesicle budding from the plasma or Golgi membranes, synaptic vesicle recycling, post-synaptic receptor internalization, neurosecretion, and neuronal process extension.
Dynamin-2 on its own is now further shown to be a therapeutic target for treating or preventing a Coronaviridae infection, through the development of so-called dynamin inhibitors (i.e. dynamin-2 inhibitors), such as phenothiazine and phenothiazine-derived drugs.
Indeed, although compounds belonging to the phenothiazine class, such as chlorpromazine, are known to exert anti-prion effects, applications of this particular class of active agents have not yet been reported for the treatment or prevention of a Coronaviridae infection.
While, on one side, ABX464 is known to decrease inflammation, it is now further demonstrated that uncontrolled pulmonary inflammation can be critical for the prognosis and death of SARS-CoV-2 infections. Indeed, many COVID-19 patients develop acute respiratory distress syndrome (ARDS), which leads to pulmonary edema and lung failure. Without wishing to be bound by the theory, the inventors thus propose that elevated pro inflammatory cytokines involved in Th17 responses in COVID-19 infected patients may be the cause of vascular permeability and leakage.
Some infected patients have partially reduced lung function. There is furthermore a suspicion of occurrence of pulmonary fibrosis in some cases, which is now under investigation but at least scars in the lung or lung lesions may be observed. Over time, tissue destroying makes it hard for oxygen to get into the blood. Low oxygen levels (and the stiff scar tissue itself) can cause shortness of breath, particularly during physical exertion. And recovery from lung tissue damage or destruction, after infection, may take time.
Vasculitis or at least mimicry of vasculitis has further been reported on COVID-19 patients.
Additionally, a suspected association between HCoVs and Kawasaki disease was raised even if its confirmation is also under investigation.
Hence, the inventors propose that attenuation of Th17 proliferation by ABX464, or its N-glucuronide metabolite (ABX464-N-Glu), may treat or prevent a Coronaviridae infection, and especially, the severe acute respiratory syndrome caused by SARS-CoV-2 infection.
The inventors also propose that the dual ability of ABX464, or its N-glucuronide metabolite, to dampen inflammation and reducing viral load by controlling viral RNA biogenesis or viral particle endocytosis have applicability for the treatment or prevention of Coronaviridae, including the COVID-19. Furthermore, miR-124 can promote tissue repair that may be beneficial to limit pulmonary and broncho-alveolar damage.
Indeed, Coronaviridae viruses such as SARS-Cov2 (e.g. in COVID19) contain a non-segmented, positive-sense RNA genome of ˜30 kb. The genome contains a 5′ cap structure along with a 3′ poly (A) tail, allowing it to act as a mRNA for translation of the replicase polyproteins. The 5′ cap can be recognized by eIF4E and/or CBC complex to initiate translation and/or RNA quality control, respectively. Like in HIV, CBC-ABX464 may favour the RNA quality control of COVID-19 RNA genome and block the production of RdRp polymerase thereby interfering with viral replication.
In addition, it is proposed herein that miR-124 up-regulation may directly interfere with the entry of the Coronaviridae virus (e.g. in COVID-19) to tracheobronchial tissue. As virions, after binding to ACE2 receptor and the action of serine protease TMPRSS2 for S protein priming, indeed require clathrin-mediated endocytosis for successful entry, and subsequent vesicle scission by dynamin 2, which is a direct target of miR-124.
Thus, ABX464 and its N-glucuronide metabolite may tackle both the Coronaviridae infection and the induced inflammation. Advantageously, the safety profile of ABX-464 is also very favourable with no drug related serious adverse reactions.
Furthermore, by its mode of action, compound of formula (I) or (II) as defined herein after contributes to repairing and remodeling tissues, and in particular lung tissue.
Thus, as illustrated in
Compound (I) or (II) as defined herein after, or any one of their prodrugs or pharmaceutically acceptable salts, is moreover particularly useful for treating and/or preventing severe forms of SARS-CoV-2 infections: anti-inflammatory effects to fight the cytokine storm, mucosal effectiveness, promotion of tissue repair to avoid long-term post-ventilation sequelae. As illustrated more in details in example 1 and 3, the added anti-viral effect may even contribute to prevent viral replication, spreading and an increased clearance of the virus and help mitigate control the cytokine storm that acute anti-inflammatory drugs might induce. For its anti-inflammatory properties ABX464 may be positioned as alternative to IL-6R and IL-6 inhibitors that have already shown partial clinical benefits, but it offers the advantages of acting on multiple cytokines involved in the cytokine storm, having anti-viral effects and promoting tissue repair. Finally, ABX464 results in a good bioavailability, with a rapid and high systemic and pulmonary exposure as illustrated in example 2.
According to a particular embodiment, the compound (I) or (II) as defined herein after, or any one of their prodrugs or pharmaceutically acceptable salts may be used at an early stage of a condition related to a Coronaviridae infection; in particular the COVID-19.
According to a particular embodiment, the compound (I) or (II) as defined herein after, or any one of their prodrugs or pharmaceutically acceptable salts may be used at a later stage of a condition related to a Coronaviridae infection; in particular the COVID-19.
Indeed, clinically, SARS-CoV-2 infection can lead to a cytokine storm syndrome, acute respiratory distress syndrome (ARDS) and multiple organ failure. Notably, cytokine storm (i.e. hyperinflammatory syndrome) has been associated with COVID-19 disease severity (including increased MCP1, IL-1β, TNFα, IL-17, G-CSF and IL-6). Early treatment and acting on viral replication and on the various cytokine pathways allow to successfully reduce the cytokine storm syndrome and “hyper-inflammation” and to prevent ARDS and multi-organ failure.
Accordingly, in one embodiment, the present invention relates to a compound of formula (I) or (II) as defined herein after, or any one of their prodrugs or pharmaceutically acceptable salts, for use in a method for treating a group of patients prior to the occurrence of a respiratory distress syndrome related to a Coronaviridae infection. Said patients may or not be hospitalized. Accordingly, in one embodiment, the present invention relates to a compound of formula (I) or (II) as defined herein after, or any one of their prodrugs or pharmaceutically acceptable salts, for use in a method for treating or preventing the occurrence of a respiratory distress syndrome or long-term complications related to a Coronaviridae infection.
According to particular embodiments, the compound of formula (I) or (II) as defined herein after, or any one of its prodrugs or any one of its pharmaceutically acceptable salts, is for use in a method for treating or preventing a Coronaviridae infection, is for treating or preventing the occurrence of a vascular, a cardiovascular, a neurological, pulmonary or a gastrointestinal condition related to a Coronaviridae infection.
Advantageously, ABX464 and its N-glucuronide metabolite may be considered either alone or in combination with any other active agent, in particular any other dynamin inhibitor, especially any dynamin-2 inhibitor, which is reported herein, for use in the treatment of prevention of a Coronaviridae infection.
In particular, ABX464 and its N-glucuronide metabolite may be considered either alone or in combination with Remdesivir, which is reported herein, for use in the treatment of prevention of a Coronaviridae infection.
Although not limited to one specific strain, mutant or variant, the Coronaviridae infection which are particularly considered throughout the present application include those attributed to Severe acute respiratory syndrome-related coronaviruses, especially SARS-Cov-2 and conditions related thereto.
According to another particular embodiment, the compound (I) or (II) as defined herein after, or any one of their prodrugs or pharmaceutically acceptable salts may be used at a recovery or chronic, or non-acute stage of a Coronaviridae infection, or a condition related thereto; and in particular of a condition related to COVID-19.
According to a particular embodiment, the compound (I) or (II) as defined herein after, or any one of their prodrugs or pharmaceutically acceptable salts may thus be used at a recovery or chronic, or non-acute stage of a condition related a Coronaviridae infection, selected from: a respiratory distress syndrome, such as a severe respiratory distress syndrome, a cardiovascular condition, a vascular condition, a gastrointestinal condition, a pulmonary condition or a neurological condition.
In one embodiment, the present invention relates to a compound of formula (I) or (II) as defined herein after, or any one of their prodrugs or pharmaceutically acceptable salts, for use in a method for treating a patient during or after the occurrence of a cardiovascular condition related to a Coronaviridae infection.
In one embodiment, the present invention relates to a compound of formula (I) or (II) as defined herein after, or any one of their prodrugs or pharmaceutically acceptable salts, for use in a method for treating a patient during or after the occurrence of a vascular condition related to a Coronaviridae infection.
In one embodiment, the present invention relates to a compound of formula (I) or (II) as defined herein after, or any one of their prodrugs or pharmaceutically acceptable salts, for use in a method for treating a patient during or after the occurrence of a gastrointestinal condition related to a Coronaviridae infection.
In one embodiment, the present invention relates to a compound of formula (I) or (II) as defined herein after, or any one of their prodrugs or pharmaceutically acceptable salts, for use in a method for treating a patient during or after the occurrence of a neurological condition related to a Coronaviridae infection.
In one embodiment, the present invention relates to a compound of formula (I) or (II) as defined herein after, or any one of their prodrugs or pharmaceutically acceptable salts, for use in a method for treating a patient during or after the occurrence of a respiratory distress syndrome related to a Coronaviridae infection.
In one embodiment, the present invention relates to a compound of formula (I) or (II) as defined herein after, or any one of their prodrugs or pharmaceutically acceptable salts, for use in a method for treating a patient during or after the occurrence of a pulmonary condition related to a Coronaviridae infection.
The proposed embodiments may thus further apply at a recovery stage, for instance after hospitalization and/or after an acute phase of the Coronaviridae infection.
Thus, according to another particular embodiment, the present invention relates to a compound (I) or (II) as defined herein after, or any one of their prodrugs or pharmaceutically acceptable salts, for use in a method for treating or prevention a condition related to a Coronaviridae infection, in a subject with low or no detectable presence of the said Coronaviridae infection.
According to a first main embodiment, the invention relates to a compound of formula (I)
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts, for use in a method for treating or preventing a Coronaviridae infection, and conditions related thereto. In particular, the invention relates to a compound of formula (I)
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts, for use in a method for treating or preventing a Coronaviridae infection.
As used herein, the term “ABX464” refers to such compound of formula (I) or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts.
In particular, the invention relates to a compound of formula (I)
or any one of its pharmaceutically acceptable salts, for use in a method for treating or preventing a Coronaviridae infection.
According to a second main embodiment, the invention relates to a compound of formula (II)
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts for use in a method for treating or preventing a Coronaviridae infection, and conditions related theto.
In particular, the invention relates to a compound of formula (II)
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts for use in a method for treating or preventing a Coronaviridae infection.
As used herein, the term “ABX464-N-Glu” refers to such compound of formula (II) or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts.
In particular, the invention relates to a compound of formula (II)
or any one of its pharmaceutically acceptable salts for use in a method for treating or preventing a Coronaviridae infection.
According to a third main embodiment, the invention relates to a pharmaceutical composition comprising a compound of formula (I) or (II) as defined above or any one of its prodrugs or any one of its pharmaceutically acceptable salts, and at least one pharmaceutically acceptable excipient, for use in a method for treating or preventing a Coronaviridae infection, and conditions related thereto.
In particular, the invention relates to a pharmaceutical composition comprising a compound of formula (I) or (II) as defined above or any one of its prodrugs or any one of its pharmaceutically acceptable salts, and at least one pharmaceutically acceptable excipient, for use in a method for treating or preventing a Coronaviridae infection.
According to a fourth main embodiment, the invention relates to a pharmaceutical composition comprising a compound of formula (I) or (II) as defined above, for use in a method for treating or preventing a Coronaviridae infection, and conditions related thereto.
In particular, the invention relates to a medicament comprising a compound of formula (I) or (II) as defined above, for use in a method for treating or preventing a Coronaviridae infection.
According to a fifth main embodiment, the invention relates to an in vitro or ex vivo use of at least one miRNA, said at least one miRNA being miR-124, as a biomarker of a Coronaviridae infection, or of an efficacy of a therapeutic treatment of said Coronaviridae infection, and conditions related thereto.
In particular, the invention relates to an in vitro or ex vivo use of at least one miRNA, said at least one miRNA being miR-124, as a biomarker of a Coronaviridae infection, or of an efficacy of a therapeutic treatment of said Coronaviridae infection.
According to a sixth main embodiment, the invention relates to an in vitro or ex vivo method for assessing a Coronaviridae infection in a patient presumed to be infected with a virus, comprising at least the steps of:
a—measuring a presence or an expression level of at least one miRNA, said at least one miRNA being miR-124, in a biological sample previously obtained from said patient; and
b—comparing said presence or expression level to a control reference value, wherein a modulated presence or level of expression of said miRNA relative to said control reference value is indicative of a Coronaviridae infection.
According to a seventh main embodiment, the invention relates to a dynamin inhibitor for use in a method for treating or preventing a Coronaviridae infection; in particular for reducing a Coronaviridae viral load. In particular, the Coronaviridae may be COVID-19 or any one of its mutants.
According to an eighth embodiment, the invention relates to a compound of formula (I) or (II) as defined above, or any one of its prodrugs or any one of its pharmaceutically acceptable salts, for use in a method for treating or preventing Kawasaki disease or tissue damage or destruction, in particular lung tissue damage and destruction.
As used herein, the term “patient” refers to either an animal, such as a valuable animal for breeding, company or preservation purposes, or preferably a human or a human child, which is afflicted with, or has the potential to be afflicted with one or more diseases and conditions described herein.
In particular, as used in the present application, the term “patient” refers to a mammal, including a non-human mammal such as a rodent, cat, dog, or primate, or a human; preferably said subject is a human and also extends to birds.
The identification of those patients who are in need of treatment of herein-described diseases and conditions is well within the ability and knowledge of one skilled in the art. A veterinarian or a physician skilled in the art can readily identify, by the use of clinical tests, physical examination, medical/family history or biological and diagnostic tests, those patients who are in need of such treatment.
In the context of the invention, the term “treating” or “treatment”, as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disease resulting from RNA virus infection, and more particularly RNA virus infection from group IV or V, or one or more symptoms of such disease.
As used herein, an “effective amount” refers to an amount of a compound of the present invention which is effective in preventing, reducing, eliminating, treating or controlling the symptoms of the herein-described diseases and conditions, i.e. RNA virus infection, and more particularly RNA virus infection from group IV and V. The term “controlling” is intended to refer to all processes wherein there may be a slowing, interrupting, arresting, or stopping of the progression of the diseases and conditions described herein, but does not necessarily indicate a total elimination of all diseases and condition symptoms, and is intended to include prophylactic treatment.
The term “effective amount” includes “prophylaxis-effective amount” as well as “treatment-effective amount”.
The term “preventing”, as used herein, means reducing the risk of onset or slowing the occurrence of a given phenomenon, namely in the present invention, a disease resulting from a RNA virus infection, and more particularly a RNA virus infection from group IV or V.
As used herein, «preventing» also encompasses «reducing the likelihood of occurrence» or «reducing the likelihood of reoccurrence».
The term “prophylaxis-effective amount” refers to a concentration of compound of this invention that is effective in inhibiting, preventing, decreasing the likelihood of the disease by RNA viruses, and more particularly by a RNA virus from group IV or V of the Baltimore classification, or preventing the RNA virus infection and in particular a RNA virus infection from group IV or preventing the delayed onset of the disease by the RNA virus, and more particularly by a RNA virus from group IV, when administered before infection, i.e. before, during and/or slightly after the exposure period to the RNA virus, and in particular to the RNA virus from group IV.
Likewise, the term “treatment-effective amount” refers to a concentration of compound that is effective in treating the RNA virus infection, e.g. leads to a reduction in RNA viral infection, following examination when administered after infection has occurred.
As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, excipients, compositions or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problem complications commensurate with a reasonable benefit/risk ratio.
As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.
The term “pharmaceutically acceptable carrier, adjuvant, or vehicle” may refer to any pharmaceutically acceptable excipient, such as a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
A “biological sample” suitable for the present invention can be a biological fluid, such as a blood, a plasma, or a serum, a saliva, an interstitial, fluid, or a urine sample; a cell sample, such as a cell culture, a cell line, or a PBMC sample, a tissue biopsy, such as an oral tissue, a gastrointestinal tissue, a skin, an oral mucosa sample, a pharyngeal, tracheal, bronchoalveolar sample, or a plurality of samples from a clinical trial.
A biological sample can be a crude sample, or can be purified to various degrees prior to storage, processing, or measurement. In some embodiments, a biological sample is selected from the group consisting of a biological tissue sample, a whole blood sample, a swab sample, a plasma sample, a serum sample, a saliva sample, a vaginal fluid sample, a sperm sample, a pharyngeal fluid sample, a synovial sample, a bronchial or pleural fluid sample, a fecal fluid sample, a cerebrospinal fluid sample, a lacrymal fluid sample and a tissue culture supernatant sample.
As used herein, the term “miR-124” refers to either one of the 9 haplotypes of miR-124 precursors have been identified so far (Guo et al., PLoS ONE, 2009, 4(11):e7944), from which 3 are present in the Human, hsa-miR-124-1, hsa-miR-124-2 and hsa-miR-124-3. The miR-124 microRNA precursor is a small non-coding RNA molecule. The mature ˜21 nucleotide microRNAs are processed from hairpin precursor sequences by the Dicer enzyme. The mature sequences are reported in WO2014111892.
As used herein, a “viral infection or related condition” refers to an infection of condition related to a virus, more particularly said virus having a RNA genome, and especially a RNA virus belonging to group IV according to the Baltimore classification. Viruses may be further classified in distinct families, orders and genus.
For reference, the content of the “Baltimore classification” which is reported herein further references to the virus taxonomy as set forth in the database of the International Committee of Taxonomy of Viruses (ICTV) as available online on Mar. 20, 2020 (Email ratification February 2019 & MSL #34) at https://talk.ictvonline.org/taxonomy/. This taxonomy is incorporated herein in its entirety.
Accordingly, this classification clusters viruses into families (or “groups”) depending on their type of genome. The present virus classification, as in 2018, comprises seven different groups:
As used herein, a “condition related to a Coronaviridae infection”, especially a condition related to a Severe acute respiratory syndrome-related coronavirus, such as SARS-CoV2, may be selected from a list comprising, or consisting of: severe respiratory distress syndrome, a cardiovascular condition, a vascular condition, a gastrointestinal condition or a neurological condition.
Advantageously, the patients having, or being at risk of a having a condition related to a Coronaviridae infection can also be considered.
According to exemplary embodiments, the condition related to a Coronaviridae infection which are particularly considered include: pulmonary fibrosis, vasculitis, Kawasaki disease and tissue damage or destruction, in particular lung tissue damage and destruction.
As used herein, “repairing and remodeling tissue” means promoting healing of tissues that have been damaged or destroyed by a disease, and namely lung tissue devastated by Coronaviridae infection or gastrointestinal tissue devasted by Coronaviridae infection, at least by not delaying the tissue repair, as usually stated in the framework of treatments with classical anti-inflammatory diseases, as for example corticosteroids which are the best representative of this class of drugs.
The compounds of formula (I) and (II), and their corresponding prodrugs, or anyone of their pharmaceutical salts are reported herein for the treatment or prevention of infections against the following viruses: HSV, CMV, EBV, Adenoviruses, Pox viruses, HPV (human papilloma virus), Parvovirus, Reoviruses, Hepatitis A virus, Rubella virus, Hepatitis C virus (HCV), Hepatitis E virus, Dengue virus, Chikungunya virus, Zika virus, Enteroviruses, Rhinoviruses, poliovirus, foot-and-mouth virus, yellow fever virus, Paramyxoviruses, Influenza viruses Retroviruses including HTLV-1, HTLV-2, HIV and Hepatitis B (HBV), due to their reliance on Dynamin 2-mediated endocytosis.
Unless instructed otherwise, all the disclosed compounds are specifically considered herein for the treatment or prevention of Coronaviridae, which may thus refer indifferently to any member of the said Coronaviridae family in the sense of the Baltimore convention, although particular selections of viruses will be considered hereafter as preferred embodiments.
The same applies to the uses & methods which are considered as part of the present invention, including biomarkers uses and methods for assessing a Coronaviridae infection or the efficacy of a particular therapy directed against said Coronaviridae infection.
As used herein, the term “Coronaviridae” refers to the corresponding family of RNA viruses belonging to the group IV of the Baltimore classification, which is it itself par of the Cornidovirineae suborder and of the Nidovirales Order. The Coronaviridae family includes both the Letovirinae and Orthocoronavirinae subfamilies.
As used herein, the term “Letovirinae” refers to the corresponding family of the Baltimore classification, which includes the Alphaletovirus genus, the Milecovirus subgenus, which includes (in a non-exhaustive manner) the Microhyla letovirus 1 species.
As used herein, the term “Orthocoronavirinae” refers to the corresponding family of the Baltimore classification, which includes the Alphacoronavirus, Betacoronavirus, Deltacoronavirus, and Gammacoronavirus genus.
As used herein, the term “Alphacoronavirus” refers to the corresponding family of the Baltimore classification, which includes the Colacovirus, Decacovirus, Duvinacovirus, Luchacovirus, Minacovirus, Minunacovirus, Myotacovirus, Myctacovirus, Pedacovirus, Rhinacovirus, Setracovirus, and Tegacovirus subgenus. In a non-exhaustive manner, this includes the following species: Bat coronavirus CDPHE15, Bat coronavirus HKU10, Rhinolophus ferrumequinum alphacoronavirus HuB-2013, Human coronavirus 229E, Lucheng Rn rat coronavirus, Ferret coronavirus, Mink coronavirus 1, Miniopterus bat coronavirus 1, Miniopterus bat coronavirus HKU8, Myotis ricketti alphacoronavirus Sax-2011, Nyctalus velutinus alphacoronavirus SC-2013, Porcine epidemic diarrhea virus, Scotophilus bat coronavirus 512, Rhinolophus bat coronavirus HKU2, Human coronavirus NL63, NL63-related bat coronavirus strain BtKYNL63-9b, Alphacoronavirus 1.
As used herein, the term “Betacoronavirus” refers to the corresponding family of the Baltimore classification, which includes the Embecovirus, Hibecovirus, Merbecovirus, Nobecovirus, and Sarbecovirus subgenus. In a non-exhaustive manner, this includes the following species: Betacoronavirus 1, China Rattus coronavirus HKU24, Human coronavirus HKU1, Murine coronavirus, Bat Hp-betacoronavirus Zhejiang2013, Hedgehog coronavirus 1, Middle East respiratory syndrome-related coronavirus, Pipistrellus bat coronavirus HKU5, Tylonycteris bat coronavirus HKU4, Hedgehog coronavirus 1, Middle East respiratory syndrome-related coronavirus, Pipistrellus bat coronavirus HKUS, Tylonycteris bat coronavirus HKU4, Rousettus bat coronavirus GCCDC1, Rousettus bat coronavirus HKUS, Severe acute respiratory syndrome-related coronavirus.
As used herein, the term “Severe acute respiratory syndrome-related coronavirus”, or SARS virus, includes, in a non-exhaustive manner, the SARS-CoV, SARSr-CoV WIV1, SARSr-CoV HKUS, SARSr-CoV RP3, and SARS-CoV-2; including strains responsible for COVID-19 and their mutants.
As used herein, the term “Deltacoronavirus” refers to the corresponding family of the Baltimore classification, which includes the Andecovirus, Buldecovirus, Herdecovirus, and Moordecovirus subgenus. In a non-exhaustive manner, this includes the following species: Wigeon coronavirus HKU20, Bulbul coronavirus HKU11, Coronavirus HKU15, Munia coronavirus HKU13, White-eye coronavirus HKU16, Night heron coronavirus HKU19, Common moorhen coronavirus HKU21.
As used herein, the term “Gammacoronavirus” refers to the corresponding family of the Baltimore classification, which includes the Cegacovirus and Igacovirus subgenus. In a non-exhaustive manner, this includes the following species: Beluga whale coronavirus SW1 and Avian coronavirus.
As used herein, the term “a phenothiazine” refers to a heterocyclic compound of the thiazine class, including phenothiazine and phenothiazine-derivatives, in particular those which are characterized by the following formula
wherein
R1 can be any chemical substituent, in particular any chemical substituent selected from: a halogen, an alkyl group, a substituted alkyl group, an alkoxy group, a substituted alkoxy group, a thioether or an acetyl group
R2 can be selected from acyclic groups, piperidine-derived groups and piperazine-derived groups.
Compounds for Use
According to a first main embodiment, the invention relates to a compound of formula (I)
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts, for use in a method for treating or preventing a Coronaviridae infection, and conditions related thereto.
In particular, the invention relates to a compound of formula (I)
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts, for use in a method for treating or preventing a Coronaviridae infection.
Said compound is 8-Chloro-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine.
According to a second main embodiment, the invention relates to a compound of formula (II)
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts for use in a method for treating or preventing a Coronaviridae infection, and conditions related thereto.
In particular, the invention relates to a compound of formula (II)
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts for use in a method for treating or preventing a Coronaviridae infection.
The compounds of the present invention (of formula (I) or (II) can be prepared by conventional methods of organic synthesis practiced by those skilled in the art. The general reaction sequences outlined below represent a general method useful for preparing the compounds of the present invention and are not meant to be limiting in scope or utility.
The man skilled in the Art may, for instance, refer to the content of WO2016135052 and WO2016135055 for that matter.
The compounds of the invention may exist in the form of free bases or of addition salts with pharmaceutically acceptable acids.
In particular, «Pharmaceutically acceptable salt thereof» refers to salts which are formed from acid addition salts formed with inorganic acids (e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), as well as salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, fumaric acid, maleic acid, ascorbic acid, benzoic acid, tannic acid, palmoic acid, alginic acid, polyglutamic acid, naphthalene sulfonic acid, naphthalene disulfonic acid, and poly-galacturonic acid.
Suitable physiologically acceptable acid addition salts of compounds of formula (I) or (II), or prodrugs thereof, may include hydrobromide, tartrate, citrate, trifluoroacetate, ascorbate, hydrochloride, tosylate, triflate, maleate, mesylate, formate, acetate and fumarate.
The compounds of formula (I) or (II), and their prodrugs, or any of their pharmaceutically acceptable salts may form solvates or hydrates and the invention include all such solvates and hydrates.
The terms “hydrates” and “solvates” simply mean that the compounds according to the invention can be in the form of a hydrate or solvate, i.e. combined or associated with one or more water or solvent molecules. This is only a chemical characteristic of such compounds, which can be applied for all organic compounds of this type.
Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, persulfuric acid, boric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzoate, edetate, gluceptate, bisulfate, borate, butyrate, camphorate, cyclopentaneproprionate, citrate, glycerophosphoric acid, nitric acid, cyclopentanepropionate, digluconate, dodecylsulfate, formate, acetate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, glucoheptonate, heptanoate, hexanoate, hydroiodide, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, salicylate, disalicylate, picrate, mucate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, dodecylsulfate, 3-phenylpropionate, phosphate, pivalate, propionate, undecanoate stearate, succinate, bitartrate, sulfate, tartrate, trifluoroacetate, triflate, thiocyanate, undecanoate, valerate salts, pantothenate, dodecylsulfatesulfonate, in particular alkylsufonate such as methanesulfonate (or mesylate), esylate, edisylate, estolate, ethanesulfonate, 2-hydroxy-ethanesulfonate or arylsulfonate, such as 2-naphthalenesulfonate, napadisylate, napsylate, camphorsulfonate, benzenesulfonate (or besylate), p-toluenesulfonate (or tosylate), and the like.
In particular, the pharmaceutically acceptable salts are selected from the group consisting of:
More particularly, the pharmaceutically acceptable salts are selected from sulfate, hydrobromide, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, triflate, maleate, mesylate, formate, acetate, fumarate and sulfonate, in particular alkylsufonate or arylsulfonate, and more particularly mesylate, triflate, edisylate, besylate and tosylate.
According to one embodiment, ABX464 and its metabolites, and more particularly N-glucuronide metabolites of ABX464, including compound of formula (1) as defined above is in a salt form selected from lactate, oleate, oxalate, palmitate, stearate, valerate, butyrate, malonate, succinate, malate, benzoate, gluconate, lactobionate, pamoate, adipate, alginate, aspartate, camphorate, diduconate, heptanoate, hexanoate, laurate, nicotinate, pivalate, propionate, and the like, phosphate and the like, camphorsulfonate, 2-hydroxy-ethanesulfonate, esylate, napadisylate, and the like, perchloric acid, and the like, and is particularly selected from esylate and napadisylate, even more particularly is selected from anhydrous crystalline ABX464 hemi-napadisylate salt, anhydrous crystalline ABX464 esylate salt, and crystalline hemi-THF solvate of ABX464 hemi-napadisylate.
In some embodiments, the compound ABX464, or a pharmaceutically acceptable salt thereof, the compound ABX464, or a pharmaceutically acceptable salt thereof, is under a crystallized form. In some embodiments, a crystallized form of the compound ABX464, or a pharmaceutically acceptable salt thereof, has a melting point at 120.5° C. (±2° C.).
In some embodiments, a crystallized form of the compound ABX464, or a pharmaceutically acceptable salt thereof, shows peaks in an x-ray powder diffractogram (XRPD) at angles 7.3, 14.6, 18.4, and 24.9. In some embodiments, a crystallized form of the compound ABX464, or a pharmaceutically acceptable salt thereof, shows one or more XRPD peaks at angles selected from 18.0, 24.2, 28.3, and 29.5. In some embodiments, a crystallized form of the compound ABX464, or a pharmaceutically acceptable salt thereof, shows one or more XRPD peaks at angles selected from 18.6, 22.3, 23.0, and 23.5.
According to a particular embodiment, the crystalline polymorphic form of 8-Chloro-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine is characterized by the following main peaks expressed as degree 2-Theta angles by a XRPD analysis: 7.3, 14.6, 23.5, and 28.4 (each time±0.2) and may further show the following additional peaks expressed as degree 2-Theta angles: 12.1, 17.3, 18.4, 23.0; 24.2, 24.9, 27.4 and 29.1 (each time±0.2) and even optionally further the following additional peaks expressed as degree 2-Theta angles: 13.7, 16.3, 16.9, 18.1, 22.4, and 29.6 (each time±0.2).
According to one more particular embodiment, ABX464 is in a crystalline salt form selected from:
According to one embodiment, ABX464 and its metabolites, and more particularly N-glucuronide metabolites of ABX464, including compound of formula (1) as defined above is in a co-crystal form with a co-crystal selected from: L-Proline, Gentisic acid, Malonic acid and 4, 4′-Bipyridine.
According to one more particular embodiment, ABX464 is in a co-crystal form selected from:
All the salt and crystalline form thereof may be obtained according to usual techniques known to the man skilled in the art.
According to one particular embodiment, the compound of formula (I) or ABX464 or a pharmaceutically acceptable salt thereof may be in an amorphous form. More particularly, the compound of formula (I) may be administered under the form of an amorphous solid dispersion. Said amorphous solid dispersion advantageously comprises at least one pharmaceutically acceptable carrier. In the framework of the present invention, the amorphous solid dispersion is a glass solution forming a homogeneous one-phase system, and the compound of formula (I) or a pharmaceutically acceptable salt thereof is under an amorphous form.
The pharmaceutically acceptable carrier may be selected from a polymer, a sugar, an acid, a surfactant, a cyclodextrin or a cyclodextrin derivative, pentaerythritol, pentaerythrityl tetraacetate, urea, urethane, hydroxy alkyl xanthins and mixtures thereof, in particular selected from a polymer, an acid, a surfactant, urea and mixtures thereof, more particularly selected from a polymer, an acid, a surfactant, and mixtures thereof.
Even more particularly, the pharmaceutically acceptable carrier may be:
According to a particular embodiment, the polymers suitable for use in an amorphous solid dispersion may be selected from homopolymers of N-vinyl lactams, copolymers of N-vinyl lactams, and mixtures thereof.
According to another particular embodiment, the polymers suitable for use in an amorphous solid dispersion may be selected from povidone, copovidone, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol, hydroxypropylmethylcellulose acetate succinate, methacrylic acid/ethyl acrylate copolymers, and mixtures thereof.
Among the homopolymers of N-vinyl lactams can be cited polyvinylpyrrolidone (also named povidone or PVP) which can be the ones sold for example by BASF under the name of Kollidon® 30 (also named PVP K30), PVP K17, PVP K25, or PVP K90.
Among the copolymers of N-vinyl lactams can be cited copolymers of N-vinyl pyrrolidone and vinyl acetate (also named copovidone or PVP-VA) which such as the one sold for example by BASF under the name of Kollidon® VA64 by BASF or copolymers of N-vinyl caprolactam, vinyl acetate, and ethylene glycol such as the one sold for example by BASF under the name of Soluplus®.
The weight ratio of the compound of formula (I) or a pharmaceutically acceptable salt thereof and the pharmaceutically acceptable carrier(s) may be in the range of from 1:20 to 1:0.5, particularly of from 1:10 to 1:1, more particularly of from 1:2 to 1:1.5.
Thus, in the framework of the present invention, the compound of formula (I) may be administered within a pharmaceutical composition comprising the amorphous solid dispersion as defined above, and at least one pharmaceutically acceptable excipient, in particular under the form of tablets, capsules, pills, lozenges, chewing gums, powders, granules, suppositories, emulsions, microemulsions, solutions such as aqueous solutions, suspensions such as aqueous suspensions, syrups, elixirs, ointments, drops, pastes, creams, lotions, gels, sprays, inhalants or patches.
According to particular embodiments, the compound or any one of its prodrugs or any one of its pharmaceutically acceptable salts, for use in a method for treating or preventing a Coronaviridae infection, is for reducing inflammation associated with the Coronaviridae infection.
According to particular embodiments, the compound or any one of its prodrugs or any one of its pharmaceutically acceptable salts, for use in a method for treating or preventing a Coronaviridae infection, is for reducing the Coronaviridae viral load.
According to particular embodiments, the compound or any one of its prodrugs or any one of its pharmaceutically acceptable salts, for use in a method for treating or preventing a Coronaviridae infection, is in combination with:
According to some particular embodiments, the invention thus relates to a combination of a compound of formula (I) or (II):
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts, in combination with remdesivir or any one of its pharmaceutically acceptable salts; for use as a medicament, and in particular for use in a method for treating or preventing a Coronaviridae infection, and conditions related thereto.
According to some more particular embodiments, the invention thus relates to a combination of a compound of formula (I):
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts, in combination with remdesivir or any one of its pharmaceutically acceptable salts; for use as a medicament, and in particular for use in a method for treating or preventing a Coronaviridae infection, and conditions related thereto.
It will be understood that the active ingredients which are part of the above-mentioned combinations may be administered simultaneously or sequentially; by the same route of administration or by a different route. For example, the compound of formula (I) or
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts, may be administered by the oral or nasal and/or pulmonary administration route, whereas remdesivir or any one of its pharmaceutically acceptable salts may be administrable by the parental route.
Alternatively, both the compound of formula (I) or (II)
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts, and remdesivir or any one of its pharmaceutically acceptable salts, may be administrable by the oral or nasal and/or pulmonary administration route.
According to some particular embodiments, the Coronaviridae is selected from Letovirinae and Orthocoronavirinae.
According to some particular embodiments, the Coronaviridae is an Alphacoronavirus or a Betacoronavirus or a Deltacoronavirus or a Gammacoronavirus.
According to some particular embodiments, the Coronaviridae is an Embecovirus or a Hibecovirus or a Merbecobivirus or a Nobecovirus or a Sarbecovirus.
According to some particular embodiments, the Coronaviridae is a Sarbecovirus selected from Severe Acute Respiratory Syndrome-related coronaviruses.
According to some particular embodiments, the Severe Acute Respiratory Syndrome (SARS)-related coronaviruses are selected from the group consisting of: SARS-CoV, SARSr-CoV WIV1, SARSr-CoV HKU3, SARSr-CoV RP3, SARS-CoV-2.
According to some preferred embodiments, the Severe Acute Respiratory Syndrome (SARS)-related coronaviruses are selected from SARS-CoV and SARS-CoV-2; including strains responsible for COVID-19 and their mutants.
According to some embodiments, the compounds of formula (I) or (II) or any one of their prodrugs or pharmaceutically acceptable salts are used in a method for treating or preventing a Coronaviridae infection, wherein the level of the compound, in a blood, plasma, tissue, saliva, pharyngeal, tracheal, bronchoalveolar, and/or serum sample of the patient, is measured during the use.
According to some of those embodiments, a presence and/or expression level of miR-124 is measured prior to and during the use.
Dynamin Inhibitors for Use
As used herein, “dynamin” may refer to any polypeptide, natural or recombinant, which belongs to the “dynamin superfamily”, including Dynamin I, Dynamin II and Dynamin III, in particular Dynamin II also referred herein as dynamin-2 and which is encoded in humans by the DNM2 gene.
As used herein a “dynamin inhibitor” may refer to any compound causing a decrease of the cellular content of the dynamin polypeptide, and/or the expression of the dynamin polypeptide, or the activity of the dynamin polypeptide, or the stability of the dynamin polypeptide. In particular, such a dynamin inhibitor is a direct inhibitor, meaning that it interacts directly with either the Dynamin protein or a nucleic acid encoding said Dynamin. In a particular embodiment, the Dynamin 2 inhibitor is selected from the group consisting of a nucleic acid molecule interfering specifically with Dynamin 2 expression. Still according to a more particular embodiment, the Dynamin 2 Inhibitor is a RNAi, an antisense nucleic acid or a ribozyme interfering specifically with Dynamin 2 expression. Non-limitative examples of inhibitors of dynamin expression encompass siRNAs or shRNAs, miRNAs, piRNAs that specifically bind to the dynamin-encoding nucleic acid or its corresponding mRNA, or alternatively, to a regulator of dynamin-expression. Examples of such inhibitors of dynamin expression encompass siRNAs or shRNAs, miRNAs, piRNAs that are complementary to such dynamin-encoding nucleic acid or its corresponding mRNA, or alternatively, to a regulator of dynamin-expression. Within the scope of the present invention, the term “complementary” is intended to mean that a first nucleic acid is complementary to a second nucleic acid when these nucleic acids have the base on each position which is the complementary (i.e. A to T, C to G) and in the reverse order. For example, the complementary sequence to TTAC is GTAA. If one strand of the double-stranded DNA is considered the sense strand, then the other strand, considered the antisense strand, will have the complementary sequence to the sense strand.
According to exemplary embodiments, the dynamin inhibitor may be selected from those described in EP2862928A1.
Within the scope of the present invention, the term “dynamin stability”, such as in “stability of the dynamin polypeptide” or “stability of the dynamin-encoding nucleic acid”, is intended to refer to the equilibrium reached between the synthesis and the degradation of the dynamin polypeptide or the dynamin-encoding nucleic acid.
Within the scope of the present invention “an activator of dynamin activity” is intended to refer to a compound able to increase, at least in part, the ability of the dynamin polypeptide to promote its physiological role in the cell; in particular its role in clathrin-mediated endocytosis.
Within the scope of the present invention “an inhibitor of dynamin activity” is intended to refer to a compound able to decrease, at least in part, the ability of the dynamin polypeptide to promote its physiological role in the cell; in particular its role in clathrin-mediated endocytosis.
According to one embodiment, the dynamin inhibitor may be an antibody directed against dynamin, a nucleic acid molecule interfering specifically with dynamin expression, and a small molecule inhibiting the dynamin enzymatic activity (inhibition of the GTPase activity), expression (inhibiting promoter, splicing or translation), or function (inhibition of oligomerization, activation, lipid binding or partner binding).
According to another embodiment, the dynamin inhibitor may be selected from the group consisting of an antibody directed against Dynamin 2, or a nucleic acid molecule interfering specifically with Dynamin 2 expression.
Within the scope of the present invention, a “small molecule inhibiting the dynamin enzymatic activity” is intended to refer to small molecules that can be an organic or inorganic compound, usually less than 1000 daltons, able to inhibit the dynamin enzymatic activity. Such molecules can be extracted or derived from nature or be synthetic molecules.
According to one embodiment, the dynamin inhibitor may be selected from: 3-Hydroxynaphthalene-2-carboxylic acid (3,4-dihydroxybenzylidene)hydrazide, 3-Hydroxy-N′-[(2,4,5-trihydroxyphenyl)methylidene]naphthalene-2-carbohydrazide tetradecyltrimethylammonium bromide, 4-Chloro-2-((2-(3-nitrophenyl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carbonyl)-amino)-benzoic acid, 2-Cyano-N-octyl-3-[1-(3-dimethylaminopropyl)-1 H-indol-3-yl]acrylamide, 3 (2,4-Dichloro-1-methoxyphenyl) sulfanylquinazolin-4(3H)-one, N,N′-(Propane-1,3-diyl)bis(7,8-dihydroxy-2-imino-2H-chromene-3-carboxamide), N,N′-(Ethane-1,2-diyl)bis(7,8-dihydroxy-2-imino-2H-chromene-3-carboxamide), OctadecylTriMethylAmmonium Bromide, Dynamin inhibitory peptide with aminoacid sequence: QVPS PNRAP, Myr-QVPSRPNRAP (myristolated form of the preceding aminoacid), 3-Hydroxy-N-[(2,4,5-trihydroxyphenyl)methylidene]naphthalene-2-carbohydrazide, and 4-(N,N-Dimethyl-N-octadecyl-N-ethyl)-4-aza-10-oxatricyclo-[5.2.1]decane-3,5-dione bromide.
In some embodiments, inhibitors of dynamin are inhibitors of receptor-mediated endocytosis which can be identified by methods that assay dynamin ring stabilization. These methods may comprise incubating a test agent with a dynamin polypeptide under conditions suitable for the formation of dynamin rings; and evaluating whether the test agent promotes accumulation of dynamin rings and/or inhibits disassembly of dynamin rings, the accumulation of dynamin rings and/or inhibition of disassembly of dynamin rings increasing basal dynamin GTPase activity. The evaluation of whether the test agent promotes the accumulation of dynamin rings or inhibits disassembly of dynamin rings can involve assaying for an increase in basal dynamin GTPase activity, and/or release of dynamin that is indicative of dynamin ring disassembly.
In some embodiments, dynamin-dependent endocytosis inhibitor is a dynamin GTPase inhibitor, illustrative examples of which are selected from compounds described U.S. Pat. Appl. Pub. No. 2007/0225363
Other representative compounds are selected from helical dynamin GTPase inhibitors, dimeric tyrphostins, dimeric benzylidenemalonitrile tyrphostins, iminochromenes, monomeric tyrphostins and 3-substituted naphthalene-2-carboxylic acid (benzylidene) hydrazides
According to a main embodiment, the invention relates to a dynamin inhibitor or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts, for use in a method for treating or preventing a Coronaviridae infection.
According to a particular embodiment, the dynamin inhibitor is a polypeptide.
In one framework of the present invention, the dynamin inhibitor is a dynamin inhibitor that targets the pleckstrin homology domain of dynamin.
As reported in Singh et al “dynamin functions and ligands: classical mechanism behind”, Molecular pharmacology, 91:123-134, February 2017 various dynamin ligands have been reported such as dynasore, Napthohydrazide of formula (1)
Napthoamide of formula (2)
LRRK2IN1, 1,8-Nphthalimides of formula (3)
Pyrimdyn compound-6 of formula (4)
Rhodadyn A1, Compound-5, DYRK1a inhibitor of formula (5)
sertraline, indole-24 of formula (6)
Phthaladyn-1 of formula (7)
dynole-34 and dimethyl dynole of formula (8)
A further dynamin ligand may be cited having formula (9)
All of said dynamin ligands may be used in the framework of the present invention, alone or in combination, and as well as in combination with ABX464 or its N-glucuronide metabolite, as described above.
The chemical name for Dynasore is 3-Hydroxynaphthalene-2-carboxylic acid (3,4-dihydroxybenzylidene)hydrazide.
Among further dynamin inhibitor compounds, the following may be cited:
In one embodiment, the dynamin inhibitor is a dynamin 2 inhibitor.
MiTMAB, OcTMAB, Dynasore and derivatives of Dynosore such as DD-6 or DD-11 are typical dynamin 2 inhibitors.
Long-chain acids, amines and ammonium salts are typical dynamin 1 inhibitors. 2-(dimethyl amino) ethyl myristate, tetradecylamin, DoTMAB, MiTMAB and OcTMAB may be cited.
According to one embodiment, the dynamin inhibitor is MiTMAB or Myristyl Trimethyl Ammonium Bromide, which is a dynamin 1 and 2 inhibitor of formula (10)
According to one embodiment, the dynamin inhibitor is DoTMAB of formula
According to one embodiment, the dynamin inhibitor is OcTMAB or Octadecyl Trimethyl Ammonium Bromide of formula (12)
Further dynamin inhibitors may be cited such as an ammonium salt having formula (13)
and compound having formula (14)
Still, further dynamin inhibitors may be cited, as disclosed in K. A. Mac Gregor et al, “development of quinone analogues as dynamin GTPase inhibitors”, European Journal of Medicinal Chemistry 85 (2014) 191-206, and namely 2,5-bis-(4-hydroxyanilino)-1,4-benzoquinone, as compound (45) in said article, 2,5-bis(4-carboxyanilino)-1,4-benzoquinone, as compound (49) in said article, 2,5-Bis(3-hydroxyanilino)-1,4-benzoquinone as compound (50) in said article and 2,5-Bis(3-carboxyanilino)-1,4-benzoquinone, as compound (48) in said article.
Still, further dynamin inhibitors may be cited, as disclosed in James A. Daniel et al, “Phenothiazine-Derived Antipsychotic Drugs Inhibit Dynamin and Clathrin-Mediated Endocytosis” Traffic 2015; 16: 6354-654, as namely Calmidazolium, more particularly reported as dynamin 2 inhibitor and Flunarizine, both more particularly reported as dynamin 2 inhibitor.
According to a further embodiment, the dynamin inhibitor is a phenothiazine derivative, well known in the pharmaceutical field.
Among such derivatives, the following may be cited calmidazzine, promethazine and methylene blue, including 4-MB.
More generally, representative phenothiazine derivatives are:
Another phenothiazine may be cited: methotrimeprazine.
Quinacrine and acridine may also be cited.
According to a particular embodiment, the dynamin inhibitor is selected from phenothiazine, Iminodyn-17, Iminodyn-22, Chlorpromazine, Dynasore, long chain amines and ammonium salts, such as MiTMABs and OcTMAB, dynoles, DD-6, desipramine, fluoxetine, reboxetine, fluphenazine, haloperidol and clozapine.
A dynamin inhibitor may comprise a mixture of dynamin inhibitors as described above.
According to a particular embodiment, the dynamin inhibitor for use in a method for treating or preventing a Coronaviridae infection, is a phenothiazine, or any one of its pharmaceutically acceptable salts.
According to a particular embodiment, the dynamin inhibitor for use in a method for treating or preventing a Coronaviridae infection, is aphenothiazine selected from the group consisting of: chlorpromazines, pecazines, and perphenazines, or any one of their pharmaceutically acceptable salts.
According to a particular embodiment, the dynamin inhibitor for use in a method for treating or preventing a Coronaviridae infection, is a chlorpromazine selected from the group consisting of: acepromazine, chlorpromazine, cyamemazine, levomepromazine, oxomemazine, promazine, promethazine, triflupromazine, or any one of their pharmaceutically acceptable salts.
According to a particular embodiment, the dynamin inhibitor for use in a method for treating or preventing a Coronaviridae infection, is a pecazine selected from the group consisting of: mesoridazine, metopimazine, pecazine, thioridazine, or any one of their pharmaceutically acceptable salts.
According to a particular embodiment, the dynamin inhibitor for use in a method for treating or preventing a Coronaviridae infection, is a perphenazine selected from the group consisting of: carfenazine, fluphenazine, perazine, perphenazine, prochlorperazine, trifluoperazine, or any one of their pharmaceutically acceptable salts.
According to a particular embodiment, the dynamin inhibitor for use in a method for treating or preventing a Coronaviridae infection, is a phenothiazine, Iminodyn-17, Iminodyn-22, Chlorpromazine, Dynasore, long chain amines, long chain ammonium salts, dynoles, DD-6, desipramine, fluoxetine, reboxetine, fluphenazine, haloperidol, clozapine, methylene blue, or any one of their pharmaceutically acceptable salts.
According to a particular embodiment, any one of the dynamin inhibitors reported herein for use in a method for treating or preventing a Coronaviridae infection, may be considered in combination with a compound of formula (I) or (II), or any one of their prodrugs or pharmaceutically acceptable salts; wherein compound (I) and compound (II) are respectively of formula (I) and (II)
According to a particular embodiment, the dynamin inhibitor for use in a method for treating or preventing a Coronaviridae infection, or a condition related thereto, is in combination with a cholesterol ester modulating agent.
According to a particular embodiment, the dynamin inhibitor for use in a method for treating or preventing a Coronaviridae infection, or a condition related thereto, is in combination with chloroquine of hydroxychloroquine.
According to a particular embodiment, the dynamin inhibitor for use in a method for treating or preventing a Coronaviridae infection, or a condition related thereto, is in combination with at least one compound selected from (1) a compound of formula (I) or (II), or any one of their prodrugs or pharmaceutically acceptable salts; wherein compound (I) and compound (II) are respectively of formula (I) and (II)
and/or (2) a cholesterol ester modulating agent and (3) chloroquine or hydroxychloroquine.
Pharmaceutical Combinations with Dynamin Inhibitor According to another main embodiment, the invention relates to a pharmaceutical composition comprising a dynamin inhibitor or one of its pharmaceutically acceptable salts; for use in a method for treating or preventing a Coronaviridae infection, and conditions related thereto.
In particular, the invention relates to a pharmaceutical composition comprising a dynamin inhibitor or one of its pharmaceutically acceptable salts; for use in a method for treating or preventing a Coronaviridae infection.
According to a particular embodiment of the invention, the dynamin inhibitor may be administered in combination with other compounds.
Thus, the pharmaceutical composition may further comprise at least one of compound (I) and (II), or any one of their prodrugs or pharmaceutically acceptable salts.
According to one embodiment, the dynamin inhibitor may be administered in combination with ABX464 or its N-glucuronide metabolite ABX464-N-Glu.
According to another embodiment, the dynamin inhibitor may be administered in combination with a cholesterol ester modulating agent, for example for increasing its stability.
Among such cholesterol ester modulating agent one may cite everolimus, pioglitazone, progesterone, verapamil and everolimus.
According to another embodiment, the dynamin inhibitor may be administered in combination with chloroquine or hydroxychloroquine.
In the framework of the present invention, a combination may comprise a dynamin inhibitor in combination with at least one compound selected from (1) ABX464 or its N-glucuronide metabolite ABX464-N-Glu, (2) a cholesterol ester modulating agent and (3) chloroquine or hydroxychloroquine.
According to one embodiment, such a combination may further comprise at least a compound selected from an antibiotic, such as selected from the group consisting of beta-lactams, fluoroquinolones, and macrolides, such as azythromicin; remdesivir; ribavirin; ritonavir; lopanivir; beta-interferon; an anti-inflammatory compound, such as one selected from the group consisting of: anti-TNF, Jak inhibitors, anti-IL6 antibodies, IL6 receptor antagonists and a calcium inhibitor, such as diltiazem.
Such combination may be suitable for separate administration, administration spread out over time or simultaneous administration to patients in need thereof.
The separate administration, simultaneous administration or administration spread out over time of a medicinal combination means that the elementary constituents of the combination, can be administered at the same time, each in one go at distinct moments, or repeatedly, or else at different moments, in particular during cycles. The elementary constituents can, in order to do this, be formulated as mixtures, only if they are administered simultaneously, or else formulated separately for the other administration schemes.
Triple combination comprising a dynamin inhibitor, ABX464 or its N-glucuronide metabolite and chloroquine or hydroxychloroquine are thus encompassed within the scope of the present invention.
Pharmaceutical Compositions & Medicaments
According to a third main embodiment, the invention relates to a pharmaceutical composition comprising a compound of formula (I) or (II) as defined above or any one of its prodrugs or any one of its pharmaceutically acceptable salts, and at least one pharmaceutically acceptable excipient, for use in a method for treating or preventing a Coronaviridae infection, or a condition related thereto.
In certain embodiments, a composition of this invention is formulated for administration to a patient in need of such composition. In some embodiments, a composition of this invention is formulated for oral administration or injectable, IV, IM, SC or sustained release or for inhalation to a patient.
Accordingly, the invention relates to a use of a compound of formula (I) or (II) as defined above or any one of its prodrugs or any one of its pharmaceutically acceptable salts, for the preparation of a medicament for treating or preventing a Coronaviridae infection, or a condition related thereto.
According to a fourth main embodiment, the invention relates to a medicament comprising a compound of formula (I) or (II) as defined above, for use in a method for treating or preventing a Coronaviridae infection, or a condition related thereto.
Alternatively, the invention relates to the use of a compound of formula (I) or (II) as defined above or any one of its prodrugs or any one of its pharmaceutically acceptable salts, for the preparation of a pharmaceutical composition or medicament for the treatment or prevention of a Coronaviridae infection, or a condition related thereto.
Compositions of the present invention may be administered orally, parenterally, by inhalation, aerosol, by spray, topically, rectally, nasally, buccally, vaginally, ophtalmologically or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intra-tracheal, and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally, intravenously or by inhalation. Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.
For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
Pharmaceutically acceptable compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
Alternatively, pharmaceutically acceptable compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.
Pharmaceutically acceptable compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
Topical application for the lower intestinal tract can be performed in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically transdermal patches may also be used.
For topical applications, provided pharmaceutically acceptable compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, provided pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
For ophthalmic use, provided pharmaceutically acceptable compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutically acceptable compositions may be formulated in an ointment such as petrolatum.
According to a particular embodiment, pharmaceutically acceptable compositions of this invention may also be administered by nasal aerosol or inhalation.
Hence, according to a particular embodiment, the pharmaceutical composition is under an inhalation dosage form, a intraperitoneal dosage form or a intramuscular dosage form.
Hence, according to a particular embodiment, the pharmaceutical composition of the invention may be in the form of an intraperitoneal dosage form or an intramuscular dosage form.
According to a particular embodiment, the pharmaceutical composition takes the form of eye drops or is under dermatological preparation form.
Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents. Most preferably, pharmaceutically acceptable compositions of this invention are formulated for oral administration. Such formulations may be administered with or without food.
In some embodiments, pharmaceutically acceptable compositions of this invention are administered without food. In other embodiments, pharmaceutically acceptable compositions of this invention are administered with food.
The amount of compounds of the present invention that may be combined with the carrier materials to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration. Preferably, provided compositions should be formulated so that a dosage of between 0.01-100 mg/kg body weight/day of the inhibitor can be administered to a patient receiving these compositions.
It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a compound of the present invention in the composition will also depend upon the particular compound in the composition.
Such pharmaceutically acceptable compositions may also be considered in combination with other active compounds, or alternatively may include the compounds according to the invention in combination with other active agents.
In a non-limitative manner, such combinations with active agent(s) may thus consist of combinations with:
According to a particular embodiment, the invention thus also relates to a pharmaceutical composition or kit comprising:
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts; and
remdesivir or any one of its pharmaceutically acceptable salts.
According to a particular embodiment, the invention thus also relates to a pharmaceutical composition or kit comprising:
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts; and
According to a particular embodiment, the invention thus also relates to a pharmaceutical composition or kit comprising:
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts; and
for use as a medicament; in particular for use in a method for treating or preventing a Coronaviridae infection and conditions related thereto; and more particularly for use in a method for treating or preventing a SARS-CoV or SARS-CoV-2 infection and conditions related thereto.
According to a particular embodiment, the invention thus also relates to a pharmaceutical composition or kit comprising:
or any one of its or any one of its prodrugs or any one of its pharmaceutically acceptable salts; and
for use as a medicament; in particular for use in a method for treating or preventing a Coronaviridae infection and conditions related thereto; and more particularly for use in a method for treating or preventing a SARS-CoV or SARS-CoV-2 infection and conditions related thereto.
Treatment Monitoring and miR-124 as a Biomarker & Uses & Methods Thereof
In some embodiments, a method of the present invention for treating or preventing an infection of condition related to a virus, more particularly said virus having a RNA genome, and especially a RNA virus belonging to group IV according to the Baltimore classification, and more particularly for treating or preventing a Coronaviridae infection further comprises measuring a level of a compound or any one of its prodrugs or a pharmaceutically acceptable salt thereof as described herein, in a patient. In some embodiments, a level of a compound or any one of its prodrugs or a pharmaceutically acceptable salt thereof as described herein, is measured in a patient's biological sample. In some embodiments, a patient's biological sample is a blood, plasma, tissue, saliva, pharyngeal, tracheal, broncho alveolar and/or serum sample.
In a further embodiment, the invention provides the compound or any one of its prodrugs or any one of its pharmaceutically acceptable salts, for use in a method as defined above, wherein the level of a compound or any one of its prodrugs or a pharmaceutically acceptable salt thereof as described herein, in a blood, plasma, tissue, saliva, pharyngeal, tracheal, broncho alveolar and/or serum sample of the patient is measured during the use.
In some embodiments, a method of the present invention for treating an inflammatory disease, disorder or condition further comprises measuring a total level of compounds of formulas (I) and (II) as defined above, or pharmaceutically acceptable salts thereof, in a patient. In some embodiments, a method of the present invention for treating or preventing an infection of condition related to a virus, more particularly said virus having a RNA genome, and especially a RNA virus belonging to group IV according to the Baltimore classification, and more particularly for treating or preventing a Coronaviridae infection further comprises measuring a total level of compounds of formulas (I) and (II), or pharmaceutically acceptable salts thereof, in a patient.
In some embodiments, a method of the present invention for treating or preventing an infection of condition related to a virus, more particularly said virus having a RNA genome, and especially a RNA virus belonging to group IV according to the Baltimore classification, and more particularly for treating or preventing a Coronaviridae infection further comprises measuring and/or monitoring a presence and/or level of a biomarker in a patient. In some embodiments, a presence and/or level of a biomarker is measured in a patient's biological sample. In some embodiments, a patient's biological sample is a blood sample. In some embodiments, a patient's biological sample is a tissue sample. In some embodiments, a patient's biological sample is a pharyngeal, tracheal and/or broncho alveolar sample. In some embodiments, a biomarker measured and/or monitored in a method of the present invention is miR-124, as described in WO 2014/111892, the entire content of which is incorporated herein by reference. In some embodiments, a method of the present invention for treating or preventing an infection of condition related to a virus, more particularly said virus having a RNA genome, and especially a RNA virus belonging to group IV according to the Baltimore classification, and more particularly for treating or preventing a Coronaviridae infection further comprises measuring and/or monitoring a presence and/or expression level of miR-124 in a patient prior to administering a compound or a pharmaceutically acceptable salt or composition thereof as described herein. In some embodiments, a method of the present invention for treating or preventing an infection of condition related to a virus, more particularly said virus having a RNA genome, and especially a RNA virus belonging to group IV according to the Baltimore classification, and more particularly for treating or preventing a Coronaviridae infection further comprises measuring and/or monitoring a presence and/or expression level of miR-124 in a patient during the course of a treatment with a compound or a pharmaceutically acceptable salt or composition thereof as described herein. In some embodiments, a method of the present invention for treating or preventing an infection of condition related to a virus, more particularly said virus having a RNA genome, and especially a RNA virus belonging to group IV according to the Baltimore classification, and more particularly for treating or preventing a Coronaviridae infection further comprises selecting a patient for a treatment with a compound or a pharmaceutically acceptable salt or composition thereof as described herein, by measuring and/or monitoring a presence and/or expression level of miR-124 in the patient. In some embodiments, a method of the present invention treating or preventing an infection of condition related to a virus, more particularly said virus having a RNA genome, and especially a RNA virus belonging to group IV according to the Baltimore classification, and more particularly for treating or preventing a Coronaviridae infection further comprises excluding a patient from a treatment with a compound or a pharmaceutically acceptable salt or composition thereof as described herein, by measuring and/or monitoring a presence and/or expression level of miR-124 in the patient. In some embodiments, a method of the present invention treating or preventing an infection of condition related to a virus, more particularly said virus having a RNA genome, and especially a RNA virus belonging to group IV according to the Baltimore classification, and more particularly for treating or preventing a Coronaviridae infection further comprises adjusting (such as increasing or decreasing) dosage regimen (such as dose amount and/or dose schedule) of a compound or a pharmaceutically acceptable salt or composition thereof as described herein to be administered to a patient, by measuring and/or monitoring a presence and/or expression level of miR-124 in the patient.
In some embodiments, a method of the present invention for treating or preventing an infection of condition related to a virus, more particularly said virus having a RNA genome, and especially a RNA virus belonging to group IV according to the Baltimore classification, and more particularly for treating or preventing a Coronaviridae infection comprises comparing a measured expression level of miR-124 in a patient to a control reference value. A control reference value to be used for comparing a measured expression level of miR-124 in a patient is obtained from a control sample. A control sample can be taken from various sources. In some embodiments, a control sample is taken from a patient prior to treatment or prior to the presence of a disease (such as an archival blood sample, pharyngeal, tracheal, broncho alveolar or tissue sample). In some embodiments, a control sample is taken from a set of normal, non-diseased members of a population. In some embodiments, a control sample is taken from a patient prior to treatment with a compound or a pharmaceutically acceptable salt or composition thereof as described herein. In some embodiments, a cell assay can be performed on a biological sample.
In some embodiments, a modulated presence and/or expression level of miR-124 in a patient compared to a control reference value indicates an infection of condition related to a virus, more particularly said virus having a RNA genome, and especially a RNA virus belonging to group IV according to the Baltimore classification, and more particularly a Coronaviridae infection. In some embodiments, a modulated presence and/or expression level of miR-124 in a patient compared to a control reference value indicates an efficacy of a treatment with a compound or a pharmaceutically acceptable salt or composition thereof as described herein, which is administered to the patient. The terms “modulation” or “modulated presence and/or expression level” means the presence or expression level of a biomarker is either induced or increased, or alternatively is suppressed or decreased.
In some embodiments, a measured reduced or suppressed presence, or a decreased expression level, of miR-124 relative to a control reference value indicates an infection of condition related to a virus, more particularly said virus having a RNA genome, and especially a RNA virus belonging to group IV according to the Baltimore classification, and more particularly for treating or preventing a Coronaviridae infection. In some embodiments, a measured induced or increased presence, or an increased expression level, of miR-124 relative to a control reference value indicates an efficacy of a compound or a pharmaceutically acceptable salt or composition thereof as described herein. In some embodiments, a measured expression level of miR-124 in a patient treated with a compound or a pharmaceutically acceptable salt or composition thereof as described herein is a two-fold, four-fold, six-fold, eight-fold, or ten-fold increase relative to a control reference value.
Thus, according to a particular embodiment, the present invention further provides the compound or any one of its prodrugs or any one of its pharmaceutically acceptable salts, for use in a method for treating or preventing a Coronaviridae infection, as defined above, wherein a presence and/or expression level of miR-124 in a blood and/or tissue sample of the patient is measured prior to and during the use.
According to a fifth main embodiment, the invention relates to an in vitro or ex vivo use of at least one miRNA, said at least one miRNA being miR-124, as a biomarker of a Coronaviridae infection, or of an efficacy of a therapeutic treatment of said Coronaviridae infection.
According to a sixth main embodiment, the invention relates to an in vitro or ex vivo method for assessing a Coronaviridae infection in a patient presumed to be infected with a virus, comprising at least the steps of:
a—measuring a presence or an expression level of at least one miRNA, said at least one miRNA being miR-124, in a biological sample previously obtained from said patient; and
b—comparing said presence or expression level to a control reference value, wherein a modulated presence or level of expression of said miRNA relative to said control reference value is indicative of a Coronaviridae infection.
According to one embodiment, uses and methods according to the invention may, in particular, allow for the determining of a Coronaviridae infection in a patient, and in particular for the follow-up of such infection.
According to one embodiment, a presence or a level of expression of miR-124 is measured into an isolated biological sample, and then is compared to a control reference value.
A modulation of the presence or level of expression of miR-124 relative to the control reference value may be indicative of a viral infection. In particular a reduced or suppressed presence, or a decreased level of expression, of said miRNA relative to a control reference value may be indicative of a viral infection.
In one embodiment, a use of the invention may comprise obtaining of a measured level of expression of said miR-124 into an isolated biological sample and comparing said measured level of expression to a control reference value. An observation of a modulation of said measured level relative to said control reference value may be indicative of a viral infection, or of an efficacy of a therapeutic treatment of said viral infection.
When miR-124 from a sample is “decreased” or “down-regulated” in a biological sample isolated from a patient, as compared to a control reference value, this decrease can be, for example, of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 100%, 200%, 300%, 500%, 1,000%, 5,000% or more of the comparative control reference value (i.e., without the treatment by the quinoline derivative).
In particular, the measured level expression of miR-124 may be at least a two-fold, preferably at least a four-fold, preferably at least a six-fold, preferably at least an eight-fold, and more preferably at least a ten-fold decrease relative to said control reference value.
According to one embodiment, uses of and methods implementing miR-124 as a biomarker for a Coronaviridae infection, may be combined with the determination of others biomarkers specific from said infection such as the determination of the presence or level of expression of peptides, proteins or nucleic acid sequences specific from said virus.
According to one embodiment, the increase of the presence or level of expression of miR-124 in a biological sample taken from a patient suffering from a Coronaviridae infection and receiving a treatment for this infection relative to a biological sample taken from the same patient before initiating said treatment may be indicative of the severity of the disease or efficacy of said treatment.
According to one embodiment, the uses and methods of the invention may be for assessing a responsiveness of a patient to a treatment with said compounds of formula (I) or (II).
According to another embodiment, the uses and methods of the invention may be for assessing an effectiveness of a treatment with said compounds of formula (I) or (II).
According to another embodiment, the uses and methods of the invention may be for assessing a therapeutic efficacy of said compounds of formula (I) or (II) as a therapeutic agent for preventing and/or treating a Coronaviridae infection.
According to one embodiment, the uses and methods of the invention may be for assessing a patient compliance with a treatment with said compounds of formula (I) or (II).
The miR-124 biomarker may be used to monitor or manage compounds of formula (I) or (II) activity during patient treatment of a Coronaviridae infection
According to one embodiment, a use or a method according to the invention may be implemented for optimizing the dosing regimen of a patient. Patients may respond differently to a given compound of formula (I) or (II), depending on such factors as age, health, genetic background, presence of other complications, disease progression, and the co-administration of other drugs. It may be useful to utilize the miR-124 biomarker to assess and optimize the dosage regimen, such as the dose amount and/or the dose schedule, of a quinoline derivative in a patient. In this regard, miR-124-based biomarker can also be used to track and adjust individual patient treatment effectiveness over time. The biomarker can be used to gather information needed to make adjustments in a patient's treatment, increasing or decreasing the dose of an agent as needed. For example, a patient receiving a compound of formula (I or (II) can be tested using the miR-124-based biomarker to see if the dosage is becoming effective, or if a more aggressive treatment plan needs to be put into place. The amount of administered drug, the timing of administration, the administration frequency, the duration of the administration may be then adjusted depending on the miR-124 biomarker measurement.
The miR-124 biomarker may also be used to track patient compliance during individual treatment regimes, or during clinical trials. This can be followed at set intervals to ensure that the patients included in the trial are taking the drugs as instructed. Furthermore, a patient receiving a quinoline derivative can be tested using the miR-124 biomarker to determine whether the patient complies with the dosing regimen of the treatment plan. An increased expression level of the biomarker compared to that of an untreated control sample is indicative of compliance with the protocol.
A biomarker of the invention may be implemented to assess and follow the efficacy of compounds of formula (I) or (II). Accordingly, a presence or level of expression of miR-124 may be measured into an isolated biological sample obtained from a patient previously treated with compounds of formula (I) or (II). Then, the measured presence or level expression of miR-124 into an isolated biological sample may be compared to a control reference value.
When an increase of the measured level relative to the control reference value is observed, then the measure is indicative of an activity of said compounds of formula (I) or (II).
In another embodiment, when an increase of the measured level relative to the control reference value is observed, then the measure may be indicative of a responsiveness of a patient to a treatment with said compounds of formula (I) or (II).
In another embodiment, when an increase of the measured level relative to the control reference value is observed, then the measure may be indicative of an effectiveness of a treatment with said compounds of formula (I) or (II).
In another embodiment, when an increase of the measured level of expression relative to the control reference value is observed, then the measure may be indicative a therapeutic efficacy of said compounds of formula (I) or (II) as a therapeutic agent for preventing and/or treating a Coronaviridae infection.
When miR-124 from a sample is “increased” or “up-regulated” after a treatment with a quinoline derivative, as compared to a non-treated control reference value, this increase can be, for example, of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 100%, 200%, 300%, 500%, 1,000%, 5,000% or more of the comparative control reference value (i.e., without the treatment by the compounds of formula (I) or (II).
In particular, the measured level expression of miR-124 may be at least a two-fold, preferably at least a four-fold, preferably at least a six-fold, preferably at least an eight-fold, and more preferably at least a ten-fold increase relative to said control reference value.
According to another embodiment of the invention, when monitoring a Coronaviridae infection or assessing an efficacy of a Coronaviridae infection treatment, in particular with a compound of formula (I) or (II), a patient may be tested with a method or a use of the invention at a time interval selected from the group consisting of hourly, twice a day, daily, twice a week, weekly, twice a month, monthly, twice a year, yearly, and every other year. The then collected sample can be tested immediately, or can be stored for later testing.
According to another embodiment, use and methods according to the invention may, in particular, allow for the screening, identification or evaluation of potential active agents as a drug candidate.
In particular, use and methods according to the invention are particularly advantageous for the screening, identification or evaluation of potential active agents, such as a drug candidate or a vaccine presumed effective towards a Coronaviridae infection.
According to another embodiment of the invention, a miR-124 biomarker may be implemented to screen a drug candidate or a vaccine candidate presumed effective for preventing and/or treating a Coronaviridae infection. In such embodiment, a presence or level of expression of miR-124 may be measured into an isolated biological sample or isolated cell previously contacted with the drug or vaccine to be screened. Then, the obtained measure may be compared to a control reference value.
When an increase of the measured level into an isolated biological sample or isolated cell, previously contacted with the compound, drug or vaccine candidate to be screened, relative to a control reference value is observed, then the measure may be indicative of said candidate to have a biological effect and in particular to be efficient for altering the physiological activity of a cell.
In particular, a drug candidate or vaccine candidate may be characterized as being efficient in preventing and/or treating the Coronaviridae infection
When miR-124 from a sample is “increased” or “up-regulated” after treatment with a drug candidate, as compared to a non-treated control reference value, this increase can be, for example, of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 100%, 200%, 300%, 500%, 1,000%, 5,000% or more of the comparative control reference value i.e., without the treatment by the compound of formula (I) or (II).
In particular, the measured level expression of miR-124 may be at least a two-fold, preferably at least a four-fold, preferably at least a six-fold, preferably at least an eight-fold, and more preferably at least a ten-fold increase relative to said control reference value.
The uses and methods of the invention may comprise measuring a level of expression of miR-124 into an isolated biological sample. Any suitable sample may be used to assess the miR-124 biomarker.
The step of collecting biological samples for the uses and methods of the invention is performed before carrying out the invention and is not a step of a use or a method in accordance with the invention.
Samples for miRNA assessment can be taken during any desired intervals. For example, samples can be taken hourly, twice per day, daily, weekly, monthly, every other month, yearly, or the like. The sample can be tested immediately, or can be stored for later testing.
The samples can be purified prior to testing. In some embodiments, the miR-124 can be isolated from the remaining cell contents prior to testing. Further, the miR-124 molecules can be separated from the rest of the mRNA in the sample, if desired. For example, the miR-124 can be separated from the mRNA based on size differences prior to testing.
Control reference value to be used for comparing the measured level of expression of miR-124 in a tested biological sample is obtained from a control sample.
Control samples can be taken from various sources. In some embodiments, control samples are taken from the patient prior to treatment or prior to the presence of the disease (such as an archival blood sample). In other embodiments, the control samples are taken from a set of normal, non-diseased members of a population. In another embodiment, a cell assay can be performed on a control cell culture, for example, that has not been treated with the test compound or has been treated with a reference compound, such as the 8-chloro-N-[4-(trifluoromethoxy)phenyl]quinolin-2-amine.
According to one embodiment, for the determination or monitoring of a viral infection in a patient, a control reference value may be obtained from an isolated biological sample obtained on an individual or group of individuals known to not suffer from such condition.
According to another embodiment, for the determination or monitoring of an efficacy of a treatment of a viral infection into a patient, a control reference value may be obtained from an isolated biological sample obtained from an individual or group of individuals known to not suffer from such condition, and not receiving the treatment the efficacy of which is to be determined or monitored. Alternatively, a control reference value may be obtained from an isolated biological sample obtained from a patient suffering from a viral infection and receiving a treatment the efficacy of which being to be determined or monitored, the isolated biological sample being taken from the patient before administration of the treatment.
Numerous methods are available to the skilled man to measure a presence or level of expression of the miR-124 biomarker.
For example, nucleic acid assays or arrays can be used to assess the presence and/or expression level of miR-124 in a sample.
The sequence of the miR-124 may be used to prepare a corresponding nucleotide acting as complementary probe or primer to be used in different nucleic acid assays for detecting the expression or presence of the miR-124 biomarker in the sample, such as, but not limited to, Northern blots and PCR-based methods (e.g., Real-Time Reverse Transcription-PCR or qRT-PCR). Methods such as qRT-PCR may be used to accurately quantitate the amount of the miRNA in a sample.
Sense and anti-sense probes or primers according to the invention may be obtained using every process known to the man skilled in the art, in particular those that are described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 3rd ED., 2001, Cold Spring Harbour, N.Y.).
Methods related to the detection and quantification of RNA or DNA are well known in the art. The man skilled in the art may for instance refer to Wang et al. (1989, Proc Natl Acad Sci USA, Vol. 86: 917-921), de Wong et al. (2005, Bio Techniques, Vol. 39 (1): 75-85), de Nolan et al. (2006, Nat Protoc, Vol. 1(3): 1559-1582) et de Klinck et al. (2008, Cancer Research, Vol. 68: 657-663), or also to a general review published by Bustin (2000, Journal of Molecular Endocrinology, Vol. 25: 169-193).
In one embodiment, a method for the detection and quantification of nucleic acids may be a fluorescent-dye-based method, wherein nucleic acid concentration is assessed by measuring the fluorescence intensity of ligands, such as dyes, that bind to said nucleic acids. Fluorescent dyes are well known in the art.
Alternatively, said nucleic acid may be quantified using spectrophotometry.
In another embodiment, a method for the detection and quantification of nucleic acids may be a hybridation-based method. Said hybridation-based methods may include PCR and quantitative-PCR (qRT-PCR or q-PCR) techniques or reverse transcriptase/polymerase based techniques. Advantageously, said method may comprise, or be further combined, with a sequencing step.
Those methods may comprise (i) a step of extraction of cellular mRNAs, (ii) a step of reverse transcription of mRNA to DNA using a reverse transcriptase and (iii) a step of DNA amplification from DNA obtained on the previous step. Usually, starting from the same sample, the following nucleic acids are amplified: (a) DNA obtained after a reverse transcription step of the target mRNA and (b) a DNA or a plurality of DNAs obtained after reverse transcription of mRNAs which are constitutively and constantly expressed by cells («housekeeping genes»), such as RNAs coded by genes MRPL19, PUM1 and GADPH.
The amplified DNA can be quantified, after separation by electrophoresis, and measure of DNA bands. Results related to the target mRNA(s) are expressed as relative units in comparison to mRNAs coded by «housekeeping» genes. In some embodiments, the step of separation of amplified DNAs is achieved after agarose gel electrophoresis, and then coloration of DNA bands with ethidium bromide, before quantification of DNA contained in those migration bands with densitometry. In other embodiments, one may use a micro-channel device in which amplified DNA is separated by capillar electrophoresis, before quantification of the emitted signal using a laser beam. Such a device may be a LabChip® device, for instance from the «GX» series, commercialized by the company Caliper LifeSciences (Hopkinton, Mass., USA).
Quantitative results obtained by qRT-PCR can sometimes be more informative than qualitative data, and can simplify assay standardization and quality management. Thus, in some embodiments, qRT-PCR-based assays can be useful to measure miRNA levels during cell-based assays. The qRT-PCR method may be also useful in monitoring patient therapy. Commercially available qRT-PCR based methods (e.g., TaqmanR Array™)
Any suitable assay platform can be used to determine the expression or presence of the miRNA in a sample. For example, an assay may be in the form of a dipstick, a membrane, a chip, a disk, a test strip, a filter, a microsphere, a slide, a multiwell plate, or an optical fiber. An assay system may have a solid support on which an oligonucleotide corresponding to the miRNA is attached. The solid support may comprise, for example, a plastic, silicon, a metal, a resin, glass, a membrane, a particle, a precipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, a capillary, a film a plate, or a slide. The assay components can be prepared and packaged together as a kit for detecting an miRNA.
In some embodiments, an oligonucleotide array for testing for the compound or drug candidate activity in a biological sample can be prepared or purchased. An array typically contains a solid support and at least one oligonucleotide contacting the support, where the oligonucleotide corresponds to at least a portion of the miR-124 biomarker. In some embodiments, the portion of the miR-124 biomarker comprises at least 5, 10, 15, 20 or more bases.
According to one embodiment, the presence or expression of miR-124 may be assayed in combination with others miRNA also used as biomarkers. In such an embodiment, an array can be used to assess the expression or presence of multiple miRNAs in a sample, including miRNA-124. In general, the method comprises the following steps: a) contacting the sample with an array comprising a probe set under conditions sufficient for specific binding to occur; and b) examining the array to detect the presence of any detectable label, thereby evaluating the amount of the respective target miRNAs in the sample. The use of an expression array allows obtaining a miRNA expression profile for a given sample.
Methods of preparing assays or arrays for assaying miRNAs are well known in the art and are not needed to be further detailed here.
Nucleic acid arrays can be used to detect presence or differential expression of miRNAs in biological samples. Polynucleotide arrays (such as DNA or RNA arrays) typically include regions of usually different sequence polynucleotides (“capture agents”) arranged in a predetermined configuration on a support. The arrays are “addressable” in that these regions (sometimes referenced as “array features”) have different predetermined locations (“addresses”) on the support of array. The region (i.e., a “feature” or “spot” of the array) at a particular predetermined location (i.e., an “address”) on the array will detect a particular miRNA target. The polynucleotide arrays typically are fabricated on planar supports either by depositing previously obtained polynucleotides onto the support in a site specific fashion or by site specific in situ synthesis of the polynucleotides upon the support. Arrays to detect miRNA expression can be fabricated by depositing (e.g., by contact- or jet-based methods or photolithography) either precursor units (such as nucleotide or amino acid monomers) or pre-synthesized capture agent. After depositing the polynucleotide capture agents onto the support, the support is typically processed (e.g., washed and blocked for example) and stored prior to use.
An array to detect miRNA expression has at least two, three, four, or five different subject probes. However, in certain embodiments, a subject array may include a probe set having at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1,000 or more probes that can detect a corresponding number of miRNAs. In some embodiments, the subject arrays may include probes for detecting at least a portion or all of the identified miRNAs of an organism, or may include orthologous probes from multiple organisms.
A nucleic acid array may be contacted with a sample or labeled sample containing miRNA analytes under conditions that promote specific binding of the miRNA in the sample to one or more of the capture agents present on the array to exhibit an observed binding pattern. This binding pattern can be detected upon interrogating the array. For example, the target miRNAs in the sample can be labeled with a suitable label (such as a fluorescent compound), and the label then can be accurately observed (such as by observing the fluorescence pattern) on the array after exposure of the array to the sample. The observed binding pattern can be indicative of the presence and/or concentration of one or more miRNA components of the sample.
The labeling of miRNAs may be carried using methods well known in the art, such as using DNA ligase, terminal transferase, or by labeling the RNA backbone, etc. In some embodiments, the miRNAs may be labeled with fluorescent label. Exemplary fluorescent dyes include but are not limited to xanthene dyes, fluorescein dyes, rhodamine dyes, fluorescein isothiocyanate (FITC), 6 carboxyfluorescein (FAM), 6 carboxy-2 1,4 1,7′,4,7-hexachlorofluorescein (HEX), 6 carboxy 4′, 5′ dichloro 2′, 7′ dimethoxyfluorescein (JOE or J), N,N,N′,N′ tetramethyl 6 carboxyrhodamine (TAMRA or T), 6 carboxy X rhodamine (ROX or R), 5 carboxyrhodamine 6G (R6G5 or G5), 6 carboxyrhodamine 6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; Alexa dyes, e.g. Alexa-fluor-555; coumarin, Diethylaminocoumarin, umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, BODIPY dyes, quinoline dyes, Pyrene, Fluorescein Chlorotriazinyl, R1 10, Eosin, JOE, R6G, Tetramethylrhodamine, Lissamine, ROX, Naptho fluorescein, and the like.
In some embodiments, an oligonucleotide array for assessing immunomodulatory activity can be prepared or purchased, for example from Affymetrix. The array may contain a solid support and a plurality of oligonucleotides contacting the support. The oligonucleotides may be present in specific, addressable locations on the solid support; each corresponding to at least a portion of miRNA sequences which may be differentially expressed upon treatment of a quinoline derivative or a drug candidate in a cell or a patient. The miRNA sequences comprise at least one miR-124 sequence.
When an array is used to assess miRNAs, a typical method can contain the steps of 1) obtaining the array containing surface-bound subject probes; 2) hybridization of a population of miRNAs to the surface-bound probes under conditions sufficient to provide for specific binding (3) post-hybridization washes to remove nucleic acids not bound in the hybridization; and (4) detection of the hybridized miRNAs. The reagents used in each of these steps and their conditions for use may vary depending on the particular application.
Hybridization can be carried out under suitable hybridization conditions, which may vary in stringency as desired. Typical conditions are sufficient to produce probe/target complexes on an array surface between complementary binding members, i.e., between surface-bound subject probes and complementary miRNAs in a sample. In certain embodiments, stringent hybridization conditions may be employed. Hybridization is typically performed under stringent hybridization conditions. Standard hybridization techniques which are well-known in the art (e.g. under conditions sufficient to provide for specific binding of target miRNAs in the sample to the probes on the array) are used to hybridize a sample to a nucleic acid array. Selection of appropriate conditions, including temperature, salt concentration, polynucleotide concentration, hybridization time, stringency of washing conditions, and the like will depend on experimental design, including source of sample, identity of capture agents, degree of complementarity expected, etc., and may be determined as a matter of routine experimentation for those of ordinary skill in the art. In general, a “stringent hybridization” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization are typically sequence dependent, and are different under different experimental conditions. Hybridization may be done over a period of about 12 to about 24 hours. The stringency of the wash conditions can affect the degree to which miRNA sequences are specifically hybridized to complementary capture agents. Those of ordinary skill will readily recognize that alternative but comparable hybridization and wash conditions can be utilized to provide conditions of similar stringency.
As an illustration, in one embodiment, the miRNA expression profiling experiments may be conducted using the Affymetrix Genechip miRNA Array 2.0 and following the protocols described in the instruction manual.
In one particular embodiment, said hybridization can be performed using the GeneChip® Hybridization, Wash, and Stain Kit (Affymetrix Ref. #900720). Advantageously, said hybridization is performed by following the protocols of the manufacturer.
After the miRNA hybridization procedure, the array-surface bound polynucleotides are typically washed to remove unbound nucleic acids. Washing may be performed using any convenient washing protocol, where the washing conditions are typically stringent, as described above. For instance, a washing step may be performed using washing buffers sold by the company Affymetrix (Ref. #900721 and #900722). The hybridization of the target miRNAs to the probes is then detected using standard techniques of reading the array. Reading the resultant hybridized array may be accomplished, for example, by illuminating the array and reading the location and intensity of resulting fluorescence at each feature of the array to detect miRNA/probe binding complexes.
ABX464 and its N-Glucuronide Derivative Inhibit SARS-CoV2 Replication
Supported by the fact that coronavirus RNAs are capped and that their nucleoprotein (N) interacts with UPF1, a direct binding partner of the CBC complex, and also that cell entry is mediated by dynamin 2, a protein which is itself downregulated by miR124, we examined whether ABX464 or its N-glucuronide could have antiviral effects.
ABX464 was tested in a human reconstituted airway epithelial model of bronchial origin that sustains SARS-CoV2 infection. The viral genome was quantified by RTqPCR. The daily treatment with increasing concentrations of ABX464 led to a dose-dependent inhibition of SARS-CoV2 replication.
ABX464 was tested in a human reconstituted airway epithelial model of bronchial origin that sustains SARS-CoV2 infection. Trans-epithelial electrical resistance (TEER), was used to measure epithelium integrity, while the viral genome was quantified by RTqPCR.
Material & Methods
Tested Concentrations
Compound powder is resuspended in DMSO to make a 10 mM stock solution.
Evaluation of Antiviral Activity of Selected Compounds in Differenciated Primary Cells (MucilAir™ Epithelix)
Cells are pre-incubated during 48h with the corresponding drug concentration (no change of the media until day 0). According to the lab protocol, the drugs are added 1h post-infection. On day 1, the media is changed with fresh drug. On day 2, the cells are harvested and a Total RNA extraction is performed (PPE supernatant) (BSL-3) for viral quantification by RT-qPCR (BSL-2) and depending on the results, confirmation by infectious titration (BSL-3).
Cytotoxicity studies are achieved according to the Cytotoxicity kit Detection KitPLUS LDH from Roche (Roche, ref Merck 4744926001).
Total RNA extraction was achieved according to the ML buffer Macherey-Nagel Nucleospin miRNeasy kit.
The daily treatment with increasing concentrations of AB X464 led to a dose-dependent inhibition of SARS-CoV2 replication. The effect of ABX464 on viral replication is consistent with its protective effect on the integrity of bronchial epithelium measured by TEER. By contrast, high viral replication, correlated with a reduction in epithelium integrity at 48h post-infection were compared in this assay with a control compound (“ABX300”) that does not increase miR-124, of formula:
as disclosed in patent application published under WO2010/143170.
Reagents & Cells
The differenciated primary cells used for the experiment are MucilAir™ Epithelix human respiratory epithelial cells.
Clinical Samples, Viral Isolation and Sequencing
The SARS-CoV-2 strain used in this study was isolated by directly inoculating VeroE6 cell monolayers with a nasal swab sample collected from a one of the first COVID-19 cases confirmed in France: a 47y-o female patient hospitalized in January 2020 in the Department of Infectious and Tropical Diseases, Bichat Claude Bernard Hospital, Paris (11). Once characteristic CPE was observable in more than 50% of the cell monolayer, supernatants were collected and immediately stored at −80° C. for subsequent viral RNA extraction using the QiAmp viral RNA Kit (Qiagen).
The complete viral genome sequence was obtained using Illumina MiSeq sequencing technology, was then deposited after assembly on the GISAID EpiCoV platform (Accession ID EPLISL_411218) under the name BetaCoV/France/IDF0571/2020.
Viral Quantification Viral stocks and collected samples were titrated by tissue culture infectious dose 50% (TCID50/ml) in VeroE6 cells, using the Reed & Muench statistical method. In parallel, relative quantification of viral genome was performed by one-step real-time quantitative reverse transcriptase and polymerase chain reaction (RT-qPCR) from viral or total RNA extracted using QiAmp viral RNA or RNeasy Mini Kit (Qiagen) in the case of supernatants/apical washings or cell lysates, respectively. Primer and probe sequences were selected from those designed by the School of Public Health/University of Hong Kong (Leo Poon, Daniel Chu and Malik Peiris) and synthetized by Eurogentec.
Real-time one-step RT-qPCR was performed using the EXPRESS One-Step Superscript™ qRT-PCR Kit (Invitrogen, reference 1178101K), in a 20 μl reaction volume containing 10 μl of Express qPCR supermix at 2×, 1 μl of forward primer at 10 μM, 1 μl of reverse primer at 10 μM, 0.5 μl of probe at 10 μM, 3.1 μl of PCR-water (Qiagen, reference 17000-10), 0.4 μl of Rox dye at 25 μM, and 2 μl of vRNA template.
Thermal cycling was performed in a StepOnePlus™ Real-Time PCR System (Applied Biosystems) in MicroAmp™ Fast Optical 96-well reaction plates (Applied Biosystems, reference 4346907).
Cycling conditions were as follows: reverse transcription at 50° C. during 15 min, followed by initial polymerase activation at 95° C. for 2 min, and then 40 cycles of denaturation at 95° C. for 15 sec and annealing/extension at 60° C. for 1 minute. The SARS-CoV-2-specific primer and probes used for viral genome quantification were as follows:
Viral Infection and Treatment in Reconstituted Human Airway Epithelia (HAE)
MucilAir™ HAE reconstituted from human primary cells obtained from nasal or bronchial biopsies, were provided by Epithelix SARL (Geneva, witzerland) and maintained in air-liquid interphase with specific culture medium in Costar Transwell inserts (Corning, N.Y., USA) according to the manufacturer's instructions. For infection experiments, apical poles were gently washed twice with warm OptiMEM medium (Gibco, ThermoFisher Scientific) and then infected directly with nasal swab samples or a 150 μl dilution of virus in OptiMEM medium, at a multiplicity of infection (MOI) of 0.1. For mock infection, the same procedure was performed using OptiMEM as inoculum. Samples collected from apical washes or basolateral medium at different time-points were separated into 2 tubes: one for TCID50 viral titration and one RT-qPCR.
HAE cells were harvested in RLT buffer (Qiagen) and total ARN was extracted using the RNeasy Mini Kit (Qiagen) for subsequent RT-qPCR and Nanostring assays. Treatments with specific dilutions of candidate molecules alone or in combination in MucilAir® culture medium were applied through basolateral poles. All treatments were initiated on day 0 (5 1h after viral infection) and continued once daily at 24 and 48 hpi (2 and 3 treatments in total for samples collected at 48 and 72 hpi, respectively). Variations in transepithelial electrical resistance (ΔTEER) were measured using a dedicated volt-ohm meter (EVOM2, Epithelial Volt/Ohm Meter for TEER) and expressed as Ohm/cm2.
Results
The following results were obtained, and expressed in % inhibition of viral infection, based on RTqPCR results (experiments in duplicates, marked as N1 and N2).
Of note, the % inhibition results obtained with ABX300 were all of 0%, which indicates a lack of % inhibition of viral replication and thus a lack of efficacy toward this strain of the virus.
Overall, those results show a dose-dependent effect which applies both to the ABX464 compound and its N-glucuronide metabolite, although the dose necessary to reach a detectable effect in terms of percentage inhibition is slightly higher for the N-glucuronide in this experiment.
In contrast, the same experiment using ABX300, which does not increase miR-124, fails to show a marked effect on viral replication at the same concentrations. Again, this suggests that the effect observed in SARS-CoV2 inhibition is linked to the regulation of miR-124.
Interestingly, the antiviral effect of ABX464 and the N-glucuronide also appear to be similar to what is observed with Remdesivir.
In summary, ABX464 is an orally deliverable molecule for which the clinical profile appears suited to satisfy the needs of severe forms of SARS-CoV-2 infections: anti-inflammatory effects to fight the cytokine storm, mucosal effectiveness, promotion of tissue repair to avoid long-term post-ventilation sequelae.
The added anti-viral effect may thus also contribute to an increased clearance of the virus and help mitigate control the cytokine storm that acute anti-inflammatory drugs might induce. For its anti-inflammatory properties ABX464 can be positioned as alternative to IL-6R and IL-6 inhibitors that have already shown partial clinical benefits, but it offers the advantages of acting on multiple cytokines involved in the cytokine storm, having anti-viral effects and promoting tissue repair. Finally, ABX464 results in a good bioavailability, with a rapid and high systemic and pulmonary exposure.
Among the long list of candidates to treat various presentations of Covid-19, the unique properties of ABX464 and its already proven efficacy in a severe inflammatory disease may result in clinical benefits in Covid-19 patients.
ABX464 Possesses Excellent Systemic and Tissue Bioavailability by the Oral Route and Rapidly Reaches the Lungs
This study was conducted in order to determine the tissue distribution and rates and routes of excretion of radioactivity in the rat following a single oral administration of [14C]-ABX464. Analysis was performed using liquid scintillation counting (plasma and excreta) and quantitative whole-body autoradiography.
Material & Methods
Species, Specification and Supplier
Sufficient albino rats of the Sprague Dawley strain were obtained from Charles River Limited (UK), to provide 9 male study animals, respectively. All animals were examined on arrival for external signs of ill health and were acclimatised in an experimental room for 7 days. During this time the health status of the animals was reassessed and their suitability for experimental purposes confirmed.
The animals were housed up to 5 per cage, according to strain, in suitable solid floor cages containing suitable bedding. They were kept in rooms thermostatically maintained at a temperature of 20 to 24° C., with a relative humidity of between 45 to 70%, and exposed to fluorescent light (nominal 12 hours) each day. Temperature and relative humidity was recorded on a daily basis. The facility is designed to give 15 to 20 air-changes/hour. In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and polycarbonate tunnels. The supplier provided certificates of analysis for each batch of blocks used and these were maintained in a central file at Covance.
To reduce the chance of animals re-ingesting radioactivity from faecal material, the bedding, chew blocks and tunnels in the cages was changed at the end of the dosing day and again the following day.
All animals were allowed free access to commercial pellet diet, SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services). The diet supplier provided an analysis of the concentration of certain contaminants and some nutrients for each batch used. The animals were allowed free access to mains water from bottles attached to the cages.
Dose Formulation
The radiochemical purity of [14C]-ABX464 ABX464 was determined by high performance liquid chromatography (HPLC). The identity of the test substance was also confirmed by co-elution with the non-radiolabelled material by HPLC.
Radiolabelled ABX464 was then prepared for administration in a 0.5% (w/v) carboxymethylcellulose (CMC, 400-800 centipoises) and 0.5% Tween 80 in water for injection. The concentration of SPL464 in the final formulation was targeted at 4 mg/mL.
On the day of dosing, the radiolabelled (4.90 mg) and non-radiolabelled (74.6 mg) test substances were dissolved in 1 mL of acetonitrile into a pre-weighed formulation vessel. The solvent was removed to near dryness under stream of nitrogen. A volume of 0.5% (w/v) CMC and 2.5% Tween 80 in water for injection (3.96 mL), equating to 20% of the target final volume was added. The suspension was mixed by a magnetic stirrer for ca 5 minutes and sonicated for ca 1 minute. The suspension was made up to final volume (19.8 mL) using 0.5% (w/v) CMC in water for injection. The suspension was continually stirred during pre and post dose analysis and the dosing procedure.
The radiochemical purity of the formulated test substance was confirmed by injecting portions of the formulation before and after dose administration onto the HPLC system.
The radioactivity concentration/homogeneity of the formulation prepared was determined before and after dosing. Triplicate weighed portions (100 μL) were diluted to 100 mL with deionised water/acetonitrile, triplicate aliquots of the resulting solutions (1.0 mL) were taken whilst stirring and subjected to liquid scintillation counting. Where possible, aliquots were taken from the top, middle and bottom of the prepared formulation.
Dose Administered
All animals received a single oral administration of [14C]-ABX464 by oral gavage, at a nominal dose level of 20 mg/kg body weight and a dose volume of approximately 5 mL/kg. The target radioactive dose administered was ca 8 MBq/kg body weight.
Radioactivity in Blood and Plasma Whilst under terminal anaesthesia, but before freezing, up to 2 mL of blood was obtained from all animals by cardiac puncture and transferred to tubes pre-coated with K2-EDTA. The residual sample was centrifuged in order to obtain plasma. The radioactivity concentration was measured in blood and plasma by liquid scintillation counting.
Whole Body Autoradiography
For each animal, the legs, tail and whiskers were trimmed off and the frozen carcass set in a block of aqueous 2% (w/v) carboxymethylcellulose. The block was mounted onto the stage of a Leica CM3600 cryomicrotome maintained at ca −20° C. (Leica Microsystems (UK) Ltd) and sagittal sections (nominal thickness 30 μm) were obtained through the carcass to include the following tissues: exorbital lachrymal gland, intra-orbital lachrymal gland, Harderian gland, adrenal gland, thyroid, brain and spinal cord. The sections, mounted on Filmolux 610 Tape (Neschen UK), were freeze-dried in a GVD03 bench-top freeze-drier (Girovac Ltd) and placed in contact with FUJI imaging plates (type BAS-MS, Raytek Scientific Ltd). [14C]-Blood standards of appropriate activity (also sectioned at a nominal thickness of 30 μm) were placed in contact with all imaging plates.
Image Analysis of Whole-Body Autoradiograms
After exposure in a copper-lined, lead exposure box for 7 days, the imaging plates were processed using a FUJI FLA-5000 radiography system (Raytek Scientific Ltd). Electronic images were analysed using a PC-based image analysis package (Seescan 2 software, LabLogic Ltd). The carbon-14 standards included with each autoradiogram were used to construct calibration lines over a range of radioactivity concentrations.
For the purposes of quantification, it was assumed that all tissues analysed had a similar density and quench characteristics to blood (used as calibration standards). Wherever possible, the maximum area within a single autoradiogram was defined for each tissue for measurement.
Liquid Scintillation Counting
A suitable scintillation counter was used. Radioassays were performed at least in duplicate. Efficiency correlation curves were prepared and routinely checked by the use of [14-C]toluene and Ultima Gold™ quenched standards (PerkinElmer LAS (UK) Ltd).
The limit of quantification for each batch of samples analysed by direct counting was taken as twice the mean background disintegration rate obtained from vials containing an equivalent volume of an appropriate solvent in liquid scintillant.
The limit of quantification of each batch of samples analysed by combustion was taken as twice the mean background disintegration rate obtained when Combusto-Cones™ containing ashless floc are combusted.
Calculation of μg Equivalents of ABX464 in Tissues
Concentration of radioactivity in sample=C (dpm/g)
Specific radioactivity of test substance=S (MBq/mg)
Concentration of radioactivity in sample=(C/60000)/S (μg equiv/g)
Calculation of μg equivalents of ABX464 in plasma
Weight of aliquot of sample assayed W (g)
Radioactivity (dpm−background value) in aliquot of
sample analysed R (dpm)
Concentration of radioactivity in sample C=R/W (dpm/g)
Specific radioactivity of test substance S (MBq/mg)
Concentration of radioactivity in sample (C/60000)/S (μg equiv/g)
Results
Tissue concentration data are reported in terms of μg equivalents of [14C]-ABX464. Results are provided in Table 1 and discussed hereafter.
The plasma and blood levels were measured by liquid scintillation counting. The numbers are in μg equivalents per gram for all measurements.
Radioactivity was rapidly and widely distributed following oral administration. All investigated tissues, with the exception of the lens of the eye contained quantifiable drug-related radioactivity at 2 or more sampling times.
No trend in partitioning of radioactivity into tissues from the blood was apparent, with typically half of the investigated tissues having a tissue: plasma concentration ratio of greater than 1:1 at each sampling time.
In blood, plasma ratios increased over time from 0.7 to 7, suggested that the affinity for [14C]-ABX-464 related radioactivity to bind to the cellular partition of blood increased 10-fold over the study duration. The detection of low levels of radioactivity in the central nervous system suggested that drug related material crossed the blood-brain barrier, but was subsequently eliminated. There was no evidence for the binding of [14C]-ABX464 related material to melanin. Elimination of drug related radioactivity was rapid. Although approximately a third of investigated tissues in albino rats contained quantifiable radioactivity at the final sampling time of 168 hours, analysis of the carcasses in the excretion balance phase of the study showed this equated to less than 0.5% of the administered dose. This indicated that excretion was essentially complete. Elimination of radioactivity was primarily via voiding in faeces (88%), with less than 5% excreted via the renal system. This suggests that at least 5% of the orally administered dose was absorbed. Total mean recovery was 93%±1.8%. All study objectives were achieved, with the tissue distribution and routes and rates of excretion following an oral dose of [14C]-ABX464 targeted at 20 mg/kg bodyweight, fully investigated.
Overall, this study illustrates the excellent systemic and tissue bioavailability of ABX464 by the oral route, and the fact that ABX464 rapidly reaches the lungs. This provides evidence of the efficacy of ABX464 or ABX-464-N-Glu in the early treatment of high-risk patients infected with SARS-CoV2.
Effect of ABX464 on Infectious Titers
The following experiment measures the variation of TCID50. The TCID50 is determined in replicate cultures of serial dilutions of the infected supernatants treated with the candidate molecules from Example 1.
Material & Methods
Details on viral isolation, sequencing and viral quantification are as described in Example 1.
Viral Replication Kinetics and Antiviral Treatment in VeroE6 Cells
In Example 1, cells were treated with ABX464, ABX300, ABX-464-N-Glu or remdesivir 48h prior to infection, on the day of infection and 24h post infection. Supernatant samples were collected 48 hours post infection to determine TCID50
VeroE6 cells were seeded 24 h in advance in multi-well 6 plates, washed twice with PBS and then infected with serial dilutions of supernatants described above. Cells were incubated for 96h. The cytopathic effect (CPE) was monitored and the number of positive (i.e. with CPE) and negative (i.e. without CPE) wells were recorded and TCID50 was determined.
Results
The results are indicated in
Overall, the data show that ABX464 provides a dose-dependent decrease of the TCID50 at 48 hours post-infection (hpi). This decrease is at least comparable to what is observed with remdesivir at the same molar concentration.
Interestingly, a decrease is also observed with the N-glucuronide form of ABX464, which agains shows that it is also potent on its own as an antiviral compound.
As expected the samples treated with compound ABX300 show a very modest effect on the TCID50.
Thus, this experiment fully validates the original conclusions observed in Example 1 with RTqPCR.
Effect of ABX464 in Combination with Remdesivir on an In Vitro Model of Reconstructed Human Respiratory Epithelium.
The following experiment measures the toxicity of a combination of ABX464 and Remdesivir in an in vitro model of epithelium membrane (
Material & Methods
Reconstitution of Human Airway Epithelial (HAE)
MucilAir™ HAE is reconstituted from human primary cells obtained from nasal or bronchial biopsies, provided by Epithelix SARL (Geneva, Switzerland) and maintained in air-liquid interphase with specific culture medium in Costar Transwell inserts (Corning, N.Y., USA) according to the manufacturer's instructions.
For infection experiments, apical poles were gently washed twice with warm OptiMEM medium (Gibco, ThermoFisher Scientific) and then inoculated directly with a 150 μl dilution of virus in OptiMEM medium, at a multiplicity of infection (MOI) of 0.1, as described by Pizzorno et al. (“Characterization and treatment of SARS-CoV2 in nasal and bronchial human airway epithelial” (2020), Cell Reports Medicine, Volume 1, Issue 4). For mock infection, the same procedure was performed using OptiMEM™ as inoculum.
Samples collected from apical washes or basolateral medium at different time-points were separated into 2 tubes: one for TCID50 viral titration and one RT-qPCR. HAE cells were harvested in RLT buffer (Qiagen) and total RNA was extracted using the Rneasy Mini Kit (Qiagen) for subsequent RT-qPCR. Treatments with specific dilutions of candidate molecules in MucilAir™ culture medium were applied through basolateral poles.
All treatments were initiated 48 hours prior to viral infection and continued once daily at 1 and 24 hpi (3 treatments in total). Samples were collected as following:
Assessment of Monolayer Integrity
Monolayer integrity was assessed through measuring variations in transepithelial electrical resistance (ΔTEER) using a dedicated vol-ohm meter (EVOM2, Epithelial Vol/Ohm Meter for TEER) and expressed as Ohm/cm2. Cellular viability was assessed through lactate dehydrogenase (LDH) measurement, using the Cytotoxicity Detection Kit LDH (Roche, ref 11644793001).
Results
The combination of ABX464 and Remdesivir (REM) had no significant effect on TEER values meaning there was no toxicity on epithelium membrane (
Altogether, those data suggest that association of ABX464 with remdesivir is more potent to reduce viral RNA in HEA cells, when compared to ABX464 or remdesivir alone. Moreover, those results suggest that ABX464 treatment of infected HAE can lead to less infectious SARS-CoV2 viral particles in comparison to remdesivir treatment outcome.
Conversely, remdesivir antiviral effect against SARS-CoV2 is potentiated by the combination with ABX464 in reconstructed human respiratory epithelium (based on the assessment of viral genome relative quantification by RT-qPCR).
| Number | Date | Country | Kind |
|---|---|---|---|
| 20305299.8 | Mar 2020 | WO | international |
| 20305327.7 | Mar 2020 | WO | international |
| 20305482.0 | May 2020 | WO | international |
| 20306483.7 | Dec 2020 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/057123 | 3/19/2021 | WO |
