Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds
  • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

• the invention relates to novel compositions and methods for the treatment of COVID- 19 disease. More specifically, the invention relates to the use of at least one antagonist or inhibitor of chemokine receptor CXCR4 for use in the treatment of COVID-19 particularly in patients at moderate to severe stages of the disease.
• Coronaviruses are known to cause severe respiratory and gastrointestinal diseases in animals.
• the infection of humans with coronavirus strains have been described for many years to be associated with respiratory tract infections, Le ⁇ common cold-like diseases.
• SARS-CoV severe Acute Respiratory Syndrome-Corona Virus
• ARDS acute respiratory distress syndrome
• This virus appeared as an epidemic in 2003 after having crossed the species barriers from bats to civet cats and humans demonstrating the potential of coronaviruses to cause high morbidity and mortality in humans.
• the strains HCoV-NL63 and HCoV-HKUl were discovered in 2004 and 2005, respectively.
• Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV -2) has been identified as the emerging virus responsible for the pandemic COVID-19 now declared as a “global threat for public health” by the WHO. In most cases, clinical features of COVID-19 remain benign. However, COVID-19 condition can be much more severe and may require for oxygen support at hospital in 16 to 20% of patients. It can cause acute respiratory distress syndrome (ARDS) in 5 to 10% of patients with admission to intensive care unit (ICU) and invasive mechanical ventilation. Long ICU length of stay participates in healthcare resources overwhelming. ARDS is responsible for 99% of admission to ICU and COVID-19 related deaths. ARDS is characterized by a respiratory worsening triggered by gas exchange impairment secondary to alveolar-capillary barrier oedema. This lung injury results from a massive dysregulated inflammatory response to lung epithelial invasion by the virus.
• SARS-CoV -2 Severe acute respiratory syndrome-coronavirus 2
• SARS-Cov-2 has infected hundreds of millions of people since 2019 and represent a global health emergency requiring the development of potent antiviral treatments.
• the present application relates to a composition for use in a method of treating and to a method of treating respiratory viral disease caused by SARS-Cov-2 virus such as in particular COVID-19 comprising administering to a patient a therapeutically effective amount of at least one antagonist or inhibitor of chemokine receptor CXCR4, said antagonist or inhibitor of chemokine receptor CXCR4 being distinct from hydroxychloroquine.
• compositions and methods of treatment according to the present invention are particularly effective in improving survival in patients suffering from acute respiratory distress syndrome (ARDS) occurring in moderate to severe cases of respiratory viral infections caused by SARS-Cov-2 virus.
• ARDS acute respiratory distress syndrome
• Said compositions and methods of treatment are thus useful in for treating moderate to severe cases of COVID-19, and/or in cases of co-morbidity or multiple co-morbidities.
• Figure 1 is a graph showing murine lung function measured by the FEV0.05/FVC after daily subcutaneous injections of 1 mg/kg plerixafor during the 5 last weeks of the protocol. Mice tested presented lung injury induced by cigarette smoke exposure with poly-IC instillations, and either PBS injections (gray squares) or 1 mg/kg plerixafor injections (black squares). *: PO.05.
• Figures 2A-B are graphs showing cardiac remodeling, quantified by Fulton index, calculated as RV/(LV+S), with LV: left ventricle, RV: right ventricle, S: septum.
• A Fulton index. Control mice (black circles) and mice with lung injury induced by cigarette smoke exposure, with poly-IC instillations (gray squares).
• B Fulton index. Tested mice presented lung injury induced by cigarette smoke exposure with poly-IC instillations, and either PBS injection (gray squares) or 1 mg/kg plerixafor injection (black squares).
• Figures 3A-B are graphs showing the effect of plerixafor on K18-hACE2 mice infected with SARS-CoV-2.
• A shows the individual percentage of body weight loss of vehicle-treated and plerixafor-treated mice on day 4 and 5 post infection with SARS-CoV-2.
• B shows the percentage of vehicle-treated and plerixafor-treated mice survival after infection with SARS-CoV-2.
• Figures 5A-C show comparison of (A) DAPI CD16 + , (B) DAPI CD 16' CXCR4 , and (C) DAPI CD 16' CXCR4 1 cell migration in response to sera from patients with severe COVID-19. Results are expressed with symbols indicating individual subject values. * P ⁇ 0.05, Wilcoxon matched pair test.
• Figures 6 A-C show comparison of (A) DAPI CD16 + , (B) DAPI CD16 + CXCR4 , and (C) DAPI CD16 + CXCR4 + cell migration in response to sera from patients with severe COVID-19. Results are expressed with symbols indicating individual subject values. * P ⁇ 0.05, Wilcoxon matched pair test. DETAILED DESCRIPTION
• the present invention thus provides compounds, compositions, pharmaceutical compositions and methods of use of certain compounds that are antagonists or inhibitors of chemokine receptor CXCR4, for treating coronavirus infections caused by SARS-CoV2.
• Antagonists or inhibitors of chemokine receptor CXCR4 according to the present invention are distinct from hydroxychloroquine.
• the present invention also relates to compounds, compositions, pharmaceutical compositions and methods of use of certain compounds that are antagonists or inhibitors of chemokine receptor CXCR4, for treating COVID-19, comprising administering to a patient a therapeutically effective amount at least one antagonist or inhibitor of chemokine receptor CXCR4.
• the present invention specifically provides a composition for use in a method of treating and a method of treating COVID-19 with or without multiple comorbidities comprising administering to said patient a therapeutically effective amount at least one antagonist or inhibitor of chemokine receptor CXCR4.
• the present invention further relates to compounds, compositions, pharmaceutical compositions and methods of decrease the NETosis and/or mitigating pneumonia, acute respiratory failure and ARDS in SARS-Cov-2 infected patients comprising administering to said patient a therapeutically effective amount at least one antagonist or inhibitor of chemokine receptor CXCR4, with the proviso that said antagonists or inhibitors of chemokine receptor CXCR4 are preferably distinct from hydroxychloroquine.
• treatment refers to curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition and includes suppression of clinical relapse.
• the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
• Acute respiratory failure or severe acute respiratory syndrome may affect persons of all ages, with a higher likelihood for the first responders, ej., nurses, physicians, etc...
• Initial symptoms include fever, chills, myalgia, cough, but also shortness of breath and/or tachypnea.
• Two third of the SARS-Cov-2 infected patients however developed complications such as ARDS.
• antagonist or inhibitor of chemokine receptor CXCR4 may be chosen among indole-based compound, a N-substituted indole compound, a bicyclam compound, a cyclam mimetic compound, a para-xylyl-enediamine-based compound, a guanidine-based antagonist compound, a tetrahydroquinolines-based compound, or a 1,4- phenylenebis(methylene) compound.
• Said antagonists or inhibitors of chemokine receptor CXCR4 according to the present invention are distinct from hydroxychloroquine and/or do not include hydroxychloroquine.
• Preferred antagonist or inhibitor of chemokine receptor CXCR4 is chosen among plerixafor (AMD3100), Burixafor (TG-0054), JM1657, AMD3329, AMD3465, AMD070, MSX-122, CTCE-9908, WZ811, or BKT-140. Most preferred antagonist or inhibitor of chemokine receptor CXCR4 according to the present invention is plerixafor.
• Plerixafor is well known drug and a CXCR4 antagonist, which has originally been developed as an anti-HIV drug. Also, the combination plerixafor and granulocyte-colony stimulating factor (G-CSF) was approved in 2008, by the US Food and Drug Administration to mobilize hematopoietic stem cells (HSCs) to the peripheral blood for collection and subsequent autologous transplantation in patients with non-Hodgkin’s lymphoma and multiple myeloma. Plerixafor has been validated thru several clinical trials. Toxicology, pharmacokinetics as well as any potential adverse events are thus fully known, thereby allowing possible fast track of the registration for COVID-19 condition.
• G-CSF granulocyte-colony stimulating factor
• Applicants have showed in the Examples below that the administration of a therapeutically effective amount of CXCR4 antagonist, such as for example plerixafor (1 mg/kg) during the 5 weeks improved lung function of mice having lung injury induced by cigarette smoke exposure with poly-IC instillations, which mimic viral infection, and either PBS injection ( Figures 1 and 2).
• a therapeutically effective amount of CXCR4 antagonist such as for example plerixafor (1 mg/kg) during the 5 weeks improved lung function of mice having lung injury induced by cigarette smoke exposure with poly-IC instillations, which mimic viral infection, and either PBS injection ( Figures 1 and 2).
• plerixafor may significantly improve respiratory conditions, and lung disease evolution of COVID-19 in patients at moderate or severe stages.
• NETosis was significantly reduced in RNA virus infected mice after treatment with an effective amount of CXCR4 antagonist such as plerixafor ( Figure 4).
• CXCR4 antagonist such as plerixafor
• NETs in the alveolar space has been local inflammation and viral burden to the lungs, causing acute respiratory distress syndrome (ARDS) in infected patients.
• ARDS acute respiratory distress syndrome
• Administration of a CXCR4 antagonist in patients is expected to result in significant inhibition of migration of neutrophils in COVID-19 patients.
• the authors performed a virtual screen to identify FDA-approved small molecules that present an inhibitory potential of the SARS- CoV-2 protease, based on a simulation of molecular docking and predicted binding energy within the binding pocket of the virus’ protease.
• the authors expressly rejected clinical exploration of plerixafor for patients with COVID-19 given plerixafor known side effect of bone marrow and strong immune system suppression which was expected to lead to a worsening of the condition of the patients.
• compositions and methods according to the present invention are particularly efficient for treating COVID-19 patients at moderate to advanced or severe stage of the disease.
• Moderate COVID-19 cases are those with inflammation lower down in the lungs, wherein lung symptoms like cough are more marked.
• the lungs consist of large airways (bronchi), smaller airways (bronchioles) and the tiny air sacs on the end (alveoli) where oxygen is extracted from the air. They contain a fluid called surfactant which keeps the lungs stretchy and compliant and helps keep the air sacs open.
• Patients with moderate COVID-19 may have inflammation moving down into the bronchioles. They are more breathless and tend to have an increased heart rate, particularly if they are moving around.
• COVID-19 appears to damage the vasculature of the lungs. Particularly, it may induce capillary endothelial cell/microvascular dysfunction which may cause individual cell necrosis. Histopathologic basis of COVID-19 severe disease cases has been analyzed and showed in all cases diffuse alveolar damages involving activation of megakaryocytes, with platelet aggregation and platelet-rich clot formation, in addition to fibrin deposition.
• COVID-19 patients are detected by a consistent clinical history, epidemiological contact, and a positive SARS-CoV-2 test.
• SARS-CoV-2 infection can be confirmed by positive detection of viral RNA in nasopharyngeal secretions using a specific PCR test.
• COVID-19 ARDS is diagnosed when a COVID-19 patient presents (1) acute hypoxemic respiratory failure, (2) within 1 week of worsening respiratory symptoms; (3) bilateral airspace disease on chest x- ray, computed tomography, or ultrasound that is not fully explained by effusions, lobar or lung collapse, or nodules; and (4) cardiac failure is not the primary cause of acute hypoxemic respiratory failure. It also found that shortness of breath - also known as dyspnoea - is the only symptom of COVID-19 that is significantly associated with severe cases and with patients requiring admission to ICU.
• COVID-19 ARDS follows a predictable time course over days, with median time to intubation of 8.5 days after symptom onset. It is therefore important to monitor patients for the development of ARDS as their COVID-19 progresses.
• the respiratory rate and Sp02 are two important parameters for judging patients’ clinical condition and allowing early recognition of ARDS.
• a patient who fits any one of the following conditions may have severe disease and requires further evaluation/Respiratory rate >30 breaths/min; Sp0 2 ⁇ 92 %; Pa0 2 /Fi0 2 ⁇ 300 mmHg.
• methods of treating and compositions for use in said method may comprise further administering an anti-IL6 monoclonal antibody or an anti- IL6 receptor monoclonal antibody.
• anti-IL6 monoclonal antibody or an anti-IL6 receptor monoclonal antibody which can be administered to infected patients, preferably COVID-19 patients, in combination with at least antagonist or inhibitor of chemokine receptor CXCR4, we may cite siltuximab (an anti-IL6 chimeric monoclonal antibody for treating Castleman’s disease, and marketed under the tradename Sylvant®), olokizumab (an anti-IL6 humanized monoclonal antibody under development by the company R-Pharm for treating rheumatoid arthritis and also called CDP6038 or OKZ), sirukumab (an anti-IL6 human monoclonal antibody under development by the company Centocor for treating rheumatoid arthritis and also called CNT0136), tocilizum
• Methods of treating and compositions for use in a method according to the present invention may further comprise administering a pharmaceutically acceptable vehicle, antiviral drugs, antibacterial drugs, antibiotics, PPI Inhibitors, quinoline compounds, zinc compounds, gamma globulin, hematopoietic cells, mesenchymal cells, anti-inflammatory drugs, vaccines, small interfering RNAs and microRNAs, immunomodulators, or plasma from convalescent patients.
• said methods and compositions for use according to the present invention do not comprise further administration of any hydroxychloroquine or chloroquine to said COVID-19 patients.
• Anti-viral drugs may be selected from the following drugs: nucleotide analogues, nucleoside analogues, nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), neuraminidase inhibitors, endonuclease inhibitors, adamantanes, protease inhibitors (Pis), integrase inhibitors (INSTIs), fusion inhibitors (FIs), chemokine receptor antagonists (CCR5 antagonists), or siRNA.
• NRTIs nucleoside reverse transcriptase inhibitors
• NRTIs non-nucleoside reverse transcriptase inhibitors
• neuraminidase inhibitors neuraminidase inhibitors
• endonuclease inhibitors adamantanes
• protease inhibitors Pis
• INSTIs integrase inhibitors
• FIs fusion inhibitors
• anti-viral drug which may be administered to a COVID-19 patient in combination with the antagonist or inhibitor of chemokine receptor CXCR4 as described above, we may cite abacavir, acyclovir, adefovir, amantadine, ampligen, amprenavir (AgeneraseTM), arbidol, atazanavir, atripla, balavir, baloxavir marboxil (XofluzaTM), biktarvy, boceprevir (VictrelisTM), cidofovir, cobicistat (TybostTM), combi vir, daclatasvir (DaklinzaTM), darunavir, delavirdine, descovy, didanosine, docosanol, dolutegravir, doravirine (PifeltroTM), ecoliever, edoxudine, efavirenz, elvitegravir, emtricitabine
• compositions and methods as described above may be administered via parenteral, oral, nasal, ocular, transmucosal, or transdermal. Most preferred routes of administration are intravenous, intramuscular or subcutaneous routes.
• the specific therapeutic dose to be administered to a COVID-19 patients is the dose required to obtain therapeutic and/or prophylactic effects. This dose may be determined by the physician depending on the conditions of the patients, weight, age and sex, compound administered, the route of administration, etc...
• the dosage may be by a single dose, divided daily dose, or multiple daily doses.
• continuous dosing, such as for example, via a controlled (e.s.. slow) release dosage form can be administered on a daily basis or for more than one day at a time.
• plerixafor may be selected as antagonist or inhibitor of chemokine receptor CXCR4. It is then administered via the subcutaneous route at a dosage of around 10 to 40 pg/kg bid (20 to 80 pg/kg/day) and continuous intra-venous route at a dosage of around 10 to 120 pg/kg/hour. According to a most preferred embodiment, plerixafor is administered to COVID patients via intra-venous perfusion route a dosage of 30 pg/kg/day.
• compositions are particularly useful for treating most vulnerable patients that have health conditions or comorbidities, such as hypertension, obesity, and/or diabetes.
• health conditions or comorbidities such as hypertension, obesity, and/or diabetes.
• patients having history of myocardial infarction as well as cancer patients are excluded and thus are not treated with the compositions and methods according to the present invention.
• compositions and methods as described above are expected to have a protective effect on the heart, since Applicants have showed that administration for example of plerixafor improves cardiac remodeling quantified by Fulton index.
• Said patients include patients selected from the group consisting of an end stage renal disease (ESRD) patient, a patient having chronic obstructive pulmonary disease (COPD), an AIDS patient, a diabetic patient, a patient subject to obesity, a patient subject to hypertension, a neonate, a transplant patient, a patient on immunosuppression therapy, a patient with malfunctioning immune system, an autoimmune disease patient, an elderly person in an extended care facility, a patient with autoimmune disease on immunosuppressive therapy, a bum patient, and a patient in an acute care setting.
• ESRD end stage renal disease
• COPD chronic obstructive pulmonary disease
• CXCR4 antagonists suitable for use in accordance with the present invention can be administered alone but, in human therapy, will generally be administered in admixture with a suitable pharmaceutically acceptable vehicle, excipient, diluent, or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
• a suitable pharmaceutically acceptable vehicle or excipient may be present in an amount between 0.1% and less than 100% by weight.
• Optimizing drug-excipient ratios are with the reach of a person with ordinary skill in art for instance the desired weight ratio of drug/excipient in the composition could be less than or equal to the ratio of solubilities of drug/excipient, in a suitable medium.
• compositions of the present invention may be administered parenterally, for example, intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intrastemally, intracranially, intramuscularly, subcutaneously, or they may be administered by infusion or needle-free techniques.
• a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
• the aqueous solutions should be suitably buffered (preferably, to a pH of from about 3 to 9), if necessary.
• the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
• compositions for use in a method of treating and a method of treating COVID-19 disease in a subject in need thereof comprising: i) determining a viral load in a sample obtained from the subject by RT- PCR or other equivalent techniques; ii) comparing the viral load determined at step i) with a predetermined reference value; and iii) administering to the subject a therapeutically effective amount of at least one antagonist or inhibitor of chemokine receptor CXCR4 as described above.
• the sample is preferably a blood sample or a mucus sample.
• mice which presented lung injury induced by cigarette smoke exposure with poly-IC instillations received daily subcutaneous injections of either PBS injection or 1 mg/kg plerixafor during the 5 weeks. Cardiac function and remodeling were then quantified by Fulton index, calculated as RV/(LV+S), with LV: left ventricle, RV: right ventricle, S: septum.
• Fulton index calculated as RV/(LV+S)
• mice Two groups of transgenic C57BL/6 mice expressing the human ACE2 receptor (K18-hACE2 mice) were allocated for this study. Infection with 5x10 2 TCID50 of a clinical SARS-CoV-2 isolate SARS-CoV-2 was performed on 15 mice on day 0 by intranasal (IN) administration. The same day, infected mice were treated twice daily until termination with water for injection (group 1M) for 6 mice or Plerixafor at 10 mg/kg/day (group 2M) for 9 mice. During the study, mortality and morbidity observation and body weight measurements were performed. Mice were euthanized for ethical reasons when they achieved greater than 20% body weight loss.
• Example 3.1 Body weight loss
• mice treated with Plerixafor demonstrated a significant reduction of body weight loss kinetic in comparison to vehicle mice (pO.0011). Indeed, at day 5, Plerixafor treated mice lost in average 17,6 % ⁇ 1,28 % of their body weight while vehicle treated mice lost in average 20,43% ⁇ 2,88 % of their body weight.
• mice reaching a body weight loss superior to 20% were euthanized and the time of death was precisely recorded.
• Figure 6B all mice from the vehicle group were euthanized on day 5 in the morning.
• Plerixafor treated mice were euthanized on day 5 (2 mice in the morning and 2 mice in the afternoon) and on day 6 (4 mice in the morning).
• Significant difference in time of death was observed between vehicle and plerixafor treated mice (Wilcoxon test p ⁇ 0.05).
• EXAMPLE 4 Clinical Trial 1 “LEONARDO” pLErixafOr iN Acute Respiratory Distress syndrome related to cOvid-19 (Phase lib)
• Example 4.1 Design
• Percentage of patients alive and who did not require invasive mechanical ventilation (composite primary endpoint including mortality and or time to require invasive mechanical ventilation, whichever occurs first) from D1 to D28.
• Respiratory function including Forced Expiratory Volume in one sec (FEV1), Forced Vital Capacity (FVC), Partial arterial pressure in Oxygen (Pa0 2 ) and Transfer Lung Capacity for carbon monoxide (TLCO) [Time Frame: D90]
• ICU • Recently admitted in ICU (within 48 hours) for COVID-19 related respiratory failure.
• ICU or equivalent medical structure according to country specificities e.g., Acute Respiratory Care Unit, High Dependency Care Unit if they can provide:
• COVID-19 Vaccinated patients can be also included in the study.
• Cardio-vascular co-morbidity o History of vascular ischemic events (myocardial infarction or stroke) or congestive heart failure or peripheral arterial disease, o History or current significant cardiac rhythm disorders (e.g., ventricular tachycardia), o Known medical history of proven symptomatic postural hypotension,
• EXAMPLE 5 Clinical Trial 1 “LEONARDO- 1” pLErixafOr iN Acute Respiratory Distress syndrome related to cOvid-19 (Phase lib)
• Example 5.1 Design
• VFDs Ventilator-free days at 28 days are one of several organ failure-free outcome measures to quantify the efficacy of therapies and interventions.
• DNR order do-not-resuscitate order
• plerixafor was administered for 10 days by continuous intravenous infusion in an open-label dose escalation study from 2.5 to 160 pg/kg/h.
• plerixafor was administered for 6 months by subcutaneously injection (10 to 20 pg/kg bid for 6 months).
• moderate form of COVID-19 defined by the need for oxygen therapy between 3 and 5 L/min to obtain a percutaneous oxygen saturation greater than 97% and a respiratory rate ⁇ 25 breaths/min without the need for invasive ventilation.
• plerixafor In HIV clinical trials, plerixafor (AMD3100) was administered for 10 days by continuous intravenous infusion in an open-label dose escalation study from 2.5 to 160 pg/kg/h. In WHIM syndrome, plerixafor was administered for 6 months by subcutaneously injection (10 to 20 pg/kg bid for 6 months).
• EXAMPLE 7 Plerixafor inhibits RNA- viral infection-induced NETosis Five groups of C57B1/6 mice were allocated for this study. Infection with RNA virus was performed on 10 mice on day 0 by intranasal (IN) administration of PBS containing 50 plaque forming units (PFU).
• IN intranasal
• PFU plaque forming units
• mice were treated intranasally with 50 pi of PBS (group 1M- non infected).
• mice On day 1, infected mice were treated twice daily with water for injection (group 2M and 4M) or Plerixafor at 3 mg/kg/day (group 3M and 5M). During the study, mortality and morbidity observation and body weight measurements were performed.
• mice from group 1M, 2M and 3M were euthanized, and blood was collected for NETosis analysis in plasma (citrullinated histone H3 and DNA).
• mice from groups 1M, 2M and 3M were euthanized. Blood was collected by cardiac puncture after opening the thoracic cavity.
• Plasma samples were centrifuged for 15 minutes at 1500 g at 4°C for plasma separation. Plasma was harvested and transferred immediately to clean 1.5 ml Eppendorf tubes and stored at -70°C or in dry ice for shipment.
• Plasma samples were sent to external laboratory at Bordeaux Hospital U1034 unit for NETosis analysis.
• NETosis was evaluated by dosing the citrullinated histone H3 and DNA in plasma.
• citrullinated histone H3 dosing the technique is adapted from a method described by Thalin et al. (Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil cellular traps in human plasma. Immunol Res. 2017; 65: 706-712) with a partial used of the Cell death detection kit (Roche).
• the absorbance was read at 450nm (reference 620 nm).
• Citrullinated histone H3 a biomarker of neutrophil cellular trap
• Individual and group average citrullinated histone H3 levels are presented in Table 3 and Figure 3.
• Citrullinated histone H3 level non-infected mice plasma was detected at low concentration.
• the infection with RNA virus increased the level of this marker (2,124 ⁇ 0,563 for RNA virus infected group vs 0,113 ⁇ 0,008 for non-infected group).
• Plerixafor treatment at 3 mg/kg/day slightly decreased the level of citrullinated histone H3 (1,428 ⁇ 0,799 for RNA virus infected + Plerixafor 3 mg/kg/day vs 2,124 ⁇ 0,563 for RNA virus infected group).
• Table 3 Evaluation of citrullinated histone H3 in plasma (in absorbance at 450 nm)
• EXAMPLE 8 Plerixafor inhibits COVID-19 patients’ sera induced neutrophils’ migration
• Neutrophils were isolated from 6 patients of the COBRA cohort (COPD and asthma patient cohort). Neutrophils 5x 10 5 were pre-treated with plerixafor (25 pg/mL) for 15 min at 37°C or with HBSS vehicle. Cells were next plated in the upper compartment of a modified Boyden chamber for migration assay in 0.2 mL HBSS, containing 0.5% HSA medium. HBSS, 0.5% HSA or HBSS, 0.5% HSA+ sera (10% diluted) from severe COVID-19 patients were added to the bottom compartment of each well. The migration assay lasted 3hours, after which cells in the lower compartment were collected and stained with DAPI, anti-CD 16 and anti-CXCR4 antibodies. Cell identities and counts were analysed by FACS method.
• Neutrophils are isolated according to the method described by Hard, el al ( Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsiveness in chronic inflammatory lung diseases. J. Immunol. Baltim. Md 1950. 181:8053-8067). Neutrophils were freshly isolated from blood of COPD patients over isotonic Percoll density gradient. The neutrophils were recovered, washed twice with HBSS containing 20 mM HEPES and 0.1% (w/v) BSA and resuspended in HBSS, 20 mM HEPES, and 0.1% (w/v) BSA at a cell concentration of 5x 10 5 in 0,2 mL.
• Neutrophil migration was assessed using a Boy den chamber assay, using transwell inserts (pore size 8 pm). A total of 5x 10 5 neutrophils cells in 0.2 mL HBSS, containing 0.5% HSA were added to the upper compartment of each well. When indicated, neutrophils were pretreated for 15 min at 37°C with plerixafor an antagonist of CXCR4 (25 pg/mL). HBSS, 0.5% HSA and Sera (10% diluted) from severe COVID-19 patients was added to the bottom compartment of each well. After 3h, the content of bottom compartment was washed with 1 mL and lower chamber cell content was retrieved in a FACS tube (1 inferior chamber cell content per FACS tube) and process for DAPI staining was performed to exclude dying cells.
• FACS tubes were centrifuged 5 min, 4°C at 500 g. Fixation and permeabilization of cells were as follow: 0.3 mL of Cytofix/Cytoperm were added for 15 min (tube are protected from light). Next, 400 pL of permeabilization buffer was added and cells were processed for centrifugation for 5 min, 4°C, 400g. Supernatant was discarded, and cells were processed for antibody staining. Cells were incubated 40 min at 4°C with anti-CD 16 to account for all neutrophils, anti-CXCR4 to specifically account for CXCR4 expressing neutrophils.
• plerixafor 25 pg/mL was evaluated in the three neutrophils population, namely DAPI-CD16+, D API-CD 16+CXCR4- and D API-CD 16+CXCR4+.
• neutrophil migration from patients with stable COPD in response to 10% sera from with severe COVID in the presence of 25 pg/mL plerixafor (red circle symbols) or vehicle (black circle symbols).
• plerixafor had no significant effect on the migration of CXCR4 negative neutrophils (Fig. 5B). This is in keeping with the small percentage that DAPI-CD16+CXCR4+ represent (around 1%). In contrast, plerixafor strongly and reproducibly decreased the migration of CXCR4 positive neutrophils (Fig. 5C). Plerixafor decreased CXCR4+ neutrophils by 75.8% ⁇ 5.9% (Table 7).

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• 2020
  • 2020-05-07 EP EP20173595.8A patent/EP3906922A1/en not_active Withdrawn
• 2021
  • 2021-05-05 AU AU2021269090A patent/AU2021269090A1/en not_active Abandoned
  • 2021-05-05 EP EP21722910.3A patent/EP4146195A1/en not_active Withdrawn
  • 2021-05-05 JP JP2022567359A patent/JP2023539542A/ja active Pending
  • 2021-05-05 CA CA3177779A patent/CA3177779A1/en active Pending
  • 2021-05-05 KR KR1020227041380A patent/KR20240073720A/ko active Pending
  • 2021-05-05 CN CN202180033555.4A patent/CN116133657A/zh active Pending
  • 2021-05-05 US US17/922,865 patent/US20230157994A1/en active Pending
  • 2021-05-05 BR BR112022022578A patent/BR112022022578A2/pt not_active Application Discontinuation
  • 2021-05-05 WO PCT/EP2021/061919 patent/WO2021224356A1/en not_active Ceased

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