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WO2023172210A1 - Inhalateurs de dose mesurée de cannabidiol pour la protection contre un antigène covid et d'autres allergènes - Google Patents

Inhalateurs de dose mesurée de cannabidiol pour la protection contre un antigène covid et d'autres allergènes Download PDF

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Publication number
WO2023172210A1
WO2023172210A1 PCT/TH2023/050004 TH2023050004W WO2023172210A1 WO 2023172210 A1 WO2023172210 A1 WO 2023172210A1 TH 2023050004 W TH2023050004 W TH 2023050004W WO 2023172210 A1 WO2023172210 A1 WO 2023172210A1
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cbd
cannabidiol
antigen
mdi
covid
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Teerapol Srichana
Charisopon CHUNHACHAICHANA
Titpawan NAKPENG
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PRINCE OF SONGKLA UNIVERSITY
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/008—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy comprising drug dissolved or suspended in liquid propellant for inhalation via a pressurized metered dose inhaler [MDI]
    • 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
    • A61K31/658—Medicinal preparations containing organic active ingredients o-phenolic cannabinoids, e.g. cannabidiol, cannabigerolic acid, cannabichromene or tetrahydrocannabinol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof

Definitions

  • the present invention relates to formulations for inhalation of cannabidiol, particularly via the oral cavity.
  • SARS-CoV-2 is a member of the coronavirus family which can cause common colds and the more fatal Middle East respiratory syndrome (MERS) .
  • the SARS-CoV-2 is a positive- sense singlestranded RNA ( + ssRNA) virus with a single linear RNA segment.
  • the genome of CoV is the largest RNA genome (26.4-31.7 kilobases) of all known RNA viruses.
  • Each virion is from 50 to 200 nm in diameter and comprises four different structural proteins, namely S ( spike) , E (envelope), M (membrane) and N (nucleocapsid), where the N protein surrounds the RNA genome and the S, E, and M proteins form the viral envelope.
  • S protein is a glycoprotein that forms homo- trimeric spikes on the virion and is responsible for the ability of the virus to attach to and fuse with the membrane of the host cell. This is performed by engaging the cell surface receptor angiotensin- converting enzyme 2 (ACE- 2) that allows cell entry.
  • ACE- 2 cell surface receptor angiotensin- converting enzyme 2
  • SARS-CoV-2 is efficiently transmitted from person to person and therefore rapidly spreads across all continents. The transmission of the virus occurs via respiratory droplets from coughing, sneezing, and speaking and in indoor environments with air flow, which suggests the virus may be airborne as shown in Figure 1.
  • Cannabinoid is a class of chemical compounds that act on cannabinoid receptors, the active constituents of hemp or cannabis plants. In order to obtain high purity (98%) for pharmaceutical purposes it needs efficient purification steps. In addition, undesirable impurities like THC must be regulated to be less than 0.3% under the regulation of Thai FDA. CBD is a nonpsychoactive cannabinoid.
  • CB1 and CB2 Human cannabinoid system is well known and the most common receptors are CB1 and CB2. These protein receptors are embedded in the membrane of cells. These surface proteins are attached to another protein that determines the signaling activation or inhibition. CB1 receptors are primarily found in the brain and central nervous systems and to a lesser extent in the other tissues. The target activity involves motor activity, motor co-ordination, appetite, pain perception, short-term memory and thinking. CB2 receptors are mostly in the peripheral organs especially cells associated with the immune systems. CB2 targets on adipose tissue, skeletal and muscle, cardiovascular system, respiratory system, reproductive system and immune system. CBD is target for immune system especially in the respiratory.
  • SARS- CoV- 2 Inflammatory response in lung after coronavirus ( SARS- CoV- 2) infection. Lung susceptibility to SARS- CoV- 2 infection depends on viral spike proteins specificity for angiotensin- converting enzyme 2 (ACE2) receptors on alveolar epithelial cells. This interaction leads to hyper- inflammation sustained by cytokine storm, increase of pro- inflammatory Ml macrophages and T-helper cells, all associated in a vicious circle in which each event enhances the alteration of the other ones.
  • ACE2 angiotensin- converting enzyme 2
  • CBD may help to reduce cytokine storms and excessive lung inflammation in patients with COVID- 19 as shown in Figure 1.
  • Clinical reports indicate that a cytokine storm associated with acute respiratory distress syndrome (ARDS) is the leading cause of mortality in severe cases of CO VID-19.
  • ARDS acute respiratory distress syndrome
  • a cytokine storm causes tissue toxi cities affecting a wide variety of organs that lead to death.
  • Therapeutic approaches to manage a CO VID- 19 cytokine storm might improve survival rates and reduce mortality.
  • CBD attenuates ARDS by reducing the inflammatory cytokines, limiting damage to the lung, and improving the functional capacity of airways leading to improved oxygen levels.
  • Cannabidiol MDI and cannabinoid receptors a possible target in SARS-Co-V2 infection and other allergens.
  • Figure 2 The molecular structure of cannabidiol molecule and inter- molecular H- bonding between two CBD molecules.
  • FIG. 4 The thermograms of CBD together with the CBD reference standard.
  • Figure 6 The chromatographic system, it clearly indicates that the CBD was eluted at 3.66 min.
  • Figure 7 The experimental design to evaluate the potential of CBD- MDI to suppress cytokine production from macrophages.
  • A Cell line exposed to CBD-MDI 24 h prior to contact to allergen
  • B Cell line exposed to CBD-MDI 24 h, washed CBD-MDI out and challenged the cells with allergen
  • C Cell line in contact initially to allergens 24 h and later exposed to CBD-MDI.
  • A is the alveolar macrophage (NR8383) cell line and
  • B is the co- culture of alveolar macrophage (NR8383) cell line and human lung carcinoma (A549).
  • Aerosol delivery systems can be categorized into three major groups: pressurized metered dose (MDI) , dry powder inhaler, and nebulizer. Since CBD is highly oil soluble, it can be formulated as a metered dose inhaler. The delivered dose is very accurate and dose uniformity meets the USP standard. Targeting the right amount of CBD to the target organ, which is the alveoli, is feasible.
  • MDI pressurized metered dose
  • dry powder inhaler dry powder inhaler
  • nebulizer nebulizer
  • CBD is highly miscible in the solvent system.
  • the invention provides the formulations and methods for improved local lung delivery of cannabinoids and particularly CBD with proven values in terms of stability, size distribution, safety, and doses related to the activity at cellular levels.
  • the present invention has been exemplified for compositions comprising cannabidiol and resveratrol that are comparable in their physical, chemical, and biological properties. Resveratrol is able to reduce allergy symptoms in adults with seasonal allergies. This implies that this approach can be applied to other therapeutic agents, drugs, and food supplements that can improve solubility, stability, and efficacy. Formulations and methods of invention are further relevant to clinical applications which can improve stability, safety, and efficacy.
  • the present invention further provides liquid formulations essentially composed of a cosolvent, surfactant, lubricant, lipids, and pressurized gas specifically adapted to contain cannabinoid compositions in a single dosage form.
  • a cosolvent e.g., ethanol, sulfate, sulfate, sulfate, sulfate, sulfate, sulfate, sulfate, lipids, and pressurized gas specifically adapted to contain cannabinoid compositions in a single dosage form.
  • One prominent feature of the above formulation is that it is protected from light and ingress of oxygen from the environment. Upon actuation of the canister the content will be released and delivered to the respiratory airways.
  • Cannabidiol and resveratrol are both poorly water soluble; however, the present formulations and methods overcome these limitations in delivering the two compounds to the target organs, i.e., the lower airways and to the receptor sites. It is important to note that the oxidation, hydrolysis, and photolytic degradation are very low in these particular formulations, which help prolong the shelf life of the products.
  • Cannabidiol has an effect on the immunological and inflammatory cytokines and, therefore, has clinical use as shown in Figure 1.
  • the embodiment provides a method for oral inhalation of a cannabidiol composition.
  • the formulation is comprised of Cannabidiol, solvent or cosolvent, surfactant, lubricating agent, and compressed gas.
  • the composition may also contain optional components, such as flavoring agent, antioxidant agent and absorption enhancer.
  • Cannabidiol can be selected from a group of chemical compounds that includes cannabidiol and cannabinoids that can bind to cannabinoid receptors in the body. Therefore, in some instances, constituents, mixtures or combinations of cannabinoids are used artificially and which contain at least one of the above substances.
  • Solvent or cosolvent can be selected from alcohol or short chain alcohol or low alkyl chain esters.
  • Surfactant can be selected from the amphiphilic compounds were selected from negatively charged, positively charged, uncharged and switter ions. Surfactants can be sorbitan monooleates or mixtures of such compounds.
  • Lubricating agent can be selected from fatty acids such as oleic, myristic, stearic or polymers such as polyethylene glycol.
  • Compressed gas can be selected from hydrofluoroalkane such as HF Al 34a or HFA227.
  • the optional components can be selected from various sweeteners, antioxidants, preservatives, which are well known in this field of art can be used for these purposes.
  • the therapeutic agent can include the effective amount of cannabinoid, cannabidiol or resveratrol, precursors, or metabolites in a pharmaceutically acceptable carrier or excipient.
  • CBD-MDI cannabidiol metered dose inhaler
  • Such formulation upon administration, the formulation is propelled into a small droplets size of less than 3 microns, thereby increasing bioavailability of active substance in the lung.
  • the CBD-MDI has reasonably acceptable characteristics of inhalation dosage form.
  • the emitted doses of the formulation are close to theoretical dose of 250 pg.
  • the amount of absolute ethanol can be varied.
  • Table 2 shows the mass median aerodynamic diameter was about 2.37 microns with fine particle fraction about 45%.
  • MMAD Mass median aerodynamic diameter
  • ED emitted dose
  • FPF fine particle fraction
  • FPD fine particle dose
  • GSD geometric standard deviation
  • cannabidiol obtained from the biosynthesis and further purification, crystallized in pentane and analyzed by X-ray crystallography.
  • the crystallographic data is illustrated in Table 3.
  • the crystal structure of CBD showed a monoclinic space group P2i consist of 2 rings: cyclohexene and benzene ring.
  • the absolute configuration was confirmed as R,R.
  • the two CBD molecules can form H-binding as shown in Figure 1.
  • the inter-molecular hydrogen bonding of CBD represented at hydrogen atom and oxygen atom of hydroxyl group (H and O) in benzene ring.
  • cannabinoid encompasses the class of chemical compounds, cannabidiol, cannabiniods related compounds, acting with various affinities on the endogenous cannabinoids receptors.
  • the cannabinoid compositions, mixtures or combinations utilized according to the invention comprise one or more of the above.
  • methods of the invention pertain to oral inhalation of cannabinoid compositions comprising of cannabidiol or cannabidiol with resveratrol.
  • Figure 4 shows the thermograms of CBD together with the CBD reference standard.
  • the melting characteristic peak of CBD reference standard was around 64 °C and CBD sample showed melting point around 63.5 °C as shown below. The result indicates that CBD sample was pure and corresponded with the reference standard.
  • Figure 5 shows the FT-IR spectrum of CBD shows the characteristic bands of CBD at 3400 cm' 1 is hydrogen bonded O-H corresponding with crystallographic results of free O-H at 3650- 3600 cm' 1 ).
  • the complexation of CBD with cyclodextrin helps increasing the aqueous solubility. By inclusion complex, the wave number of each component was not altered.
  • Figure 6 shows the chromatographic system, it clearly indicates that the CBD was eluted at 3.66 min. The peak was symmetry without tailing. The purity of CBD powder is 99.0 ⁇ 0.12% in comparison with the reference standard by using HPLC assay method.
  • CBD is non-polar hydrophobic molecule that is insoluble in water (0.1 pg/mL).
  • the solubility of CBD in ethanol is 3.5 mg/mL and freely soluble in non-polar liquid like HFA-134a propellant.
  • the formulation development of CBD as inhaler was designed as a solution system by using ethanol as a solvent for product concentrate and other stabilizers (surfactant, lubricant, antioxidant) and enhancer and flavoring agent then it is able to completely miscible with HFA-134a propellant.
  • the alternative formulation was designed by partially dissolved CBD in minimal amount of ethanol but still completely dissolved CBD as a product concentrate. Other ingredients were added.
  • the formulation was completely dissolved and clear solution was obtained.
  • the propellant will quickly evaporate and only remaining CBD will enter and deposit to the respiratory tract. Small amount of ethanol was slowly evaporated and did not affect the performance of drug delivery to the airways.
  • CBD MDI CBD MDI
  • the example formulations were assayed to obtain the percent labelled amount. It was found 100.1% obtained from the model formulation.
  • the CBD uniformity delivered dose of the formulations was higher than 95% which is acceptable criteria in the USP that was within 80-120% of the expected amount of 250 pg per actuation.
  • the propellant When discharging the pressurized container, the propellant will evaporate and release the drug from the valve.
  • CBD sample was provided gratis by AVS Innovation (Bangkok, Thailand).
  • CBD standard was purchased from Department of Medical Sciences, Ministry of Public Health (Bangkok, Thailand).
  • HFA-134a 1,1,1,2-tetrafluoroethane
  • the alveolar macrophage (NR 8383, ATCC CRL-2192, MD, USA) a rat cell line, was cultured in F-12 Kaighn’s medium (Gibco®, USA) supplemented with 15% fetal bovine serum (FBS, Gibco®, USA), 100 U/mL penicillin/streptomycin (Gibco®, USA).
  • FBS fetal bovine serum
  • penicillin/streptomycin Gibco®, USA
  • the human lung adenocarcinoma cell line (A549, ATCC: CCL185, MD, USA) was cultured in Kaighn’s Modification of Ham’s F-12 Medium (F-12K, Gibco®, USA) containing 10% fetal bovine serum (FBS, Gibco®, USA) and antibiotics (100 U penicillin and 100 U/mL streptomycin, Gibco®, USA) under 5% CO2 at 37 °C. The media were changed every alternate day. When the cells reached confluence, they were harvested using 0.25% trypsin-EDTA (Gibco®, USA), followed by the addition of fresh culture medium to create a new single cell suspension for further testing.
  • SRBD Spike Receptor Binding Domain
  • S-RBD receptor binding domain
  • S- RBD SARS-CoV-2 spike
  • S- RBD residue 317-539
  • the coding sequence for the receptor binding domain (S-RBD) of SARS-CoV-2 spike (S- RBD, residue 317-539) was optimized for expression in mammalian cells from the sequence obtained from the reference strain (Wuhan-Hu-1) and cloned into pSecTag using restriction enzyme cloning (BsiWI and Xhol) in-frame with the IgK leader sequence at the N-terminus and the Myc-His tag at the C-terminus.
  • Recombinant tagged S-RBD protein is produced according to a previously published protocol (PMID: 32398876).
  • HEK 293T cells were maintained in OptiMEM with 10% fetal bovine serum supplement and transfected with pSecTag-SRBD using FuGENE transfection reagent according to the manufacturing’s protocol. The cell media were then changed to OptiMEM with no supplement at 6 h posttransfection. At 3 days post-transfection, supplement was harvested and conditioned with 4X binding buffer [1.2 M NaCl, 200 mM NaH2PO4, 40 mM imidazole] at the ratio 3: 1. Then, the conditioned supernatant was mixed with Ni-NTA agarose resin (Qiagen) for 1 h.
  • 4X binding buffer [1.2 M NaCl, 200 mM NaH2PO4, 40 mM imidazole]
  • the unbound proteins were washed off with the wash buffer [300 mM NaCl, 50 mM NaH2PO4, 20 mM imidazole].
  • Recombinant S-RBD was then elute with the elution buffer [300 mM NaCl, 50 mM NaH2PO4, 250 mM imidazole].
  • the eluted fractions were pooled, concentrated and buffer- exchanged into PBS [phosphate buffer saline solution] using 10 MWCO centrifugal filter unit.
  • the protein concentration was measured using the Bradford method (PMID: 942051).
  • Recombinant S- RBD was stored at -80°C in small aliquots unit use.
  • NR8383 and A549 cell lines were co-cultured, in the ratio 1 : 1, in same completed media (F-12 Kaighn’s medium supplemented with 15% FBS and 100 U/mL penicillin/streptomycin).
  • the 100 pL of 10 5 cells/mL of mixed cell lines were seeded in 96 well plate. The cells were allowed to grow until 70-80% confluence.
  • S-RBD, PM 2.5, nicotine or coal tar was used to stimulate lung cell inflammation, thus cytokine produced.
  • the concentration of S-RBD, PM 2.5, nicotine or coal tar was 10, 60, 1 and 40 pg/mL, respectively.
  • CBD-MDI was evaluated for its ability to suppress cytokine production from macrophage cell line in inflammation process induced by inflammatory stimulants.
  • Budesonide 50 pg/mL was used as a positive anti-inflammatory agent.
  • the experimental was designed to 3 groups as demonstrated in Figure 3.
  • the produced cytokine was assayed using ELISA technic as described below and the resulted cytokine level of all experimental were compared.
  • cytokines consisting of tumor necrosis factor-alpha (TNF- a), interleukin ip (IL-ip) and, interleukin-6 (IL-6) were determined using rat ELISA assay kit (R&D systems, MN, USA). Either TNF-a, IL-ip or IL-6 diluent (50 pl) was added to each well. Then, 50 pl of experimental cell supernatant was added to the well and incubated for 2 h at room temperature. Each well was washed with buffer solution for five times following by adding either a 100 pL of TNF-a, IL-ip or IL-6 conjugate to each well and incubated for another 2 h.
  • TNF-a tumor necrosis factor-alpha
  • IL-ip interleukin ip
  • IL-6 interleukin-6
  • each well was washed with a buffer (400 pL) for five times and 100 pL of substrate solution was added. The plates were incubated for 30 min at room temperature and 100 pL of stop solution was added. The reaction was recorded quantitatively at 450 nm based on a standard curve of TNF-a, IL-ip or IL-6.
  • NR8383 alveolar macrophage
  • NR 8383 co-cultured of alveolar macrophage
  • A549 human lung carcinoma
  • the toxicity of raw CBD was found at the concentration higher than 31. 25 pg/ mL. However, almost 100% cell viability was obtained with raw CBD concentration lower than 16.63 pg/mL. The obtained viability results of both single and co-cultured cells were in the similar pattern. However, the safety of CBD improved when it was formulated as MDI.
  • the 100% cell viability of CBD-MDI was with the concentration of 62.50 pg/mL for both NR8383 and cocultured of NR8383-A549. Whereas at the concentration of 62.50 pg/mL of CBD, less than 20% viability was observed for both NR8383 and co- cultured of NR8383- A549.
  • CBD-MDI is the clear liquid formulation of CBD dissolved in ethanol and HFA-134a.
  • 1 puff of CBD-MDI was sprayed into a well of 6- well plate and waited until the solvents completely evaporated.
  • Completed cell culture media 500 pL was added to produce 500 pg/ mL of CBD to give a final concentration of 250 pg/ mL in 96 well plate. After solubilizing and recrystallize, the crystal structure of CBD is expected to change and improve the safety profile of CBD.
  • CBD concentration in complete media cannot increase to higher than 125 pg/mL since this caused the cell survival less than 10%. Consequently, 50 pg/mL CBD-MDI was chosen based on safety principle to further evaluation of its potential to suppress the cytokine production from stimulated immune cells.
  • SAR-CoV-2 use angiotensin- converting enzyme 2 (ACE- 2) as the host receptor to enter into target cell.
  • SAR-CoV-2 receptor- binding domain (RBD) is the viral protein responsible to bind to the ACE 2 receptor.
  • the host immune system is the first line of defense against viral infection. The immune response resulting in excessive inflammation and caused lung damage.
  • CBD has been reported as a prophylactic treatment to reduce inflammation in a model of acute lung injury. Hence, the potential of CBD to alleviate the effects of cytokine produced from S-RBD stimulation was investigated in this work.
  • S-RBD was used as an antigen to stimulate alveolar macrophage to produce the response cytokine, in this study, including IL- ip , TNF-a and IL- 6.
  • the produced cytokine level was depicted in Figure 9.
  • NR8383 co- cultured of NR8383 with human lung adenocarcinoma cell, A549, provoked more responded cytokines than single NR8383 due to the synergist cascade binding of S-RBD to ACE- 2 receptor on A549. It may explain by the A549 may help or promote the attachment of S-RBD to both cells or enhance the transport into immune cells. Thus, higher responded degrees were obtained as the produced cytokine level was at 289. 10, 1007. 92 and 1572. 50 pg/ mL for IL- ip , TNF-a and IL-6, respectively.
  • the CBD-MDI formulation did not stimulate both NR 8383 cell and co-cultured NR8383-A549 to produce any inflammatory cytokines since the detected cytokine level was very low in the similar level to untreated control.
  • 3 different experimental groups were done as described in example 1. The results of single immune cells NR8383 and co- culture NR8383 with A549 are relatively similar.
  • Treatment A or prevention condition also demonstrated the potential of CBD-MDI to reduce all tested cytokines from the S- RBD stimulated tested cells in the lesser degree compared to treatment C. This reminded that the CBD-MDI formulation is effective in protection the cells from S-RBD. This suggested that the used of CBD-MDI to relieve the effect of inflammatory cytokine induced by S-RBD can be for treatment and prevention.
  • immune NR8383 cells or co-culture cells incubated with CBD formulation 24 h then washed out followed by the addition of S-RBD (treatment B: Binding affinity)
  • the wash out can remove the CBD from the cell surface resulting in only small activity left on immune cells.
  • the IL-ip, TNF-a, and IL-6 level was 150- 240 pg/ mL, 920- 930 pg/ mL and 1370- 1410 pg/ mL, respectively demonstrated less degree reduction of cytokine levels.
  • the binding the CBD might be surface binding on the cell surface and reversible/ weak binding. In other words, CBD exerts the activity only when it is available to binding to the cell surface.
  • CBD-MDI formulation to protect the cell from the action of S-RBD was compared to corticosteroid budesonide as shown in Figure 10.
  • Budesonide a glucocorticosteroid
  • budesonide of 50 mg/mL was used as a positive control in this study and the same 3 experimental treatments were applied.
  • the budesonide gave a relative similar result to CBD-MDI formulation although with different mode of action. The results suggested that budesonide gave the greatest efficacy when it was treated after 24 h S-RBD stimulation.
  • CBD-MDI (50 mg/ mL) was safe to respiratory cells thus this concentration was used to examine its potential to combat with bacterial or virus antigen induced immunological response. CBD-MDI did not provoke the immune cells to produce responded cytokine. CBD exhibited the potential to relieve cytokines production from both bacterial antigen (lipopolysaccharide, LPS) and viral antigen (SAR-Co-V 2 receptor binding protein, S-RBD) stimulated immune cells. The greatest efficacy of CBD-MDI was obtained when it was used as a treatment condition. The treatment condition refers to the CBD-MDI is used after the cell exposure to the viral or bacterial antigens. Also, CBD-MDI can be used for prevention but resulting in a lesser degree of cell protection. In addition, the potential of CBD-MDI to alleviate the immunological inflammation is comparable to budesonide at the equal dose although different mode of action.
  • CBD-MDI formulation 50 mg/mL was examined its potential to alleviate the cytokine production when the macrophage cell was stimulated with LPS.
  • CBD-MDI treated cell showed the very low cytokine level as similar to the untreated cell (control) . This suggested that CBD- MDI did not provoke an immunological response from alveolar macrophage.
  • the CBD-MDI treatments to the LPS stimulated alveolar macrophage were designed to 3 different experimental modes treatment A which CBD-MDI treated to the cells prior challenged with LPS represented a prevention condition.
  • Treatment B was designed to study the reversible/ irreversible binding of CBD to the CB2 receptor on immune cells and determined the remaining anti- inflammation activity after CBD was washed out and the cells later challenged with LPS.
  • treatment C represented a treatment mode which CBD-MDI treated to the cells after 24 h stimulated with LPS.
  • Column A and column C of Figure 11 demonstrated that CBD- MDI (50 pg/ mL) is a potential formulation used to alleviate the inflammation from cytokine production, both for prevention and treatment purposes, when the immune cells were stimulated with LPS.
  • the results of treatment B indicated that the binding affinity of CBD-MDI to the receptor is reversible since washing out of CBD-MDI from the well caused no CBD activity remaining to diminish the cytokine level produced from LPS- stimulated macrophage. This observed from the very high level of the produced cytokine resemble to a control LPS- stimulated macrophage. This may suggest that the binding CBD receptor is reversible binding/ not strong binding. However, study of binding affinity of CBD to the receptor requires further investigation.
  • the lung is directly exposed to environmental antigens including pathogens, allergens and toxins, such as tobacco smoke.
  • Nicotine, coal tar and particulate matter of 2. 5 mm (PM 2. 5) are hazardous components from cigarette smoke.
  • Cigarette smoke promotes inflammation by inducing the production of pro-inflammatory cytokines, such a TNF-a, IL- ip, IL-6, IL-8 and granulocytemacrophage colony- stimulating factor (GM-CSF) , and increasing the accumulation of immune cells in the airways. Nicotine, coal tar and PM 2.5, the tested samples in this study, may stimulate immunological response thus they were called as allergens.
  • Nicotine at the tested concentration (1 pg/mL) did not provoke the alveolar macrophage to produce all tested inflammatory cytokines including IL- ip, TNF-a, IL- 6.
  • Alveolar macrophage challenged with coal tar produced a significantly increased of TNF- a and IL- 6 compared to untreated control.
  • coal tar did not activate the immune cell to produce IL- lb.
  • the highest immunological response was from particulate matter of 2. 5 pm (P. M. 2. 5) which produced cytokine levels to 300.00, 130.75 and 206.17 pg/mL for IL-ip, TNF-a and IL-6, respectively. This suggested that P.M 2.5 proceeds high inflammatory allergens.
  • the CBD -MDI was examined as a potential formulation to reduce the inflammation induced by tobacco smoke allergens. Nicotine was excluded from these discussions since it did not activate the cell to produce the cytokines. IL- 1b was produced only when alveolar macrophage responded to P. M. 2. 5. Decreasing of IL- ip level was obtained when treated with CBD- MDI for all Exp. The treatment C gave the highest reduction degree of IL-ip. This accompanies with the results of TNF-a and IL-6 that treatment C demonstrated the highest CBD activity.
  • Treatment A was designed as a treatment that inflammation had induced before challenged with CBD- MDI.
  • Prevention condition was for treatment B which CBD- MDI was treated to the cells before allergens added.
  • Treatment C is designed to give the allergen simultaneously with CBD. The strongest CBD activity was attained with treatment C.
  • Alveolar macrophage represent the most abundant immune cell type in the healthy airspaces. Shortly after AMs exposure to P.M. 2.5 or coal tar, the augmented cytokines were produced and the lung inflammation initiated. Giving P.M. 2.5 or coal tar concurrently with CBD-MDI may cause competitive binding to the AMs cell surface between two challenged substances. However, binding of CBD-MDI to the cell surface did not provoke the immunological response, thus reducing of the produced cytokine level compared to stimulation by allergen alone.
  • CBD binding to CB2 receptor on immune cell also exert their anti-inflammatory activity. From dual actions of CBD result the highest activity of CBD to reduce cytokine level.
  • the used of CBD-MDI as a treatment mode is better than protection mode. This may due to CBD binding to CB2 receptor with low affinity thus the antiinflammation activity is not too high.
  • treatment condition Treatment A
  • the immediately anti- inflammation activity of CBD binding to CB2 receptor occurred after it was challenged to the already inflamed cells.
  • the treatment mode gives high efficiency to control cell inflammation better than prevention mode.

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Abstract

La présente divulgation concerne des inhalateurs de dose mesurée de cannabidiol pour la protection contre un antigène Covid et d'autres allergènes. La formulation comprend du cannabidiol, un solvant ou un cosolvant, un tensioactif, un agent lubrifiant et un gaz comprimé. La composition peut également contenir des constituants facultatifs, tels qu'un agent aromatisant, un agent antioxydant et un agent améliorant l'absorption.
PCT/TH2023/050004 2022-03-08 2023-03-08 Inhalateurs de dose mesurée de cannabidiol pour la protection contre un antigène covid et d'autres allergènes Ceased WO2023172210A1 (fr)

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TH2201001422 2022-03-08

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008019146A2 (fr) * 2006-08-04 2008-02-14 Insys Therapeutics Inc. Formulations aqueuses de dronabinol
WO2019021005A1 (fr) * 2017-07-28 2019-01-31 Mexichem Fluor S.A. De C.V. Composition pharmaceutique contenant un cannabinoïde
US20200253922A1 (en) * 2019-02-13 2020-08-13 Molecular Infusions, Llc Methods for non-irritating pulmonary administration of cannabinoids using soft mist inhalers

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008019146A2 (fr) * 2006-08-04 2008-02-14 Insys Therapeutics Inc. Formulations aqueuses de dronabinol
WO2019021005A1 (fr) * 2017-07-28 2019-01-31 Mexichem Fluor S.A. De C.V. Composition pharmaceutique contenant un cannabinoïde
US20200253922A1 (en) * 2019-02-13 2020-08-13 Molecular Infusions, Llc Methods for non-irritating pulmonary administration of cannabinoids using soft mist inhalers

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