Therapeutic nanoparticles encapsulating terpenoids and / or cannabinoids

Abstract

To provide novel therapeutic compositions for systemic administration (e.g., oral administration) and long-term release of terpenoids and cannabinoids, and methods of making and using the therapeutic compositions.SOLUTION: The present invention provides a terpenoid-encapsulating PLGA nanoparticle, comprising a PLGA nanoparticle composed of copolymer poly(lactic-co-glycolic acid) and a first terpenoid encapsulated in the PLGA nanoparticle. The nanoparticle further encapsulates a second compound composed of (i) a cannabinoid or (ii) a second terpenoid other than the first terpenoid.SELECTED DRAWING: None

Description

[Technical field]

[0001] Technical Field The present invention relates to therapeutic nanoparticles encapsulating terpenoids and / or cannabinoids.

[0002] cross references This application claims the benefit of U.S. Provisional Application No. 62 / 757,660, filed November 8, 2018, and No. 62 / 897,235, filed September 6, 2019, the contents of each of which are incorporated herein by reference. [Background technology]

[0003] Channels of the transient receptor superfamily (TRP), e.g., TRPV1, TRPM8, and TRPA1, are nonselective cation channels that conduct calcium and sodium to a range of cell types in mammals. They are present on sensory neurons and were initially identified as having a role in nociception by their responsiveness at the molecular level to nociceptive secondary plant metabolites (e.g., capsaicin) and to compounds that are otherwise pungent and mimic a burning or cooling sensation (e.g., allicin, cinnamaldehyde, menthol).

[0004] Due to their role in nociception, TRP channels have been identified as targets for treating pain disorders. Both antagonism and agonism of TRP channels have been utilized for pain management. For example, TRPV1 antagonists have utility in acute analgesia. For chronic pain management, TRPV1 agonists are typically used. This latter strategy exploits the fact that continued TRPV1 receptor agonism causes desensitization (receptor internalization, degradation, and recycling) at the cell surface. Prolonged agonism of TRPV1, in turn, leads to calcium and sodium cation overload of TRPV1-containing sensory neurons, leading to cell death.

[0005] In fact, the use of TRPV1 agonists to cause desensitization involves the repeated, time-consuming topical application of high levels of the well-known TRPV1 agonist, capsaicin, to the affected area. This therapeutic approach has the advantages of efficacy and low cost. However, it also has weaknesses.

[0006] First, high affinity and high specificity TRPV1 agonists only target TRPV1-containing nociceptors, leaving other sensory neurons and TRP channels involved in pain intact.Second, the use of high affinity and high specificity TRPV1 agonists, such as capsaicin, causes high levels of discomfort during initial treatment, in the period before desensitization.It is for this reason that postherpetic pain cannot currently be addressed using TRPV1-mediated desensitization, due to the highly irritating nature of the treatment on sensitive areas, such as gastric mucosa and genital tract mucosa.Third, capsaicin-mediated desensitization treatment is limited to topical use, and visceral pain, headache and certain musculoskeletal pain disorders are not addressed by this treatment.

[0007] Therefore, there is a need for therapeutic TRPV1 ligands, such as TRPV1 agonists, that have lower affinity than capsaicin and can be administered by mouth for systemic relief. Such lower affinity ligands should cause pain reduction during desensitization, thereby allowing for local treatment of sensitive body areas. There is a need for TRPV1 ligands with broader target specificity that can target multiple types of TRP-bearing nociceptors, thereby improving the degree of tissue desensitization. Furthermore, there is a need for pharmaceutical compositions that contain such TRPV1 ligands that are suitable for systemic administration, in addition to the local application and long-term release of such TRPV1 ligands to induce chronic downregulation of TRPV1. Oral administration is highly desirable for pain medication for systemic use.

[0008] These new oral drug therapies are also useful for treating various diseases associated with TRPV1 other than pain. While TRP channels were first shown to be involved in pain and nociception, they are now known to have a variety of other physiological roles, suggesting that they may be targets for the treatment of other diseases. For example, TRP channels have been identified as targets for the treatment of cardiovascular disease, and targeted pharmacological inhibition of TRPV1 has been shown to significantly reduce cardiac hypertrophy in mouse models. See U.S. Patent No. 9,084,786. Chronic downregulation of TRPV1 levels by receptor desensitization using TRPV1 agonists is therefore expected to protect and potentially rescue cardiac hypertrophy and its associated symptoms and prognosis (cardiac remodeling, cardiac fibrosis, apoptosis, hypertension or heart failure) as well.

[0009] However, there are currently no oral or other pharmacological compositions of TRP agonists suitable for systemic administration and chronic downregulation of TRPs in visceral organs, and therefore there is a need to develop such an approach in a manner similar to the chronic pain approaches described above.

[0010] Creating nanoparticles is an effective way to modify the surface properties of active molecules to make them more bioavailable and to modify their release kinetics systemically. Oral nanoparticles can be created that provide effective time-release formulations of various TRP channel modulators, such as terpenoids and cannabinoids. These oral formulations and pharmacological compositions of TRP ligands provide new and more effective ways to treat various diseases associated with TRP channels, including pain disorders and cardiovascular diseases. Summary of the Invention [Problem to be solved by the invention]

[0011] Applicant has recently demonstrated that myrcene is a new TRPV1 agonist that causes TRPV1 desensitization after prolonged exposure.Applicant has further demonstrated that other terpenoids and cannabinoids, including those that do not demonstrate significant TRPV1 agonist activity by themselves, act in combination to increase the efficacy of myrcene.Furthermore, multiple TRP channels in addition to TRPV1 have been identified as targets for myrcene, suggesting that the efficacy of myrcene likely extends beyond TRPV1 to other nociceptive neurons whose primary pain conduction channels are distinct TRP receptors.These findings are described in PCT application PCT / US2018 / 033956, which is incorporated herein by reference in its entirety.

[0012] Emerging findings suggest that myrcene and other terpenoids and cannabinoids may be alternative therapeutic TRP receptor ligands that affect multiple TRP channels in addition to TRPV1, induce less discomfort than capsaicin during initial treatment, and may potentially be suitable for systemic administration for chronic effects. See PCT / US2018 / 033956. Further development of pharmaceutical compositions containing myrcene and other terpenoids and cannabinoids, particularly those that allow for systemic administration and chronic and long-term release of myrcene, other terpenoids or cannabinoids, is needed for the therapeutic use of new TRPV1 agonists and agonist mixtures. This is particularly important because terpenoids and cannabinoids are highly lipophilic, have low boiling points, and are volatile, thus limiting the administration of terpenoids and cannabinoids to fast-release drug therapy or natural product applications.

[0013] The present invention addresses the need to stabilize terpenoids and cannabinoids and make them bioavailable in a sustained release manner by providing novel therapeutic compositions for systemic administration (e.g., oral administration) and extended release of terpenoids and cannabinoids, and methods of making and using the therapeutic compositions. In particular, the present invention uses PLGA nanoparticles encapsulating terpenoids and cannabinoids to deliver the terpenoids and cannabinoids to a mammalian subject. Additionally, the present disclosure provides therapeutic compositions containing multiple terpenoids or cannabinoids. The therapeutic compositions can contain multiple populations of nanoparticles, each population encapsulating an individual terpenoid or cannabinoid, or a population of nanoparticles, each nanoparticle encapsulating multiple terpenoids or cannabinoids. The terpenoids or cannabinoids encapsulated in the nanoparticles can be synthetic compounds or compounds isolated from chemical or natural mixtures. In some embodiments, the compounds are isolated from Cannabis sativa extracts. In some embodiments, delta-9 tetrahydrocannabinol is additionally or separately encapsulated in the nanoparticles. [Means for solving the problem]

[0014] Thus, in a first aspect, provided herein is a terpenoid-encapsulated PLGA nanoparticle comprising a PLGA nanoparticle and a first terpenoid encapsulated in the PLGA nanoparticle.

[0015] In some embodiments, the PLGA nanoparticles comprise a PLGA copolymer, and the weight ratio of the first terpenoid to the PLGA copolymer is between 1:50 and 1:10. In one embodiment, the weight ratio of the first terpenoid to the PLGA copolymer is about 1:14.

[0016] In some embodiments, the first terpenoid is selected from the group consisting of myrcene, β-caryophyllene, and nerolidol. In some embodiments, the first terpenoid is myrcene, and the weight ratio of the first terpenoid to the PLGA copolymer is about 1:22. In some embodiments, the first terpenoid is β-caryophyllene, and the weight ratio of the first terpenoid to the PLGA copolymer is between 1:5 and 1:7. In some embodiments, the first terpenoid is nerolidol, and the weight ratio of the first terpenoid to the PLGA copolymer is between 1:5 and 1:7.

[0017] In some embodiments, the PLGA nanoparticles have a surface modified with PEG, the molecular weight of which may range from 2,000 to 20,000 Da.

[0018] In some embodiments, the nanoparticles further encapsulate at least a second compound, the second encapsulated compound being (i) a cannabinoid or (ii) a second terpenoid other than the first terpenoid. In some embodiments, the second encapsulated compound is cannabigerolic acid (CBGA). In some embodiments, the second encapsulated compound is cannabidiol (CBD). In some embodiments, the second encapsulated compound is cannabinol (CBN). In some embodiments, the second encapsulated compound is cannabidivarin (CBDV). In some embodiments, the second encapsulated compound is cannabichromene (CBC). In some embodiments, the second encapsulated compound is cannabidiolic acid (CBDA). In some embodiments, the second encapsulated compound is cannabigerol (CBG). In some embodiments, the second encapsulated compound is myrcene, β-caryophyllene, or nerolidol. In some embodiments, the second encapsulated compound is limonene. In some embodiments, the second encapsulated compound is phytol. In some embodiments, the second encapsulation compound is pinene. In some embodiments, the second encapsulation compound is linalool.

[0019] In some embodiments, the PLGA nanoparticles have an average diameter between 200 and 350 nm.

[0020] In some embodiments, the PLGA nanoparticles comprise a PLGA copolymer having a ratio of lactic acid to glycolic acid of about 10-90% lactic acid to about 90-10% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

[0021] In another aspect, the present disclosure provides a pharmaceutical composition comprising a first population of terpenoid-encapsulated PLGA nanoparticles as described herein and a pharma- ceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition further comprises trehalose. In some embodiments, the composition is lyophilized. In some embodiments, the composition further comprises a second population of PLGA nanoparticles, the second population of nanoparticles encapsulating a third compound, the third compound being a cannabinoid or terpenoid different from the cannabinoid and terpenoid encapsulated in the first population of nanoparticles.

[0022] In some embodiments, the third encapsulation compound is cannabigerolic acid (CBGA). In some embodiments, the third encapsulation compound is cannabidiol (CBD). In some embodiments, the third encapsulation compound is cannabinol (CBN). In some embodiments, the third encapsulation compound is cannabidivarin (CBDV). In some embodiments, the third encapsulation compound is cannabichromene (CBC). In some embodiments, the third encapsulation compound is cannabidiolic acid (CBDA). In some embodiments, the third encapsulation compound is cannabigerol (CBG). In some embodiments, the third encapsulation compound is myrcene, β-caryophyllene, or nerolidol. In some embodiments, the third encapsulation compound is limonene. In some embodiments, the third encapsulation compound is phytol. In some embodiments, the third encapsulation compound is pinene. In some embodiments, the third encapsulation compound is linalool.

[0023] In some embodiments, the composition further comprises a third population of PLGA nanoparticles, the third population of nanoparticles encapsulating a fourth compound, the fourth compound being a different cannabinoid or terpenoid than the cannabinoid and terpenoid encapsulated in the first population of nanoparticles or the second population of nanoparticles. In some embodiments, the composition further comprises a fourth population of PLGA nanoparticles, the fourth population of nanoparticles encapsulating a fifth compound, the fifth compound being a different cannabinoid or terpenoid than the cannabinoid and terpenoid encapsulated in the first population of nanoparticles, the second population of nanoparticles, or the third population of nanoparticles.

[0024] In yet another aspect, the disclosure provides a method for obtaining terpenoid-encapsulated PLGA nanoparticles, the method comprising the steps of: (a) providing an organic solution comprising a terpenoid, a PLGA copolymer and a solvent, and an aqueous solution comprising a surfactant; (b) emulsifying the two solutions to form a suspension of terpenoid-encapsulated PLGA nanoparticles; (c) evaporating the solvent from the emulsion; and (d) obtaining terpenoid-encapsulated PLGA nanoparticles.

[0025] In some embodiments, the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1:5 to about 1:1. In some embodiments, the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1:5. In some embodiments, the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1:4. In some embodiments, the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1:3. In some embodiments, the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1:2. In some embodiments, the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1:1.

[0026] In some embodiments, the incorporation efficiency of the first terpenoid in step (b) is between about 4% and about 10%. In some embodiments, the incorporation efficiency is at least 4%. In some embodiments, the incorporation efficiency is at least 5%. In some embodiments, the incorporation efficiency is at least 6%. In some embodiments, the incorporation efficiency is at least 7%. In some embodiments, the incorporation efficiency is at least 8%. In some embodiments, the incorporation efficiency is at least 9%. In some embodiments, the incorporation efficiency is at least 10%.

[0027] In some embodiments, the average weight ratio of the encapsulated terpenoid to the PLGA copolymer in the terpenoid-encapsulated PLGA nanoparticles of step (d) is between about 1:50 and about 1:10. In some embodiments, the average weight ratio of the encapsulated terpenoid to the PLGA copolymer in the terpenoid-encapsulated PLGA nanoparticles is at least about 1:50. In some embodiments, the average weight ratio of the encapsulated terpenoid to the PLGA copolymer in the terpenoid-encapsulated PLGA nanoparticles is at least about 1:40. In some embodiments, the average weight ratio of the encapsulated terpenoid to the PLGA copolymer in the terpenoid-encapsulated PLGA nanoparticles is at least about 1:30. In some embodiments, the average weight ratio of the encapsulated terpenoid to the PLGA copolymer in the terpenoid-encapsulated PLGA nanoparticles is at least about 1:20. In some embodiments, the average weight ratio of the encapsulated terpenoid to the PLGA copolymer in the terpenoid-encapsulated PLGA nanoparticles is at least about 1:10.

[0028] In some embodiments, the PLGA copolymer has a ratio of lactic acid to glycolic acid that is about 10-90% lactic acid to about 90-10% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

[0029] In some embodiments, the solution of step (a) further comprises at least one cannabinoid or terpenoid other than the first terpenoid. In some embodiments, the at least one cannabinoid or terpenoid is selected from the group consisting of cannabidiol (CBD), cannabinol (CBN), cannabidivarin (CBDV), cannabichromene (CBC), cannabidiolic acid (CBDA) and cannabigerol (CBG). In some embodiments, the at least one cannabinoid or terpenoid is selected from the group consisting of myrcene, β-caryophyllene, nerolidol, phytol, limonene, linalool and pinene.

[0030] In some embodiments, the solvent is acetone, dichloromethane, or ethyl acetate. In some embodiments, the surfactant is polyethylene glycol, poloxamer, or polyvinyl alcohol (PVA). In some embodiments, the surfactant is polyvinyl alcohol (PVA).

[0031] In some embodiments, the emulsifying step comprises homogenization or sonication. In some embodiments, the emulsifying step is homogenization. In some embodiments, the homogenization is performed at 20,000 to 30,000 rpm. In some embodiments, the homogenization is performed at 24,000 rpm. In some embodiments, the solution is homogenized for 30 seconds to 10 minutes. In some embodiments, the solution is homogenized for 1 minute.

[0032] In some embodiments, evaporating the solvent comprises at least one of stirring the solvent, applying a gas flow, applying heat, maintaining a cold temperature of 10° C., or creating a vacuum. In some embodiments, evaporating the solvent comprises stirring the suspension at room temperature. In some embodiments, the suspension is stirred for between 5 minutes and 120 minutes to evaporate the solvent. In some embodiments, the suspension is stirred for 60 minutes.

[0033] In some embodiments, the step of obtaining the terpenoid-encapsulated PLGA nanoparticles comprises centrifugation, filtration, or centrifugation and filtration. In some embodiments, the step of obtaining comprises centrifugation. In some embodiments, the centrifugation is between 2,000 x g and 15,000 x g. In some embodiments, the centrifugation is at 4,000 x g.

[0034] In some embodiments, the method further comprises the subsequent step of adding a cryoprotectant to the terpenoid-encapsulated PLGA nanoparticles. In some embodiments, the cryoprotectant is trehalose. In some embodiments, the cryoprotectant is added in an amount of 1-10% (w / v) of the terpenoid-encapsulated PLGA nanoparticles. In some embodiments, the cryoprotectant is added in an amount of 5% (w / v) of the terpenoid-encapsulated PLGA nanoparticles.

[0035] In some embodiments, the method further comprises lyophilizing the terpenoid-encapsulated PLGA nanoparticles.

[0036] In one aspect, the present disclosure provides terpenoid-encapsulated PLGA nanoparticles obtainable by the methods described herein.

[0037] In another aspect, the present disclosure provides a method for causing TRPV1 desensitization in a cell of a mammalian subject, comprising administering to the mammalian subject a pharmaceutical composition as described herein in an amount, route of administration, and time sufficient to cause TRPV1 desensitization in a cell in the mammalian subject. In some embodiments, the cell is a nociceptor. In some embodiments, the nociceptor is a peripheral nociceptor. In some embodiments, the nociceptor is a visceral nociceptor.

[0038] In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered topically. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered by inhalation.

[0039] In one aspect, the disclosure provides a method of treating pain in a mammalian subject, comprising administering to the subject a pharmaceutical composition described herein in an amount, by a route of administration and for a time sufficient to cause TRPV1 desensitization in nociceptors in the subject.

[0040] In some embodiments, the nociceptor is a peripheral nociceptor and the pharmaceutical composition is administered locally, hi some embodiments, the nociceptor is a visceral nociceptor and the pharmaceutical composition is administered systemically.

[0041] In some embodiments, the pain is neuropathic pain. In some embodiments, the neuropathic pain is diabetic peripheral neuropathic pain. In some embodiments, the pain is post-herpetic neuralgia.

[0042] In some embodiments, the pharmaceutical composition is administered at least once a day for at least three days. In some embodiments, the pharmaceutical composition is administered at least once a day for at least five days. In some embodiments, the pharmaceutical composition is administered at least once a day for at least seven days.

[0043] In some embodiments, the pharmaceutical composition is administered at a dose, route and schedule sufficient to maintain levels of the first terpenoid at the nociceptor for at least 3 days that are effective to desensitize TRPV1 in the nociceptor. In some embodiments, the pharmaceutical composition is administered at a dose, route and schedule sufficient to maintain levels of the first terpenoid at the nociceptor for at least 5 days that are effective to desensitize TRPV1 in the nociceptor. In some embodiments, the pharmaceutical composition is administered at a dose, route and schedule sufficient to maintain levels of the first terpenoid at the nociceptor for at least 7 days that are effective to desensitize TRPV1 in the nociceptor.

[0044] In one aspect, the disclosure provides a method of treating cardiac hypertrophy in a mammalian subject, the method comprising administering to the subject an anti-hypertrophy effective amount of a pharmaceutical composition described herein. In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered by inhalation. In some embodiments, the pharmaceutical composition is administered orally.

[0045] In one aspect, the disclosure provides a method for the preventive treatment of cardiac hypertrophy in a mammalian subject, comprising administering to a subject at risk for cardiac hypertrophy an antihypertrophic effective amount of a pharmaceutical composition described herein.

[0046] In one aspect, the present disclosure provides a method for treating overactive bladder in a mammalian subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered by bladder irrigation.

[0047] In one aspect, the disclosure provides a method of treating refractory chronic cough in a mammalian subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered by inhalation.

[0048] In another aspect, the present disclosure provides a cannabinoid-encapsulated PLGA nanoparticle comprising a PLGA nanoparticle and a first cannabinoid encapsulated in the PLGA nanoparticle.

[0049] In some embodiments, the PLGA nanoparticles comprise a PLGA copolymer, and the weight ratio of the first cannabinoid to the PLGA copolymer is between 1:50 and 1:4. In some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer is between 1:25 and 1:5. In some embodiments, the first cannabinoid is selected from the group consisting of cannabidiol, cannabidivarin, cannabinol, cannabigerol, and cannabichromene.

[0050] In some embodiments, the first cannabinoid is cannabidiol and the weight ratio of the first cannabinoid to the PLGA copolymer is about 1:14. In some embodiments, the first cannabinoid is cannabidiol and the weight ratio of the first cannabinoid to the PLGA copolymer is between 1:5 and 1:7. In some embodiments, the first cannabinoid is cannabidiol and the weight ratio of the first cannabinoid to the PLGA copolymer is between 1:5 and 1:7.

[0051] In some embodiments, the nanoparticles further encapsulate at least a second compound, the second encapsulated compound being (i) a terpenoid, or (ii) a second cannabinoid other than the first cannabinoid. In some embodiments, the second encapsulated compound is cannabigerolic acid (CBGA). In some embodiments, the second encapsulated compound is cannabidivarin (CBV). In some embodiments, the second encapsulated compound is cannabinol (CBN). In some embodiments, the second encapsulated compound is cannabidivarin (CBDV). In some embodiments, the second encapsulated compound is cannabichromene (CBC). In some embodiments, the second encapsulated compound is cannabidiolic acid (CBDA). In some embodiments, the second encapsulated compound is cannabigerol (CBG). In some embodiments, the second encapsulated compound is myrcene, β-caryophyllene, or nerolidol. In some embodiments, the second encapsulated compound is limonene. In some embodiments, the second encapsulated compound is phytol. In some embodiments, the second encapsulation compound is pinene. In some embodiments, the second encapsulation compound is linalool. In some embodiments, the second encapsulation compound is myrcene.

[0052] In some embodiments, the PLGA nanoparticles have an average diameter between 200-350 nm. In some embodiments, the PLGA nanoparticles comprise a PLGA copolymer having a ratio of lactic acid to glycolic acid of about 10-90% lactic acid to about 90-10% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

[0053] One aspect of the present disclosure provides a pharmaceutical composition comprising a first population of cannabinoid-encapsulated PLGA nanoparticles provided herein and a pharma- ceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition further comprises trehalose.

[0054] In some embodiments, the composition is lyophilized. In some embodiments, the composition further comprises a second population of PLGA nanoparticles, the second population of nanoparticles encapsulating a third compound, the third compound being a terpenoid or cannabinoid different from the terpenoid and cannabinoid encapsulated in the first population of nanoparticles. In some embodiments, the third encapsulated compound is cannabigerolic acid (CBGA). In some embodiments, the third encapsulated compound is cannabidiol (CBD). In some embodiments, the third encapsulated compound is cannabinol (CBN). In some embodiments, the third encapsulated compound is cannabidivarin (CBDV). In some embodiments, the third encapsulated compound is cannabichromene (CBC). In some embodiments, the third encapsulated compound is cannabidiolic acid (CBDA). In some embodiments, the third encapsulated compound is cannabigerol (CBG). In some embodiments, the third encapsulated compound is myrcene, β-caryophyllene, or nerolidol. In some embodiments, the third encapsulation compound is limonene.In some embodiments, the third encapsulation compound is phytol.In some embodiments, the third encapsulation compound is pinene.In some embodiments, the third encapsulation compound is linalool.In some embodiments, the third encapsulation compound is myrcene.

[0055] In some embodiments, the composition further comprises a third population of PLGA nanoparticles, the third population of nanoparticles encapsulating a fourth compound, the fourth compound being a different cannabinoid or terpenoid than the cannabinoid and terpenoid encapsulated in the first population of nanoparticles or the second population of nanoparticles. In some embodiments, the composition further comprises a fourth population of PLGA nanoparticles, the fourth population of nanoparticles encapsulating a fifth compound, the fifth compound being a different cannabinoid or terpenoid than the cannabinoid and terpenoid encapsulated in the first population of nanoparticles, the second population of nanoparticles, or the third population of nanoparticles.

[0056] Another aspect of the present disclosure provides a method for obtaining cannabinoid-encapsulated PLGA nanoparticles, the method comprising the steps of: (a) providing an organic solution comprising a terpenoid, a PLGA copolymer and a solvent, and an aqueous solution comprising a surfactant; (b) emulsifying the two solutions to form a suspension of cannabinoid-encapsulated PLGA nanoparticles; (c) evaporating the solvent from the emulsion; and (d) obtaining cannabinoid-encapsulated PLGA nanoparticles.

[0057] In some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1:5 to about 1:1. In some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1:5. In some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1:4. In some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1:3. In some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1:2. In some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1:1.

[0058] In some embodiments, the incorporation efficiency of the first cannabinoid in step (b) is between about 4% and about 10%. In some embodiments, the incorporation efficiency is at least 4%. In some embodiments, the incorporation efficiency is at least 5%. In some embodiments, the incorporation efficiency is at least 6%. In some embodiments, the incorporation efficiency is at least 7%. In some embodiments, the incorporation efficiency is at least 8%. In some embodiments, the incorporation efficiency is at least 9%. In some embodiments, the incorporation efficiency is at least 10%.

[0059] In some embodiments, the average weight ratio of the encapsulated cannabinoid to the PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles of step (d) is between about 1:50 and about 1:10. In some embodiments, the average weight ratio of the encapsulated cannabinoid to the PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1:50. In some embodiments, the average weight ratio of the encapsulated cannabinoid to the PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1:40. In some embodiments, the average weight ratio of the encapsulated cannabinoid to the PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1:30. In some embodiments, the average weight ratio of the encapsulated cannabinoid to the PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1:20. In some embodiments, the average weight ratio of the encapsulated cannabinoid to the PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1:10.

[0060] In some embodiments, the PLGA copolymer has a ratio of lactic acid to glycolic acid that is about 10-90% lactic acid to about 90-10% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

[0061] In some embodiments, the solution of step (a) further comprises at least one cannabinoid or terpenoid other than the first cannabinoid. In some embodiments, the at least one cannabinoid or terpenoid is selected from the group consisting of cannabinol (CBN), cannabidivarin (CBDV), cannabichromene (CBC), cannabidiolic acid (CBDA) and cannabigerol (CBG). In some embodiments, the at least one cannabinoid or terpenoid is selected from the group consisting of myrcene, β-caryophyllene, nerolidol, phytol, limonene, linalool and pinene.

[0062] In some embodiments, the solvent is acetone, dichloromethane, or ethyl acetate. In some embodiments, the surfactant is polyethylene glycol, poloxamer, or polyvinyl alcohol (PVA). In some embodiments, the surfactant is polyvinyl alcohol (PVA).

[0063] In some embodiments, the emulsifying step comprises homogenization or sonication. In some embodiments, the emulsifying step is homogenization. In some embodiments, the homogenization is performed at 20,000 to 30,000 rpm. In some embodiments, the homogenization is performed at 24,000 rpm. In some embodiments, the solution is homogenized for 30 seconds to 10 minutes. In some embodiments, the solution is homogenized for 1 minute.

[0064] In some embodiments, evaporating the solvent comprises at least one of stirring the solvent, applying a gas flow, applying heat, maintaining a cold temperature of 10° C., or creating a vacuum. In some embodiments, evaporating the solvent comprises stirring the suspension at room temperature. In some embodiments, the suspension is stirred for between 5 minutes and 120 minutes to evaporate the solvent. In some embodiments, the suspension is stirred for 60 minutes.

[0065] In some embodiments, the step of obtaining the cannabinoid-loaded PLGA nanoparticles comprises centrifugation, filtration, or centrifugation and filtration. In some embodiments, the step of obtaining comprises centrifugation. In some embodiments, the centrifugation is between 2,000 x g and 15,000 x g. In some embodiments, the centrifugation is at 4,000 x g.

[0066] In some embodiments, the method further comprises the subsequent step of adding a cryoprotectant to the cannabinoid-encapsulated PLGA nanoparticles. In some embodiments, the cryoprotectant is trehalose. In some embodiments, the cryoprotectant is added in an amount of 1-10% (w / v) of the cannabinoid-encapsulated PLGA nanoparticles. In some embodiments, the cryoprotectant is added in an amount of 5% (w / v) of the cannabinoid-encapsulated PLGA nanoparticles.

[0067] In some embodiments, the method further comprises lyophilizing the cannabinoid-encapsulated PLGA nanoparticles.

[0068] In one aspect, the present disclosure provides cannabinoid-loaded PLGA nanoparticles obtainable by the methods described herein.

[0069] In another aspect, the present disclosure provides a method for causing TRPV1 desensitization in a cell of a mammalian subject, comprising administering to the mammalian subject a pharmaceutical composition as provided herein in an amount, route of administration, and time sufficient to cause TRPV1 desensitization in a cell in the mammalian subject. In some embodiments, the cell is a nociceptor. In some embodiments, the nociceptor is a peripheral nociceptor. In some embodiments, the nociceptor is a visceral nociceptor.

[0070] In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered topically. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered by inhalation.

[0071] In yet another aspect, the disclosure provides a method of treating pain in a mammalian subject, comprising administering to the subject a pharmaceutical composition provided herein in an amount, by a route of administration and for a time sufficient to cause TRPV1 desensitization in nociceptors in the subject.

[0072] In some embodiments, the nociceptor is a peripheral nociceptor and the pharmaceutical composition is administered locally, hi some embodiments, the nociceptor is a visceral nociceptor and the pharmaceutical composition is administered systemically.

[0073] In some embodiments, the pain is neuropathic pain. In some embodiments, the neuropathic pain is diabetic peripheral neuropathic pain. In some embodiments, the pain is post-herpetic neuralgia.

[0074] In some embodiments, the pharmaceutical composition is administered at least once a day for at least three days. In some embodiments, the pharmaceutical composition is administered at least once a day for at least five days. In some embodiments, the pharmaceutical composition is administered at least once a day for at least seven days. In some embodiments, the pharmaceutical composition is administered at least once a day for more than seven days. In some embodiments, the pharmaceutical composition is administered at a dose, route of administration and schedule sufficient to maintain the level of cannabinoids at the nociceptor for at least three days that are effective to desensitize TRPV1 at the nociceptor. In some embodiments, the pharmaceutical composition is administered at a dose, route of administration and schedule sufficient to maintain the level of cannabinoids at the nociceptor for at least five days that are effective to desensitize TRPV1 at the nociceptor. The cannabinoid, pharmaceutical composition is administered at a dose, route of administration and schedule sufficient to maintain the level of cannabinoids at the nociceptor for at least seven days that are effective to desensitize TRPV1 at the nociceptor.

[0075] One aspect of the present disclosure provides a method of treating cardiac hypertrophy in a mammalian subject, the method comprising administering to the subject an anti-hypertrophy effective amount of a pharmaceutical composition provided herein.

[0076] In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered by inhalation. In some embodiments, the pharmaceutical composition is administered orally.

[0077] Another aspect of the present disclosure provides a method for the prophylactic treatment of cardiac hypertrophy in a mammalian subject, comprising administering to a subject at risk for cardiac hypertrophy an antihypertrophy effective amount of a pharmaceutical composition provided herein.

[0078] In yet another aspect, the present disclosure provides a method of treating overactive bladder in a mammalian subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

[0079] In some embodiments, the pharmaceutical composition is administered systemically, hi some embodiments, the pharmaceutical composition is administered by bladder irrigation.

[0080] In one aspect, the disclosure provides a method of treating refractory chronic cough in a mammalian subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

[0081] In some embodiments, the pharmaceutical composition is administered systemically, hi some embodiments, the pharmaceutical composition is administered by inhalation.

[0082] These and other features, aspects and advantages of the present invention will become better understood with regard to the following description and accompanying drawings. [Brief description of the drawings]

[0083] [Figure 1] FIG. 1 provides a scheme of the nanoprecipitation method. [Diagram 2] FIG. 1 provides a scheme of PLGA nanocapsules obtained using a microemulsion system. [Diagram 3] FIG. 1 provides a scheme of the emulsification (high speed homogenizer) method for solvent evaporation. [Figure 4A] FIG. 1 shows a gas chromatogram of myrcene. [Figure 4B] FIG. 1 shows the mass spectrum of myrcene. [Figure 4C] FIG. 1 shows the calibration curve of myrcene with concentrations ranging between 1-60 ppm as measured and analyzed by a Thermo Scientific Instrument Delta V GC-MS instrument. [Figure 5A]FIG. 1 shows a gas chromatogram of β-caryophyllene. [Figure 5B] FIG. 1 shows a mass spectrum of β-caryophyllene. [Figure 5C] FIG. 1 shows a calibration curve of β-caryophyllene with concentrations ranging between 1-60 ppm as measured and analyzed by a Thermo Scientific Instrument Delta V GC-MS instrument. [Figure 6] FIG. 1 shows gas chromatograms of both cis- and trans-nerolidol as measured and analyzed by a Thermo Scientific Instrument Delta V GC-MS instrument. [Figure 7A] FIG. 1 shows the mass spectrum of cis-nerolidol. [Figure 7B] FIG. 1 shows the calibration curve of cis-nerolidol with concentrations between 1 and 60 ppm as measured and analyzed by a Thermo Scientific Instrument Delta V GC-MS instrument. [Figure 7C] 7C-7D: Similarly, the mass spectrum and calibration curve, respectively, of trans-nerolidol when measured and analyzed under the same conditions as the cis isomer. [Figure 7D] This is a continuation of Figure 7C. [Figure 8A] 8A, 8C and 8E) and zeta potential (FIGS. 8B, 8D and 8F) measurements of nanoparticles (NPs) prepared using nanoprecipitation. [Figure 8B] This is a continuation of Figure 8A. [Figure 8C] This is a continuation of Figure 8B. [Figure 8D] This is a continuation of Figure 8C. [Figure 8E] This is a continuation of Figure 8D. [Figure 8F] Continuation of Figure 8E. [Figure 9A] FIG. 9A shows the average diameter (FIG. 9A) and zeta potential (FIG. 9B) measurements of NPs prepared using microemulsification. [Figure 9B] This is a continuation of Figure 9A. [Figure 10A] FIG. 10A shows the average diameter (FIG. 10B) and zeta potential (FIG. 10B) measurements of NPs prepared using emulsification with a homogenizer. [Figure 10B] This is a continuation of Figure 10A. [Figure 11A] 11A and 11C) and zeta potential (FIGS. 11B and 11D) measurements of NPs prepared by emulsification (FIGS. 11A and 11B) and nanoprecipitation (FIGS. 11C and 11D). [Figure 11B] This is a continuation of Figure 11A. [Figure 11C] This is a continuation of Figure 11B. [Figure 11D] This is a continuation of Figure 11C. [Figure 12A] FIG. 12A shows the average diameter (FIG. 12A) and zeta potential (FIG. 12B) measurements of emulsions prepared by adding myrcene dissolved in acetone to a solution of PVA 0.5% (w / w). [Figure 12B] This is a continuation of Figure 12A. [Figure 13] 13A and 13B: Scanning electron microscope (SEM) images of myrcene-containing nanoparticles (NPs). [Figure 14] 14A and 14B: Scanning electron microscope (SEM) images of β-caryophyllene-containing nanoparticles (NPs). [Figure 15] 15A and 15B: Scanning electron microscope (SEM) images of nerolidol-containing nanoparticles (NPs). [Figure 16] FIG. 1 shows a schematic diagram of a nanocapsule. [Figure 17] FIG. 1 shows the fluorescence changes measured using a calcium signaling assay following treatment of HEK TRPV1 cells with both free and encapsulated terpenoids including myrcene, nerolidol and β-caryophyllene and ionomycin when presented on a single graph. [Figure 18A-B]FIG. 18 shows individual representations of fluorescence changes measured using a calcium signaling assay following treatment of HEK TRPV1 cells with unencapsulated and encapsulated myrcene (FIG. 18A), nerolidol (FIG. 18B), and β-caryophyllene (FIG. 18C). [Figure 18C] This is a continuation of Figures 18A-B. [Figure 19A] 19A-D show fluorescence changes measured using a calcium signaling assay following treatment of HEK TRPV1 cells with individual, unencapsulated terpenoids compared to their corresponding combinations: FIG. 19A shows unencapsulated myrcene, nerolidol, and combinations thereof; FIG. 19B shows unencapsulated myrcene, β-caryophyllene, and combinations thereof; FIG. 19C shows unencapsulated β-caryophyllene, nerolidol, and combinations thereof; and FIG. 19D shows unencapsulated myrcene, nerolidol, β-caryophyllene, and combinations thereof. [Fig. 19B-C] This is a continuation of Figure 19A. [Figure 19D] This is a continuation of Figures 19B-C. [Figure 20A] 20A and 20B show the fluorescence changes measured using a calcium signaling assay following treatment of HEK TRPV1 cells with individual nanoparticles (NPs) of encapsulated terpenoids compared to their corresponding combinations: FIG. 20A shows the results from myrcene NPs, nerolidol NPs, and their combinations; FIG. 20B shows the results from myrcene NPs, β-caryophyllene NPs, and their combinations; FIG. 20C shows the results from nerolidol NPs, β-caryophyllene NPs, and their combinations; and FIG. 20D shows the results from NPs containing myrcene, nerolidol and β-caryophyllene, NPs containing myrcene and nerolidol, NPs containing myrcene and β-caryophyllene, and NPs containing nerolidol and β-caryophyllene. [Fig. 20B-C] This is a continuation of Figure 20A. [Figure 20D] This is a continuation of Figures 20B-C.

[0084] [Figure 21A]Figure 21 shows the fluorescence changes measured using a calcium signaling assay following treatment of HEK TRPV1 cells with individually encapsulated terpenoid nanoparticles compared to their corresponding non-encapsulated terpenoid combinations. Figure 21A shows results from combinations of non-encapsulated and encapsulated myrcene and nerolidol, Figure 21B shows results from combinations of non-encapsulated and encapsulated myrcene and β-caryophyllene, Figure 21C shows results from combinations of non-encapsulated and encapsulated nerolidol and β-caryophyllene, and Figure 21D shows results from combinations of non-encapsulated and encapsulated myrcene, nerolidol and β-caryophyllene. [Figure 21B-C] This is a continuation of Figure 21A. [Figure 21D] This is a continuation of Figures 21B-C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] definition Terms used in the claims and specification are defined as set forth below, unless otherwise specified.

[0086] The term "myrcene" or "β-myrcene" as used herein refers to 7-methyl-3-methylideneocta-1,6-diene and has the structural formula

[0087] [ka] As exemplified by:

[0088] The term "α-ocimene" as used herein refers to cis-3,7-dimethyl-1,3,7-octatriene and has the structural formula

[0089] [ka] As exemplified by:

[0090] The term "cis-β-ocimene" as used herein refers to (Z)-3,7-dimethyl-1,3,6-octatriene and has the structural formula

[0091] [ka] As exemplified by:

[0092] The term "trans-β-ocimene" as used herein refers to (E)-3,7-dimethyl-1,3,6-octatriene and has the structural formula

[0093] [ka] As exemplified by:

[0094] The term "linalool," as used herein, refers to 3,7-dimethyl-1,6-octadien-3-ol and has the structural formula

[0095] [ka] As exemplified by:

[0096] The term "nerolidol" as used herein refers to 3,7,11-trimethyl-1,6,10-dodecatrien-3-ol and has the structural formula

[0097] [ka] As exemplified by:

[0098] The term "cis-nerolidol," as used herein, refers to the cis isomer of nerolidol and has the structural formula

[0099] [ka] As exemplified by:

[0100] The term "trans-nerolidol," as used herein, refers to the trans isomer of nerolidol and has the structural formula

[0101] [ka] As exemplified by:

[0102] The term "bisabolol" as used herein refers to 6-methyl-2-(4-methylcyclohex-3-en-1-yl)hept-5-en-2-ol and has the structural formula

[0103] [ka] As exemplified by:

[0104] The term "β-caryophyllene," as used herein, refers to (1R,4E,9S)-4,11,11-trimethyl-8-methylidenebicyclo[7.2.0]undec-4-ene and has the structural formula

[0105] [ka] As exemplified by:

[0106] The term "dimethylallyl" group has the formula

[0107] [ka] It refers to an unsaturated C5H9 alkyl substituent as exemplified by:

[0108] The term "terpenoids," as used herein, refers to a class of organic compounds produced by plants, including alpha-bisabolol (α-bisabolol), alpha-humulene (α-humulene), alpha-pinene (α-pinene), β-caryophyllene (β-caryophyllene), myrcene, (+)-beta-pinene (β-pinene), camphene, limonene, linalool, phytol, and nerolidol.

[0109] The term "cannabinoid" as used herein refers to a class of chemical compounds that include cannabigerolic acid (CBGA), cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabidiolic acid (CBDA) and cannabigerol (CBG).

[0110] "Pharmaceutically active ingredient" (synonymously, pharmaceutical active ingredient) means any substance or mixture of substances that is intended to be used in the manufacture of a drug product and that, when used in the production of a drug, becomes an active ingredient in the drug product. Such substances are intended to provide pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure and function of the body. Such substances or mixtures of substances are preferably generated in accordance with Current Good Manufacturing Practice (CGMP) regulations pursuant to section 501(a)(2)(B) of the Federal Food, Drug, and Cosmetic Act.

[0111] A pharma- ceutical active ingredient is "substantially free of THC" if it contains less than 0.3% (w / w) delta-9 tetrahydrocannabinol. A pharmaceutical composition is "substantially free of THC" if it contains less than 0.3% (w / v) delta-9 tetrahydrocannabinol.

[0112] A "cannabis extract" is a composition obtained from cannabis plant material by fluid and / or gas extraction, for example by supercritical fluid extraction (SFE) using CO2. Cannabis extracts typically contain myrcene, cannabinoids and terpenoids, and may also contain phytocannabinoids and other secondary metabolites.

[0113] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease, condition or its symptoms, and / or therapeutic in terms of partial or complete cure for a disease or condition and / or adverse effects, e.g., symptoms that may result from a disease or condition. "Treatment", as used herein, encompasses any treatment of a mammalian, particularly a human, disease or condition, including: (a) preventing the disease or condition from occurring in a subject who may be susceptible to the disease or condition but has not yet been diagnosed as having it, (b) inhibiting the disease or condition (e.g., arresting its onset), or (c) alleviating the disease or condition (e.g., causing regression of the disease or condition, providing improvement of one or more symptoms). Improvement of any condition can be readily determined according to standard methods and techniques known in the art. The population of subjects treated by the method includes subjects suffering from an undesirable condition or disease, as well as subjects at risk of developing a condition or disease.

[0114] By the term "therapeutically effective dose" or "therapeutically effective amount" is meant a dose or amount that produces the desired effect for which it is administered. The exact dose or amount will depend on the purpose of the treatment and will be ascertainable by one of skill in the art using known techniques (see, for example, Lloyd (2012) The Art, Science and Technology of Pharmaceutical Compounding, 4th Edition). A therapeutically effective amount may be a "prophylactically effective amount" such that prevention may be considered treatment.

[0115] The term "sufficient amount" means an amount sufficient to produce a desired effect.

[0116] The term "ameliorating" refers to any therapeutically beneficial outcome in the treatment of a disease state, eg, an immune disorder, including prevention, reduction in severity or progression, alleviation, or cure thereof.

[0117] The term "mammal", as used herein, includes both humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0118] The term "PLGA nanoparticles" as used herein refers to nanoparticles containing PLGA copolymers, and includes both PLGA nanocapsules and PLGA nanospheres. PLGA nanocapsules are characterized by an outer PLGA polymeric membrane (shell) surrounding an inner core of a substance, e.g., a drug compound, such as myrcene, other terpenoids, or cannabinoids. PLGA nanospheres are characterized by a PLGA polymeric spherical matrix in which a substance or drug compound is dispersed or embedded.

[0119] The term "myrcene-encapsulated PLGA nanoparticles" as used herein refers to nanoparticles made of PLGA copolymers that encapsulate or contain myrcene. The nanoparticles may be PLGA nanospheres or PLGA nanocapsules. Myrcene can be encapsulated as a liquid inner core inside a PLGA outer membrane (shell), forming a nanocapsule, or myrcene can be dispersed or embedded in a PLGA matrix, forming a nanosphere.

[0120] The term "cannabinoid-encapsulated PLGA nanoparticles" as used herein refers to nanoparticles made of PLGA copolymers that encapsulate or contain cannabinoids. The nanoparticles may be PLGA nanospheres or PLGA nanocapsules. The cannabinoids may be encapsulated as a liquid inner core inside a PLGA shell, forming nanocapsules, or the cannabinoids may be dispersed or embedded in a PLGA matrix, forming nanospheres.

[0121] The term "terpenoid-encapsulated PLGA nanoparticles" as used herein refers to nanoparticles made of PLGA copolymers that encapsulate or contain terpenoids. The nanoparticles may be PLGA nanospheres or PLGA nanocapsules. The terpenoids may be encapsulated as a liquid inner core inside a PLGA outer membrane (shell), forming nanocapsules, or the terpenoids may be dispersed or embedded in a PLGA matrix, forming nanospheres.

[0122] The term "terpenoid / cannabinoid-encapsulated PLGA nanoparticles" as used herein refers to terpene-encapsulated PLGA nanoparticles or cannabinoid-encapsulated PLGA nanoparticles.

[0123] The terms "free terpenoid" and "unencapsulated terpenoid" as used herein refer to terpenoids that are not encapsulated inside nanoparticles and are in bulk solution. These terms are used interchangeably throughout this specification.

[0124] The term "PLGA polymer," as used herein, refers to poly(lactic-co-glycolic acid).

[0125] The term "lyophilization" as used herein refers to a low temperature dehydration process in which water is removed from a material by freezing the material under low pressure or in a vacuum. The water freezes and is then removed from the material via sublimation. Alternative terms for the process include freeze-drying.

[0126] The term "entrapment efficiency" or "EE%" as used herein refers to the percentage of the mass of a compound in a nanoparticle compared to the starting mass of the compound. The EE percentage is calculated using the following formula: "CPD" is an abbreviation for "compound" and refers to a drug compound, such as myrcene or other terpenoids or cannabinoids, that is encapsulated in the nanoparticles.

[0127] The term "drug loading" or "DL%" as used herein refers to the mass of a compound incorporated into a nanoparticle compared to the total mass of the nanoparticle including the compound. The DL percentage is calculated using the following formula: "CPD" refers to a drug compound, e.g., myrcene or other terpenoid or cannabinoid, encapsulated in the nanoparticle.

[0128]

number

[0129] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0130] Ranges recited herein are understood to be shorthand for all values ​​within the range, including the recited endpoints. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0131] Unless otherwise indicated, a reference to a compound having one or more stereocenters contemplates each stereoisomer and all combinations of such stereoisomers.

[0132] Terpenoid / Cannabinoid-Encapsulated PLGA Nanoparticles Terpenoid-encapsulated PLGA nanoparticles In one aspect, the present invention provides a terpenoid-encapsulated PLGA nanoparticle comprising a PLGA nanoparticle and a first terpenoid encapsulated in the PLGA nanoparticle. The terpenoid-encapsulated PLGA nanoparticle can encapsulate a compound other than the first terpenoid, for example, another terpenoid and / or a cannabinoid.

[0133] The first terpenoid encapsulated in the PLGA nanoparticles may be a terpenoid extractable from cannabis.

[0134] The first terpenoid may be myrcene, β-caryophyllene, or nerolidol.

[0135] In various such embodiments, the first terpenoid, the optional cannabinoid, and the optional terpenoid other than the first terpenoid collectively constitute at least 75% by weight, but less than 100% by weight, of the total compounds encapsulated in the PLGA nanoparticles. In certain embodiments, the first terpenoid, the optional cannabinoid, and the optional terpenoid other than the first terpenoid collectively constitute at least 80% by weight, at least 85% by weight, at least 90% by weight, at least 91% by weight, at least 92% by weight, at least 93% by weight, at least 94% by weight, or at least 95% by weight, but less than 100% by weight, of the total compounds encapsulated in the PLGA nanoparticles. In particular embodiments, the first terpenoid, the optional cannabinoid, and the optional terpenoid other than the first terpenoid collectively constitute at least 96% by weight, at least 97% by weight, at least 98% by weight, or at least 99% by weight, but less than 100% by weight, of the total compounds encapsulated in the PLGA nanoparticles.

[0136] In embodiments where the first terpenoid, the optional cannabinoid, and the optional terpenoid other than the first terpenoid collectively constitute less than 100% by weight (wt%) of the total compounds encapsulated in the PLGA nanoparticles, the PLGA nanoparticles can further encapsulate compounds other than the first terpenoid, the optional cannabinoid, or the optional terpenoid. In such embodiments, the additional compounds can be extractable from cannabis. In certain embodiments, all or a portion of the compounds encapsulated in the PLGA nanoparticles are present in an extract made from cannabis.

[0137] The amount of the first terpenoid encapsulated in the PLGA nanoparticles can be quantified as the weight ratio of the weight of the PLGA copolymer in the nanoparticle to the amount of the first terpenoid encapsulated within the nanoparticle. The weight ratio of the first terpenoid to the PLGA copolymer can be between 1:50 and 1:1. The weight ratio of the first terpenoid to the PLGA copolymer can be between 1:100 and 1:5, 1:75 and 1:10, 1:50 and 1:10, 1:40 and 1:10, 1:30 and 1:10, 1:20 and 1:10, 1:50 and 1:40, 1:40 and 1:30, 1:30 and 1:20, 1:20 and 1:10, 1:10 and 1:5 and 1:5 and 1:1. The weight ratio of the first terpenoid to the PLGA copolymer may be 1:50, 1:45, 1:40, 1:35, 1:30, 1:25, 1:20, 1:19, 1:17, 1:18, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1. In a particular embodiment, the ratio is about 1:14. In a particular embodiment, the ratio is about 1:22.

[0138] In some embodiments, the weight ratio of the first terpenoid to the PLGA copolymer is between 1:50 and 1:10. In one embodiment, the weight ratio of the first terpenoid to the PLGA copolymer is about 1:14. In one embodiment, the weight ratio of the first terpenoid to the PLGA copolymer is about 1:22.

[0139] In one embodiment, the terpenoid encapsulated in the nanoparticle is myrcene. The amount of myrcene encapsulated in the PLGA nanoparticle can be quantified as the weight ratio of the weight of the PLGA copolymer in the PLGA nanoparticle to the amount of myrcene encapsulated in the nanoparticle. The weight ratio of myrcene to PLGA copolymer can be between 1:50 and 1:1. The weight ratio of myrcene to PLGA copolymer can be between 1:100 and 1:5, 1:75 and 1:10, 1:50 and 1:10, 1:40 and 1:10, 1:30 and 1:10, 1:20 and 1:10, 1:50 and 1:40, 1:40 and 1:30, 1:30 and 1:20, 1:20 and 1:10, 1:10 and 1:5 and 1:5 and 1:1. The weight ratio of myrcene and PLGA copolymer may be 1:50, 1:45, 1:40, 1:35, 1:30, 1:25, 1:20, 1:19, 1:17, 1:18, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1. In a particular embodiment, the ratio is about 1:22.

[0140] In some embodiments, the weight ratio of myrcene to PLGA copolymer is between 1:50 and 1: 10. In one embodiment, the weight ratio of myrcene to PLGA copolymer is about 1:14 or about 1:22.

[0141] A compound other than the first terpenoid ("second inclusion compound") can also be encapsulated in the PLGA nanoparticles. In various embodiments, the second inclusion compound is a cannabinoid and / or a terpenoid other than the first terpenoid. In one embodiment, the second inclusion compound is cannabigerolic acid (CBGA). In one embodiment, the second inclusion compound is cannabidiol (CBD). In one embodiment, the second inclusion compound is cannabinol (CBN). In one embodiment, the second inclusion compound is cannabidivarin (CBDV). In one embodiment, the second inclusion compound is cannabichromene (CBC). In one embodiment, the second inclusion compound is cannabidiolic acid (CBDA). In one embodiment, the second inclusion compound is cannabigerol (CBG). In one embodiment, the second inclusion compound is myrcene. In one embodiment, the second inclusion compound is caryophyllene. In one embodiment, the second inclusion compound is nerolidol. In one embodiment, the second inclusion compound is limonene. In one embodiment, the second inclusion compound is phytol. In one embodiment, the second inclusion compound is pinene. In one embodiment, the second inclusion compound is linalool.

[0142] Cannabinoid-loaded PLGA nanoparticles In another aspect, the present invention provides a cannabinoid-encapsulated PLGA nanoparticle comprising a PLGA nanoparticle and a first cannabinoid encapsulated in the PLGA nanoparticle. The cannabinoid-encapsulated PLGA nanoparticle can encapsulate a compound other than the first cannabinoid, for example, other terpenoids and / or cannabinoids.

[0143] The first terpenoid encapsulated in the PLGA nanoparticles may be a cannabinoid extractable from cannabis.

[0144] The first cannabinoid may be cannabidiol, cannabidivarin, cannabinol, cannabigerol or cannabichromene.

[0145] In various such embodiments, the first cannabinoid, the optional cannabinoid other than the first cannabinoid, and the optional terpenoid collectively constitute at least 75% by weight, but less than 100% by weight, of the total compounds encapsulated in the PLGA nanoparticles. In certain embodiments, the first cannabinoid, the optional cannabinoid other than the first cannabinoid, and the optional terpenoid collectively constitute at least 80% by weight, at least 85% by weight, at least 90% by weight, at least 91% by weight, at least 92% by weight, at least 93% by weight, at least 94% by weight, or at least 95% by weight, but less than 100% by weight, of the total compounds encapsulated in the PLGA nanoparticles. In particular embodiments, the first cannabinoid, the optional cannabinoid other than the first cannabinoid, and the optional terpenoid collectively constitute at least 96% by weight, at least 97% by weight, at least 98% by weight, or at least 99% by weight, but less than 100% by weight, of the total compounds encapsulated in the PLGA nanoparticles.

[0146] In embodiments where the first cannabinoid, the optional cannabinoid other than the first cannabinoid, and the optional terpenoid collectively constitute less than 100% by weight (wt%) of the total compounds encapsulated in the PLGA nanoparticles, the PLGA nanoparticles may further encapsulate compounds other than the first cannabinoid, the optional cannabinoid, or the optional terpenoid. In such embodiments, the additional compounds may be extractable from cannabis. In certain embodiments, all or a portion of the compounds encapsulated in the PLGA nanoparticles are present in an extract made from cannabis.

[0147] The amount of the first cannabinoid encapsulated in the PLGA nanoparticles can be quantified as the weight ratio of the weight of the PLGA copolymer in the PLGA nanoparticle shell to the amount of the first cannabinoid encapsulated within the nanoparticles. The weight ratio of the first cannabinoid to the PLGA copolymer can be between 1:50 and 1:1. The weight ratio of the first cannabinoid to the PLGA copolymer can be between 1:100 to 1:5, 1:75 to 1:10, 1:50 to 1:10, 1:40 to 1:10, 1:30 to 1:10, 1:20 to 1:10, 1:50 to 1:40, 1:40 to 1:30, 1:30 to 1:20, 1:20 to 1:10, 1:10 to 1:5, and 1:5 to 1:1. The weight ratio of the first cannabinoid to the PLGA copolymer may be 1:50, 1:45, 1:40, 1:35, 1:30, 1:25, 1:20, 1:19, 1:17, 1:18, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2 or 1:1. In a particular embodiment, the ratio is about 1:14. In a particular embodiment, the ratio is about 1:22.

[0148] In some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer is between 1:50 and 1:10. In one embodiment, the weight ratio of the first cannabinoid to the PLGA copolymer is about 1:14.

[0149] A compound other than the first cannabinoid ("second inclusion compound") can also be encapsulated in the PLGA nanoparticles. In various embodiments, the second inclusion compound is a cannabinoid and / or a terpenoid other than the first cannabinoid. In one embodiment, the second inclusion compound is cannabigerolic acid (CBGA). In one embodiment, the second inclusion compound is cannabidiol (CBD). In one embodiment, the second inclusion compound is cannabinol (CBN). In one embodiment, the second inclusion compound is cannabidivarin (CBDV). In one embodiment, the second inclusion compound is cannabichromene (CBC). In one embodiment, the second inclusion compound is cannabidiolic acid (CBDA). In one embodiment, the second inclusion compound is cannabigerol (CBG). In one embodiment, the second inclusion compound is myrcene. In one embodiment, the second inclusion compound is caryophyllene. In one embodiment, the second inclusion compound is nerolidol. In one embodiment, the second inclusion compound is limonene. In one embodiment, the second inclusion compound is phytol. In one embodiment, the second inclusion compound is pinene. In one embodiment, the second inclusion compound is linalool.

[0150] Delta-9 Tetrahydrocannabinol (THC) Content In an exemplary embodiment, the terpenoid / cannabinoid-encapsulated PLGA nanoparticles are either completely or substantially free of delta-9 tetrahydrocannabinol (THC) and therefore lack the psychotropic effects that provide certain regulatory and other physiological benefits.

[0151] In certain embodiments, the terpenoid / cannabinoid-encapsulated PLGA nanoparticles are not substantially free of delta-9 THC. In certain of these embodiments, the terpenoid / cannabinoid-encapsulated PLGA nanoparticles contain 1-10 weight percent (wt%) THC. In certain embodiments, the terpenoid / cannabinoid-encapsulated PLGA nanoparticles contain 2-9 wt% THC, 3-8 wt% THC, 4-7 wt% THC. In certain embodiments, the terpenoid / cannabinoid-encapsulated PLGA nanoparticles contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 wt% THC.

[0152] PLGA Nanoparticles The nanoparticles comprise the copolymer poly(lactic-co-glycolic acid) (PLGA), which is known to have high biocompatibility, low toxicity and high control of drug delivery. PLGA copolymers suitable for use in the nanoparticles described herein are commercially available, including, for example, from commercial sources, including Sigma-Aldrich under the Resomer brand.

[0153] The ratio of lactic acid and glycolic acid in the PLGA copolymer can be adjusted, resulting in varying amounts of each component in the PLGA polymer and in the PLGA nanoparticle composition. The weight percentage (w / w) of lactic acid in the PLGA polymer can be between about 10%-90%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90%. The weight percentage (w / w) of lactic acid in the PLGA polymer can be about 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. The weight percentage (w / w) of glycolic acid in the PLGA polymer may be between about 10%-90%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90%. The weight percentage (w / w) of glycolic acid in the PLGA polymer may be about 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. The weight ratio (w / w) of lactic acid to glycolic acid in the polymer may be between about 10% lactic acid to about 90% glycolic acid to about 90% lactic acid to about 10% glycolic acid. The weight ratio (w / w) of lactic acid to glycolic acid in the polymer may be about 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% lactic acid to about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10% glycolic acid.

[0154] In various embodiments, the weight ratio (w / w) of lactic acid to glycolic acid is about 10-90% lactic acid to about 90-10% glycolic acid. In some embodiments, the weight ratio (w / w) of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid. In some embodiments, the weight ratio (w / w) of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid. In some embodiments, the weight ratio (w / w) of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid. In some embodiments, the weight ratio (w / w) of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid. In some embodiments, the weight ratio (w / w) of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

[0155] In addition, PLGA nanoparticles can be surface modified or functionalized with additional polymers, such as polyethylene glycol (PEG) or copolymers, such as poloxamers. A variety of PEG and poloxamer reagents commercially available from a variety of sources, including Sigma-Aldrich and ThermoFisher Scientific, are suitable for this use.

[0156] The size and diameter of PLGA nanoparticles may depend on the method used to prepare the nanoparticles, such as nanoprecipitation or emulsion.In all cases, the size and surface charge of the nanoparticles can be quantified through dynamic light scattering (DLS) and zeta potential (ζ potential) measurements, respectively.The common instrument used to perform these measurements is the Malvern Zetasizer Nano ZS, and unless otherwise specified, all measurements listed herein are obtained using this device.These measurements can be used to determine the average diameter of particles, the surface charge of particles (ζ potential), and the polydispersity of particles in a population (polydispersity index, PdI).

[0157] In various embodiments, the myrcene-loaded PLGA nanoparticles have an average diameter of about 150-500 nm, 150-200 nm, 200-250 nm, 200-350 nm, 250-300 nm, 300-350 nm, 350-400 nm, 400-450 nm, or 450-500 nm. In some embodiments, the nanoparticles have an average diameter of about 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 450 nm, 475 nm, or 500 nm. In some embodiments, the nanoparticles have an average diameter of about 150-500 nm, 150-200 nm, 200-250 nm, 250-300 nm, 300-350 nm, 350-400 nm, 400-450 nm, or 450-500 nm or less. In some embodiments, the nanoparticles have an average diameter of about 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 450 nm, 475 nm, or 500 nm or less.

[0158] The diameter of the nanoparticles in a population can also be described using a particle size distribution (D value). The D value reflects the mass of the nanoparticles as a percentage when the particles in the population are arranged on an ascending mass basis. For example, the D value is the diameter at which 10% of the nanoparticle population mass is composed of particles less than the indicated diameter value. In such a case, the nanoparticle population is composed primarily of particles larger than the indicated diameter value. The D value is the diameter at which 50% of the nanoparticle population mass is composed of particles less than the indicated diameter value and 50% of the nanoparticle population mass is composed of particles larger than the indicated value. In such a case, the nanoparticle population is composed equally of particles larger than the indicated diameter value and particles smaller than the indicated diameter value. The D value is the diameter at which 90% of the nanoparticle population mass is composed of particles less than the indicated diameter value. In such a case, the nanoparticle population is composed primarily of particles smaller than the indicated diameter value.

[0159] In various embodiments, the terpenoid / cannabinoid loaded PLGA nanoparticles have a D10 diameter value of about 150nm, 175nm, 200nm, 225nm, 250nm, 275nm, 300nm, 325nm, 350nm, 375nm, 400nm, 450nm, 475nm or 500nm. The terpenoid / cannabinoid loaded PLGA nanoparticles may have a D50 diameter value of about 150nm, 175nm, 200nm, 225nm, 250nm, 275nm, 300nm, 325nm, 350nm, 375nm, 400nm, 450nm, 475nm or 500nm. The D90 diameter value of the terpenoid / cannabinoid loaded PLGA nanoparticles may be about 150nm, 175nm, 200nm, 225nm, 250nm, 275nm, 300nm, 325nm, 350nm, 375nm, 400nm, 450nm, 475nm or 500nm.

[0160] In certain embodiments, the nanoparticles include additional polymers known in the art. Such polymers include poly(lactic acid), poly(glycolic acid), gelatin, dextran, chitosan, lipids, phospholipids, polycyanoacrylates, polyesters, and poly(ε-caprolactone). The polymers can be selected based on their degradation characteristics, resulting in the formulation of the pharmaceutical composition for long-term drug delivery. Polymers can be selected for their ability to affect retention in the subject over time, allowing for timed release of the drug during treatment.

[0161] Pharmaceutical Compositions In another aspect, provided herein is a pharmaceutical composition comprising a population of terpenoid / cannabinoid-loaded PLGA nanoparticles as described herein and a pharma- ceutically acceptable carrier or diluent.In some embodiments, the pharmaceutical composition is lyophilized.In some embodiments, the pharmaceutical composition is lyophilized in the presence of trehalose.

[0162] In some embodiments, the pharmaceutical composition further comprises another population of PLGA nanoparticles (i.e., a "second population of PLGA nanoparticles"), the second population of nanoparticles encapsulating at least one compound (i.e., a "third encapsulated compound"). The compound encapsulated in the second population of nanoparticles may be a cannabinoid or a terpenoid. In some embodiments, the cannabinoid or terpenoid in the second population of nanoparticles is different from the cannabinoid and terpenoid encapsulated in the first population of terpenoid / cannabinoid-encapsulated PLGA nanoparticles.

[0163] In one embodiment, the third inclusion compound is cannabigerolic acid (CBGA). In one embodiment, the third inclusion compound is cannabidiol (CBD). In one embodiment, the third inclusion compound is cannabinol (CBN). In one embodiment, the third inclusion compound is cannabidivarin (CBDV). In one embodiment, the third inclusion compound is cannabichromene (CBC). In one embodiment, the third inclusion compound is cannabidiolic acid (CBDA). In one embodiment, the third inclusion compound is cannabigerol (CBG). In one embodiment, the third inclusion compound is myrcene. In one embodiment, the third inclusion compound is β-caryophyllene. In one embodiment, the third inclusion compound is nerolidol. In one embodiment, the third inclusion compound is limonene. In one embodiment, the third inclusion compound is phytol. In one embodiment, the third inclusion compound is pinene. In one embodiment, the third inclusion compound is linalool. In some embodiments, the second population of nanoparticles encapsulates a compound other than a cannabinoid or a terpenoid.

[0164] In some embodiments, the pharmaceutical composition further comprises an additional population of PLGA nanoparticles (i.e., a "third population of PLGA nanoparticles"), the third population of nanoparticles encapsulating at least one compound (i.e., a "fourth encapsulated compound"). The compound encapsulated in the third population of nanoparticles may be a cannabinoid or a terpenoid. In some embodiments, the cannabinoid or terpenoid in the third population of nanoparticles is different from the cannabinoid and terpenoid encapsulated in the first or second population of terpenoid / cannabinoid-encapsulating PLGA nanoparticles.

[0165] In some embodiments, the pharmaceutical composition further comprises one or more additional populations of PLGA nanoparticles (i.e., a "fourth population of PLGA nanoparticles," a "fifth population of PLGA nanoparticles," etc.), wherein the one or more additional populations of nanoparticles encapsulate at least one compound. The compound encapsulated in the one or more additional populations of nanoparticles may be a cannabinoid or a terpenoid. In some embodiments, the cannabinoid or terpenoid in the one or more additional populations of nanoparticles is different from the cannabinoids and terpenoids encapsulated in other populations of terpenoid / cannabinoid-encapsulating PLGA nanoparticles.

[0166] In some embodiments, the pharmaceutical composition comprises multiple populations of nanoparticles, each population of nanoparticles comprising a unique terpenoid or cannabinoid. In one embodiment, the pharmaceutical composition comprises a first population of nanoparticles encapsulating a terpenoid and a second population of nanoparticles encapsulating a cannabinoid. In one embodiment, the pharmaceutical composition comprises a first population of nanoparticles encapsulating a first terpenoid and a second population of nanoparticles encapsulating a second terpenoid. The pharmaceutical composition may further comprise a third population of nanoparticles encapsulating a third terpenoid, a fourth population of nanoparticles encapsulating a fourth terpenoid, etc. In another embodiment, the pharmaceutical composition comprises a first population of nanoparticles encapsulating a first cannabinoid and a second population of nanoparticles encapsulating a second cannabinoid. The pharmaceutical composition may further comprise a third population of nanoparticles encapsulating a third cannabinoid, a fourth population of nanoparticles encapsulating a fourth cannabinoid, etc.

[0167] In one particular embodiment, the pharmaceutical composition comprises nanoparticles encapsulating β-myrcene. In one embodiment, the pharmaceutical composition comprises nanoparticles encapsulating β-caryophyllene. In one embodiment, the pharmaceutical composition comprises nanoparticles encapsulating nerolidol. In one embodiment, the pharmaceutical composition comprises a first population of nanoparticles encapsulating β-myrcene, a second population of nanoparticles encapsulating β-caryophyllene, and / or a third population of nanoparticles encapsulating nerolidol.

[0168] In one embodiment, the pharmaceutical composition comprises nanoparticles encapsulating cannabidiol. In one embodiment, the pharmaceutical composition comprises a first population of nanoparticles encapsulating cannabidiol and a second population of nanoparticles encapsulating terpenoids, such as β-myrcene, β-caryophyllene, nerolidol, and / or other terpenoids. In one embodiment, each of the terpenoids is encapsulated in a separate population of nanoparticles.

[0169] Dose range, general In vivo and / or in vitro assays can be optionally used to help identify optimal dosage ranges for use. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the condition, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0170] Unit dosage form Pharmaceutical compositions comprising terpenoid / cannabinoid-containing PLGA nanoparticles may conveniently be presented in unit dosage form.

[0171] The unit dosage form is typically adapted for one or more particular routes of administration of the nanoparticle pharmaceutical composition.

[0172] In various embodiments, the unit dosage form is adapted for administration by inhalation. In certain of these embodiments, the unit dosage form is adapted for administration by a vaporizer. In certain of these embodiments, the unit dosage form is adapted for administration by a nebulizer. In certain of these embodiments, the unit dosage form is adapted for administration by an aerosolizer.

[0173] In various embodiments, the unit dosage form is adapted for oral, buccal, or sublingual administration.

[0174] In some embodiments, the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration.

[0175] In some embodiments, the unit dosage form is adapted for intrathecal or intraventricular administration.

[0176] In some embodiments, the unit dosage form is adapted for topical administration, including, for example, transdermal administration.

[0177] Methods for obtaining terpenoid / cannabinoid-loaded PLGA nanoparticles In another aspect, a method for obtaining terpenoid / cannabinoid-loaded PLGA nanoparticles is provided. In various embodiments, the method for obtaining terpenoid / cannabinoid-loaded PLGA nanoparticles includes the steps of (a) providing an organic solution comprising a first terpenoid or a first cannabinoid, a PLGA copolymer and a solvent, and an aqueous solution comprising a surfactant, (b) emulsifying the two solutions to form a suspension of terpenoid / cannabinoid-loaded PLGA nanoparticles, (c) evaporating the solvent from the emulsion, and (d) obtaining terpenoid / cannabinoid-loaded PLGA nanoparticles.

[0178] Solutions to obtain terpenoid / cannabinoid-loaded PLGA nanoparticles Varying ratios of terpenoid / cannabinoid to PLGA copolymer can be used in the solution of step (a). These ratios can be equal amounts of the first terpenoid or cannabinoid and PLGA copolymer (e.g., 1:1 weight ratio, or 100% (w / w) of the first terpenoid / cannabinoid to PLGA copolymer), lower amounts of the first terpenoid / cannabinoid compared to the PLGA copolymer (e.g., 1:2, 1:3, or 1:5 weight ratio, or 50%, 33%, 20% (w / w) of the first terpenoid / cannabinoid to PLGA copolymer), or higher amounts of the first terpenoid / cannabinoid compared to the PLGA copolymer (e.g., 2:1, 3:1, or 5:1 weight ratio, or 200%, 300%, 500% (w / w) of the first terpenoid / cannabinoid to PLGA copolymer). The weight ratio of the terpenoid / cannabinoid to the PLGA copolymer may be between about 1:1 to 1:20, 1:1 to 1:10, 1:1 to 1:5, 1:5 to 1:10, 1:10 to 1:15, or 1:15 to 1:20. The weight ratio of the first terpenoid / cannabinoid to the PLGA copolymer may be at least about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20. In various embodiments, the weight ratio of the first terpenoid / cannabinoid to the PLGA copolymer in the solution is about 1:5 to about 1:1. In various embodiments, the weight ratio of the first terpenoid / cannabinoid to the PLGA copolymer in the solution is about 1:5. In various embodiments, the weight ratio of the first terpenoid / cannabinoid to the PLGA copolymer in the solution is about 1:4. In various embodiments, the weight ratio of the first terpenoid / cannabinoid to the PLGA copolymer in the solution is about 1:3. In various embodiments, the weight ratio of the first terpenoid / cannabinoid to the PLGA copolymer in the solution is about 1:2. In various embodiments, the weight ratio of the first terpenoid / cannabinoid to the PLGA copolymer in the solution is about 1:1.

[0179] The solution may contain additional terpenoids and / or cannabinoids other than the first terpenoid or cannabinoid. In one embodiment, the solution contains at least one cannabinoid or terpenoid other than the first terpenoid or cannabinoid, and is substantially free of THC. In some embodiments, the at least one cannabinoid or terpenoid other than the first terpenoid or cannabinoid is selected from the group consisting of cannabidiol (CBD), cannabinol (CBN), cannabidivarin (CBDV), cannabichromene (CBC), cannabidiolic acid (CBDA) and cannabigerol (CBG). In other embodiments, the at least one cannabinoid or terpenoid other than the first terpenoid or cannabinoid is selected from the group consisting of myrcene, β-caryophyllene, nerolidol, phytol, limonene, linalool and pinene.

[0180] The solution used in step (a) of the method for producing terpenoid / cannabinoid-loaded PLGA nanoparticles also comprises a solvent and a surfactant.

[0181] A variety of solvents are known in the art. Any suitable solvent can be used to prepare terpenoid / cannabinoid-loaded PLGA nanoparticles. Illustrative solvents include, but are not limited to, acetone, ethyl acetate, ethanol, methanol, diethyl ether, toluene, hexane, benzene, dichloromethane, tetrahydrofuran, acetonitrile, dimethylformamide, dimethylsulfoxide, acetic acid, n-butanol, isopropanol, n-propanol and formic acid, or any combination thereof. In one embodiment, the solvent is acetone, dichloromethane or ethyl acetate. In another embodiment, the solvent is ethyl acetate.

[0182] A variety of surfactants are known in the art, and any suitable surfactant can be used to prepare terpenoid / cannabinoid-loaded PLGA nanoparticles. Illustrative surfactants include, but are not limited to, anionic surfactants, such as ammonium lauryl sulfate, sodium lauryl sulfate or sodium dodecyl sulfate, sodium laureth sulfate, sodium myreth sulfate, sodium stearate, and other carboxylate salts; cationic surfactants, such as compounds using primary, secondary, tertiary, or quaternary amines, such as octenidine dihydrochloride; cetrimonium bromide, benzalkonium chloride, and benzethonium chloride; or nonionic surfactants, such as ethoxylates, such as fatty acid ethoxylates, alkylphenol ethoxylates, such as nonoxynol, poloxamer, fatty acid esters of glycerol, such as glycerol monostearate and glycerol monolaurate, polyethylene glycol (PEG), and polyvinyl alcohol (PVA), or any combination thereof. Surfactants can be hydrophilic or lyophilic. In one embodiment, the surfactant is polyethylene glycol, poloxamer or polyvinyl alcohol (PVA).In one embodiment, the surfactant is polyvinyl alcohol (PVA).

[0183] emulsification Terpenoid / cannabinoid-encapsulated nanoparticles are formed in step (b) by mixing two solvent phases to produce either nanocapsules or nanospheres depending on the method selected: methods such as nanoprecipitation result in the formation of nanospheres, while microemulsion and emulsification using high speed homogenizers produce nanocapsules.

[0184] In various embodiments, the emulsification method is homogenization or sonication. In certain embodiments, the emulsifying step is performed using homogenization.

[0185] Homogenization can be performed using a high speed homogenizer, such as a Polytron homogenizer available from Thomas Scientific. Homogenization can be performed at about 10,000 to 50,000 rpm. In some embodiments, homogenization is performed at about 10,000 to 50,000 rpm, 10,000 to 15,000 rpm, 15,000 to 20,000 rpm, 20,000 to 25,000 rpm, 25,000 to 30,000 rpm, 30,000 to 35,000 rpm, 35,000 to 40,000 rpm, 40,000 to 45,000 rpm, or 45,000 to 50,000 rpm. In some embodiments, homogenization is performed at about 10,000 rpm, 15,000 rpm, 20,000 rpm, 21,000 rpm, 22,000 rpm, 23,000 rpm, 24,000 rpm, 25,000 rpm, 26,000 rpm, 27,000 rpm, 28,000 rpm, 29,000 rpm, 30,000 rpm, 35,000 rpm, 40,000 rpm, 45,000 rpm, or 50,000 rpm. In one embodiment, homogenization is performed at 20,000 to 30,000 rpm. In another embodiment, homogenization is performed at 24,000 rpm.

[0186] In some embodiments, the solution is homogenized for at least about 10 seconds to 10 minutes. In some embodiments, the solution is homogenized for at least about 10 seconds, 15 seconds, 20 seconds, 30 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, 5.6 minutes, 6 minutes, 6.5 minutes, 7 minutes, 7.5 minutes, 8 minutes, 8.5 minutes, 9 minutes, 9.5 minutes, or 10 minutes. In one embodiment, the solution is homogenized for about 30 seconds to 10 minutes. In one embodiment, the solution is homogenized for 1 minute.

[0187] Solvent evaporation After the emulsification step, the solution contains a suspension of terpenoid / cannabinoid-loaded PLGA nanoparticles. In step (c), the solvent in the nanoparticle suspension is then removed via evaporation. Evaporation techniques include, but are not limited to, stirring the suspension and solvent, applying a gas stream, applying heat, maintaining the temperature at 10°C, and creating a vacuum. A rotary evaporator can be used to evaporate the solvent in the nanoparticle suspension at room temperature or lower. In such cases, the sample can be placed on ice during evaporation in a refrigerator or cold room, or the rotary evaporator can have an attached cooling unit. The solvent can also be evaporated by stirring the nanoparticle suspension. In some embodiments, evaporating the solvent includes stirring the suspension at room temperature. The suspension can be stirred for about 5 minutes to 120 minutes. In some embodiments, the suspension is stirred for about 5-10 minutes, 10-15 minutes, 15-30 minutes, 30-45 minutes, 45-60 minutes, 60-75 minutes, 75-90 minutes, 90-105 minutes, or 105-120 minutes. In some embodiments, the suspension is stirred for about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, or 120 minutes. In one embodiment, the suspension is stirred for 5 minutes to 120 minutes to evaporate the solvent. In one embodiment, the suspension is stirred for 60 minutes.

[0188] Centrifugation In some embodiments, evaporation of the solvent in step (c) is sufficient to allow PLGA nanoparticles to be obtained. In a particular embodiment, the centrifugation is performed at 10°C.

[0189] In some embodiments, after evaporating the solvent, the terpenoid / cannabinoid-loaded PLGA nanoparticles are obtained by further centrifugation, filtration, or centrifugation and filtration. In a particular embodiment, obtaining the nanoparticles includes centrifugation. The nanoparticle suspension can be centrifuged between 2,000 x g and 15,000 x g. In some embodiments, the nanoparticle suspension is centrifuged between 2,000 and 4,000 x g, 2,000 and 10,000 x g, 2,000 and 15,000 x g, 4,000 and 10,000 x g, 4,000 and 15,000 x g, 7,000 and 10,000 x g, 7,000 and 15,000 x g, or 10,000 and 15,000 x g. The nanoparticle suspension can be centrifuged between 2,000 rpm and 15,000 rpm. In some embodiments, the nanoparticle suspension is centrifuged at between 2,000 rpm and 4,000 rpm, 2,000 rpm and 10,000 rpm, 2,000 rpm and 15,000 rpm, 4,000 rpm and 10,000 rpm, 4,000 rpm and 15,000 rpm, 7,000 rpm and 10,000 rpm, 7,000 rpm and 15,000 rpm, or 10,000 rpm and 15,000 rpm. In one embodiment, the centrifugation is at 4,000×g.

[0190] The suspension can be centrifuged for about 5 to 60 minutes. The suspension can be centrifuged for about 5 to 10 minutes, 10 to 15 minutes, 15 to 20 minutes, 20 to 25 minutes, 25 to 30 minutes, 30 to 35 minutes, 35 to 40 minutes, 40 to 45 minutes, 45 to 50 minutes, 50 to 55 minutes, or 55 to 60 minutes. The suspension can be centrifuged for about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes. In some embodiments, the centrifugation is performed for 10 minutes, 15 minutes, 30 minutes, 45 minutes, or 10 to 45 minutes. In one embodiment, the centrifugation is performed for 30 minutes. In some embodiments, the suspension can be centrifuged for longer. For example, the suspension can be centrifuged for about 30 minutes to 1 hour, 30 minutes to 2 hours, or 3 minutes to 3 hours.

[0191] Freeze drying After the terpenoid / cannabinoid-encapsulated nanoparticles are obtained, the nanoparticles can be optionally lyophilized. In this case, a cryoprotectant or cryopreservation agent can be added to the terpenoid / cannabinoid-encapsulated PLGA nanoparticles. Any suitable cryoprotectant known in the art can be used, including but not limited to trehalose, ethylene glycol, propylene glycol, glycerol, and dimethyl sulfoxide, and any combination thereof. In one embodiment, the cryoprotectant is trehalose. The cryoprotectant can be added at a concentration of between about 1-25% (w / v) of the terpenoid / cannabinoid-encapsulated PLGA nanoparticles. The cryoprotectant can be added to the nanoparticles at a concentration of between about 1-2% (w / v), 2-5% (w / v), 5-10% (w / v), 10-15% (w / v), 15-20% (w / v), or 20-25% (w / v). The cryoprotectant may be added to the nanoparticles at a concentration of at least about 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), 10% (w / v), 12% (w / v), 15% (w / v), 17% (w / v), 20% (w / v), 22% (w / v), or 25% (w / v). In some embodiments, the cryoprotectant is added at a concentration of 1-10% (w / v) of the terpenoid / cannabinoid-encapsulated PLGA nanoparticles. In one embodiment, the cryoprotectant is added at a concentration of 5% (w / v) of the terpenoid / cannabinoid-encapsulated PLGA nanoparticles.

[0192] The nanoparticles can be lyophilized for between about 10 to 240 minutes, 10 to 30 minutes, 30 to 45 minutes, 45 to 60 minutes, 60 to 75 minutes, 75 to 90 minutes, 90 to 105 minutes, 105 to 120 minutes, 120 to 135 minutes, 135 to 150 minutes, 150 to 165 minutes, 165 to 180 minutes, 180 to 195 minutes, 195 to 210 minutes, 210 to 225 minutes, 22 minutes to 240 minutes, or for more than 240 minutes, e.g., overnight. The nanoparticles may be lyophilized for about 10, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210, 225, 240 minutes, or overnight. In some embodiments, the terpenoid / cannabinoid-loaded PLGA nanoparticles are lyophilized for 30, 60, 90, 120, 150, 180 minutes, or between 30 and 180 minutes. In one embodiment, the terpenoid / cannabinoid-loaded PLGA nanoparticles are lyophilized for 120 minutes.

[0193] In some embodiments, the nanoparticles are freeze-dried for longer periods of time, for example, the nanoparticles are freeze-dried for about 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer (e.g., 24-72 hours), depending on the volume of water used.

[0194] Drug loading and encapsulation efficiency The amount of terpenoid / cannabinoid or other compound encapsulated in the nanoparticles can be quantified. Drug compound content can be expressed as entrapment efficiency (EE, %) and drug loading (DL, %) as defined in Section 4.1 above.

[0195] In some embodiments, the entrapment efficiency (EE,%) of the method is between about 1% and 100%. In some embodiments, the entrapment efficiency is between about 1-5%, 5-10%, 4-7%, 4-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, or 95-100%. In some embodiments, the incorporation efficiency is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. In some embodiments, the incorporation efficiency is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. In one embodiment, the incorporation efficiency is at least 4%. In one embodiment, the incorporation efficiency is at least 5%. In one embodiment, the incorporation efficiency is at least 6%. In one embodiment, the incorporation efficiency is at least 7%. In one embodiment, the incorporation efficiency is at least 8%. In one embodiment, the incorporation efficiency is at least 9%. In one embodiment, the incorporation efficiency is at least 10%.

[0196] In some embodiments, the drug loading (DL%) of the method is between about 1% and 40%. In some embodiments, the drug loading is between about 1-5%, 5-10%, 4-7%, 4-10%, 10-15%, 15-20%, 20-25%, or 25-30%. In some embodiments, the drug loading is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, or 30%. In some embodiments, the drug loading is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, or 30%.

[0197] In some embodiments, the drug loading is expressed as the average weight ratio between the encapsulated terpenoid / cannabinoid and the PLGA copolymer in the nanoparticle. In some embodiments, the average weight ratio between the encapsulated terpenoid / cannabinoid and the PLGA copolymer in the terpenoid / cannabinoid-encapsulated PLGA nanoparticle is between about 1:50 and about 1:10. In some embodiments, the average weight ratio is between about 1:50 and 1:40, 1:40 and 1:30, 1:30 and 1:20, and 1:20 and 1:10. In some embodiments, the average weight ratio is at least about 1:50, 1:45, 1:40, 1:35, 1:30, 1:25, 1:20, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10. In some embodiments, the average weight ratio is at least about 1:50. In some embodiments, the average weight ratio is at least about 1:40. In some embodiments, the average weight ratio is at least about 1:30. In some embodiments, the average weight ratio is at least about 1:20. In some embodiments, the average weight ratio is at least about 1:10.

[0198] Methods of Treatment Using Terpenoid / Cannabinoid-Loaded PLGA Nanoparticles In yet another aspect, methods are provided for treating various conditions or diseases in mammalian subjects.The methods include administering the terpenoid / cannabinoid-containing PLGA nanoparticles described herein.These methods are particularly intended for therapeutic and prophylactic treatment of mammals, more particularly humans.

[0199] The actual amount administered, and the rate and schedule of administration, will depend on the nature and severity of the condition or disease being treated. Decisions regarding treatment prescriptions, e.g., dosage, etc., are within the skill of general practitioners and other medical professionals and typically take into account the disorder being treated, the condition of the individual patient, the route of administration, the site to be treated, and other factors known to the practitioner.

[0200] In vivo and / or in vitro assays can be optionally used to help identify optimal dosage ranges for use and routes and times for administration. The exact dose to be used in the formulation will also depend on the route of administration and the severity of the condition, and should be determined according to the judgment of the practitioner and each subject's circumstances. Effective doses and methods of administration can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0201] In some embodiments, the encapsulated terpenoid or cannabinoid is administered in an amount of less than 1 g, less than 500 mg, less than 100 mg, or less than 10 mg per dose.

[0202] In the methods of treatment described herein, the pharmaceutical compositions comprising the terpenoid / cannabinoid-containing PLGA nanoparticles can be administered alone or in combination with other treatments administered either simultaneously or sequentially with the terpenoid / cannabinoid-containing composition.

[0203] Methods for producing TRPV1 desensitization in cells of a mammalian subject Provided is a method for producing TRPV1 desensitization in cells of a mammalian subject, comprising administering to the subject a terpenoid / cannabinoid-containing pharmaceutical composition as described herein in an amount, route of administration and time sufficient to cause TRPV1 desensitization in cells in the subject. In some embodiments, the cells that are subjected to TRPV1 desensitization are nociceptors, such as peripheral nociceptors and visceral nociceptors.

[0204] In various embodiments, the pharmaceutical composition is administered systemically, hi some embodiments, the pharmaceutical composition is administered orally, bucally, or sublingually.

[0205] In various embodiments, the pharmaceutical composition is administered topically, hi particular embodiments, the pharmaceutical composition is administered topically to effect transdermal delivery.

[0206] In some embodiments, the pharmaceutical composition is administered parenterally. In certain embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered by inhalation.

[0207] Methods for Treating Pain In some embodiments, the receptors subject to TRPV1 desensitization are nociceptive, and the method comprises administering to a subject a terpenoid / cannabinoid-containing pharmaceutical composition described herein in an amount, by a route of administration, and for a time sufficient to cause TRPV1 desensitization in nociceptors in the subject.

[0208] In some embodiments, the nociceptor is a peripheral nociceptor. In certain aspects of these embodiments, the pharmaceutical composition is administered locally. In some embodiments, the pain sensory neuron is visceral. In certain aspects of these embodiments, the pharmaceutical composition is administered systemically.

[0209] In a related aspect, a method is provided for treating pain in a mammalian subject, the method comprising administering to the subject a pharmaceutical composition described herein in an amount, by a route of administration and for a time sufficient to reduce pain.

[0210] In certain embodiments, the pain is neuropathic pain. In particular embodiments, the neuropathic pain is diabetic peripheral neuropathic pain. In particular embodiments, the pain is post-herpetic neuralgia. In particular embodiments, the pain is trigeminal neuralgia.

[0211] In some embodiments, the subject has pain associated with or caused by a strain, a sprain, arthritis or other joint pain, a bruise, back pain, fibromyalgia, endometriosis, surgery, migraine, cluster headache, psoriasis, irritable bowel syndrome, chronic interstitial cystitis, vulvodynia, trauma, a musculoskeletal disorder, shingles, sickle cell disease, heart disease, cancer, stroke, or mouth sores or ulcers due to chemotherapy or radiation.

[0212] In some embodiments, the pharmaceutical composition is administered at least once a day for at least three days. In some embodiments, the pharmaceutical composition is administered at least once a day for at least five days. In some embodiments, the pharmaceutical composition is administered at least once a day for at least seven days. In some embodiments, the pharmaceutical composition is administered at least once a day for more than seven days.

[0213] In various embodiments, the pharmaceutical composition is administered at a dose, route of administration and schedule sufficient to maintain effective levels of the terpenoid or cannabinoid at the nociceptor for at least 3 days, at least 5 days, or at least 7 days.

[0214] Methods for Treating Cardiac Hypertrophy In another aspect, there is provided a method of treating cardiac hypertrophy in a mammalian subject, the method comprising administering to the subject an anti-hypertrophy effective amount of a pharmaceutical composition described herein.

[0215] In typical embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered by inhalation.

[0216] Methods of preventive treatment for cardiac hypertrophy In another aspect, a method of preventive treatment for cardiac hypertrophy in a mammalian subject is provided, comprising administering to a subject at risk of cardiac hypertrophy an antihypertrophy effective amount of a terpenoid / cannabinoid-containing pharmaceutical composition as described herein.

[0217] Methods for Treating Overactive Bladder In another aspect, a method for treating overactive bladder in a mammalian subject is provided, comprising administering to the subject a therapeutically effective amount of a terpenoid / cannabinoid-containing pharmaceutical composition as described herein.

[0218] In typical embodiments, the pharmaceutical composition is administered systemically, hi some embodiments, the pharmaceutical composition is administered by bladder irrigation.

[0219] Methods for Treating Refractory Chronic Cough In another aspect, a method of treating refractory chronic cough is provided, the method comprising administering to a subject a therapeutically effective amount of a terpenoid / cannabinoid-containing pharmaceutical composition described herein.

[0220] In some embodiments, the pharmaceutical composition is administered systemically.

[0221] In some embodiments, the pharmaceutical composition is administered by inhalation.

[0222] Methods of Treating Disorders with TRPV1 Pathogenesis In another aspect, the disease or disorder that is treated with the pharmaceutical composition containing terpenoid / cannabinoid described herein includes diseases related to the abnormal function of TRPV1.The disease may be related to the abnormal activation, inhibition or dysregulation of TRPV1.In some embodiments, the disease is related to the abnormal expression or mutation of the gene encoding TRPV1.

[0223] In some embodiments, the disease treated with the terpenoid / cannabinoid-containing pharmaceutical compositions described herein is a disease associated with abnormal synthesis of endogenous TRPV1 agonists.

[0224] [Example] Below are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0225] The practice of the present invention employs, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology, within the skill of the art, and such techniques are fully explained in the literature. Abbreviation DCM Dichloromethane DL Drug Loading EE uptake efficiency NP Nanoparticles PEG Polyethylene glycol PLGA Copolymer of lactic acid and glycolic acid PVA Polyvinyl Alcohol ζ Zeta potential

[0226] methodology Methods for characterization of terpenoid-containing nanoparticles The mean diameter and size distribution of synthesized and purified terpenoid-containing nanoparticles were measured in triplicate by Dynamic Light Scattering (Nanosizer ZS, Malvern Instruments Ltd., UK) at 25.0 ± 0.5 °C.

[0227] The zeta potential of PLGA-PEG-based NPs (terpenoid-containing and terpenoid-free) was characterized by laser Doppler electrophoresis (Nanosizer ZS, Malvern Instruments Ltd., UK) and triplicate ζ measurements were performed on the NPs after a final wash in MQ water at 25 °C.

[0228] The morphological characterization of the nanoparticles was performed by scanning electron microscopy on a FEI TENEO microscope with image analysis. Samples were prepared by diluting the terpenoid-containing nanoparticle suspension (without trehalose) to approximately 0.8 mg / ml and covering with an 8-9 nm Pd / Pt shell under vacuum (Leica EM SCD500).

[0229] The amount of myrcene, β-caryophyllene or nerolidol encapsulated in the nanoparticles was measured by GC-MS spectrometry. The analysis was performed using a Trace 1300 GC gas chromatography system equipped with a ZB-1MS capillary column (30 m × 0.25 mm × 0.25 μm, Thermo Scientific) in parallel with a mass spectrometer TSQ8000 (Thermo Scientific), and data acquisition and analysis were performed using Xcalibur.

[0230] For mass spectrometric detection, ionization was performed by electron impact (EI) using a voltage of 70 eV and full scan mode in the m / z range of 20-300 with an ion source temperature of 200 °C in positive ion mode. For quantification of myrcene, caryophyllene and nerolidol, signature ions for terpenoids at m / z 69, 93 and 133, respectively, were monitored by selected ion monitoring (SIM) mode. For the purpose of generating calibration curves, myrcene, caryophyllene or nerolidol with a series of concentrations ranging between 1 and 60 ppm (1, 2, 5, 10, 20, 40 and 60 ppm) in dichloromethane (DCM) were prepared and measured in the GC-MS instrument.

[0231] The drug encapsulation capacity of PLGA-PEG nanoparticles can be expressed as the entrapment efficiency (EE, %) and drug loading (DL, %) according to Equations 1 and 2, respectively.

[0232]

number

[0233] cell HEK TRPV1 cells were provided by the Helen Turner laboratory at Chaminade University of Honolulu and were maintained in MEM essential medium supplemented with 10% FBS, 50 units / ml to 50 μg / ml penicillin-streptomycin, and 0.6 mg / ml geneticin.

[0234] Calcium signaling assay procedure HEK TRPV1 cells were harvested with trypsin, inactivated with medium, and counted. Cells were centrifuged at 1000 rpm for 5 min at room temperature. Cells were washed twice with 1 mM Ca Assay Buffer (Na Ringer's [140 mM NaCl, 2.8 mM KCI, 2 mM MgCl2, 11 mM glucose, 10 mM HEPES], 2 mM probenecid, 1 mM CaCl2, pH 7.4) and collected by centrifugation at 1000 rpm for 5 min at room temperature. Cells were resuspended in 1 μM Fluo-4 (1 μl of 5 mM Fluo-4 was mixed with 1 μl of 20% Pluronic® F-127 in DMSO, then 5 ml of 1 mM Ca Assay Buffer was added and the mixture was incubated at 37° C. for 10 min in the dark) and incubated at 37° C. for 30 min in the dark. Cells were then washed twice with 1 mM Ca assay buffer, then resuspended in assay buffer and pipetted into opaque-walled 96-well plates at a density of 150,000 cells / 180 μl / well. Fluorescence was measured using a plate reader (Synergy HTX, BioTek, USA) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Once a baseline was established (triplicate measurements), 20 μl of stimulus (free terpenoids, terpenoid-loaded NPs, and control) solution was added to each well and measurements continued for 1 hour, taking one reading every 40 seconds.

[0235] Preparation of stimuli solutions All solutions were prepared in 1 mM Ca Assay Buffer. Free terpenoids were first dissolved in DMSO and then a specific volume was diluted with assay buffer to produce the required concentration. Terpenoid-loaded PLGA-PEG or poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) nanoparticles (NPs) were prepared by the emulsification method and diluted directly using assay buffer. Negative controls of assay buffer containing DMSO and blank NPs were also prepared. Ionomycin 4 μM was used as a positive control. Free and encapsulated terpenoids were prepared at a concentration of 400 μg / ml (to give a final concentration of 40 μg / ml after addition to cells), either alone or in combination (e.g., myrcene plus nerolidol, myrcene plus caryophyllene, nerolidol plus caryophyllene, and myrcene plus nerolidol plus β-caryophyllene).

[0236] Experiments were performed in triplicate, negative control responses were deduced, averages were computed and used to plot calcium response profiles.

[0237] [Example 1] Nanoprecipitation method for producing myrcene-encapsulated nanoparticles A nanoprecipitation method based on solvent displacement (Figure 1) was tested for the generation of myrcene-containing PLGA nanoparticles. Particle formation by the nanoprecipitation method is regulated by the so-called Marangoni effect, which appears at the interface of the solvent and non-solvent and is subject to interfacial disturbances due to complex and accumulated phenomena such as flow, diffusion and surface tension fluctuations. The presence of a stabilizer is very important to avoid aggregate formation and to provide stability to the nanoparticles during the nanoprecipitation technique.

[0238] Formulation PLGA-based nanoparticles (NPs) were prepared using nanoprecipitation method according to formulas (F1-F11) in Table 1. A mixture of hydrophilic and lyophilic surfactants (Pluronic and Span) was used to ensure system stability. Acetone was evaporated at room temperature using different experimental conditions or in the absence or presence of tocopheryl polyethylene glycol succinate (TPGS) using a rotary evaporator.

[0239] [Table 1]

[0240] Preparation methods using evaporation of organic solvents at room temperature (F1-F6) PLGA (22.5 mg) and Span® 60 (7.5 mg) were dissolved in 1.5 mL of acetone. Myrcene (10-100% w / w) was added to the organic solution. The organic phase was added dropwise to 4.5 mL of Pluronic® F-68 aqueous solution (0.5% w / v) under continuous stirring at a rate of 5 mL / h using a syringe-pump. Stirring was continued for 2 hours at room temperature to allow the acetone to evaporate. To remove unencapsulated compounds as well as excess surfactant, the suspension was adjusted up to 45 mL with MQ water and centrifuged at 10,000 rpm for 30 minutes at 4° C. The pellet was resuspended in 45 mL of MQ water and centrifuged again using the same conditions. The NP pellet was resuspended in MQ water to make 1 mL of NP suspension. This suspension was then dried in an oven and the loaded myrcene was measured.

[0241] The same method was used to prepare NPs to measure the weight and yield of NPs produced. After the final centrifugation, the NP pellet was resuspended in 2 mL of MQ water and the preparation was freeze-dried. The lyophilized NPs were collected as a white cotton-like material. The supernatant was also collected and freeze-dried to measure unencapsulated myrcene.

[0242] Samples were prepared for GC-MS analysis. 100 μL of freshly prepared NPs were completely dried in an oven for 30-40 min at 40 °C (removal of water). DCM (2 mL) was added to dissolve the dried residue. Then, 1.5 mL of the DCM solution was transferred into a suitable vial and sealed. The lyophilized supernatant stored at -20 °C was weighed and samples were prepared for GC-MS analysis as described for NPs. Briefly, 5 mg of NP sample was dissolved in DCM and 1.5 mL of the solution was transferred into a suitable vial and sealed.

[0243] Preparation methods using evaporation of organic solvents using a rotary evaporator in the absence of TPGS (F7-8) PLGA (22.5 mg) and Span® 60 (7.5 mg) were dissolved in 1.5 mL of acetone. Myrcene (40% w / w or 60% w / w) was added to the organic solution. To 4.5 mL of Pluronic® F-68 aqueous solution (0.5% w / v), the organic phase was added dropwise using a syringe pump at a rate of 5 mL / h. During mixing, the mixture was kept in a cold water bath to reduce evaporation of the drug. Acetone was removed using a rotary evaporator without heating over a period of 20 minutes. To remove unencapsulated compounds as well as excess surfactant, the suspension was adjusted up to 45 ml with MQ water and centrifuged at 10,000 rpm for 30 minutes at 4° C. The pellet was resuspended in 45 mL of MQ water and centrifuged again using the same conditions. The NP pellet was resuspended in 2 mL of MQ water and the preparation was freeze-dried. The lyophilized NPs were collected as a white cotton-like material. The supernatant was also collected and lyophilized to determine unencapsulated myrcene.

[0244] Samples were prepared for GC-MS analysis. Lyophilized NPs were dissolved in DCM. To facilitate dissolution, samples were sonicated in a bath sonicator for 5 min. Samples were filtered using a 1 μm syringe filter (compatible with DCM). 1.5 mL of sample solution was transferred into an appropriate vial and sealed. Lyophilized supernatant stored at -20°C was weighed and samples were prepared for GC-MS analysis as described for NPs. Briefly, 5 mg of NP sample was dissolved in DCM and 1.5 mL of solution was transferred into an appropriate vial and sealed.

[0245] Preparation methods using evaporation of organic solvents using a rotary evaporator in the presence of TPGS (F9-11) Tocopheryl polyethylene glycol succinate (TPGS), Vitamin E TPGS is a water-soluble derivative of natural Vitamin E. It was selected as an emulsifier in the formulation due to its several advantages, including its bulky structure and large surface area characteristics that make it an excellent emulsifier. PLGA-based NPs were prepared using the nanoprecipitation method in the presence of tocopheryl polyethylene glycol succinate (TPGS) according to formulas, F9-F11 (Table 1).

[0246] PLGA (22.5 mg), Span® 60 (5, 2.5 or 0 mg) and TPGS (2.5, 5 or 7.5 mg) were dissolved in 1.5 mL of acetone. Myrcene (9 mg, 40% w / w) was added to the organic solution. To 4.5 mL of Pluronic® F-68 aqueous solution (0.5% w / v), the organic phase was added dropwise using a syringe pump at a rate of 5 mL / h. During mixing, the mixture was kept in a cold water bath to reduce evaporation of the drug. Acetone was removed using a rotary evaporator without heating over a period of 20 minutes. To remove unencapsulated compounds as well as excess surfactant, the suspension was adjusted up to 45 mL with MQ water and centrifuged at 10,000 rpm for 30 minutes at 4° C. The pellet was resuspended in 45 mL of MQ water and the spinning was repeated using the same conditions. The NP pellet was resuspended in 2 mL of MQ water and the preparation was freeze-dried. The lyophilized NPs were collected as a white cotton-like material. The supernatant was also collected and freeze-dried to measure unencapsulated myrcene.

[0247] Samples were prepared for GC-MS analysis. NP lyophilisates were dissolved in DCM. To facilitate dissolution, samples were sonicated in a bath sonicator for 5 min. Samples were filtered using a 1 μm syringe filter (compatible with DCM). 1.5 mL of sample solution was transferred into an appropriate vial and sealed. Lyophilised supernatant stored at -20°C was weighed and samples were prepared for GC-MS analysis as described for NP. Briefly, 5 mg of NP sample was dissolved in DCM and 1.5 mL of solution was transferred into an appropriate vial and sealed.

[0248] Characterization of nanoparticles prepared by nanoprecipitation Table 2 displays the average diameter, size distribution, zeta potential, weight, yield, amount of loaded myrcene, EE% and DL% of NPs prepared by nanoprecipitation.

[0249] [Table 2]

[0250] The particle size of the NPs produced by nanoprecipitation ranges between 200-240 nm, with good polydispersity (PdI) below 0.2 and a negative zeta potential of about -30 (Table 2). Figures 8A and 8B show the size and zeta potential distributions by intensity, respectively, for sample F6. Figures 8C and 8D show the size and zeta potential distributions by intensity, respectively, for sample F8. Figures 8E and 8F show the size and zeta potential distributions by intensity, respectively, for sample F9.

[0251] Stable NPs were successfully produced at concentrations of myrcene up to 60%. At myrcene concentrations above 60% (w / w), the NPs were not physically stable and aggregates formed during the initial mixing step.

[0252] Evaporation of acetone using a rotary evaporator without heating significantly increased the amount of loaded myrcene by more than five-fold, from 0.036 mg and 0.042 mg in F5 and F6, respectively, to 0.155 mg and 0.275 mg in F7 and F8, respectively (Table 2).

[0253] TPGS is a synthetic amphiphile of vitamin E and is FDA approved as a water-soluble vitamin E nutritional supplement and drug delivery vehicle. It functions as a surfactant, solubilizer, and has the potential to improve drug loading in NPs. In addition, α-tocopheryl derivatives may enhance the oral absorption of hydrophobic materials. The use of TPGS did not appear to improve the amount of loaded myrcene in PLGA NPs; instead, it was found that the loaded myrcene was reduced when the TPGS percentage was increased (F9 and F10, Table 2).

[0254] [Example 2] Microemulsification method for producing myrcene-loaded nanoparticles The formation of microemulsions was also used to obtain PLGA nanoparticles. A microemulsion is a system of water, oil and amphiphile, which is a single optically isotropic and thermodynamically stable solution. Its formation is spontaneous (Figure 2). Soy lecithin, a natural lipid-containing phospholipid mixture, has previously been used as an amphiphile for the preparation of various delivery nanosystems, such as nanoemulsions, liposomes, micelles and nanoparticles. This method allows the formation of spherical nanocapsules in which an oily core composed of myrcene is entrapped and held within a thin dense wall formed by PLGA polymer and phosphatidylcholine.

[0255] Formulation PLGA-based NPs were prepared using the microemulsification method according to the formula in Table 3 .

[0256] [Table 3]

[0257] Myrcene-loaded NPs were prepared according to the method previously described by Iannitelli et al. (Int. J, Mol. Sci. 2011, 12, 5039-5051). Briefly, PLGA (11 mg) and Epikuron 200 (13 mg) were dissolved in 2.5 mL of acetone. Myrcene (48 mg) was added to the organic solution. The organic phase containing the drug was added all at once to the aqueous phase (5 mL of Pluronic® F-68 (0.5% w / v or 5% w / v) or polyvinyl alcohol (PVA) (0.5% w / v)) with continuous stirring. The beaker containing the mixture was covered with parafilm to prevent evaporation of myrcene and stirring was continued for 10 min. Acetone was removed using a rotary evaporator without heating over a period of 40 min. To remove unencapsulated compounds as well as excess surfactant, the suspension was adjusted up to 45 ml with MQ water and centrifuged at 10,000 rpm for 30 min at 4° C. The pellet was resuspended in 45 mL MQ water and centrifuged again using the same conditions. The NP pellet was resuspended in 2 mL MQ water and the preparation was freeze-dried. The lyophilized NP was collected as a white cotton-like material. The supernatant was also collected and freeze-dried to measure unencapsulated myrcene.

[0258] Samples were prepared for GC-MS analysis. Lyophilized NPs were dissolved in DCM. To facilitate dissolution, samples were sonicated in a bath sonicator for 5 min. Samples were filtered using a 1 μm syringe filter (compatible with DCM). 1.5 mL of sample solution was transferred into an appropriate vial and sealed. Lyophilized supernatant stored at -20°C was weighed and samples were prepared for GC-MS analysis as described for NPs. Briefly, 5 mg of NP sample was dissolved in DCM and 1.5 mL of solution was transferred into an appropriate vial and sealed.

[0259] Characterization of nanoparticles prepared by microemulsification The microemulsion method of NP preparation involves the preparation of an intermediate microemulsion that forms spontaneously when high concentrations and the right combination of surfactants (usually a surfactant and a co-surfactant, in this work Pluronic® F-68 and Epikuron 200) are used. The solvent then diffuses from the emulsion droplets and is evaporated leaving the NPs behind.

[0260] The average diameter, size distribution, zeta potential, weight, yield, amount of loaded myrcene, EE% and DL% of NPs prepared by microemulsification are displayed in Table 4. Figure 9A shows the size distribution by intensity and Figure 9B shows the zeta potential distribution of sample F17.

[0261] [Table 4]

[0262] NPs prepared using PVA 0.5% (as aqueous phase) were not physically stable and aggregates formed during preparation (F12). The particle size of blank NPs was about 150 nm (F13, F14), whereas myrcene-loaded NPs were approximately larger than 100 nm, with a diameter of about 250 nm. All NPs showed good polydispersity below 0.2 and negative zeta potential between -30 and -35.

[0263] In all cases, particles were obtained in the nanometer range (PdI<0.2) with a narrow size distribution and a zeta potential of about -32 mV. No difference between the loaded and blank (-30 mV) nanoparticles was observed. Thus, very little drug was retained on the surface.

[0264] [Example 3] Emulsification (high speed homogenizer) method for producing myrcene-loaded nanoparticles A method based on emulsification of a polymer organic solution in an aqueous phase, followed by organic solvent evaporation (Figure 3) was used for the preparation of myrcene-encapsulated nanoparticles. The organic phase is poured into a continuous phase (aqueous phase), where a surfactant is dissolved to provide stability to the emulsion. The emulsification is carried out under high shear forces to reduce the size of the emulsion droplets. This process largely determines the final particle size.

[0265] Formulation PLGA-based NPs were prepared using a single emulsion method according to the formula in Table 5. Emulsification was performed with a Polytron® homogenizer.

[0266] [Table 5]

[0267] PLGA (40 mg) was dissolved in 1 mL of ethyl acetate. Myrcene (10-100% w / w based on PLGA) was added to the organic solution. The preparation of NPs was carried out using a high-speed homogenizer. The homogenizer probe was immersed inside a Falcon tube containing 5 mL of PVA (0.5% w / v). Using a pipette, the organic phase containing the drug was added dropwise, the homogenizer was set at 24,000 rpm, and homogenization continued for 1 min. Homogenization was carried out on ice to reduce the temperature generated during the process. The organic solvent was removed using a rotary evaporator without heating over a period of 1 h. To remove unencapsulated compounds as well as excess surfactant, the suspension was adjusted up to 45 ml with MQ water and centrifuged at 10,000 rpm for 30 min at 4 °C. The pellet was resuspended in 45 mL of MQ water and centrifuged again using the same conditions. The NP pellet was resuspended in 2 mL of MQ water and the preparation was freeze-dried. The lyophilized NPs were collected as a white cotton-like material. The supernatant was also collected and freeze-dried to measure unencapsulated myrcene.

[0268] Samples were prepared for GC-MS analysis. Lyophilized NPs were dissolved in DCM. To facilitate dissolution, samples were sonicated in a bath sonicator for 5 min. Samples were filtered using a 1 μm syringe filter (compatible with DCM). 1.5 mL of sample solution was transferred into an appropriate vial and sealed. Lyophilized supernatant stored at -20°C was weighed and samples were prepared for GC-MS analysis as described for NPs. Briefly, 5 mg of NP sample was dissolved in DCM and 1.5 mL of solution was transferred into an appropriate vial and sealed.

[0269] Characterization of nanoparticles produced by emulsification The average diameter, size distribution, zeta potential, weight, yield, amount of loaded myrcene, EE% and DL% of NPs prepared by the emulsification method are displayed in Table 6. Figure 10A shows the size distribution by intensity and Figure 10B shows the zeta potential distribution of sample F20.

[0270] [Table 6]

[0271] The particle sizes are 225 nm for blank NPs, about 265 nm for myrcene-loaded NPs with drug concentrations between 10-40%, and between 350-400 nm for myrcene-loaded NPs with drug concentrations between 80-100%. However, the NPs in F22, F23, and F24 appeared to be damaged during centrifugation, resulting in a cloudy supernatant. The polydispersity of all NPs was good (below 0.2), and the negative zeta potential was slightly lower (about -25 mV) than the other methods. No difference was observed between the loaded and blank (-25 mV) nanoparticles. Thus, very little drug on the surface is retained.

[0272] The emulsification method showed higher drug loading efficiency in comparison with other methods used in this work, with the highest DL% values ​​obtained for F23 (1.29%) and F20 (1.20%). However, there were indications of damaged or ruptured NPs during centrifugation, as indicated by the cloudy supernatant and the percentage of polymer sticking on the wall of the Falcon tube near the top of the supernatant. Damage to NPs can be reduced, leading to higher drug loading efficiency, by performing centrifugation at a lower spinning speed or by replacing centrifugation with another purification method, e.g., dialysis.

[0273] [Example 4] Solvent Optimization Next, the volume of solvent used was optimized to reduce the time of the evaporation step to minimize potential evaporation of myrcene during the solvent evaporation step. The effect of reducing the volume of solvent was investigated using two preparation methods: nanoprecipitation (acetone) and emulsification (ethyl acetate).

[0274] Emulsification method The effect of varying the volume of organic solvent was determined using the single emulsion method. Nanoparticles were prepared and lyophilized as previously described in Example 3.

[0275] [Table 7]

[0276] Nanoprecipitation method The effect of varying the volume of organic solvent was determined using the nanoprecipitation method. Nanoparticles were prepared and freeze-dried as previously described in Example 1.

[0277] [Table 8]

[0278] Characterization of nanoparticles produced using lower amounts of solvent We started with the minimum volume of solvent to dissolve the organic phase components. In general, it was found that particle size decreased as the volume of solvent increased (Table 9). Figure 11A shows the size distribution by intensity, and Figure 11B shows the zeta potential distribution of sample F28. Figure 11C shows the size distribution by intensity, and Figure 11D shows the zeta potential distribution of sample F31.

[0279] In the single emulsion method, low volumes of ethyl acetate, 0.125 ml (F25) and 0.25 ml (F26), resulted in NPs with large diameters that could not be redisposed of after centrifugation. With 0.5 ml of ethyl acetate, the NPs produced were physically stable. F27 showed the largest diameter among the stable NPs, but also the highest drug loading efficiency of 0.486 mg (EE%: 6.08, DL%: 2.02). It is important to point out that this formula was centrifuged at 6,000 rpm and could be recovered at this speed due to its large diameter ≈ 670 nm, whereas all other formulas were centrifuged at 10,000 rpm. It is possible that higher centrifugation speeds may cause deformation of the NPs, resulting in drug escape. Although myrcene is not soluble in water, it can be solubilized by surfactants, PVA or Pluronic®.

[0280] Similarly, in the nanoprecipitation method, formula F31 with a larger diameter (≈400 nm) showed a higher myrcene loading efficiency (EE%: 2.09, DL%: 1.04) up to 0.188 mg in F31. The use of a cooling bath during the addition of the organic phase appeared to increase the particle size, but it could not be concluded that it could increase the drug loading (by preventing / reducing drug evaporation during preparation).

[0281] [Table 9]

[0282] [Example 5] Determination of myrcene in the supernatant Due to the relatively low amount of loaded myrcene in the NPs, we sought to measure the drug in the supernatant to understand how the drug was lost. Table 10 shows that the total amount of myrcene detected, including both loaded myrcene in the NPs and unloaded free myrcene present in the supernatant, was between 0.06 and 3.44% of the starting amount of drug. The results shown in Table 10 indicate that the majority of myrcene is lost at some stage, during NP preparation, during sample preparation for analysis, or during storage.

[0283] Myrcene losses likely occurred through evaporation during production rather than during storage, since the freeze-dried samples were stored in the freezer in well-closed containers that should prevent evaporation of the drug.

[0284] [Table 10]

[0285] [Example 6] Factors affecting myrcene loss Filtration during sample preparation for GC-MS analysis The effect of filtration on possible myrcene losses was studied for myrcene-loaded NPs (F27, F28, F29 and F30) prepared using the emulsification method. Filtration was used to remove undissolved components of NPs in DCM, e.g., PVA, during preparation of samples for GC-MS analysis. Insoluble impurities in the samples may result in clogging of the syringe during analysis and may interfere with the measurement. 5 mg of lyophilized NPs were dissolved in DCM. To facilitate dissolution, the samples were sonicated for 5 min in a bath sonicator. The samples were filtered using a 1 μm syringe filter (compatible with DCM). The solutions of the samples (1.5 mL each) were transferred into appropriate vials and sealed. Another set of samples (each weighing 5 mg of NPs) was prepared in DCM without filtration.

[0286] The amount of loaded myrcene in the NPs before and after filtration using a 1 μm syringe filter, EE% and DL% are presented in Table 11.

[0287] [Table 11]

[0288] Filtration resulted in a relative reduction in the measured amount of myrcene. Most samples showed little concentration reduction, however, this effect was more significant in F27, which had the highest concentration of loaded myrcene. The amount of myrcene was reduced by about 25%, from 0.486 mg before filtration to 0.368 mg after filtration. This may be due to an interaction between the drug and the filter, which results in a percentage of the drug being entrapped. Based on these findings, filtration was not the primary cause of myrcene loss during the preparation.

[0289] Organic solvent evaporation To study the effect of evaporation using a rotary evaporator on the loss of myrcene, two different samples were prepared as in the table below. In the first sample, 40 mg of Resomer 502 was dissolved in 1 ml of ethyl acetate, and then 8 mg of myrcene was added. In the second sample, 8 mg of myrcene was mixed with 1 ml of ethyl acetate without polymer. The two samples were subjected to evaporation under pressure using a rotary evaporator without heating for 1 hour. The formula is shown in Table 12.

[0290] [Table 12]

[0291] The amount of myrcene retained in the samples and the percentage of myrcene loss are shown in Table 13.

[0292] [Table 13]

[0293] Rotary evaporation of myrcene and ethyl acetate samples without PLGA (F38) resulted in a complete loss of myrcene. However, when PLGA was added to myrcene (F37), about 40% of the starting drug was retained, which may be due to drug adsorption to the polymer. This result may importantly indicate that myrcene incorporated in the polymer (in the NPs) can be protected from evaporation, whereas any free drug may be subject to loss during the evaporation step.

[0294] Evaporation, centrifugation and freeze-drying To investigate the possible loss of myrcene at different steps of NP production, samples were prepared using the nanoprecipitation method as described in Example 1 and according to the formula in Table 14.

[0295] F39 was centrifuged at 10,000 rpm for 30 min at 4° C. The NP pellet was resuspended in 2 mL MQ water and the preparation was freeze-dried. F40 and F41 were freeze-dried immediately after evaporation of the solvent without centrifugation. The amount of myrcene in the samples was measured using GC-MS spectroscopy. 5 mg of each sample was dissolved in 2 mL DCM and the solution was filtered using a 1 μm syringe filter, filled into appropriate vials and sealed.

[0296] [Table 14]

[0297] The mean diameter, size distribution, zeta potential, weight of the lyophilisate, amount of myrcene remaining in the samples and percentage of myrcene loss for the different formulations are presented in Table 15. Figure 9A shows the size distribution by intensity and Figure 9B shows the zeta potential distribution for sample F41.

[0298] [Table 15]

[0299] F41 was prepared by adding myrcene dissolved in acetone without polymer to the aqueous phase. The measured size of 357 nm indicates the formation of an emulsion of myrcene in the presence of PVA as a surfactant. These are not polymeric nanoparticles, since no PLGA was used, only micelles of PVA. To check whether centrifugation could be the cause of drug loss from the NPs during centrifugation, F39 was prepared. However, even F40, in which the NPs were prepared without centrifugation, showed low retention of drug. Similarly, F3, an emulsion of myrcene (without added PLGA), also exhibited similar retention of drug after lyophilization. These findings indicate that a large proportion of the drug may be lost during lyophilization. It is possible that some of the drug escapes from the NPs during centrifugation, but this cannot be tested with the current method of preparation, since the measurement of the drug should be performed immediately after centrifugation and not after freeze-drying.

[0300] Several methods were used to encapsulate myrcene in PLGA NPs, including: (i) nanoprecipitation, (ii) emulsification using a high-speed homogenizer, and (iii) microemulsification. To improve the loading efficiency, we developed and investigated several formulations as well. To reduce the possible evaporation of myrcene, the preparation method was optimized in terms of temperature during NP production and the use of a rotary evaporator without heating the samples, which resulted in an improvement in the drug loading capacity.

[0301] We obtained particles in the nanometer range with narrow size distribution (PdI<0.2) and negative zeta potential (-25 to -35mv). Using a single emulsion method, we achieved the highest loading capacity with up to 6% of the incorporation efficiency of the starting drug (F27). The low amount of myrcene measured in the supernatant collected after centrifugation of the NPs indicates that the drug is lost during the production process and not due to the failure of PLGA to encapsulate myrcene.

[0302] Myrcene has a low molecular weight (136.23 g / mol), low vapor pressure (2.09 mm Hg at 25°C), but even with a boiling point of 167°C, it is a volatile liquid at room temperature, which makes it difficult to keep myrcene in the NPs and prevent its evaporation. Although myrcene can be evaporated during the evaporation step of the production process, which is carried out in a rotary evaporator without heating, its evaporation is significantly reduced by the presence of PLGA. Thus, the drug loading in the NPs can largely protect myrcene from evaporation. The amount of solvent and the time of evaporation were also optimized, reduced from 1 hour to 25 minutes, minimizing possible evaporation of the drug. Myrcene was also lost during freeze-drying. It is unclear whether the loss is due to evaporation or sublimation. However, myrcene was not frozen even at -80°C.

[0303] [Example 7] Optimization of freeze-drying and centrifugation of nanoparticles Freeze drying To investigate whether myrcene is lost during lyophilization, three samples of either free myrcene, a mixture of myrcene and water, or a mixture of myrcene, water and PLGA were prepared in duplicate as illustrated in Table 16. The samples were lyophilized for 24 hours. The vials containing the samples were weighed before and after lyophilization to calculate the weight of the dried sample.

[0304] [Table 16]

[0305] The results of the lyophilization assay are shown in Table 17.

[0306] [Table 17]

[0307] The results demonstrate that most of the myrcene, both free and in combination with the polymer and water, is lost during the 24 hour freeze-drying process. Myrcene does not freeze at the freeze-drying temperature (approximately -80°C), which may indicate that it is lost by evaporation under vacuum in the freeze dryer rather than by sublimation.

[0308] Since freeze-drying was a crucial step in the loss of myrcene from the NPs, we hypothesized that reducing the freeze-drying time could reduce the loss of myrcene during this process. Aliquots of 100 μL of NP suspensions of formula F49 (myrcene: 100% w / w, centrifugation: Falcon tube, 10,000 rpm, 30 min, 4° C.) and F54 (myrcene: 100% w / w, centrifugation: Amicon tube, 4,000×g, 30 min, 12° C.) were transferred into Eppendorfs, frozen, and freeze-dried for only 2 h, which was sufficient to remove 100 μL of water. The dried products were then dissolved in DCM and analyzed for myrcene content using a GC-MS instrument as previously described. Samples were prepared in duplicate.

[0309] Centrifugation After confirming that myrcene is lost during lyophilization, we measured the amount of myrcene loaded in the NPs prior to the lyophilization step. Two different centrifugation protocols were compared to determine the optimal conditions. PLGA NPs were prepared using the single emulsion homogenization method described in Example 3. Sample formulas are presented in Tables 18 and 19. Formulas F45-F49 were centrifuged in Falcon tubes at 10,000 rpm for 30 minutes at 4°C to recover the NPs. Formulas F50-F54 were spun in Amicon tubes at 4,000 x g for 30 minutes at 12°C. Samples were prepared in duplicate.

[0310] [Table 18]

[0311] [Table 19]

[0312] The results of increasing the starting amount of myrcene are presented in Table 20. Increasing the starting amount of myrcene was found to enhance the amount of loaded drug in the NPs to approximately 5% drug loading when 100% (w / w, myrcene vs PLGA, 40 mg myrcene and 40 mg PLGA) myrcene was used. The amount of loaded myrcene is slightly higher when the nanoparticles are recovered by centrifugation in Falcon tubes at 10,000 rpm compared to those recovered by Amicon tubes at 4,000×g.

[0313] [Table 20]

[0314] Comparison of generation methods Once the myrcene loss during lyophilization was confirmed, we repeated the preparation of NPs using the three preparation methods previously described herein to select the best method for optimal drug loading. To select the method that provided the highest myrcene loading efficiency and amount, the myrcene content in the NPs was measured after centrifugation but not before lyophilization.

[0315] NPs were prepared according to the formulas presented in Table 21 using high-speed homogenizer-induced emulsification as described in Example 3, nanoprecipitation as described in Example 1, and microemulsion as described in Example 2. After centrifugation of the preparations, the NPs were resuspended in a total volume of 1 mL MQ water. Samples were prepared for GC-MS analysis as previously described. 100 μL of each NP suspension was transferred into an Eppendorf tube and centrifuged at 10,000 rpm at 4° C. for 30 minutes. The supernatant was discarded and 1 mL of DCM was added to dissolve the NPs and myrcene. The DCM solution was then diluted and analyzed for myrcene content using a GC-MS instrument as previously described. Samples were prepared in duplicate.

[0316] [Table 21]

[0317] As demonstrated in Table 22, emulsification using homogenizer resulted in the highest drug loading with EE% of 9%, especially after centrifugation using Falcon tube. Nanoprecipitation also produced NPs with relatively good loading, but the maximum starting myrcene concentration was 60%, because higher concentrations promoted NP aggregation. In contrast, microemulsion gave NPs with poor drug loading. In both homogenizer emulsification and microemulsion methods, liquid drug is expected to be incorporated inside the NP, resulting in so-called nanocapsules, which are nanoparticles with a liquid core surrounded by a polymer shell. In the nanoprecipitation method, solid NPs known as nanospheres, which are polymer matrices in which the drug is dispersed, are produced. In the case of liquid myrcene, a higher drug loading can be expected in comparison with nanospheres, when it is the loading in nanocapsules. This can explain the higher drug loading achieved by homogenizer emulsification and can suggest that the formula used in microemulsion preparation may need optimization to produce stable particles with the ability to incorporate drugs.

[0318] [Table 22]

[0319] Use of cryoprotectants to stabilize nanoparticles It was believed that myrcene-loaded NPs were damaged during freezing and freeze-drying, resulting in increased drug loss during the process of freeze-drying. To test the effect of the cryoprotectant trehalose on stabilizing the NPs, samples were prepared using the two centrifugation methods described in section 5.8.2 and the emulsification method as previously described. 50 μL of NP suspension was mixed with 50 μL of 10% trehalose solution (to obtain a final mixture of 5% trehalose). The product was frozen and subsequently freeze-dried for 2 hours. The dried product was then dissolved in DCM and analyzed for myrcene content using a GC-MS instrument as previously described. Samples were prepared in duplicate. The results of the assay are shown in Table 23.

[0320] [Table 23]

[0321] conclusion Myrcene was found to be lost during lyophilization. Measurement of myrcene in NP suspensions before lyophilization indicates that the best method of preparation is emulsification using a high-speed homogenizer. This method can produce nanocapsules that incorporate liquid myrcene in the core. Optimization of lyophilization conditions, including duration and addition of cryoprotectants, resulted in optimal conditions for enhancing drug loading. Factors such as centrifugation speed and freezing can cause damage to NPs and contribute significantly to the loss of myrcene during lyophilization. The duration of lyophilization also affects myrcene loss.

[0322] NPs prepared by emulsification using a homogenizer, centrifuged at 4,000×g in Amicon tubes, cryoprotected with 5% trehalose, and lyophilized for 2 h successfully demonstrated the highest drug loading among the lyophilized NPs, approximately 7%. These conditions allow the production of lyophilized NPs with relatively high drug loading, up to 8% when 100% (w / w, myrcene to PLGA, 40 mg myrcene and 40 mg PLGA) myrcene was used.

[0323] [Example 8] Synthesis and characterization of nanoparticles containing β-myrcene, β-caryophyllene, or nerolidol This example illustrates the synthesis and characterization of PLGA-PEG nanoparticles containing β-myrcene, β-caryophyllene, or nerolidol.

[0324] β-Myrcene, β-caryophyllene and nerolidol-containing PLGA-PEG or poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) nanoparticles were prepared by an emulsification and solvent evaporation method using a high-speed homogenizer (Kinematica Polytron™ PT 2500E homogenizer, Fisher Scientific). The method is based on emulsification of a polymer organic solution in an aqueous phase followed by organic solvent evaporation, as illustrated in FIG. 3. The organic phase is poured into a continuous or aqueous phase, where a surfactant is dissolved to provide stability to the emulsion. Emulsification is carried out under high shear forces to reduce the size of the emulsion droplets. This process mainly determines the final particle size of the nanoparticles. This emulsification step is followed by evaporation of the solvent under reduced pressure to obtain the desired nanoparticles.

[0325] Synthesis of terpenoid-containing PLGA-PEG nanoparticles The formulas of blank and terpenoid-containing PLGA-PEG nanoparticles as prepared using the emulsification method are summarized in Table 24.

[0326] [Table 24]

[0327] In the container, 40 mg of PLGA-PEG (PEG average M n is 2,000, and the average M of PLGA nOrganic phase solutions were obtained by dissolving the terpenoids (11,500 for 100 mL, 50:50 for lactide:glycolide) in 1 mL of ethyl acetate followed by the addition of 10 mg of either myrcene, β-caryophyllene or nerolidol (based on 12.5% ​​w / w PLGA weight). In a separate Falcon tube containing 5-mL PVA (0.5% w / v) and the homogenizer probe, the terpenoid-containing organic solution was added dropwise using a pipette over 1 min under homogenization conditions operating at 24,000 rpm and 0° C. The organic solvent was then removed using a rotary evaporator without heating for at least 30 min to obtain a mixture containing terpenoid-containing nanoparticles. To remove unencapsulated terpenoids and residual surfactant (PVA), the mixture was diluted with 10 mL of deionized and purified water, transferred to an Amicon tube (Ultracel-100 kDa regenerated cellulose membrane, 15 mL sample volume) and evaporated under centrifugation conditions (4,000 x g, 12 °C for 30 min), this process was repeated a total of three times. The resulting nanoparticle concentrated suspension was further mixed with 1 mL of trehalose solution (10 w / v) as a mixture with at least 1 mL of deionized and purified water, and the resulting mixture was lyophilized or freeze-dried at -80 °C and < 0.100 mbar (TESLAR Cryodos). The lyophilized terpenoid-containing nanoparticles were collected as a white cotton-like solid. For each terpenoid, three samples of nanoparticles were prepared in triplicate for further testing and study.

[0328] Sample preparation for GC-MS analysis For GC-MS analysis and characterization, the terpenoid-containing nanoparticle lyophilisates were dissolved in 1 mL of dichloromethane (DCM) and the resulting mixture was sonicated for at least 5 minutes. The samples were then centrifuged for 4 minutes to precipitate undissolved materials, such as trehalose and traces of PVA surfactant. From the supernatant, either 0.25 or 0.5 mL of this solution was then diluted with dichloromethane (DCM) to a final volume of 2 mL, of which 1.5 mL of the final solution was transferred into a suitable vial for GC-MS analysis.

[0329] Average diameter and size distribution The average diameter, size distribution, zeta potential, weight, and yield of terpenoid-loaded PLGA-PEG NPs prepared by emulsification are presented in Table 25. Encapsulation of terpenoids inside the nanoparticles resulted in an increase in particle size. This increase in particle size was more significant for β-caryophyllene and nerolidol-containing nanoparticles, which had particle sizes of approximately 350 and 323 nm, respectively, when compared to myrcene-containing nanoparticles, which had a diameter of 266 nm.

[0330] [Table 25]

[0331] Although the inclusion of terpenoids increased the polydispersity of the nanoparticles, the polydispersity index (PDI) was <0.3 for all nanoparticles. The nanoparticles furthermore exhibited a negative surface charge with a measured zeta potential of approximately -35 mV. Furthermore, between 77 and 92% of the starting material was recovered as measured by gravimetry.

[0332] Scanning Electron Microscope (SEM) Images SEM images confirmed the spherical structure of the terpenoid-containing nanoparticles as illustrated in Figures 13A-B (myrcene), Figures 14A-B (β-caryophyllene) and Figures 15A-B (nerolidol). Furthermore, similar structure and size distribution of the nanoparticles was observed regardless of which terpenoid was encapsulated. As illustrated in Figures 13A and 15B, ruptured large nanoparticles could be detected, providing evidence that the terpenoid-containing nanoparticles were of the core-shell nanocapsule class as shown in Figure 16. One possible explanation is that the nanoparticles ruptured while under vacuum during sample preparation for SEM imaging.

[0333] Gas chromatography-mass spectrometry (GC-MS) analysis Using the GC-MS method and conditions as described herein, the retention times for myrcene (Figure 4A), β-caryophyllene (Figure 5A), cis-nerolidol (Figure 6), and trans-nerolidol (Figure 7) were measured to be 6.93, 5.72, 6.77, and 7.16 minutes, respectively, and their mass spectra are shown in Figures 4B, 5B, 6A, and 6C, respectively. The calibration curves for myrcene (Figure 4C) and β-caryophyllene (Figure 5C) in the concentration range of 1 to 60 ppm were linear, with r values ​​of 0.9990 and 0.9993, respectively. 2 The ratio of cis to trans isomers of nerolidol was found to be 1.02:0.98, or approximately 1:1, and the calibration curves for the cis (FIG. 7B) and trans (FIG. 7D) isomers of nerolidol within the concentration range of 0.5 to 30 ppm were linear, with r values ​​of 0.9998 and 0.9997, respectively. 2 was the value.

[0334] Terpenoids Encapsulated The results of terpenoid encapsulation are presented in Table 26. The starting mass for all terpenoids was 10 mg. The amount of encapsulated myrcene was 1.8 mg, with EE=18.1% and DL=4.7%, likely due to its low boiling point and volatile nature. Both β-caryophyllene and nerolidol showed higher encapsulation amounts of 6.3 (EE=64.9% and DL=15.1%) and 5.7 mg (EE=55.7% and DL=13.9%), respectively. The increased encapsulation efficiency of β-caryophyllene and nerolidol compared to myrcene may be due to the increased diameter of the respective terpenoid-containing nanoparticles, as summarized in Table 25.

[0335] [Table 26]

[0336] [Example 9] Calcium responses in HEK TRPV1 cells induced by terpenoid-loaded PLGA nanoparticles This example illustrates the investigation of the ability of terpenoid-containing PLGA-PEG or poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) nanoparticles (NPs) to improve the interaction of encapsulated species with the TRPV1 receptor as an in vitro assessment of their potential use in pain management.

[0337] A calcium signaling assay was used to assess ion influx into HEK TRPV1 cells. Cells were dispersed in 1 mM calcium assay buffer and intracellular calcium levels were measured using a plate reader in the presence of Fluo-4 as a fluorophore. Fluo-4 is a Ca 2+ It exhibits increased fluorescence upon binding of ions and is a potent signaling regulator of intracellular Ca in living cells. 2+ The concentration of terpenoids was measured using a 1-hour experiment to provide sufficient time for the encapsulated terpenoids to be released from the nanoparticles. The fluorescence changes in response to free terpenoids, encapsulated terpenoids, and combinations of free and encapsulated terpenoids were detected and compared.

[0338] Non-encapsulated versus encapsulated individual terpenoids As shown in Figure 17, all non-encapsulated terpenoids induced a calcium response, with free myrcene giving the strongest response, followed by free nerolidol and free β-caryophyllene. Surprisingly, all nanoparticle formulations containing terpenoids significantly increased the intensity of fluorescence in comparison to the non-encapsulated terpenoids in bulk solution. Both free and encapsulated β-caryophyllene exhibited the lowest calcium response compared to the rest (Figure 18C), while nerolidol demonstrated a significant but delayed increase in its calcium response at TRPV1 when encapsulated inside the nanoparticles compared to in bulk solution (Figure 18B). The calcium responses of free and encapsulated myrcene are shown in Figure 18A.

[0339] Combination of non-encapsulated terpenoids The calcium response in HEK TRPV1 cells to combinations of non-encapsulated terpenoids was also investigated. A total of four combinations were performed, namely myrcene plus nerolidol (FIG. 19A), myrcene plus β-caryophyllene (FIG. 19B), nerolidol plus β-caryophyllene (FIG. 19C), and myrcene plus nerolidol plus β-caryophyllene (FIG. 19D), using a concentration of 40 μg / ml of each terpenoid. Surprisingly, all combinations of non-encapsulated terpenoids improved calcium influx in comparison to the use of the individual terpenoids in bulk solution.

[0340] Combination of different terpene-loaded nanoparticles Combinations of various populations of nanoparticles, each population of nanoparticles encapsulating a different terpenoid, were also tested and compared to treatment with a single population of nanoparticles, at a concentration of 40 μg / ml of each terpenoid. As illustrated by Figures 20A-20D, combinations of different populations of nanoparticles led to higher and improved calcium responses when compared to single populations of nanoparticles containing individual terpenoids.

[0341] Encapsulated terpenoid combinations compared to non-encapsulated terpenoid combinations Finally, terpenoid-encapsulated nanoparticle combinations were tested and compared to non-encapsulated terpenoid combinations at a concentration of 40 μg / ml of each terpenoid. Across all combinations tested, myrcene plus nerolidol (FIG. 21A), myrcene plus β-caryophyllene (FIG. 21B), nerolidol plus β-caryophyllene (FIG. 21C), and myrcene plus β-caryophyllene plus nerolidol (FIG. 21D), the terpenoid-encapsulated nanoparticle combinations led to greatly improved calcium responses when compared to the non-encapsulated terpenoid combinations.

[0342] conclusion Using the calcium signaling assay described herein, calcium responses of HEK TRPV1 cells were examined in the presence of free terpenoids in bulk solution, terpenoid-containing PLGA-PEG nanoparticles, and combinations of both non-encapsulated and nanoparticle-encapsulated terpenoids. As demonstrated in Figures 17, 18A-18C, 19A-19D, 20A-20D, 21A-21D and described above, terpenoid combinations improved calcium responses regardless of encapsulation within nanoparticles. More importantly, terpenoid-containing nanoparticles, whether administered individually or in combination, were found to generate higher and improved calcium cellular influx when compared to non-encapsulated terpenoids in bulk solution. These data and results provide strong evidence that encapsulation of terpenoids within nanoparticles significantly improves the in vitro pharmacological activity of said terpenoids. Furthermore, these results highlight the advantages of formulations and pharmaceutical compositions containing terpenoid combinations for their use in future in vivo studies and as potential therapeutics.

[0343] While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

[0344] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety and for all purposes.

[0345] It will be understood that when one aspect of the invention is depicted as a method of treating a disease or condition comprising administering to a subject a pharmaceutical composition of the invention, this is also intended to encompass the pharmaceutical composition of the invention for use in treating said disease or condition.

Claims

1. A terpenoid-encapsulated PLGA nanoparticle comprising a PLGA nanoparticle and a first terpenoid encapsulated in the PLGA nanoparticle.

2. The terpenoid-encapsulated PLGA nanoparticle of claim 1, wherein the PLGA nanoparticle comprises a PLGA copolymer and the weight ratio of the first terpenoid to the PLGA copolymer is between 1:50 and 1:

4.

3. The terpenoid-encapsulated PLGA nanoparticle of claim 2, wherein the weight ratio of the first terpenoid to the PLGA copolymer is between 1:25 and 1:

5.

4. The terpenoid-encapsulated PLGA nanoparticle of any one of claims 1 to 3, wherein the first terpenoid is selected from the group consisting of myrcene, β-caryophyllene and nerolidol.

5. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the first terpenoid is myrcene and the weight ratio of the first terpenoid to the PLGA copolymer is about 1:

22.

6. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the first terpenoid is β-caryophyllene, the weight ratio of the first terpenoid to the PLGA copolymer is between 1:5 and 1:7, and optionally the nanoparticle further comprises PEG.

7. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the first terpenoid is nerolidol, the weight ratio of the first terpenoid to the PLGA copolymer is between 1:5 and 1:7, and optionally the nanoparticle further comprises PEG.

8. The terpenoid-encapsulated PLGA nanoparticle of any one of claims 1 to 7, wherein the nanoparticle further encapsulates at least a second compound, the second encapsulated compound being (i) a cannabinoid or (ii) a second terpenoid other than the first terpenoid.

9. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the second encapsulated compound is cannabigerolic acid (CBGA).

10. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the second encapsulated compound is cannabidiol (CBD).

11. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the second encapsulated compound is cannabinol (CBN).

12. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the second encapsulated compound is cannabidivarin (CBDV).

13. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the second encapsulated compound is cannabichromene (CBC).

14. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the second encapsulated compound is cannabidiolic acid (CBDA).

15. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the second encapsulated compound is cannabigerol (CBG).

16. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the second encapsulated compound is myrcene, β-caryophyllene or nerolidol.

17. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the second encapsulated compound is limonene.

18. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the second encapsulation compound is phytol.

19. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the second encapsulated compound is pinene.

20. The terpenoid-encapsulated PLGA nanoparticle of claim 4, wherein the second encapsulated compound is linalool.

21. The terpenoid-encapsulated PLGA nanoparticles of any one of claims 1 to 20, wherein the PLGA nanoparticles have an average diameter between 200 and 350 nm.

22. A terpenoid-encapsulated PLGA nanoparticle according to any one of claims 1 to 21, wherein the PLGA nanoparticle comprises a PLGA copolymer having a ratio of lactic acid to glycolic acid of about 10-90% lactic acid to about 90-10% glycolic acid.

23. The terpenoid-encapsulated LGA nanoparticle of claim 22, wherein the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid.

24. The terpenoid-encapsulated LGA nanoparticle of claim 22, wherein the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid.

25. The terpenoid-encapsulated PLGA nanoparticle of claim 22, wherein the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid.

26. The terpenoid-encapsulated PLGA nanoparticle of claim 22, wherein the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid.

27. The terpenoid-encapsulated PLGA nanoparticle of claim 22, wherein the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

28. 28. A pharmaceutical composition comprising a first population of terpenoid-encapsulated PLGA nanoparticles according to any one of claims 1 to 27, and a pharma- ceutically acceptable carrier or diluent.

29. 29. The pharmaceutical composition of claim 28, further comprising trehalose.

30. 30. The pharmaceutical composition of any one of claims 28 to 29, which is lyophilized.

31. 31. The pharmaceutical composition of any one of claims 28 to 30, further comprising a second population of PLGA nanoparticles, the second population of nanoparticles encapsulating a third compound, the third compound being a cannabinoid or terpenoid different from the cannabinoid and terpenoid encapsulated in the first population of nanoparticles.

32. 32. The pharmaceutical composition of claim 31, wherein the third inclusion compound is cannabigerolic acid (CBGA).

33. 32. The pharmaceutical composition of claim 31 , wherein the third encapsulation compound is cannabidiol (CBD).

34. 32. The pharmaceutical composition of claim 31, wherein the third encapsulated compound is cannabinol (CBN).

35. 32. The pharmaceutical composition of claim 31, wherein the third encapsulated compound is cannabidivarin (CBDV).

36. 32. The pharmaceutical composition of claim 31 , wherein the third inclusion compound is cannabichromene (CBC).

37. 32. The pharmaceutical composition of claim 31, wherein the third encapsulation compound is cannabidiolic acid (CBDA).

38. 32. The pharmaceutical composition of claim 31 , wherein the third inclusion compound is cannabigerol (CBG).

39. 32. The pharmaceutical composition of claim 31 , wherein the third inclusion compound is myrcene, β-caryophyllene or nerolidol.

40. 32. The pharmaceutical composition of claim 31 , wherein the third inclusion compound is limonene.

41. 32. The pharmaceutical composition of claim 31 , wherein the third inclusion compound is phytol.

42. 32. The pharmaceutical composition of claim 31 , wherein the third inclusion compound is pinene.

43. 32. The pharmaceutical composition of claim 31, wherein the third inclusion compound is linalool.

44. 44. The pharmaceutical composition of any one of claims 31 to 43, further comprising a third population of PLGA nanoparticles, the third population of nanoparticles encapsulating a fourth compound, the fourth compound being a cannabinoid or terpenoid different from the cannabinoid and terpenoid encapsulated in the first population of nanoparticles or the second population of nanoparticles.

45. 45. The pharmaceutical composition of claim 44, further comprising a fourth population of PLGA nanoparticles, the fourth population of nanoparticles encapsulating a fifth compound, the fifth compound being a cannabinoid or terpenoid different from the cannabinoid and terpenoid encapsulated in the first population of nanoparticles, the second population of nanoparticles or the third population of nanoparticles.

46. A method for obtaining terpenoid-encapsulated PLGA nanoparticles, comprising: (a) providing an organic solution comprising a terpenoid, a PLGA copolymer and a solvent, and an aqueous solution comprising a surfactant; (b) emulsifying the two solutions to form a suspension of terpenoid-loaded PLGA nanoparticles; (c) evaporating the solvent from the emulsion; and (d) Obtaining terpenoid-encapsulated PLGA nanoparticles The method includes:

47. 47. The method of claim 46, wherein the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is from about 1:5 to about 1:

1.

48. 48. The method of claim 47, wherein the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1:

5.

49. 48. The method of claim 47, wherein the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1:

4.

50. 48. The method of claim 47, wherein the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1:

3.

51. 48. The method of claim 47, wherein the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1:

2.

52. 48. The method of claim 47, wherein the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1:

1.

53. 53. The method of any one of claims 46 to 52, wherein the incorporation efficiency of the first terpenoid in step (b) is between about 4% and about 10%.

54. 54. The method of claim 53, wherein the incorporation efficiency is at least 4%.

55. 54. The method of claim 53, wherein the incorporation efficiency is at least 5%.

56. 54. The method of claim 53, wherein the incorporation efficiency is at least 6%.

57. 54. The method of claim 53, wherein the incorporation efficiency is at least 7%.

58. 54. The method of claim 53, wherein the incorporation efficiency is at least 8%.

59. 54. The method of claim 53, wherein the incorporation efficiency is at least 9%.

60. 54. The method of claim 53, wherein the incorporation efficiency is at least 10%.

61. 61. The method of any one of claims 46 to 60, wherein the average weight ratio of encapsulated terpenoid to PLGA copolymer in the terpenoid-encapsulated PLGA nanoparticles of step (d) is between about 1:50 and about 1:

10.

62. The method of claim 61, wherein the average weight ratio of encapsulated terpenoid to PLGA copolymer in the terpenoid-encapsulated PLGA nanoparticles is at least about 1:

50.

63. The method of claim 61, wherein the average weight ratio of encapsulated terpenoid to PLGA copolymer in the terpenoid-encapsulated PLGA nanoparticles is at least about 1:

40.

64. The method of claim 61, wherein the average weight ratio of encapsulated terpenoid to PLGA copolymer in the terpenoid-encapsulated PLGA nanoparticles is at least about 1:

30.

65. The method of claim 61, wherein the average weight ratio of encapsulated terpenoid to PLGA copolymer in the terpenoid-encapsulated PLGA nanoparticles is at least about 1:

20.

66. The method of claim 61, wherein the average weight ratio between the encapsulated terpenoid and the PLGA copolymer in the terpenoid-encapsulated PLGA nanoparticles is at least about 1:

10.

67. 67. The method of any one of claims 46 to 66, wherein the PLGA copolymer has a ratio of lactic acid to glycolic acid that is about 10-90% lactic acid to about 90-10% glycolic acid.

68. 68. The method of claim 67, wherein the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid.

69. 68. The method of claim 67, wherein the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid.

70. 68. The method of claim 67, wherein the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid.

71. 68. The method of claim 67, wherein the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid.

72. 68. The method of claim 67, wherein the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

73. 73. The method of any one of claims 46 to 72, wherein the solution of step (a) further comprises at least one cannabinoid or terpenoid other than the first terpenoid.

74. 74. The method of claim 73, wherein the at least one cannabinoid or terpenoid is selected from the group consisting of cannabidiol (CBD), cannabinol (CBN), cannabidivarin (CBDV), cannabichromene (CBC), cannabidiolic acid (CBDA), and cannabigerol (CBG).

75. 74. The method of claim 73, wherein the at least one cannabinoid or terpenoid is selected from the group consisting of myrcene, beta-caryophyllene, nerolidol, phytol, limonene, linalool and pinene.

76. 76. The process according to any one of claims 46 to 75, wherein the solvent is acetone, dichloromethane or ethyl acetate.

77. 77. The method of any one of claims 46 to 76, wherein the surfactant is polyethylene glycol, poloxamer or polyvinyl alcohol (PVA).

78. 78. The method of claim 77, wherein the surfactant is polyvinyl alcohol (PVA).

79. 79. The method of any one of claims 46 to 78, wherein the emulsifying step comprises homogenization or sonication.

80. 80. The method of claim 79, wherein the emulsifying step is homogenization.

81. 81. The method of claim 80, wherein the homogenization is carried out at 20,000 to 30,000 rpm.

82. 81. The method of claim 80, wherein the homogenization is carried out at 24,000 rpm.

83. 83. The method of any one of claims 80 to 82, wherein the solution is homogenized for between 30 seconds and 10 minutes.

84. 84. The method of claim 83, wherein the solution is homogenized for 1 minute.

85. 85. The method of any one of claims 46 to 84, wherein the step of evaporating the solvent comprises at least one of stirring the solvent, applying a gas flow, applying heat, maintaining a cold temperature of 10°C, or creating a vacuum.

86. 86. The method of claim 85, wherein the step of evaporating the solvent comprises stirring the suspension at room temperature.

87. 87. The method of claim 86, wherein the suspension is stirred for between 5 and 120 minutes to evaporate the solvent.

88. 88. The method of claim 87, wherein the suspension is stirred for 60 minutes.

89. 89. The method of any one of claims 46 to 88, wherein the step of obtaining terpenoid-encapsulated PLGA nanoparticles comprises centrifugation, filtration, or centrifugation and filtration.

90. 90. The method of claim 89, wherein the obtaining step comprises centrifugation.

91. 91. The method of claim 90, wherein the centrifugation is performed at between 2,000 x g and 15,000 x g.

92. 92. The method of claim 91, wherein the centrifugation is at 4,000 x g.

93. 93. The method of any one of claims 46 to 92, further comprising a subsequent step of adding a cryoprotectant to the terpenoid-encapsulated PLGA nanoparticles.

94. 94. The method of claim 93, wherein the cryoprotectant is trehalose.

95. 95. The method of any one of claims 93 to 94, wherein the cryoprotectant is added in an amount of 1 to 10% (w / v) of the terpenoid-encapsulated PLGA nanoparticles.

96. The method of claim 95, wherein the cryoprotectant is added in an amount of 5% (w / v) of the terpenoid-encapsulated PLGA nanoparticles.

97. 97. The method of any one of claims 44 to 96, further comprising freeze-drying the terpenoid-encapsulated PLGA nanoparticles.

98. 98. Terpenoid-encapsulated PLGA nanoparticles obtainable by the method according to any one of claims 46 to 97.

99. A pharmaceutical composition described in any one of claims 28 to 45 for use in desensitizing TRPV1 in cells of a mammalian subject, administered to a mammalian subject in an amount, by a route of administration and for a time sufficient to cause TRPV1 desensitization in cells within the mammalian subject.

100. 100. The pharmaceutical composition for use according to claim 99, wherein the cell is a nociceptor.

101. 101. The pharmaceutical composition for use according to claim 100, wherein the nociceptor is a peripheral nociceptor.

102. 101. The pharmaceutical composition for use according to claim 100, wherein the nociceptor is a visceral nociceptor.

103. 103. The pharmaceutical composition for use according to any one of claims 99 to 102, wherein the pharmaceutical composition is administered orally.

104. 103. A pharmaceutical composition for use according to any one of claims 99 to 102, which is administered topically.

105. 103. The pharmaceutical composition for use according to any one of claims 99 to 102, which is administered systemically.

106. 103. A pharmaceutical composition for use according to any one of claims 99 to 102, which is administered intravenously.

107. 103. A pharmaceutical composition for use according to any one of claims 99 to 102, which is administered subcutaneously.

108. 103. A pharmaceutical composition for use according to any one of claims 99 to 102, to be administered by inhalation.

109. 46. ​​A pharmaceutical composition according to any one of claims 28 to 45, for use in treating pain in a mammalian subject, administered to the subject in an amount, by a route of administration and for a time sufficient to cause TRPV1 desensitization in nociceptors within the subject.

110. 110. The pharmaceutical composition for use according to claim 109, wherein the nociceptor is a peripheral nociceptor and the pharmaceutical composition is administered locally.

111. 110. The pharmaceutical composition for use according to claim 109, wherein the nociceptor is a visceral nociceptor and the pharmaceutical composition is administered systemically.

112. 112. A pharmaceutical composition for use according to any one of claims 109 to 111, wherein the pain is neuropathic pain.

113. 113. The pharmaceutical composition for use according to claim 112, wherein the neuropathic pain is diabetic peripheral neuropathic pain.

114. 112. The pharmaceutical composition for use according to any one of claims 109 to 111, wherein the pain is post-herpetic neuralgia.

115. 115. The pharmaceutical composition for use according to any one of claims 109 to 114, which is administered at least once daily for at least three days.

116. 116. The pharmaceutical composition for use according to claim 115, which is administered at least once daily for at least five days.

117. 116. The pharmaceutical composition for use according to claim 115, which is administered at least once daily for at least 7 days.

118. A pharmaceutical composition for use according to any one of claims 109 to 117, administered at a dose, route of administration and schedule sufficient to maintain levels of the first terpenoid in the nociceptor for at least three days that are effective in desensitizing TRPV1 in the nociceptor.

119. The pharmaceutical composition for use according to claim 118, administered at a dose, route of administration and schedule sufficient to maintain levels of the first terpenoid in the nociceptor for at least 5 days that are effective in desensitizing TRPV1 in the nociceptor.

120. The pharmaceutical composition for use according to claim 118, administered at a dose, route of administration and schedule sufficient to maintain levels of the first terpenoid in the nociceptor for at least 7 days that are effective in desensitizing TRPV1 in the nociceptor.

121. 46. ​​A pharmaceutical composition according to any one of claims 28 to 45 for use in the treatment of cardiac hypertrophy in a mammalian subject, wherein an anti-hypertrophic effective amount is administered to the subject.

122. 122. A pharmaceutical composition for use according to claim 121, which is administered orally.

123. 122. The pharmaceutical composition for use according to claim 121, which is administered systemically.

124. 122. The pharmaceutical composition for use according to claim 121, which is administered intravenously.

125. 122. The pharmaceutical composition for use according to claim 121, which is administered subcutaneously.

126. 122. The pharmaceutical composition for use according to claim 121, which is administered by inhalation.

127. 122. A pharmaceutical composition for use according to claim 121, which is administered orally.

128. 46. ​​The pharmaceutical composition according to any one of claims 28 to 45, for use in the prophylactic treatment of cardiac hypertrophy in a mammalian subject, wherein an antihypertrophic effective amount is administered to a subject at risk of cardiac hypertrophy.

129. 46. ​​A pharmaceutical composition according to any one of claims 28 to 45 for use in the treatment of overactive bladder in a mammalian subject, wherein a therapeutically effective amount is administered to the subject.

130. 130. The pharmaceutical composition for use according to claim 129, which is administered systemically.

131. 130. The pharmaceutical composition for use according to claim 129, which is administered by bladder irrigation.

132. 46. ​​A pharmaceutical composition according to any one of claims 28 to 45 for use in the treatment of refractory chronic cough in a mammalian subject, wherein a therapeutically effective amount is administered to the subject.

133. 133. The pharmaceutical composition for use according to claim 132, which is administered systemically.

134. 133. The pharmaceutical composition for use according to claim 132, which is administered by inhalation.

135. A cannabinoid-encapsulated PLGA nanoparticle comprising a PLGA nanoparticle and a first cannabinoid encapsulated in the PLGA nanoparticle.

136. A cannabinoid-encapsulated PLGA nanoparticle as described in claim 135, comprising a PLGA copolymer, wherein the weight ratio of the first cannabinoid to the PLGA copolymer is between 1:50 and 1:

4.

137. A cannabinoid-encapsulated PLGA nanoparticle as described in claim 136, wherein the weight ratio of the first cannabinoid to the PLGA copolymer is between 1:25 and 1:

5.

138. 138. A cannabinoid-encapsulated PLGA nanoparticle as claimed in any one of claims 135 to 137, wherein the first cannabinoid is selected from the group consisting of cannabidiol, cannabidivarin, cannabinol, cannabigerol and cannabichromene.

139. The cannabinoid-encapsulated PLGA nanoparticle of claim 138, wherein the first cannabinoid is cannabidiol and the weight ratio of the first cannabinoid to the PLGA copolymer is about 1:

14.

140. The cannabinoid-encapsulated PLGA nanoparticle of claim 138, wherein the first cannabinoid is cannabidiol and the weight ratio of the first cannabinoid to the PLGA copolymer is between 1:5 and 1:

7.

141. The cannabinoid-encapsulated PLGA nanoparticle of claim 138, wherein the first cannabinoid is cannabidiol and the weight ratio of the first cannabinoid to the PLGA copolymer is between 1:5 and 1:

7.

142. 142. A cannabinoid-encapsulated PLGA nanoparticle as described in any one of claims 135 to 141, wherein the nanoparticle further encapsulates at least a second compound, the second encapsulated compound being (i) a terpenoid or (ii) a second cannabinoid other than the first cannabinoid.

143. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulated compound is cannabigerolic acid (CBGA).

144. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulated compound is cannabidivarin (CBV).

145. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulated compound is cannabinol (CBN).

146. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulated compound is cannabidivarin (CBDV).

147. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulated compound is cannabichromene (CBC).

148. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulated compound is cannabidiolic acid (CBDA).

149. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulated compound is cannabigerol (CBG).

150. 143. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulated compound is myrcene, beta-caryophyllene or nerolidol.

151. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulated compound is limonene.

152. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulation compound is phytol.

153. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulated compound is pinene.

154. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulated compound is linalool.

155. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulation compound is myrcene.

156. 156. A cannabinoid-encapsulated PLGA nanoparticle according to any one of claims 135 to 155, having an average diameter between 200 and 350 nm.

157. 157. A cannabinoid-encapsulated PLGA nanoparticle as described in any one of claims 135 to 156, comprising a PLGA copolymer having a ratio of lactic acid to glycolic acid that is about 10-90% lactic acid to about 90-10% glycolic acid.

158. The cannabinoid-encapsulated PLGA nanoparticle of claim 157, wherein the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid.

159. The cannabinoid-encapsulated PLGA nanoparticle of claim 157, wherein the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid.

160. Cannabinoid-encapsulated PLGA nanoparticles as described in claim 157, wherein the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid.

161. The cannabinoid-encapsulated PLGA nanoparticle of claim 157, wherein the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid.

162. The cannabinoid-encapsulated PLGA nanoparticle of claim 157, wherein the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

163. 163. A pharmaceutical composition comprising a first population of cannabinoid-encapsulated PLGA nanoparticles according to any one of claims 135 to 162 and a pharma- ceutically acceptable carrier or diluent.

164. 164. The pharmaceutical composition of claim 163, further comprising trehalose.

165. 165. The pharmaceutical composition of any one of claims 163 to 164, which is lyophilized.

166. 166. The pharmaceutical composition of any one of claims 163 to 165, further comprising a second population of PLGA nanoparticles, the second population of nanoparticles encapsulating a third compound, the third compound being a terpenoid or cannabinoid different from the terpenoid and cannabinoid encapsulated in the first population of nanoparticles.

167. The pharmaceutical composition of claim 166, wherein the third encapsulated compound is cannabigerolic acid (CBGA).

168. The pharmaceutical composition of claim 166, wherein the third encapsulated compound is cannabidiol (CBD).

169. The pharmaceutical composition of claim 166, wherein the third encapsulated compound is cannabinol (CBN).

170. The pharmaceutical composition of claim 166, wherein the third encapsulated compound is cannabidivarin (CBDV).

171. The pharmaceutical composition of claim 166, wherein the third inclusion compound is cannabichromene (CBC).

172. The pharmaceutical composition of claim 166, wherein the third encapsulated compound is cannabidiolic acid (CBDA).

173. The pharmaceutical composition of claim 166, wherein the third inclusion compound is cannabigerol (CBG).

174. 167. The pharmaceutical composition of claim 166, wherein the third inclusion compound is myrcene, beta-caryophyllene or nerolidol.

175. The pharmaceutical composition of claim 166, wherein the third inclusion compound is limonene.

176. 167. The pharmaceutical composition of claim 166, wherein the third inclusion compound is phytol.

177. 167. The pharmaceutical composition of claim 166, wherein the third inclusion compound is pinene.

178. The pharmaceutical composition of claim 166, wherein the third inclusion compound is linalool.

179. The pharmaceutical composition of claim 166, wherein the third inclusion compound is myrcene.

180. 180. The pharmaceutical composition of any one of claims 163 to 179, further comprising a third population of PLGA nanoparticles, the third population of nanoparticles encapsulating a fourth compound, the fourth compound being a cannabinoid or terpenoid different from the cannabinoid and terpenoid encapsulated in the first population of nanoparticles or the second population of nanoparticles.

181. 181. The pharmaceutical composition of claim 180, further comprising a fourth population of PLGA nanoparticles, wherein the fourth population of nanoparticles encapsulates a fifth compound, the fifth compound being a cannabinoid or terpenoid different from the cannabinoid and terpenoid encapsulated in the first population of nanoparticles, the second population of nanoparticles, or the third population of nanoparticles.

182. A method for obtaining cannabinoid-loaded PLGA nanoparticles, comprising: (a) providing an organic solution comprising a first cannabinoid, a PLGA copolymer and a solvent, and an aqueous solution comprising a surfactant; (b) emulsifying the two solutions to form a suspension of cannabinoid-loaded PLGA nanoparticles; (c) evaporating the solvent from the emulsion; and (d) Obtaining cannabinoid-loaded PLGA nanoparticles The method includes:

183. 183. The method of claim 182, wherein the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is from about 1:5 to about 1:

1.

184. The method of claim 182, wherein the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1:

5.

185. The method of claim 182, wherein the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1:

4.

186. The method of claim 182, wherein the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1:

3.

187. The method of claim 182, wherein the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1:

2.

188. The method of claim 182, wherein the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1:

1.

189. 189. The method of any one of claims 182 to 188, wherein the incorporation efficiency of the first cannabinoid in step (b) is between about 4% and about 10%.

190. 189. The method of claim 189, wherein the incorporation efficiency is at least 4%.

191. 189. The method of claim 189, wherein the incorporation efficiency is at least 5%.

192. 189. The method of claim 189, wherein the incorporation efficiency is at least 6%.

193. 189. The method of claim 189, wherein the incorporation efficiency is at least 7%.

194. 189. The method of claim 189, wherein the incorporation efficiency is at least 8%.

195. 189. The method of claim 189, wherein the incorporation efficiency is at least 9%.

196. 189. The method of claim 189, wherein the incorporation efficiency is at least 10%.

197. 197. A method according to any one of claims 182 to 196, wherein the average weight ratio of encapsulated cannabinoid to PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles of step (d) is between about 1:50 and about 1:

10.

198. The method of claim 197, wherein the average weight ratio of encapsulated cannabinoid to PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1:

50.

199. The method of claim 197, wherein the average weight ratio of encapsulated cannabinoid to PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1:

40.

200. The method of claim 197, wherein the average weight ratio of encapsulated cannabinoid to PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1:

30.

201. The method of claim 197, wherein the average weight ratio of encapsulated cannabinoid to PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1:

20.

202. The method of claim 197, wherein the average weight ratio of encapsulated cannabinoid to PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1:

10.

203. 203. The method of any one of claims 182-202, wherein the PLGA copolymer has a ratio of lactic acid to glycolic acid that is about 10-90% lactic acid to about 90-10% glycolic acid.

204. 204. The method of claim 203, wherein the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid.

205. 204. The method of claim 203, wherein the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid.

206. 204. The method of claim 203, wherein the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid.

207. 204. The method of claim 203, wherein the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid.

208. 204. The method of claim 203, wherein the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

209. 209. The method of any one of claims 182 to 208, wherein the solution of step (a) further comprises at least one cannabinoid or terpenoid other than the first cannabinoid.

210. 209. The method of claim 209, wherein the at least one cannabinoid or terpenoid is selected from the group consisting of cannabinol (CBN), cannabidivarin (CBDV), cannabichromene (CBC), cannabidiolic acid (CBDA) and cannabigerol (CBG).

211. 210. The method of any one of claims 208 to 209, wherein the at least one cannabinoid or terpenoid is selected from the group consisting of myrcene, beta-caryophyllene, nerolidol, phytol, limonene, linalool and pinene.

212. 212. The method of any one of claims 182 to 211, wherein the solvent is acetone, dichloromethane or ethyl acetate.

213. 213. The method of any one of claims 182 to 212, wherein the surfactant is polyethylene glycol, poloxamer or polyvinyl alcohol (PVA).

214. 214. The method of claim 213, wherein the surfactant is polyvinyl alcohol (PVA).

215. 215. The method of any one of claims 182 to 214, wherein the emulsifying step comprises homogenization or sonication.

216. 216. The method of claim 215, wherein the emulsifying step is homogenization.

217. 217. The method of claim 216, wherein the homogenization is carried out at 20,000 to 30,000 rpm.

218. 218. The method of claim 217, wherein the homogenization is carried out at 24,000 rpm.

219. 219. The method of any one of claims 216 to 218, wherein the solution is homogenized for between 30 seconds and 10 minutes.

220. 220. The method of claim 219, wherein the solution is homogenized for 1 minute.

221. 221. The method of any one of claims 182 to 220, wherein the step of evaporating the solvent comprises at least one of stirring the solvent, applying a gas flow, applying heat, maintaining a cold temperature of 10°C, or creating a vacuum.

222. 222. The method of claim 221, wherein the step of evaporating the solvent comprises stirring the suspension at room temperature.

223. 223. The method of claim 222, wherein the suspension is stirred for between 5 and 120 minutes to evaporate the solvent.

224. 224. The method of claim 223, wherein the suspension is stirred for 60 minutes.

225. 225. A method according to any one of claims 182 to 224, wherein the step of obtaining cannabinoid-encapsulated PLGA nanoparticles comprises centrifugation, filtration, or centrifugation and filtration.

226. 226. The method of claim 225, wherein the obtaining step includes centrifugation.

227. 227. The method of claim 226, wherein centrifugation is performed at between 2,000 x g and 15,000 x g.

228. The method of claim 224, wherein the centrifugation is at 4,000 x g.

229. 229. The method of any one of claims 182 to 228, further comprising the subsequent step of adding a cryoprotectant to the cannabinoid-encapsulated PLGA nanoparticles.

230. 230. The method of claim 229, wherein the cryoprotectant is trehalose.

231. 231. A method according to any one of claims 229 to 230, wherein the cryoprotectant is added in an amount of 1 to 10% (w / v) of the cannabinoid-encapsulated PLGA nanoparticles.

232. The method of claim 231, wherein the cryoprotectant is added in an amount of 5% (w / v) of the cannabinoid-encapsulated PLGA nanoparticles.

233. 233. The method of any one of claims 182 to 232, further comprising freeze-drying the cannabinoid-encapsulated PLGA nanoparticles.

234. 234. Cannabinoid-loaded PLGA nanoparticles obtainable by the method according to any one of claims 182 to 233.

235. A pharmaceutical composition described in any one of claims 163 to 181 for use in desensitizing TRPV1 in cells of a mammalian subject, administered to a mammalian subject in an amount, by a route of administration and for a time sufficient to cause TRPV1 desensitization in cells within the mammalian subject.

236. 236. The pharmaceutical composition for use according to claim 235, wherein the cell is a nociceptor.

237. 237. The pharmaceutical composition for use according to claim 236, wherein the nociceptor is a peripheral nociceptor.

238. 237. The pharmaceutical composition for use according to claim 236, wherein the nociceptor is a visceral nociceptor.

239. 239. A pharmaceutical composition for use according to any one of claims 235 to 238, which is administered orally.

240. 239. A pharmaceutical composition for use according to any one of claims 235 to 238, which is administered topically.

241. 239. The pharmaceutical composition for use according to any one of claims 235 to 238, which is administered systemically.

242. 239. The pharmaceutical composition for use according to any one of claims 235 to 238, which is administered intravenously.

243. 239. A pharmaceutical composition for use according to any one of claims 235 to 238, which is administered subcutaneously.

244. 239. A pharmaceutical composition for use according to any one of claims 235 to 238, administered by inhalation.

245. 182. A pharmaceutical composition according to any one of claims 163 to 181 for use in treating pain in a mammalian subject, administered to the subject in an amount, by a route of administration and for a time sufficient to cause TRPV1 desensitization in nociceptors in the subject.

246. 246. The pharmaceutical composition for use according to claim 245, wherein the nociceptor is a peripheral nociceptor and the pharmaceutical composition is administered locally.

247. 246. The pharmaceutical composition for use according to claim 245, wherein the nociceptor is a visceral nociceptor and the pharmaceutical composition is administered systemically.

248. 248. A pharmaceutical composition for use according to any one of claims 245 to 247, wherein the pain is neuropathic pain.

249. 249. The pharmaceutical composition for use according to claim 248, wherein the neuropathic pain is diabetic peripheral neuropathic pain.

250. 248. The pharmaceutical composition for use according to any one of claims 245 to 247, wherein the pain is post-herpetic neuralgia.

251. 251. The pharmaceutical composition for use according to any one of claims 245 to 250, administered at least once daily for at least three days.

252. 252. The pharmaceutical composition for use according to claim 251, administered at least once daily for at least five days.

253. 253. The pharmaceutical composition for use according to claim 252, administered at least once daily for at least 7 days.

254. 254. The pharmaceutical composition for use according to claim 253, administered at least once daily for more than 7 days.

255. A pharmaceutical composition for use as described in any one of claims 245 to 254, administered at a dose, route of administration and schedule sufficient to maintain cannabinoid levels in the nociceptor for at least three days that are effective in desensitizing TRPV1 in the nociceptor.

256. A pharmaceutical composition for use as described in claim 255, administered at a dose, route of administration and schedule sufficient to maintain cannabinoid levels in the nociceptor for at least 5 days that are effective in desensitizing TRPV1 in the nociceptor.

257. A pharmaceutical composition for use as described in claim 256, administered at a dose, route of administration and schedule sufficient to maintain cannabinoid levels in the nociceptor for at least 7 days that are effective in desensitizing TRPV1 in the nociceptor.

258. 182. A pharmaceutical composition according to any one of claims 163 to 181 for use in the treatment of cardiac hypertrophy in a mammalian subject, wherein an anti-hypertrophic effective amount is administered to the subject.

259. 259. A pharmaceutical composition for use according to claim 258, which is administered orally.

260. 259. The pharmaceutical composition for use according to claim 258, which is administered systemically.

261. 259. The pharmaceutical composition for use according to claim 258, which is administered intravenously.

262. 259. The pharmaceutical composition for use according to claim 258, which is administered subcutaneously.

263. 259. The pharmaceutical composition for use according to claim 258, administered by inhalation.

264. 259. A pharmaceutical composition for use according to claim 258, which is administered orally.

265. 182. The pharmaceutical composition of any one of claims 163 to 181 for use in the preventive treatment of cardiac hypertrophy in a mammalian subject, wherein an anti-hypertrophy effective amount is administered to a subject at risk of cardiac hypertrophy.

266. 182. A pharmaceutical composition according to any one of claims 163 to 181 for use in treating overactive bladder in a mammalian subject, wherein a therapeutically effective amount is administered to the subject.

267. 267. The pharmaceutical composition for use according to claim 266, which is administered systemically.

268. 267. The pharmaceutical composition for use according to claim 266, administered by bladder irrigation.

269. 182. A pharmaceutical composition according to any one of claims 163 to 181 for use in the treatment of refractory chronic cough in a mammalian subject, wherein a therapeutically effective amount is administered to the subject.

270. 270. The pharmaceutical composition for use according to claim 269, which is administered systemically.

271. 270. The pharmaceutical composition for use according to claim 269, administered by inhalation.

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