Patent Application
20210348204
The current inventive technology relates to the chemical modification of plant derived terpenes, and in particular the in vivo bioconversion of Cannabis-derived terpenes into water-soluble terpene glycosides.
Terpenes, which may be used interchangeably with the term terpenoids, are a large and diverse class of naturally occurring organic chemicals. Terpenes are essential for plant metabolism, influencing general development, herbivory defense, pollination and stress response. These compounds have been extensively used as flavoring and scenting agents in cosmetics, detergents, food and pharmaceutical products. They also display multiple biological activities in humans, such as anti-inflammatory, anti-microbial, antifungal and antiviral.
Cannabis terpenes profiles define the aroma of each plant and share the same precursor (geranyl pyrophosphate) and the same synthesis location (glandular trichomes) as phytocannabinoids. While over 400 terpenoids have been identified in Cannabis, a few of the most common include: limonene, myrcene, alpha-pinene, linalool, beta-caryophyllene, caryophyllene oxide, nerol and alpha-terpineol.
As shown in
As highlighted in table 6, a survey of the terpenoid profiles of several Cannabis varieties. Additional studies indicate that these terpenoids express at high enough levels to potentially have their own pharmacological effects and also to act in synergy with cannabinoids. The biochemical and phenomenological differences between different varieties of Cannabis, which are attributed to their unique relative cannabinoid and terpene ratios is known as the “entourage effect,” and is generally considered to result in plants providing advantages over only using the natural products that are isolated from them. These advantages include synergy with THC, the primary active ingredient, and also mitigation of side effects from THC.
However, terpenes are generally insoluble in water making them difficult to use in various consumer products and formulations that require water-solubility. On strategy to solubilize terpenes is through glycosylation which involves chemically modifying metabolites with glucose molecules. Glycosylation and is generally carried out by the activity of UDP-glycosyltransferase (UGTs) enzymes. glycosyltransferases are UGTs which display high activity against monoterpenes, leading to the accumulation of monoterpene glycosides. Some UGTs display a large range of acceptors, such as the grape VvGT14 and VvGT7, which glycosylate geraniol, citronellol, and nerol, among other compounds. These promiscuous terpene UGTs could be incorporated into systems for the enzymatic conversion of Cannabis terpenes into terpene glycosides either in planta by means of integrating these genes into the plant genome or expressing them into cell cultures (yeast, tobacco, hops) that could be used for post-harvest processing of full-spectrum Cannabis extracts.
The glycosylation of terpenes in an in vivo system may enable enhanced accumulation, storage and transport of hydrophilic substances. Moreover, the glycosylation of terpenes may lead to higher solubility in water, reduced volatility, and lesser reactivity, which could improve pharmacokinetic parameters and therefore, could be used as a method for pro-drug preparations. Importantly, the glycosyl moieties can be subsequently cleaved by naturally occurring glycosidases, regenerating the parent terpene compound without generation of harmful metabolites to humans.
The glycosylation of terpenes would also enable the simultaneous water-based extraction of terpene glycosides and/or cannabinoid glycosides. This co-glycosylation may result in an enhanced reconstitution of the compounds found in Cannabis extracts, known to produce the entourage effect. In fact, many animal studies indicate that the pharmacological potential of cannabinoids combined with certain terpenes is superior to cannabinoids alone. Alternatively, the glycosylation of terpenes would also enable the generation of a number of compositions that might incorporate one or more glycosylated terpene, in some instances with one or more cannabinoids or glycosylated cannabinoids.
As detailed below, the present inventors have developed novel systems, methods, and compositions for glycosylating Cannabis derived terpenes in yeast and tobacco cell suspension cultures (BY2), as well as an in planta system using transgenic Cannabis or hemp plants. In certain preferred embodiment, cell suspension cultures are contained, sterile and can be scaled up in large-fermenters. Based on the reduced retention time on an HPLC gradient from 85% water to 85% acetonitrile, the present inventors show that the terpene glycosides produced in these novel systems elute earlier than their non-modified forms and are therefore more water-soluble. The present inventors have identified multiple glycosyltransferases with activity against diverse terpenes naturally present in different Cannabis cultivars.
In one preferred embodiment discussed below, a Cannabis extract, which preferably may include a “full spectrum” Cannabis extract containing both cannabinoids and terpenes may be introduced into one of the systems described herein for the production of water-soluble terpenes/terpene along with cannabinoids, preserving the “entourage effect.” Moreover, some of the enzymes with activity against terpenes can also glycosylate cannabinoids (such as NtGT3 and NtGT4), making the heterologous expression in transgenic hemp a novel strategy to generate a water-soluble extract that better represents the full spectrum of compounds observed in Cannabis plants, which would encompass hemp plants. Consumer product incorporating water-soluble cannabinoids and terpenes could have a more predictable onset time and could incorporate full, or approximately full Cannabis extracts, as opposed to purified cannabinoids, preserving the desired “entourage effect.”
One aspect of the current inventive technology may encompass systems, methods and compositions for the in vivo production, modification and isolation of terpene compounds from Cannabis or other plants. In particular, the invention provides systems and methods for high level in vivo biosynthesis of water-soluble terpene compounds. In one preferred embodiment, the present invention generally relates to the conversion of Cannabis sativa or hemp terpenes into water-soluble compounds (terpene glycosides). In one preferred aspect, the invention may include system for glycosylating Cannabis derived terpenes in yeast and tobacco cell suspension cultures (BY2), as well as an in planta system using transgenic Cannabis or hemp plants.
Another aspect of the invention may include the production of terpene glycosides in multiple platforms, such as in planta or in plant cell suspension cultures. In this embodiment, such terpene glycosides may be generated in vivo by over-expressing certain glycosyltransferases (UGTs) displaying terpene glycosyltransferase activity. In this aspect, such water-soluble terpenes a may be generated in vivo by over-expressing certain exogenous and/or endogenous genes that may chemically modify said terpenes, preferably by glycosylation. In a preferred embodiment, such glycosylated terpene and cannabinoids compounds may be isolated and further included in one or more compositions as generally described herein.
Another aspect of the invention may include the production of terpene glycosides in multiple platforms, such as cultures of microorganism. In this embodiment, such terpene glycosides may be generated in vivo by over-expressing certain glycosyltransferases (UGTs) displaying terpene glycosyltransferase activity in yeast and adding one or more terpenes, or Cannabis or hemp extracts to the yeast culture wherein one or more terpenes may be bio-converted into a water-soluble terpene glycoside. Another embodiment is removal of the glycosyl moiety from the glycosylated terpenes using glycosidases to regenerate the parent compound.
Another aspect of the current inventive technology includes systems and methods for enhanced production and/or accumulation of terpene glycosides. In one embodiment, the invention may include systems and methods for enhanced production and/or accumulation of terpene glycosides in an in vivo system, such as a plant, or plant cell culture. Another aspect of the current inventive technology includes systems and methods for enhanced production of terpene glycosides in a yeast culture system. Another aspect of the current inventive technology includes systems and methods for enhanced production of terpene glycosides in a bacterial culture system. Another aspect of the current inventive technology includes systems and methods for enhanced production of terpene glycosides in a fungal culture system.
Another aspect of the current invention may include the generation of genetically modified plants overexpressing certain endogenous/exogenous genes that result in the production of terpene glycosides. Another aspect of the current inventive technology includes the generation of genetically modified yeast, or other eukaryotic/prokaryotic cells that overexpress certain endogenous/exogenous genes that result in the production of terpene glycosides. Additional aspects of this invention may include polynucleotide constructs that may be used to transform plant, bacterial, and/or yeast cell to express one or more endogenous/exogenous genes that result in the production of terpene glycosides. In certain systems, substrate terpenes may be endogenously produced, or added to the in vivo system.
Another aspect of the current invention may include the application of one or more glycosidases or other enzymes that may remove the sugar group from a terpene glycoside reconstituting it back to the parent terpene compound.
Another aim of the current inventive technology may include the generation of one or more of the above referenced genetically modified plant or plant cell cultures utilizing Agrobacterium Ti-plasmid mediated transformation. Another aim of the current inventive technology may include the generation of one or more of the above referenced genetically modified cells, such as a plant cell, a yeast cell, a bacterial cell, or a fungi cell, for example through techniques such as viral, electroporation, biolistic, and glass bead transformation techniques among others.
Additional aspects of the invention may include delivery systems and compositions that include water-soluble terpenes, preferably water-soluble glycosylated terpenes. Additional embodiments may further include methods and systems for the production of compositions that include water-soluble terpenes, preferably water-soluble glycosylated terpenes. Additional embodiments may include the isolation of these water-soluble terpenes followed by enzymatic conversion or reconstitution to their original or parent compound.
Another aspect of the current invention may include systems, methods and compositions for the delivery of water-soluble terpenes, preferably glycosylated and/or acetylated glycoside terpenes as a prodrug. Included in this invention may include novel prodrug compositions. Additional aspects may include one or more compositions of matter that may include water-soluble terpenes and cannabinoids, preferably glycosylated and/or acetylated glycoside terpenes and cannabinoids. In certain aspects, such compositions may include beverages and/or food additives that may impart an “entourage effect” on the user through the synergistic action of said terpenes and cannabinoids profiles present in the composition(s).
Additional aspects include isolated nucleotides sequences encoding one or more UGTs operably linked to a promoter including: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); UGT94P1 (SEQ ID NO. 7); NtGt3 (SEQ ID NO. 11); NtGt4 (SEQ ID NO. 13); Bs-YjiC (SEQ ID NO. 29); and VvGT14 (SEQ ID NO. 31), and homologous sequences thereof. Additional aspects include isolated nucleotides sequences encoding one or more UGTs operably linked to a promoter including amino acid sequences: UGT58A1 (SEQ ID NO. 2); MhGt1 (SEQ ID NO. 4); VvGt7 (SEQ ID NO. 10); UGT85K11 (SEQ ID NO. 6); UGT94P1 (SEQ ID NO. 8); NtGt3 (SEQ ID NO. 12); NtGt4 (SEQ ID NO. 14); Bs-YjiC (SEQ ID NO. 30); and VvGT14 (SEQ ID NO. 32), and homologs thereof.
Another aspect of the invention includes systems, methods and compositions for the in vivo formulation of terpene glycosides and/or cannabinoid glycosides. In this preferred aspect, UGTs may be selectively expressed in one or more of the in vivo systems described herein, such as an in planta, yeast, or cell culture system. Such selectively expressed UGTs may have glycosylation activity towards some terpenes or cannabinoids, and not others. By selectively co-expressing combinations of UGTs in an in vivo system, the water-solubilized glycosylated compounds may be selectively removed allowing for isolation of selective formulations of water-soluble terpenes and/or cannabinoid or a mixture of the two. Such selective formulations of water-soluble terpenes and/or cannabinoid or a mixture of the two may be selected for flavor, texture, and taste among other attributes and may further be incorporated into one or more consumer products as described herein.
Another aspect of the invention may include the composition according to Formula I:
Another aspect of the invention may include the composition according to Formula II:
Additional embodiments may include the following preferred embodiments:
1. An in vivo method for producing water-soluble terpenes comprising the steps:
or
47. The method of embodiment 34 wherein the nucleotide sequence selected from the group consisting of: SEQ ID NO. 1; SEQ ID NO. 3; SEQ ID NO. 9; SEQ ID NO. 5; SEQ ID NO. 7; SEQ ID NO. 11; SEQ ID NO. 13; SEQ ID NO. 29; and SEQ ID NO. 31, is operably linked to a promoter to produce an expression vector and wherein said expression vector is configured to be introduced via transformation to a Cannabis sativa or hemp plant.
48. A genetically modified Cannabis sativa or hemp plant or part thereof produced by the method of embodiment 47.
49. A genetically modified Cannabis sativa or hemp cell or part thereof produced by the method of embodiment 47, wherein said Cannabis sativa or hemp cell is further used to generate a cell suspension culture.
50. An in vivo method for producing water-soluble terpenes comprising the steps of:
78. A compound having the chemical structure of Formula (II) or a prodrug, therapeutically active metabolite, hydrate, solvate, or pharmaceutically acceptable salt thereof:
79. A pharmaceutical composition comprising a compound of embodiment 77, or embodiment 78 and at least one pharmaceutically acceptable additive.
80. A pharmaceutical kit containing a pharmaceutical composition of embodiment 79, prescribing information for the composition, and a container.
81. A composition comprising a compound of embodiment 77, or embodiment 78, wherein the compound is co-administered to the subject with at least one cannabinoid.
82. A composition of embodiment 81 wherein said at least one cannabinoid comprises at least one glycosylated cannabinoid.
83. The compositions of embodiments 77, or embodiment 78, wherein said compositions are administered to a subject as pro-drugs.
84. At composition of embodiments 77, 78, and, 82, wherein said compositions are administered to the subject as pro-drugs.
Additional aspect will become apparent from the figures and discussion below.
Generally, the inventive technology relates to the field of chemical modification and isolation of Cannabis terpenes in planta, as well as in plant and yeast suspension cultures. The present inventive technology further relates to improved systems and methods for the modification and isolation of pharmaceutically active terpenes from plant materials. In one embodiment, the inventive technology may encompass a novel system for the generation of chemically modified terpene compounds in planta, as well as in plant and yeast suspension cultures. The inventive technology may include systems and methods for high-efficiency chemical modification and isolation of terpene compounds in planta, as well as in plant and yeast suspension cultures. In this embodiment, various select terpene compounds may be chemically modified into soluble configurations. In other embodiments, the invention may also include the coupled chemical modification of Cannabis derived cannabinoids in planta, as well as in plant and yeast suspension cultures as generally described in U.S. application Ser. Nos. 16/110,728, and 16/110,954. (Both of which are hereby incorporated in their entity by reference).
In one embodiment the current inventive technology includes improved systems and methods for the modification of terpenes in planta, or in a sterile yeast and/or plant culture system (such systems being generally referred to as in vivo systems). In other embodiments the current inventive technology includes improved systems and methods for the modification of terpenes in vitro. Such methods including, for example incubating one or more terpenes with a sugar substrate and a glycosyltransferase or other sufficiently promiscuous UGT that may catalyze the glycosylation of the subject terpene in vitro.
In one embodiment, the inventive technology may include the production of genetically modified plant, or a sterile yeast and/or plant cell suspension culture. The inventive technology may allow for certain transgenes to be introduced into these yeast and/or plant strains to transiently modify the chemical structure of the terpene compounds. This transient modification may render the terpene soluble in water. Such modifications may also alter the rate at which the terpenes are metabolized generating a modified terpene with enhanced kinetics that may be used in certain therapeutic applications or as a prodrug. These modified terpenes, aided by their modified chemical structure, may be allowed to accumulate at higher than native levels without having a deleterious effect on the cultured yeast and/or plant cells. Being soluble, such Cannabis derived terpenes may also be secreted through endogenous and/or exogenous ABC or other trans-membrane protein transporters into the culture medium for later harvesting and isolation. These transiently modified terpenes may further be harvested and isolated from the aforementioned in vivo systems, and then enzymatically restored to their original chemical structure. Other embodiments may allow for the regulation of terpene modification and isolation. In such embodiments, discreet and known amounts of terpenes may be introduced into a yeast and/or plant suspension culture and modified into a water-soluble form. Later, such modified terpenes may be extracted from the cell culture and isolated such that the quantity and relative ratios of the various terpenes is known and quantifiable. In this manner the isolated terpene extract may be chemically consistent and as such, easily dosable for both pharmaceutical and/or other commercial applications.
In more specific embodiments, the inventive technology may include systems and methods for the in vitro and more preferably in vivo chemical modification of terpene compounds. In one preferred embodiment, a suspension or hairy root or cell suspension culture may be established in a fermenter or other similar apparatus. It should be noted that the use of suspension culture may be broadly interpreted to include both Cannabis plant strains, such C. sativa as well as non-Cannabis plants such as tobacco plants among others. For example, in certain other embodiments, various Cannabis strains, mixes of strains, hybrids of different strains or clones, as well as different varieties may be used to generate a suspension or hairy root culture. In certain embodiments, cell cultures of terpene producing as well as non-terpene producing cell cultures may be established in a fermenter. Such fermenters may include large industrial-scale fermenters allowing for a large quantity of terpene producing C. sativa cells to be cultured. In this embodiment, it may be possible to culture a large quantity of unadulterated cells from a single-strain of, for example, tobacco or C. sativa, which may establish a cell culture having a consistent production and/or modification of terpene compounds in both quantity and type. Such cultured growth may be continuously sustained with the supplementation of nutrient and other growth factors to the culture. Such features may be automated or accomplished manually.
Another embodiment of the inventive technology may include systems and methods for the production of modified terpene compounds in non-Cannabis plants. In one embodiment, a suspension or hairy root culture of one or more tobacco plant strains may be established. It should be noted that the term strain may refer to a plant strain, as well as a cell culture, or cell line derived from a tobacco plant. In one preferred embodiment, a suspension or hairy root culture of Nicotiana benthamiana plant may be established in a fermenter or other similar apparatus. It should be noted that the use of N. benthamiana in this embodiment is exemplary only. For example, in certain other embodiments, various Nicotiana strains, mixes of strains, hybrids of different strains or clones, as well as different varieties may be used to generate a cell suspension or hairy root culture.
In one embodiment, a suspension culture of one or more yeast strains may be established. In one preferred embodiment, culture, and more preferably a suspension culture of Saccharomyces cerevisiae and/or Pichia pastoris or other suitable yeast species may be established in a fermenter or other similar apparatus. It should be noted that the use of the above identified example in this embodiment is exemplary only, as various yeast strains, mixes of strains, hybrids of different strains or clones may be used to generate a suspension culture. For example, Pichia pastoris or any other appropriate yeast strain, including but not limited to all strains of yeast deposited with the ATCC. (The yeast strain deposit database(s) being incorporated by reference in its entirety.) In certain cases, such fermenters may include large industrial-scale fermenters allowing for a large quantity of yeast cells to be grown. In this embodiment, it may be possible to culture a large quantity of cells from a single-strain of, for example, P. pastoris or K. marxianus, which may establish a cell culture having a consistent or predictable rate of terpene modification. Such cultured growth may be continuously sustained with the continual addition of nutrient and other growth factors being added to the culture. Such features may be automated or accomplished manually. As noted above, terpene producing strains of Cannabis, as well as other plants may be utilized with the inventive technology. In certain preferred embodiments, Cannabis plant material containing one or more terpenes of interest may be harvested and undergo extraction through one or more of the methods generally known in the art. These extracted terpenes may be introduced into genetically modified yeast or plant suspension cell culture to be further modified as described below.
The inventive technology may include the generation of transgenic plant and yeast strains having artificial genetic constructs that may express/overexpress one or more glycosyltransferases, or other enzymes capable of glycosylating terpene compounds. In one preferred embodiment, artificial genetic constructs having genes encoding one or more UDP- and/or ADP-glycosyltransferases, including non-human analogues of those described above, as well as other isoforms, may be expressed in a genetically modified plant/plant cell and/or yeast cells and may be grown in suspension or other cell cultures. Additional embodiments may include genetic control elements such as promotors and/or enhancers as well as post-transcriptional regulatory control elements that may also be expressed in a transgenic plant or cells such that the presence, quantity and activity of any glycosyltransferases present in the suspension culture may be regulated. Additional embodiments may include artificial genetic constructs having one or more genes encoding one or more UDP- and/or ADP-glycosyltransferases having tags that may assist in the movement of the gene product to a certain portion of the cell, such as the cellular locations were terpenes and/or glycosylated terpenes may be stored, and/or excreted from the cell.
In one specific preferred embodiment, the invention may include the generation of transgenic or genetically modified strains of Cannabis, or other plants such as tobacco, having artificial genetic constructs that may express one or more genes that may glycosylate terpenes. For example, the inventive technology may include the generation of transgenic plant strains or cell lines having artificial genetic constructs that may express one or more endogenous/or exogenous glycosyltransferases or other enzymes capable of glycosylating terpene compounds. For example, in one embodiment one or more glycosyltransferases from N. benthamiana, or other non-Cannabis plants may be introduced into a Cannabis plant or cell culture and configured to glycosylate terpenes in vivo. In other embodiments, endogenous glycosyltransferases from N. benthamiana may be over-expressed so to as to increase in vivo terpene glycosylation.
An additional embodiment of the invention may include artificial genetic constructs having one or more genes encoding one or more UDP- and/or ADP-glycosyltransferases being co-expressed with one or more exogenous genes that may assist in the movement of the protein to a certain portion of the cell, such as the cellular locations were terpenes and/or glycosylated terpenes may be stored, and/or excreted from the cell.
In yet another separate embodiment, the now soluble transiently modified terpenes may be passively and/or actively excreted from a plant or yeast cell. In one exemplary model, an ATP-binding cassette transporter (ABC transporters) or other similar molecular structure may recognize the functional group (conjugate) on the glycosylated terpenes, for example and actively transport it into the surrounding media. In this embodiment, an in-vivo cell culture may be allowed to grow until an output parameter is reached. In one example, an output parameter may include allowing the cell culture to grow until a desired cell/optical density is reached, or a desired level of modified terpenes is reached. In this embodiment, the culture media containing the modified, and preferably water-soluble glycosylated terpenes, may be harvested for later extraction and/or isolation. Additionally, the modified terpenes present in the raw and/or treated media may be isolated and purified, for example, through affinity chromatography in a manner similar to that described above. The term “recovering” may generally encompass extracting and/or isolating one or more compounds of interest, and in particular one or more glycosylated compounds of interest.
In certain embodiments, this purified isolate may contain a mixture of primary and secondary glycosylated terpenes. As noted above, such purified glycosylated terpenes may be water-soluble and metabolized slower than unmodified terpenes providing a slow-release capability that may be desirable in certain pharmaceutical applications, such as for use in tissue-specific applications or as a prodrug. In this embodiment, purified glycosylated terpenes may be incorporated into a variety of pharmaceutical and/or nutraceutical applications as described in greater detail below. For example, the purified glycosylated terpenes may be incorporated into various solid and/or liquid delivery vectors for use in pharmaceutical applications. Additional therapeutic applications may include the administration of a therapeutic dose of an “entourage” of isolated and purified glycosylated terpenes and glycosylated cannabinoids.
The inventive technology may also include a system to convert or reconstitute glycosylated terpenes. In one preferred embodiment, glycosylated terpenes may be converted into non-glycosylated terpenes through their treatment with one or more generalized or specific glycosidases. In this embodiment, these glycosidase enzymes may remove a sugar moiety. Specifically, these glycosidases may remove the glucuronic acid moiety reconstituting the terpene compound. In one embodiment, glycosylated terpenes may be isolated to generate a highly purified mixture post-glycosylation, which may further include, or be combined with a highly purified mixture of glycosylated cannabinoids. These reconstituted terpene compounds may also be incorporated into various solid and/or liquid delivery vectors for use in a variety of pharmaceutical and other commercial applications. In certain embodiments, modified terpenes may be reconstituted through incubation with one or more generalized or specific glycosidases in an in vitro system.
In one embodiment, a polynucleotide may be generated that expresses one or more of the SEQ ID NOs identified herein, or any SEQ ID NOs. incorporated specifically by reference from priority document U.S. Provisional Patent Application No. 62/746,053, filed Oct. 16, 2018. In certain preferred embodiments, the proteins of the invention may be expressed using any of a number of systems to obtain the desired quantities of the protein. Typically, the polynucleotide that encodes the protein or component thereof is placed under the control of a promoter that is functional in the desired host cell. An extremely wide variety of promoters may be available, and can be used in the expression vectors of the invention, depending on the particular application. Ordinarily, the promoter selected depends upon the cell in which the promoter is to be active. Other expression control sequences such as ribosome binding sites, transcription termination sites and the like are also optionally included. Constructs that include one or more of these control sequences are termed “expression cassettes” or “constructs.” Accordingly, the nucleic acids that encode the joined polypeptides are incorporated for high level expression in a desired host cell. Exemplary promotors may include, but not be limited to: a non-constitutive promotor; an inducible promotor, a tissue-preferred promotor; a tissue-specific promotor, a plant-specific promotor, or a constitutive promotor. In a preferred embodiment, one or more select genes may be operably linked to a leaf-specific gene promotor, such as Cab1. Additional promoters and operable configurations for expression, as well as co-expression of one or more of the selected genes are generally known in the art.
Another embodiment comprises a tissue culture comprising a plurality of the genetically altered plant cells.
Another embodiment provides for a method for constructing a genetically altered plant or part thereof having increased cannabinoid/terpene production compared to a non-genetically altered plant or part thereof, the method comprising the steps of: introducing a polynucleotide encoding a protein into a plant or part thereof to provide a genetically altered plant or part thereof, wherein said protein comprising at least one UGT having glycosylation activity towards one or more terpenes.
In one embodiment, the invention may include an in vivo production, accumulation and modification system that may generate water-soluble terpene. In one preferred embodiment, a plant, such as Cannabis or tobacco, may be genetically modified to express one or more heterologous glycosyltransferase genes, such as UDP glycosyltransferase. In this preferred embodiment, UDP glycosyltransferase (76G1) from Stevia rebaudiana may be expressed in terpene producing plant or cell suspension culture. In a preferred embodiment, the terpene producing plant or cell suspension culture may be Cannabis. In another embodiment, one or more glycosyltransferase from Nicotiana tabacum and/or a homologous glycosyltransferase from Nicotiana benthamiana, may be expressed in a terpene-producing plant, such as Cannabis, or may be over-expressed in an endogenous plant and/or plant cell culture system. In a preferred embodiment, a glycosyltransferase gene and/or protein may be selected from the exemplary plant, such as Nicotiana tabacum. Such glycosyltransferase gene and/or protein may include, but not limited to: Glycosyltransferase (NtGT5a) Nicotiana tabacum; Glycosyltransferase (NtGT5a) Nicotiana tabacum; Glycosyltransferase (NtGT5b) Nicotiana tabacum; Glycosyltransferase (NtGT5b) Nicotiana tabacum; UDP-glycosyltransferase 73C3 (NtGT4) Nicotiana tabacum; UDP-glycosyltransferase 73C3 (NtGT4) Nicotiana tabacum; Glycosyltransferase (NtGT1b) Nicotiana; Glycosyltransferase (NtGT1b) Nicotiana tabacum; Glycosyltransferase (NtGT1a) Nicotiana tabacum (Glycosyltransferase (NtGT1a) Nicotiana tabacum; Glycosyltransferase (NtGT3) Nicotiana tabacum; Glycosyltransferase (NtGT3) Nicotiana tabacum; Glycosyltransferase (NtGT2) Nicotiana tabacum; and/or Glycosyltransferase (NtGT2) Nicotiana tabacum. The sequences from Nicotiana tabacum are exemplary only as other tobacco and non-tobacco glycosyltransferase may be used. Such glycosyltransferase may be expressed in an in planta system, as well as an in vivo yeast or bacterial system as generally described herein. As noted above, such glycosyltransferases may glycosylate the terpene in a plant or cell culture system as generally described here. Naturally, other glycosyltransferase genes from alternative sources may be included in the current invention.
It should be noted that a number of combinations and permutations of the genes/proteins described herein may be co-expressed and thereby accomplish one or more of the goals of the current invention. Such combinations are exemplary of preferred embodiments only, and not limiting in any way.
In one preferred embodiment, the present inventors demonstrate the generation of a genetically modified plant configured to produce water-soluble, and preferably glycosylated terpenes. In one preferred embodiment a cannabinoid-producing plant, such as a Cannabis sativa plant, may be transformed to over-express one or more select UGTs. In one preferred embodiment, a Cannabis plant or cell may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: alpha-Pinene, Camphene, beta-Myrcene, beta-Pinene, delta-3-Carene, alpha-Terpine, Ocimene 1, d-Limonene, p-Cymene, Ocimene 2, Eucalyptol, gamma-Terpinene, Terpinoline, Linalool, Isolpulegol, Geraniol, beta-Caryophyllene, alpha-Humulene, Nerolidol 1, Nerolidol 2, Guaiol, Caryophyllene oxide, and alpha-Bisabolol.
In another preferred embodiment, a Cannabis plant or cell may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); UGT94P1 (SEQ ID NO. 7); NtGt3 (SEQ ID NO. 11); NtGt4 (SEQ ID NO. 13); Bs-YjiC (SEQ ID NO. 29); and VvGT14 (SEQ ID NO. 31). In this embodiment, one or more of the above UGTs may glycosylate at higher then wild-type levels, in vivo, one or more terpenes selected from the group consisting of: alpha-Pinene, Camphene, beta-Myrcene, beta-Pinene, delta-3-Carene, alpha-Terpine, Ocimene 1, d-Limonene, p-Cymene, Ocimene 2, Eucalyptol, gamma-Terpinene, Terpinoline, Linalool, Isolpulegol, Geraniol, beta-Caryophyllene, alpha-Humulene, Nerolidol 1, Nerolidol 2, Guaiol, Caryophyllene oxide, and alpha-Bisabolol.
In another preferred embodiment, a Cannabis plant or cell may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); UGT94P1 (SEQ ID NO. 7); NtGt3 (SEQ ID NO. 11); NtGt4 (SEQ ID NO. 13); Bs-YjiC (SEQ ID NO. 29); and VvGT14 (SEQ ID NO. 31), wherein at least one glycosylated terpene is produced, and preferably produced at higher than wild-type levels, selected from the group consisting of: alpha-Bisabolyl monoglucoside, alpha-Bisabolyl O-acetyl glucoside, alpha-Bisabolyl oxidized glucoside, alpha-Bisabolyl diglucoside, Citronellyl monoglucoside, Geranyl O-acetyl glucoside, Geranyl monoglucoside, Linalyl monoglucoside, Linalyl glucoside-xyloside, Linalyl diglucoside, Neryl monoglucoside, Neryl diglucoside and Terpineol diglucoside.
In another preferred embodiment, expression of one or more UGTs may further be in conjunction with the expression of a heterologous glycosyltransferase that has glycosylation activity directed to cannabinoids as well as terpenes. In this preferred embodiment, a Cannabis plant may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding at least one the following UGTs having glycosylation activity directed to cannabinoids as well as terpenes selected from the group consisting of: NtGt3 (SEQ ID NO. 11); and NtGt4 (SEQ ID NO. 13), in addition to the heterologous expression of one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); and UGT94P1 (SEQ ID NO. 7). Additional UGTs that have glycosylation activity towards cannabinoids are incorporated by reference from Sayre et al. in PCT/US18/24409.
In additional embodiments, multiple UGTs having activity toward different terpenes and/or cannabinoids may be co-expressed in one or more expression cassettes operably linked to a promoter generating an expression vector. Genes encoding one or more polynucleotides and/or a homologue thereof of the invention may be introduced into a plant, and/or plant cell, and preferably a Cannabis plant or cell, using several types of transformation approaches developed for the generation of transgenic plants. Standard transformation techniques, such as Ti-plasmid Agrobacterium-mediated transformation, particle bombardment, microinjection, and electroporation may be utilized to construct stably transformed transgenic plants.
In one preferred embodiment, the present inventors demonstrate the generation of a genetically modified yeast cell configured to produce water-soluble, and preferably glycosylated terpene compounds. In one preferred embodiment a genetically modified yeast cell, such as Saccharomyces cerevisiae, K. marxianus, and Pichia pastoris or other suitable yeast species may be transformed to express one or more heterologous UGTs having activity towards at least one terpene. Select, or full spectrum terpenes, as well as cannabinoid compounds generated by the Cannabis sativa plant may be harvested, for example as an oil or other extract and may fed to such a genetically modified yeast cell culture in a fermenter. Such terpenes, as well as cannabinoid compounds may be efficiently glycosylated through interaction with the overexpressed endogenous and/or heterologous glycosyltransferase enzymes. In this embodiment, the synthesis of water-soluble cannabinoids, terpenes and/or terpenes in the yeast cells may be harvested and isolated separately or together as a “full-spectrum” water-based extract. The post-harvest conversion of terpenes into terpene glycosides may allow their inclusion in one or more of the compositions described herein, or reconstituted to their original structure through the action of one or more glycosidases. Additional embodiments include the addition of one or more “glycosidase inhibitor” to a yeast culture which may inhibit glycosidase enzymes which catalyze the hydrolysis of glyosidic bonds.
In one preferred embodiment, a yeast cell may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: alpha-Pinene, Camphene, beta-Myrcene, beta-Pinene, delta-3-Carene, alpha-Terpine, Ocimene 1, d-Limonene, p-Cymene, Ocimene 2, Eucalyptol, gamma-Terpinene, Terpinoline, Linalool, Isolpulegol, Geraniol, beta-Caryophyllene, alpha-Humulene, Nerolidol 1, Nerolidol 2, Guaiol, Caryophyllene oxide, and alpha-Bisabolol.
In another preferred embodiment, a yeast cell may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); UGT94P1 (SEQ ID NO. 7); NtGt3 (SEQ ID NO. 11); NtGt4 (SEQ ID NO. 13); Bs-YjiC (SEQ ID NO. 29); and VvGT14 (SEQ ID NO. 31). In this embodiment, one or more of the above UGTs may glycosylate one or more terpenes selected from the group consisting of: alpha-Pinene, Camphene, beta-Myrcene, beta-Pinene, delta-3-Carene, alpha-Terpine, Ocimene 1, d-Limonene, p-Cymene, Ocimene 2, Eucalyptol, gamma-Terpinene, Terpinoline, Linalool, Isolpulegol, Geraniol, beta-Caryophyllene, alpha-Humulene, Nerolidol 1, Nerolidol 2, Guaiol, Caryophyllene oxide, and alpha-Bisabolol.
In another preferred embodiment, a yeast cell may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); UGT94P1 (SEQ ID NO. 7); NtGt3 (SEQ ID NO. 11); NtGt4 (SEQ ID NO. 13); Bs-YjiC (SEQ ID NO. 29); and VvGT14 (SEQ ID NO. 31), wherein at least one glycosylated terpene is selected from the group consisting of: alpha-Bisabolyl monoglucoside, alpha-Bisabolyl O-acetyl glucoside, alpha-Bisabolyl oxidized glucoside, alpha-Bisabolyl diglucoside, Citronellyl monoglucoside, Geranyl O-acetyl glucoside, Geranyl monoglucoside, Linalyl monoglucoside, Linalyl glucoside-xyloside, Linalyl diglucoside, Neryl monoglucoside, Neryl diglucoside and Terpineol diglucoside.
In another preferred embodiment, expression of one or more UGTs may further be in conjunction with the expression of a heterologous glycosyltransferase that has glycosylation activity directed to cannabinoids as well as terpenes. In this preferred embodiment, a yeast cell may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding at least one the following UGTs having glycosylation activity directed to cannabinoids as well as terpenes selected from the group consisting of: NtGt3 (SEQ ID NO. 11); and NtGt4 (SEQ ID NO. 13), in addition to the heterologous expression of one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); and UGT94P1 (SEQ ID NO. 7). Additional UGTs that have glycosylation activity towards cannabinoids are incorporated by reference from Sayre et al. in PCT/US18/24409.
In one preferred embodiment, the present inventors demonstrate the generation of a genetically modified non-cannabinoid producing plant cell culture configured to produce water-soluble, and preferably glycosylated terpenes. Examples of such non-cannabinoid producing plant cells may include, but not be limited to tobacco plant cells, Humulus lupulus (hops) plant cells, Eucalyptus perriniana, Mentha piperita, Averrhoa carambola, Ocimum basilicum, and Arabidopsis thaliana.
In one preferred embodiment a tobacco cell may be transformed to over-express one or more select UGTs that have activity towards one or more terpenes which may further be in conjunction with the endogenous glycosyltransferase from a tobacco plant that exhibits activity toward both cannabinoids and terpene compounds. In one preferred embodiments, a tobacco cell may be transformed to over-express one or more select UGTs that have activity towards one or more terpenes which may further be in conjunction with the endogenous glycosyltransferases NtGt3 (SEQ ID NO. 12); and NtGt4 (SEQ ID NO. 14) from a tobacco plant that exhibits activity toward both cannabinoids and terpene compounds. Select, or full spectrum terpenes, as well as cannabinoid compounds generated by the Cannabis sativa plant may be harvested, for example as an oil or other extract and may fed to such a genetically modified non-cannabinoid producing plant suspension cell culture, and preferably a tobacco suspension cell culture. Such terpenes, as well as cannabinoid compounds may be efficiently glycosylated through interaction with the endogenous and/or heterologous glycosyltransferase enzymes. In this embodiment, the synthesis of water-soluble cannabinoids and terpenes in the tobacco cell suspension cell culture may be harvested and isolated separately or together as a “full-spectrum” water-based extract. The post-harvest conversion of terpenes into terpene glycosides may allow their inclusion in one or more of the compositions described herein, or reconstituted to their original structure through the action of one or more glycosidases. Additional embodiments include the addition of one or more “glycosidase inhibitor” to a cell culture which may inhibit glycosidase enzymes which catalyze the hydrolysis of glyosidic bonds.
In one preferred embodiment, a tobacco cell may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: alpha-Pinene, Camphene, beta-Myrcene, beta-Pinene, delta-3-Carene, alpha-Terpine, Ocimene 1, d-Limonene, p-Cymene, Ocimene 2, Eucalyptol, gamma-Terpinene, Terpinoline, Linalool, Isolpulegol, Geraniol, beta-Caryophyllene, alpha-Humulene, Nerolidol 1, Nerolidol 2, Guaiol, Caryophyllene oxide, and alpha-Bisabolol.
In another preferred embodiment, a tobacco cell may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); UGT94P1 (SEQ ID NO. 7). In this embodiment, one or more of the above UGTs may glycosylate one or more terpenes selected from the group consisting of: alpha-Pinene, Camphene, beta-Myrcene, beta-Pinene, delta-3-Carene, alpha-Terpine, Ocimene 1, d-Limonene, p-Cymene, Ocimene 2, Eucalyptol, gamma-Terpinene, Terpinoline, Linalool, Isolpulegol, Geraniol, beta-Caryophyllene, alpha-Humulene, Nerolidol 1, Nerolidol 2, Guaiol, Caryophyllene oxide, and alpha-Bisabolol.
In another preferred embodiment, a tobacco cell may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); UGT94P1 (SEQ ID NO. 7). 13), wherein at least one glycosylated terpene is selected from the group consisting of: alpha-Bisabolyl monoglucoside, alpha-Bisabolyl O-acetyl glucoside, alpha-Bisabolyl oxidized glucoside, alpha-Bisabolyl diglucoside, Citronellyl monoglucoside, Geranyl O-acetyl glucoside, Geranyl monoglucoside, Linalyl monoglucoside, Linalyl glucoside-xyloside, Linalyl diglucoside, Neryl monoglucoside, Neryl diglucoside and Terpineol diglucoside.
In another preferred embodiment, expression in a tobacco cell, and preferably BY2 tobacco cells, of one or more UGTs may further be in conjunction with the expression of an endogenous glycosyltransferase that has glycosylation activity directed to cannabinoids as well as terpenes. In this preferred embodiment, a tobacco cell may express, or overexpress an endogenous nucleotide sequence, operably linked to a promoter, encoding at least one the following UGTs having glycosylation activity directed to cannabinoids as well as terpenes selected from the group consisting of: NtGt3 (SEQ ID NO. 11); and NtGt4 (SEQ ID NO. 13), in addition to the heterologous expression of one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); and UGT94P1 (SEQ ID NO. 7). Additional UGTs that have glycosylation activity towards cannabinoids are incorporated by reference from Sayre et al. in PCT/US18/24409.
In another preferred embodiment, in a Cannabis, or alternatively a non-cannabinoid producing cell, may express one or more endogenous glycosyltransferases that have activity toward one or more terpenes, and in some embodiments one or more terpenes and cannabinoids. In a preferred embodiment, cells expressing one or more endogenous glycosyltransferases having activity toward one or more terpenes may be established in a suspension cell culture. Select, or full spectrum terpenes, as well as cannabinoid compounds generated by the Cannabis sativa plant may be harvested, for example as an oil or other extract and may fed to such a genetically modified cell culture in a fermenter. Such terpenes, as well as cannabinoid compounds may be efficiently glycosylated through interaction with the endogenous glycosyltransferase enzymes. In this embodiment, the synthesis of water-soluble cannabinoids, terpenes and/or terpenes in the non-genetically modified cells may be harvested and isolated separately or together as a “full-spectrum” water-based extract. The post-harvest conversion of terpenes into terpene glycosides may allow their inclusion in one or more of the compositions described herein, or reconstituted to their original structure through the action of one or more glycosidases. Additional embodiments include the addition of one or more “glycosidase inhibitor” to a cell culture which may inhibit glycosidase enzymes which catalyze the hydrolysis of glyosidic bonds.
In another preferred embodiment, a non-genetically modified tobacco cell and preferably BY2 tobacco cells, expressing one or more endogenous glycosyltransferases such as NtGt3 (SEQ ID NO. 11), and/or NtGt4 (SEQ ID NO. 13) or a homolog thereof, may be established in a suspension cell culture. Select, or full spectrum terpenes, as well as cannabinoid compounds generated by the Cannabis sativa plant may be harvested, for example as an oil or other extract and may fed to such a genetically modified cell culture in a fermenter. Such terpenes, as well as cannabinoid compounds may be efficiently glycosylated through interaction with the endogenous glycosyltransferase enzymes NtGt3 (SEQ ID NO. 14), and/or NtGt4 (SEQ ID NO. 14). In this embodiment, the synthesis of water-soluble cannabinoids, terpenes and/or terpenes in the non-genetically modified tobacco cells may be harvested and isolated separately or together as a “full-spectrum” water-based extract. The post-harvest conversion of terpenes into terpene glycosides may allow their inclusion in one or more of the compositions described herein, or reconstituted to their original structure through the action of one or more glycosidases. Additional embodiments include the addition of one or more “glycosidase inhibitor” to a yeast culture which may inhibit glycosidase enzymes which catalyze the hydrolysis of glyosidic bonds.
The present inventors may demonstrate the generation of a mom-GMO plant and/or cell culture configured to produce water-soluble, and preferably converted to a water-soluble terpene glycosides utilizing endogenous UGTs in wild-type yeast, such as Pichia pastoris. In this embodiment, a wild-type cell suspension culture of yeast cells may be generated. Select, or full spectrum terpenes, as well as cannabinoid compounds generated by the Cannabis sativa plant may be harvested, for example as an oil or other extract and may fed to such a genetically modified cell culture in a fermenter. Such terpenes, as well as cannabinoid compounds may be efficiently glycosylated through interaction with the endogenous glycosyltransferase enzymes. In this embodiment, the synthesis of water-soluble cannabinoids, terpenes and/or terpenes in the non-genetically modified cells may be harvested and isolated separately or together as a “full-spectrum” water-based extract. The post-harvest conversion of terpenes into terpene glycosides may allow their inclusion in one or more of the compositions described herein, or reconstituted to their original structure through the action of one or more glycosidases. Additional embodiments include the addition of one or more “glycosidase inhibitor” to a yeast culture which may inhibit glycosidase enzymes which catalyze the hydrolysis of glyosidic bonds.
In one preferred embodiment, the present inventors demonstrate the generation of a genetically modified prokaryotic organism configured to produce water-soluble, and preferably glycosylated terpene compounds. In one preferred embodiment a genetically modified bacterial cell, or other suitable bacterial species may be transformed to express one or more heterologous UGTs having activity towards at least one terpene. Select, or full spectrum terpenes, as well as cannabinoid compounds generated by the Cannabis sativa plant may be harvested, for example as an oil or other extract and may fed to such a genetically modified bacterial cell culture in a fermenter. Such terpenes, as well as cannabinoid compounds may be efficiently glycosylated through interaction with the overexpressed endogenous and/or heterologous glycosyltransferase enzymes. In this embodiment, the synthesis of water-soluble cannabinoids, terpenes and/or terpenes in the bacterial cells may be harvested and isolated separately or together as a “full-spectrum” water-based extract. The post-harvest conversion of terpenes into terpene glycosides may allow their inclusion in one or more of the compositions described herein, or reconstituted to their original structure through the action of one or more glycosidases. Additional embodiments include the addition of one or more “glycosidase inhibitor” to a cell culture which may inhibit glycosidase enzymes which catalyze the hydrolysis of glyosidic bonds.
In one preferred embodiment, a bacterial cell may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: alpha-Pinene, Camphene, beta-Myrcene, beta-Pinene, delta-3-Carene, alpha-Terpine, Ocimene 1, d-Limonene, p-Cymene, Ocimene 2, Eucalyptol, gamma-Terpinene, Terpinoline, Linalool, Isolpulegol, Geraniol, beta-Caryophyllene, alpha-Humulene, Nerolidol 1, Nerolidol 2, Guaiol, Caryophyllene oxide, and alpha-Bisabolol.
In another preferred embodiment, a bacterial cell, such as a high-protein production species of E. coli, may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); UGT94P1 (SEQ ID NO. 7); NtGt3 (SEQ ID NO. 11); NtGt4 (SEQ ID NO. 13); Bs-YjiC (SEQ ID NO. 29); and VvGT14 (SEQ ID NO. 31). In this embodiment, one or more of the above UGTs may glycosylate one or more terpenes selected from the group consisting of: alpha-Pinene, Camphene, beta-Myrcene, beta-Pinene, delta-3-Carene, alpha-Terpine, Ocimene 1, d-Limonene, p-Cymene, Ocimene 2, Eucalyptol, gamma-Terpinene, Terpinoline, Linalool, Isolpulegol, Geraniol, beta-Caryophyllene, alpha-Humulene, Nerolidol 1, Nerolidol 2, Guaiol, Caryophyllene oxide, and alpha-Bisabolol.
In another preferred embodiment, a bacterial cell may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); UGT94P1 (SEQ ID NO. 7); NtGt3 (SEQ ID NO. 11); NtGt4 (SEQ ID NO. 13); Bs-YjiC (SEQ ID NO. 29); and VvGT14 (SEQ ID NO. 31), wherein at least one glycosylated terpene is selected from the group consisting of: alpha-Bisabolyl monoglucoside, alpha-Bisabolyl O-acetyl glucoside, alpha-Bisabolyl oxidized glucoside, alpha-Bisabolyl diglucoside, Citronellyl monoglucoside, Geranyl O-acetyl glucoside, Geranyl monoglucoside, Linalyl monoglucoside, Linalyl glucoside-xyloside, Linalyl diglucoside, Neryl monoglucoside, Neryl diglucoside and Terpineol diglucoside.
In another preferred embodiment, expression of one or more UGTs may further be in conjunction with the expression of a heterologous glycosyltransferase that has glycosylation activity directed to cannabinoids as well as terpenes. In this preferred embodiment, a bacterial cell may be genetically modified to express a heterologous nucleotide sequence, operably linked to a promoter, encoding at least one the following UGTs having glycosylation activity directed to cannabinoids as well as terpenes selected from the group consisting of: NtGt3 (SEQ ID NO. 11); and NtGt4 (SEQ ID NO. 13), in addition to the heterologous expression of one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); and UGT94P1 (SEQ ID NO. 7). Additional UGTs that have glycosylation activity towards cannabinoids are incorporated by reference from Sayre et al. in PCT/US18/24409.
In one preferred embodiment, the present inventors demonstrate the generation of water-soluble, and preferably glycosylated terpene compounds in an in vitro or ex vivo system, such as a bioreactor. In one preferred embodiment, one or more UGTs having activity towards at least one terpene may be introduced in vitro, or ex vivo, for example in a cell-free bioreactor, to a select, or full spectrum terpenes, as well as cannabinoid compounds generated by the Cannabis sativa or hemp plant. Such terpenes, as well as cannabinoid compounds may be efficiently glycosylated through interaction with the glycosyltransferase enzymes. In this embodiment, the in vitro or ex vivo synthesis of water-soluble cannabinoids, terpenes and/or terpenes may be harvested and isolated separately or together as a “full-spectrum” water-based extract. In certain embodiments, such in vitro, and ex vivo system may be supplemented with an appropriate reaction buffer, as well as UDP-glucose or other appropriate glycosylation substrate. The post-harvest conversion of terpenes into terpene glycosides may allow their inclusion in one or more of the compositions described herein, or reconstituted to their original structure through the action of one or more glycosidases.
In one preferred embodiment, one or more UGTs having in vitro or ex vivo glycosylation activity towards one or more terpenes may be selected from the group consisting of: alpha-Pinene, Camphene, beta-Myrcene, beta-Pinene, delta-3-Carene, alpha-Terpine, Ocimene 1, d-Limonene, p-Cymene, Ocimene 2, Eucalyptol, gamma-Terpinene, Terpinoline, Linalool, Isolpulegol, Geraniol, beta-Caryophyllene, alpha-Humulene, Nerolidol 1, Nerolidol 2, Guaiol, Caryophyllene oxide, and alpha-Bisabolol.
In another preferred embodiment, one or more UGTs having glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); UGT94P1 (SEQ ID NO. 7); NtGt3 (SEQ ID NO. 11); NtGt4 (SEQ ID NO. 13); Bs-YjiC (SEQ ID NO. 29); and VvGT14 (SEQ ID NO. 31). In this embodiment, one or more of the above UGTs may glycosylate one or more terpenes—in vitro or ex vivo—selected from the group consisting of: alpha-Pinene, Camphene, beta-Myrcene, beta-Pinene, delta-3-Carene, alpha-Terpine, Ocimene 1, d-Limonene, p-Cymene, Ocimene 2, Eucalyptol, gamma-Terpinene, Terpinoline, Linalool, Isolpulegol, Geraniol, beta-Caryophyllene, alpha-Humulene, Nerolidol 1, Nerolidol 2, Guaiol, Caryophyllene oxide, and alpha-Bisabolol.
In another preferred embodiment, one or more UGTs having in vitro or ex vivo glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); UGT94P1 (SEQ ID NO. 7); NtGt3 (SEQ ID NO. 11); NtGt4 (SEQ ID NO. 13); Bs-YjiC (SEQ ID NO. 29); and VvGT14 (SEQ ID NO. 31), wherein at least one glycosylated terpene is selected from the group consisting of: alpha-Bisabolyl monoglucoside, alpha-Bisabolyl O-acetyl glucoside, alpha-Bisabolyl oxidized glucoside, alpha-Bisabolyl diglucoside, Citronellyl monoglucoside, Geranyl O-acetyl glucoside, Geranyl monoglucoside, Linalyl monoglucoside, Linalyl glucoside-xyloside, Linalyl diglucoside, Neryl monoglucoside, Neryl diglucoside and Terpineol diglucoside.
In another preferred embodiment, one or more UGTs may further include glycosyltransferase that has in vitro or ex vivo glycosylation activity directed to cannabinoids as well as terpenes. Such UGTs having in vitro or ex vivo glycosylation activity directed to cannabinoids as well as terpenes selected from the group consisting of: NtGt3 (SEQ ID NO. 11); and NtGt4 (SEQ ID NO. 13), in addition to one or more UGTs having in vitro or ex vivo glycosylation activity towards one or more terpenes selected from the group consisting of: UGT58A1 (SEQ ID NO. 1); MhGt1 (SEQ ID NO. 3); VvGt7 (SEQ ID NO. 9); UGT85K11 (SEQ ID NO. 5); UGT94P1 (SEQ ID NO. 7), Bs-YjiC (SEQ ID NO. 29); and VvGT14 (SEQ ID NO. 31). Additional UGTs that have glycosylation activity towards cannabinoids are incorporated by reference from Sayre et al. in PCT/US18/24409.
On one embodiment, the invention may include the biosynthesis of a novel terpene compound according to Formula I:
In one preferred embodiment, the compound of Formula I may be generated in vivo, and preferably in planta. In this embodiment, a Cannabis plant or cell may transformed to with an expression vector co-expressing nucleotide sequences encoding UGTs enzymes according to nucleotide sequences SEQ ID NO. 5, and SEQ ID NO. 7. In this embodiment, the co-expressed nucleotide sequences may be operably linked to a promoter, and encode UGTs enzymes according to amino acid sequences SEQ ID NO. 6, and SEQ ID NO. 8 that may convert endogenous geraniol into geranyl O-acetyl glucoside as shown in Formula I.
In another preferred embodiment, the compound of Formula I may be generated in vivo, and preferably in a yeast or cell suspension culture system as generally described herein. In this preferred embodiment, a yeast, bacterial or cell of suspension cell cultures, such as a tobacco cell suspension culture, may transformed to with an expression vector co-expressing nucleotide sequences encoding UGTs enzymes according to nucleotide sequences SEQ ID NO. 5, and SEQ ID NO. 7. In this embodiment, the co-expressed nucleotide sequences may be operably linked to a promoter, and encode UGTs enzymes according to amino acid sequences SEQ ID NO. 6, and SEQ ID NO. 8 that may convert endogenous geraniol into geranyl O-acetyl glucoside as shown in Formula I.
In another preferred embodiment, the compound of Formula I may be generated in vitro, or ex vivo such as a bioreactor. In this preferred embodiment, a quantity of geraniol may be introduced to UGTs enzymes according to amino acid sequences SEQ ID NO. 6, and SEQ ID NO. 8 that may convert the geraniol into geranyl O-acetyl glucoside as shown in Formula I. In certain embodiments, such in vitro, and ex vivo system may be supplemented with an appropriate reaction buffer, as well as UDP-glucose or other appropriate glycosylation substrate.
On one embodiment, the invention may include the biosynthesis of a novel terpene compound according to Formula III:
In one preferred embodiment, the compound of Formula I may be generated in vivo, and preferably in planta. In this embodiment, a Cannabis plant or cell may transformed to with an expression vector co-expressing nucleotide sequences encoding UGTs enzymes according to nucleotide sequences SEQ ID NO. 5, and SEQ ID NO. 7. In this embodiment, the co-expressed nucleotide sequences may be operably linked to a promoter, and encode UGTs enzymes according to amino acid sequences SEQ ID NO. 6, and SEQ ID NO. 8 that may convert endogenous geraniol into geranyl O-acetyl glucoside as shown in Formula II.
In another preferred embodiment, the compound of Formula II may be generated in vivo, and preferably in a yeast or cell suspension culture system as generally described herein. In this preferred embodiment, a yeast, bacterial or cell of suspension cell cultures, such as a tobacco cell suspension culture, may transformed to with an expression vector co-expressing nucleotide sequences encoding UGTs enzymes according to nucleotide sequences SEQ ID NO. 5, and SEQ ID NO. 7. In this embodiment, the co-expressed nucleotide sequences may be operably linked to a promoter, and encode UGTs enzymes according to amino acid sequences SEQ ID NO. 6, and SEQ ID NO. 8 that may convert endogenous Linalool into Linalyl glucoside-xyloside as shown in Formula II.
In another preferred embodiment, the compound of Formula II may be generated in vitro, or ex vivo such as a bioreactor. In this preferred embodiment, a quantity of geraniol may be introduced to UGTs enzymes according to amino acid sequences SEQ ID NO. 6, and SEQ ID NO. 8 that may convert the Linalool into Linalyl glucoside-xyloside as shown in Formula II. In certain embodiments, such in vitro, and ex vivo system may be supplemented with an appropriate reaction buffer, as well as UDP-glucose or other appropriate glycosylation substrate.
The present inventors may utilize one or more of the following exemplary UGT genes/proteins to glycosylate one or more terpenes. Such UGTs may be endogenously or heterologously expressed in plants, such as a Cannabis plant, as well as and in vivo cultured systems such as plant, yeast or bacterial cell cultures. Polynucleotide as well as amino acid sequences of the following may be ascertained without undue experimentation by one of ordinary skill in the art. As such, all sequences identified herein are specifically incorporated by reference. Codon-optimized versions of each of the aforementioned genes are also included in the current invention. Such codon-optimized genes may be configured for optimized expression in heterologous systems, such as yeast or bacterial systems. Sequences identified herein as “codon-optimized” may be codon optimized for expression in yeast.
Examples may include: Vitis vinifera: VvGT7 (XM_002276510.3), VvGT14 (XM_002285734.4), VvGT15 (XM_002281477.2), VvGT16 (XM_002263122.1), VvGT17 (XM_002285743.2), VvGT18 (XM_002285372.1), VvGT19 (XM_002285744.1), VvGT20 (XM_002266592.2). Nicotiana tobaccum: NtGT1, NtGT2, NtGT3, NtGT4, and NtGT5. (Nicotiana sequences found in Sayre et al. in PCT/US18/24409 are hereby incorporated by reference.) Mucor hiemalis: a phenolic glycosyltransferase. Pichia etchellsii: beta-glucosidase II (XM_020688260.1). Prunus dulcis: almond beta-glucosidase (S17766). Actinidia deliciosa: kiwi fruit AdGT1 (KF954941.1), AdGT2 (KF954942.1), AdGT3 (KF954943.1), AdGT4 (KF954944.1). Arabidopsis thaliana: UGT76E2 (NM_125351.3), UGT76E11 (NM_114534.3), UGT76E12 (NM 114533.2), UGT76D1 (NM_128205.3), UGT84A3 (NM_117639.3), UGT84A4 (NM_117640.4), UGT84B2 (NM_127889.1), UGT84B1 (NM_127890.3), UGT75B2 (NM_100432.2), UGT75B1 (NM_001331554.1), UGT75D1, UGT74E2 (NM_100448.4), UGT74F1 (NM_129946.3), UGT74F2 (NM_129944.3), UGT74D1 (NM_128733.5), UGT74B1 (NM_102256.3), UGT85A1 (NM_102089.5), UGT85A5 (NM 202156.2), UGT85A4 (NM_106476.3), UGT85A2 (NM_102086.3), UGT85A7 (NM_102085.3), UGT73C3, UGT73C6 (NM_129234.2), UGT73C1 (NM_129230.3), UGT71C2 (NM_128528.4), UGT88A1 (NM_112524.4). (Arabidopsis thaliana sequences found in Sayre et al. in PCT/US18/24409 are hereby incorporated by reference.)
Another aspect of the invention includes systems, methods and compositions for the in vivo formulation of terpene glycosides and/or cannabinoid glycosides. In one preferred embodiment, UGTs may be selectively expressed in one or more of the in vivo systems described herein, such as an in planta, yeast, or cell culture system. Such selectively expressed UGTs may have glycosylation activity towards some terpenes or cannabinoids, and not others. By selectively co-expressing combinations of UGTs in an in vivo system, the water-solubilized glycosylated compounds may be selectively removed allowing for isolation of selective formulations of water-soluble terpenes and/or cannabinoid or a mixture of the two. Such selective formulations of water-soluble terpenes and/or cannabinoid or a mixture of the two may be selected for flavor, texture, and taste among other attributes and may further be incorporated into one or more consumer products as described herein.
For example, in one embodiment, the expression of the UGT UGT85K11 (SEQ ID NO. 6) in a yeast or plant system may selectively glycosylate nerol to neryl monoglucoside. The glycosylated form, being water-soluble may not be selectively removed and isolated in a water-fraction. In another preferred embodiment, additional UTGs may be identified, for example through bioinformatic methods known by those of ordinary skill in the art, as having homology with other identified UTGs that preferably that have glycosylation activity towards one or more terpenes and/or cannabinoids. Such newly identified UGTs may be tested in one or more of the in vivo yeast, plant or cells systems discussed herein to determine if they exhibit selective glycosylation activity towards one or more terpenes and/or cannabinoids. Such combinations of selective UGTs may be co-expressed to generate a specific profile of terpenes and/or cannabinoid or mixtures of both that may be selectively solubilized and isolated from a water fraction.
In one embodiment, one of the aforementioned compositions may act as a prodrug. The term “prodrug” is taken to mean compounds according to the invention which have been modified by means of, for example, sugars and which are cleaved in the organism to form the effective compounds according to the invention. The terms “therapeutically effective amount” or “effective dose” or “dose” are interchangeably used herein and denote an amount of the pharmaceutical compound having a prophylactically or therapeutically relevant effect on a disease or pathological conditions, i.e. which causes in a tissue, system, animal or human a biological or medical response which is sought or desired, for example, by a researcher or physician. Pharmaceutical formulations can be administered in the form of dosage units which comprise a predetermined amount of active ingredient per dosage unit. The concentration of the prophylactically or therapeutically active ingredient in the formulation may vary from about 0.1 to 100 wt %. Preferably, the compound of formula (I) or the pharmaceutically acceptable salts thereof are administered in doses of approximately 0.5 to 1000 mg, more preferably between 1 and 700 mg, most preferably 5 and 100 mg per dose unit. Generally, such a dose range is appropriate for total daily incorporation. In other terms, the daily dose is preferably between approximately 0.02 and 100 mg/kg of body weight. The specific dose for each patient depends, however, on a wide variety of factors as already described in the present specification (e.g. depending on the condition treated, the method of administration and the age, weight and condition of the patient). Preferred dosage unit formulations are those which comprise a daily dose or part-dose, as indicated above, or a corresponding fraction thereof of an active ingredient. Furthermore, pharmaceutical formulations of this type can be prepared using a process which is generally known in the pharmaceutical art.
In the meaning of the present invention, the compound is further defined to include pharmaceutically usable derivatives, solvates, prodrugs, tautomers, enantiomers, racemates and stereoisomers thereof, including mixtures thereof in all ratios.
As noted above, the present invention allows the scaled production of water-soluble terpenes. Because of this enhanced solubility, the invention allows for the addition of such water-soluble terpene to a variety of compositions without requiring oils and/or emulsions that are generally required to maintain the non-modified terpenes in suspension. As a result, the present invention may allow for the production of a variety of compositions for both the food and beverage industry, as well as pharmaceutical applications that do not required oils and emulsion suspensions and the like.
In one embodiment the invention may include aqueous compositions containing one or more water-soluble terpenes that may be introduced to a food or beverage. In a preferred embodiment, the invention may include an aqueous solution containing one or more dissolved water-soluble terpenes. In this embodiment, such water-soluble terpenes may include a glycosylated terpene, and/or an acetylated glycoside terpene (such as Geranyl O-acetyl glucoside), and/or a mixture of both. Here, the glycosylated terpene, and/or an acetylated glycoside terpene, and/or glycosylated-xylosylated terpene were generated in vivo as generally described herein, or in vitro. Moreover, in this embodiment, the aqueous may contain one or more of the following: saline, purified water, propylene glycol, deionized water, and/or an alcohol such as ethanol as well as a pH buffer that may allow the aqueous solution to be maintained at a pH below 7.4. Additional embodiments may include the addition of an acid of base, such as formic acid, or ammonium hydroxide.
In another embodiment, the invention may include a consumable food additive having at least one water-soluble terpene, such as a glycosylated and/or an acetylated glycoside terpene, and/or a mixture of both, where such water-soluble terpenes may be generated in vivo and/or in vitro. This consumable food additive may further include one or more a food additive polysaccharides, such as dextrin and/or maltodextrin, as well as an emulsifier. Example emulisifiers may include, but not be limited to: gum arabic, modified starch, pectin, xanthan gum, gum ghatti, gum tragacanth, fenugreek gum, mesquite gum, mono-glycerides and di-glycerides of long chain fatty acids, sucrose monoesters, sorbitan esters, polyethoxylated glycerols, stearic acid, palmitic acid, mono-glycerides, di-glycerides, propylene glycol esters, lecithin, lactylated mono- and di-glycerides, propylene glycol monoesters, polyglycerol esters, diacetylated glycoside tartaric acid esters of mono- and di-glycerides, citric acid esters of monoglycerides, stearoyl-2-lactylates, polysorbates, succinylated monoglycerides, acetylated glycoside monoglycerides, ethoxylated monoglycerides, quillaia, whey protein isolate, casein, soy protein, vegetable protein, pullulan, sodium alginate, guar gum, locust bean gum, tragacanth gum, tamarind gum, carrageenan, furcellaran, Gellan gum, psyllium, curdlan, konjac mannan, agar, and cellulose derivatives, or combinations thereof.
The consumable food additive of the invention may be a homogenous composition and may further comprise a flavoring agent. Exemplary flavoring agents may include: sucrose (sugar), glucose, fructose, sorbitol, mannitol, corn syrup, high fructose corn syrup, saccharin, aspartame, sucralose, acesulfame potassium (acesulfame-K), neotame. The consumable food additive of the invention may also contain one or more coloring agents. Exemplary coloring agents may include: FD&C Blue Nos. 1 and 2, FD&C Green No. 3, FD&C Red Nos. 3 and 40, FD&C Yellow Nos. 5 and 6, Orange B, Citrus Red No. 2, annatto extract, beta-carotene, grape skin extract, cochineal extract or carmine, paprika oleoresin, caramel color, fruit and vegetable juices, saffron, Monosodium glutamate (MSG), hydrolyzed soy protein, autolyzed yeast extract, disodium guanylate or inosinate.
The consumable food additive of the invention may also contain one or more surfactants, such as glycerol monostearate and polysorbate 80. The consumable food additive of the invention may also contain one or more preservatives. Exemplary preservatives may include ascorbic acid, citric acid, sodium benzoate, calcium propionate, sodium erythorbate, sodium nitrite, calcium sorbate, potassium sorbate, BHA, BHT, EDTA, tocopherols. The consumable food additive of the invention may also contain one or more nutrient supplements, such as: thiamine hydrochloride, riboflavin, niacin, niacinamide, folate or folic acid, beta carotene, potassium iodide, iron or ferrous sulfate, alpha tocopherols, ascorbic acid, Vitamin D, amino acids, multi-vitamin, fish oil, co-enzyme Q-10, and calcium.
In one embodiment, the invention may include a consumable fluid containing at least one dissolved water-soluble terpene. In one preferred embodiment, this consumable fluid may be added to a drink or beverage to infuse it with the dissolved water-soluble terpene generated in an in vivo system as generally herein described, or through an in vitro process. As noted above, such water-soluble terpene may include a water-soluble glycosylated terpene and/or a water-soluble acetylated glycoside terpene (such as Geranyl O-acetyl glucoside) and/or a mixture of both. The consumable fluid may include a food additive polysaccharide such as maltodextrin and/or dextrin, which may further be in an aqueous form and/or solution. For example, in one embodiment, and aqueous maltodextrin solution may include a quantity of sorbic acid and an acidifying agent to provide a food grade aqueous solution of maltodextrin having a pH of 2-4 and a sorbic acid content of 0.02-0.1% by weight.
In certain embodiments, the consumable fluid may include water, as well as an alcoholic beverage; a non-alcoholic beverage, a noncarbonated beverage, a carbonated beverage, a cola, a root beer, a fruit-flavored beverage, a citrus-flavored beverage, a fruit juice, a fruit-containing beverage, a vegetable juice, a vegetable containing beverage, a tea, a coffee, a dairy beverage, a protein containing beverage, a shake, a sports drink, an energy drink, and a flavored water.
The consumable fluid may further include at least one additional ingredients, including but not limited to: xanthan gum, cellulose gum, whey protein hydrolysate, ascorbic acid, citric acid, malic acid, sodium benzoate, sodium citrate, sugar, phosphoric acid, and water.
In one embodiment, the invention may include a consumable gel having at least one water-soluble terpene and gelatin in an aqueous solution. In a preferred embodiment, the consumable gel may include a water-soluble glycosylated terpene and/or a water-soluble acetylated glycoside terpene, or a mixture of both, generated in an in vivo system, such as a whole plant or cell suspension culture system as generally herein described.
Additional embodiments may include a liquid composition having at least one water-soluble terpene solubilized in a first quantity of water; and at least one of: xanthan gum, cellulose gum, whey protein hydrolysate, ascorbic acid, citric acid, malic acid, sodium benzoate, sodium citrate, sugar, phosphoric acid, and/or a sugar alcohol. In this embodiment, a water-soluble terpene may include a glycosylated water-soluble terpene, an acetylated glycoside water-soluble terpene, or a mixture of both. In one preferred embodiment, the composition may further include a quantity of ethanol. Here, the amount of water-soluble terpene may include: less than 10 mass % water; more than 95 mass % water; about 0.1 mg to about 1000 mg of the water-soluble terpene; about 0.1 mg to about 500 mg of the water-soluble terpenes; about 0.1 mg to about 200 mg of the water-soluble terpene; about 0.1 mg to about 100 mg of the water-soluble terpene; about 0.1 mg to about 100 mg of the water-soluble terpene; about 0.1 mg to about 10 mg of the water-soluble terpene; about 0.5 mg to about 5 mg of the water-soluble terpene; about 1 mg/kg to 5 mg/kg (body weight) in a human of the water-soluble terpene.
In alternative embodiments, the composition may include at least one water-soluble terpene in the range of 50 mg/L to 300 mg/L; at least one water-soluble terpene in the range of 50 mg/L to 100 mg/L; at least one water-soluble terpene in the range of 50 mg/L to 500 mg/L; at least one water-soluble terpene over 500 mg/L; at least one water-soluble terpene under 50 mg/L. Additional embodiments may include one or more of the following additional components: a flavoring agent; a coloring agent; a coloring agent; and/or caffeine.
In one embodiment, the invention may include a liquid composition having at least one water-soluble terpene solubilized in said first quantity of water and a first quantity of ethanol in a liquid state. In a preferred embodiment, a first quantity of ethanol in a liquid state may be between 1% to 20% weight by volume of the liquid composition. In this embodiment, a water-soluble terpene may include a glycosylated water-soluble terpene, an acetylated glycoside water-soluble terpene, or a mixture of both. Such water-soluble terpenes may be generated in an in vivo and/or in vitro system as herein identified. In a preferred embodiment, the ethanol, or ethyl alcohol component may be up to about ninety-nine point nine-five percent (99.95%) by weight and the water-soluble terpene about zero point zero five percent (0.05%) by weight.
Examples of the preferred embodiment may include liquid ethyl alcohol compositions having one or more water-soluble terpenes wherein said ethyl alcohol has a proof greater than 100, and/or less than 100. Additional examples of a liquid composition containing ethyl alcohol and at least one water-soluble terpene may include, beer, wine and/or distilled spirit.
Additional embodiments of the invention may include a chewing gum composition having a first quantity of at least one water-soluble terpene. In a preferred embodiment, a chewing gum composition may further include a gum base comprising a buffering agent selected from the group consisting of acetates, glycinates, phosphates, carbonates, glycerophosphates, citrates, borates, and mixtures thereof. Additional components may include at least one sweetening agent; and at least one flavoring agent. As noted above, in a preferred embodiment,
In one embodiment, the chewing gum composition described above may include:
Here, such flavoring agents may include: menthol flavor, Eucalyptus, mint flavor and/or L-menthol. Sweetening agents may include one or more of the following: xylitol, sorbitol, isomalt, aspartame, sucralose, acesulfame potassium, and saccharin. Additional preferred embodiments may include a chewing gum having a pharmaceutically acceptable excipient selected from the group consisting of: fillers, disintegrants, binders, lubricants, and antioxidants. The chewing gum composition may further be non-disintegrating and also include one or more coloring and/or flavoring agents.
The invention may further include a composition for a water-soluble terpene infused solution comprising essentially of: water and/or purified water, at least one water-soluble terpene, and at least one flavoring agent. A water-soluble terpene infused solution of the invention may further include a sweetener selected from the group consisting of: glucose, sucrose, invert sugar, corn syrup, Stevia extract powder, stevioside, steviol, aspartame, saccharin, saccharin salts, sucralose, potassium acetosulfam, sorbitol, xylitol, mannitol, erythritol, lactitol, alitame, miraculin, monellin, and thaumatin or a combination of the same. Additional components of the water-soluble terpene infused solution may include, but not be limited to: sodium chloride, sodium chloride solution, glycerin, a coloring agent, and a demulcent. As to this last potential component, in certain embodiments, a demulcent may include: pectin, glycerin, honey, methylcellulose, and/or propylene glycol. As noted above, in a preferred embodiment, a water-soluble terpene may include at least one water-soluble acetylated glycoside terpene, and/or at least one water-soluble glycosylated terpene, and/or at least one glycosylated-xylosylated terpene or a mixture thereof. In this embodiment, such water soluble glycosylated terpene, and/or an acetylated glycoside terpene, and/or glycosylated-xylosylated terpene may have been glycosylated and/or acetylated glycoside in vivo respectively.
The invention may further include a composition for a water-soluble terpene infused anesthetic solution having water, or purified water, at least one water-soluble terpene, and at least one oral anesthetic. In a preferred embodiment, an anesthetic may include benzocaine, and/or phenol in a quantity of between 0.1% to 15% volume by weight.
Additional embodiments may include a water-soluble terpene infused anesthetic solution having a sweetener which may be selected from the group consisting of: glucose, sucrose, invert sugar, corn syrup, Stevia extract powder, stevioside, steviol, aspartame, saccharin, saccharin salts, sucralose, potassium acetosulfam, sorbitol, xylitol, mannitol, erythritol, lactitol, alitame, miraculin, monellin, and thaumatin or a combination of the same. Additional components of the water-soluble terpene infused solution may include, but not be limited to: sodium chloride, sodium chloride solution, glycerin, a coloring agent a demulcent. In a preferred embodiment, a demulcent may selected from the group consisting of: pectin, glycerin, honey, methylcellulose, and propylene glycol. As noted above, in a preferred embodiment, a water-soluble terpene may include at least one water-soluble acetylated glycoside terpene, and/or at least one water-soluble glycosylated terpene, and/or at least one glycosylated-xylosylated terpene or a mixture thereof. In this embodiment, such water soluble glycosylated terpene, and/or an acetylated glycoside terpene, and/or glycosylated-xylosylated terpene may have been glycosylated and/or acetylated glycoside in vivo respectively.
The invention may further include a composition for a hard lozenge for rapid delivery of water-soluble terpenes through the oral mucosa. In this embodiment, such a hard lozenge composition may include: a crystalized sugar base, and at least one water-soluble terpene, wherein the hard lozenge has a moisture content between 0.1 to 2%. In this embodiment, the water-soluble terpene may be added to the sugar base when it is in a liquefied form and prior to the evaporation of the majority of water content. Such a hard lozenge may further be referred to as a candy.
In a preferred embodiment, a crystalized sugar base may be formed from one or more of the following: sucrose, invert sugar, corn syrup, and isomalt or a combination of the same. Additional components may include at least one acidulant. Examples of acidulants may include, but not be limited to: citric acid, tartaric acid, fumaric acid, and malic acid. Additional components may include at least one pH adjustor. Examples of pH adjustors may include, but not be limited to: calcium carbonate, sodium bicarbonate, and magnesium trisilicate.
In another preferred embodiment, the composition may include at least one anesthetic. Example of such anesthetics may include benzocaine, and phenol. In this embodiment, first quantity of anesthetic may be between 1 mg to 15 mg per lozenge. Additional embodiments may include a quantity of menthol. In this embodiment, such a quantity of menthol may be between 1 mg to 20 mg. The hard lozenge composition may also include a demulcent, for example: pectin, glycerin, honey, methylcellulose, propylene glycol, and glycerin. In this embodiment, a demulcent may be in a quantity between 1 mg to 10 mg. As noted above, in a preferred embodiment, a water-soluble terpene may include at least one water-soluble acetylated glycoside terpene, and/or at least one water-soluble glycosylated terpene, and/or at least one glycosylated-xylosylated terpene or a mixture thereof. In this embodiment, such water soluble glycosylated terpene, and/or an acetylated glycoside terpene, and/or glycosylated-xylosylated terpene may have been glycosylated and/or acetylated glycoside in vivo respectively.
The invention may include a chewable lozenge for rapid delivery of water-soluble terpenes through the oral mucosa. In a preferred embodiment, the compositions may include: a glycerinated gelatin base, at least one sweetener; and at least one water-soluble terpene dissolved in a first quantity of water. In this embodiment, a sweetener may include sweetener selected from the group consisting of: glucose, sucrose, invert sugar, corn syrup, Stevia extract powder, stevioside, steviol, aspartame, saccharin, saccharin salts, sucralose, potassium acetosulfam, sorbitol, xylitol, mannitol, erythritol, lactitol, alitame, miraculin, monellin, and thaumatin or a combination of the same.
Additional components may include at least one acidulant. Examples of acidulants may include, but not be limited to: citric acid, tartaric acid, fumaric acid, and malic acid. Additional components may include at least one pH adjustor. Examples of pH adjustors may include, but not be limited to: calcium carbonate, sodium bicarbonate, and magnesium trisilicate.
In another preferred embodiment, the composition may include at least one anesthetic. Example of such anesthetics may include benzocaine, and phenol. In this embodiment, first quantity of anesthetic may be between 1 mg to 15 mg per lozenge. Additional embodiments may include a quantity of menthol. In this embodiment, such a quantity of menthol may be between 1 mg to 20 mg. The chewable lozenge composition may also include a demulcent, for example: pectin, glycerin, honey, methylcellulose, propylene glycol, and glycerin. In this embodiment, a demulcent may be in a quantity between 1 mg to 10 mg. As noted above, in a preferred embodiment, a water-soluble terpene may include at least one water-soluble acetylated glycoside terpene, and/or at least one water-soluble glycosylated terpene, and/or at least one glycosylated-xylosylated terpene or a mixture thereof. In this embodiment, such water soluble glycosylated terpene, and/or an acetylated glycoside terpene, and/or glycosylated-xylosylated terpene may have been glycosylated and/or acetylated glycoside in vivo respectively.
The invention may include a soft lozenge for rapid delivery of water-soluble terpenes through the oral mucosa. In a preferred embodiment, the compositions may include: polyethylene glycol base, at least one sweetener; and at least one water-soluble terpene dissolved in a first quantity of water. In this embodiment, a sweetener may include sweetener selected from the group consisting of: glucose, sucrose, invert sugar, corn syrup, Stevia extract powder, stevioside, steviol, aspartame, saccharin, saccharin salts, sucralose, potassium acetosulfam, sorbitol, xylitol, mannitol, erythritol, lactitol, alitame, miraculin, monellin, and thaumatin or a combination of the same. Additional components may include at least one acidulant. Examples of acidulants may include, but not be limited to: citric acid, tartaric acid, fumaric acid, and malic acid. Additional components may include at least one pH adjustor. Examples of pH adjustors may include, but not be limited to: calcium carbonate, sodium bicarbonate, and magnesium trisilicate.
In another preferred embodiment, the composition may include at least one anesthetic. Example of such anesthetics may include benzocaine, and phenol. In this embodiment, first quantity of anesthetic may be between 1 mg to 15 mg per lozenge. Additional embodiments may include a quantity of menthol. In this embodiment, such a quantity of menthol may be between 1 mg to 20 mg. The soft lozenge composition may also include a demulcent, for example: pectin, glycerin, honey, methylcellulose, propylene glycol, and glycerin. In this embodiment, a demulcent may be in a quantity between 1 mg to 10 mg. As noted above, in a preferred embodiment, a water-soluble terpene may include at least one water-soluble acetylated glycoside terpene, and/or at least one water-soluble glycosylated terpene, and/or at least one glycosylated-xylosylated terpene or a mixture thereof. In this embodiment, such water soluble glycosylated terpene, and/or an acetylated glycoside terpene, and/or glycosylated-xylosylated terpene may have been glycosylated and/or acetylated glycoside in vivo respectively.
In another embodiment, the invention may include a tablet or capsule consisting essentially of a water-soluble glycosylated terpene and a pharmaceutically acceptable excipient. Example may include solid, semi-solid and aqueous excipients such as: maltodextrin, whey protein isolate, xanthan gum, guar gum, diglycerides, monoglycerides, carboxymethyl cellulose, glycerin, gelatin, polyethylene glycol and water-based excipients.
In a preferred embodiment, a water-soluble terpene may include at least one water-soluble acetylated glycoside terpene, and/or at least one water-soluble glycosylated terpene, and/or at least one glycosylated-xylosylated terpene or a mixture thereof. In this embodiment, such water soluble glycosylated terpene, and/or an acetylated glycoside terpene, and/or glycosylated-xylosylated terpene may have been glycosylated and/or acetylated glycoside in vivo respectively. Examples of such in vivo systems being generally described herein, including in plant, as well as cell culture systems including Cannabis cell culture, tobacco cell culture and yeast cell culture systems. In one embodiment, a tablet or capsule may include an amount of water-soluble terpene of 5 milligrams or less. Alternative embodiments may include an amount of water-soluble terpene between 5 milligrams and 200 milligrams. Still other embodiments may include a tablet or capsule having amount of water-soluble terpene that is more than 200 milligrams.
The invention may further include a method of manufacturing and packaging a terpene dosage, consisting of the following steps: 1) preparing a fill solution with a desired concentration of a water-soluble terpene in a liquid carrier wherein said terpene solubilized in said liquid carrier; 2) encapsulating said fill solution in capsules; 3) packaging said capsules in a closed packaging system; and 4) removing atmospheric air from the capsules. In one embodiment, the step of removing of atmospheric air consists of purging the packaging system with an inert gas, such as, for example, nitrogen gas, such that said packaging system provides a room temperature stable product. In one preferred embodiment, the packaging system may include a plaster package, which may be constructed of material that minimizes exposure to moisture and air.
In one embodiment a preferred liquid carrier may include a water-based carrier, such as for example an aqueous sodium chloride solution. In a preferred embodiment, a water-soluble terpene may include at least one water-soluble acetylated glycoside terpene, and/or at least one water-soluble glycosylated terpene, and/or at least one glycosylated-xylosylated terpene or a mixture thereof. In this embodiment, such water soluble glycosylated terpene, and/or an acetylated glycoside terpene, and/or glycosylated-xylosylated terpene may have been glycosylated and/or acetylated glycoside in vivo respectively. Examples of such in vivo systems being generally described herein, including in plant, as well as cell culture systems including Cannabis cell culture, tobacco cell culture and yeast cell culture systems. In one embodiment, a desired terpene concentration may be about 1-10% w/w, while in other embodiments it may be about 1.5-6.5% w/w. Alternative embodiments may include an amount of water-soluble terpene between 5 milligrams and 200 milligrams. Still other embodiments may include a tablet or capsule having amount of water-soluble terpene that is more than 200 milligrams.
The invention may include an oral pharmaceutical solution, such as a sub-lingual spray, consisting essentially of a water-soluble terpene, 30-33% w/w water, about 50% w/w alcohol, 0.01% w/w butylated hydroxylanisole (BHA) or 0.1% w/w ethylenediaminetetraacetic acid (EDTA) and 5-21% w/w co-solvent, having a combined total of 100%, wherein said co-solvent is selected from the group consisting of propylene glycol, polyethylene glycol and combinations thereof, and wherein said water-soluble terpene is a glycosylated terpene, an acetylated glycoside terpene or a mixture of the two. In an alternative embodiment, such a oral pharmaceutical solution may consist essentially of 0.1 to 5% w/w of said water-soluble terpene, about 50% w/w alcohol, 5.5% w/w propylene glycol, 12% w/w polyethylene glycol and 30-33% w/w water. In a preferred composition, the alcohol component may be ethanol.
The invention may include an oral pharmaceutical solution, such as a sublingual spray, consisting essentially of about 0.1% to 1% w/w water-soluble terpene, about 50% w/w alcohol, 5.5% w/w propylene glycol, 12% w/w polyethylene glycol, 30-33% w/w water, 0.01% w/w butylated hydroxyanisole, having a combined total of 100%, and wherein said water-soluble terpene is a glycosylated terpene, an acetylated glycoside terpene or a mixture of the two wherein that were generated in vivo. In an alternative embodiment, such a oral pharmaceutical solution may consist essentially of 0.54% w/w water-soluble terpene, 31.9% w/w water, 12% w/w polyethylene glycol 400, 5.5% w/w propylene glycol, 0.01% w/w butylated hydroxyanisole, 0.05% w/w sucralose, and 50% w/w alcohol, wherein the a the alcohol components may be ethanol.
The invention may include a solution for nasal and/or sublingual administration of a terpene including: 1) an excipient of propylene glycol, ethanol anhydrous, or a mixture of both; and 2) a water-soluble terpene which may include glycosylated terpene an acetylated glycoside terpene or a mixture of the two generated in vivo and/or in vitro. In a preferred embodiment, the composition may further include a topical decongestant, which may include phenylephrine hydrochloride, Oxymetazoline hydrochloride, and Xylometazoline in certain preferred embodiments. The composition may further include an antihistamine, and/or a steroid. Preferably, the steroid component is a corticosteroid selected from the group consisting of: neclomethasone dipropionate, budesonide, ciclesonide, flunisolide, fluticasone furoate, fluticasone propionate, mometasone, triamcinolone acetonide. In alternative embodiments, the solution for nasal and/or sublingual administration of a terpene may further comprise at least one of the following: benzalkonium chloride solution, benzyl alcohol, boric acid, purified water, sodium borate, polysorbate 80, phenylethyl alcohol, microcrystalline cellulose, carboxymethylcellulose sodium, dextrose, dipasic, sodium phosphate, edetate disodium, monobasic sodium phosphate, propylene glycol.
The invention may further include an aqueous solution for nasal and/or sublingual administration of a terpene comprising: a water and/or saline solution; and a water-soluble terpene which may include a glycosylated terpene, an acetylated glycoside terpene or a mixture of the two generated in vivo and/or in vitro. In a preferred embodiment, the composition may further include a topical decongestant, which may include phenylephrine hydrochloride, Oxymetazoline hydrochloride, and Xylometazoline in certain preferred embodiments. The composition may further include an antihistamine, and/or a steroid. Preferably, the steroid component is a corticosteroid selected from the group consisting of: neclomethasone dipropionate, budesonide, ciclesonide, flunisolide, fluticasone furoate, fluticasone propionate, mometasone, triamcinolone acetonide. In alternative embodiments, the aqueous solution may further comprise at least one of the following: benzalkonium chloride solution, benzyl alcohol, boric acid, purified water, sodium borate, polysorbate 80, phenylethyl alcohol, microcrystalline cellulose, carboxymethylcellulose sodium, dextrose, dipasic, sodium phosphate, edetate disodium, monobasic sodium phosphate, propylene glycol.
The invention may include a topical formulation for the transdermal delivery of water-soluble terpene. In a preferred embodiment, a topical formulation for the transdermal delivery of water-soluble terpene may include a water-soluble glycosylated terpene, and/or water-soluble acetylated glycoside terpene, or a mixture of both, and a pharmaceutically acceptable excipient. Here, a glycosylated terpene and/or acetylated glycoside terpene may be generated in vivo and/or in vitro. Preferably a pharmaceutically acceptable excipient may include one or more: gels, ointments, cataplasms, poultices, pastes, creams, lotions, plasters and jellies or even polyethylene glycol. Additional embodiments may further include one or more of the following components: a quantity of capsaicin; a quantity of benzocaine; a quantity of lidocaine; a quantity of camphor; a quantity of benzoin resin; a quantity of methylsalicilate; a quantity of triethanolamine salicylate; a quantity of hydrocortisone; a quantity of salicylic acid.
The invention may include a gel for transdermal administration of a water soluble-terpene which may be generated in vitro and/or in vivo. In this embodiment, the mixture preferably contains from 15% to about 90% ethanol, about 10% to about 60% buffered aqueous solution or water, about 0.1 to about 25% propylene glycol, from about 0.1 to about 20% of a gelling agent, from about 0.1 to about 20% of a base, from about 0.1 to about 20% of an absorption enhancer and from about 1% to about 25% polyethylene glycol and a water-soluble terpene such as a glycosylated terpene, and/or acetylated glycoside terpene, and/or a mixture of the two.
In another embodiment, the invention may further include a transdermal composition having a pharmaceutically effective amount of a water-soluble terpene for delivery of the terpene to the bloodstream of a user. This transdermal composition may include a pharmaceutically acceptable excipient and at least one water-soluble terpene, such as a glycosylated terpene, an acetylated glycoside terpene, and a mixture of both, wherein the terpene is capable of diffusing from the composition into the bloodstream of the user. In a preferred embodiment, a pharmaceutically acceptable excipient to create a transdermal dosage form selected from the group consisting of: gels, ointments, cataplasms, poultices, pastes, creams, lotions, plasters and jellies. The transdermal composition may further include one or more surfactants. In one preferred embodiment, the surfactant may include a surfactant-lecithin organogel, which may further be present in an amount of between about between about 95% and about 98% w/w.
In an alternative embodiment, a surfactant-lecithin organogel comprises lecithin and PPG-2 myristyl ether propionate and/or high molecular weight polyacrylic acid polymers. The transdermal composition may further include a quantity of isopropyl myristate.
The invention may further include a transdermal composition having one or more permeation enhancers to facilitate transfer of the water-soluble terpene across a dermal layer. In a preferred embodiment, a permeation enhancer may include one or more of the following: propylene glycol monolaurate, diethylene glycol monoethyl ether, an oleoyl macrogolglyceride, a caprylocaproyl macrogolglyceride, and an oleyl alcohol. The invention may also include a liquid terpene liniment composition consisting of water, isopropyl alcohol solution and a water-soluble terpene, such as glycosylated terpene, and/or an acetylated glycoside terpene, and/or glycosylated-xylosylated terpene which may further have been generated in vivo. This liquid terpene liniment composition may further include approximately 97.5% to about 99.5% by weight of 70% isopropyl alcohol solution and from about 0.5% to about 2.5% by weight of a water-soluble terpene mixture.
Based on the improved solubility and other physical properties, as well as cost advantage and scalability of the invention's in vivo water-soluble production platform, the invention may include one or more commercial infusions. For example, commercially available products, such as lip balm, soap, shampoos, lotions, creams and cosmetics may be infused with one or more water-soluble terpenes.
As generally described herein, the invention may include one or more plants, such as a tobacco plant and/or cell culture that may be genetically modified to produce, for example water-soluble glycosylated terpenes in vivo. As such, in one preferred embodiment, the invention may include a tobacco plant and/or cell that may contain at least one water-soluble terpene. In a preferred embodiment, a tobacco plant containing a quantity of water-soluble terpenes may be used to generate a water-soluble terpene infused tobacco product such as a cigarette, pipe tobacco, chewing tobacco, cigar, and smokeless tobacco. In one embodiment, the tobacco plant may be treated with one or more glycosidase inhibitors. In a preferred embodiment, since the terpene being introduced to the tobacco plant may be controlled, the inventive tobacco plant may generate one or more selected water-terpenes. For example, in one embodiment, the genetically modified tobacco plant may be introduced to a single terpene, while in other embodiments the genetically modified tobacco plant may be introduced to a terpene extract containing a full and/or partial entourage of terpene compounds. The invention may further include a novel composition that may be used to supplement a cigarette, or other tobacco-based product. In this embodiment, the composition may include at least one water-soluble terpene dissolved in an aqueous solution. This aqueous solution may be wherein said composition may be introduced to a tobacco product, such as a cigarette and/or a tobacco leaf such that the aqueous solution may evaporate generating a cigarette and/or a tobacco leaf that contains the aforementioned water-soluble terpene(s), which may further have been generated in vivo as generally described herein.
In one embodiment the invention may include one or more methods of treating a medical condition in a mammal. In this embodiment, the novel method may include administering a therapeutically effective amount of a water-soluble terpene, such as an in vivo generated glycosylated terpene, and/or an acetylated glycoside terpene, and/or glycosylated-xylosylated terpene, and/or a mixture thereof or a pharmaceutically acceptable salt thereof, wherein the medical condition is selected from the group consisting of: obesity, post-traumatic stress syndrome, anorexia, nausea, emesis, pain, wasting syndrome, HIV-wasting, chemotherapy induced nausea and vomiting, alcohol use disorders, anti-tumor, amyotrophic lateral sclerosis, glioblastoma multiforme, glioma, increased intraocular pressure, glaucoma, Cannabis use disorders, Tourette's syndrome, dystonia, multiple sclerosis, inflammatory bowel disorders, arthritis, dermatitis, Rheumatoid arthritis, systemic lupus erythematosus, anti-inflammatory, anti-convulsant, anti-psychotic, anti-oxidant, neuroprotective, anti-cancer, immunomodulatory effects, peripheral neuropathic pain, neuropathic pain associated with post-herpetic neuralgia, diabetic neuropathy, shingles, burns, actinic keratosis, oral cavity sores and ulcers, post-episiotomy pain, psoriasis, pruritis, contact dermatitis, eczema, bullous dermatitis herpetiformis, exfoliative dermatitis, mycosis fungoides, pemphigus, severe erythema multiforme (e.g., Stevens-Johnson syndrome), seborrheic dermatitis, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, gout, chondrocalcinosis, joint pain secondary to dysmenorrhea, fibromyalgia, musculoskeletal pain, neuropathic-postoperative complications, polymyositis, acute nonspecific tenosynovitis, bursitis, epicondylitis, post-traumatic osteoarthritis, synovitis, and juvenile rheumatoid arthritis. In a preferred embodiment, the pharmaceutical composition may be administered by a route selected from the group consisting of: transdermal, topical, oral, buccal, sublingual, intra-venous, intra-muscular, vaginal, rectal, ocular, nasal and follicular. The amount of water-soluble terpenes may be a therapeutically effective amount, which may be determined by the patient's age, weight, medical condition terpene-delivered, route of delivery and the like. In one embodiment, a therapeutically effective amount may be 50 mg or less of a water-soluble terpene. In another embodiment, a therapeutically effective amount may be 50 mg or more of a water-soluble terpene. It should be noted that for any of the above composition, unless otherwise stated, an effective amount of water-soluble terpenes may include amounts between: 0.01 mg to 0.1 mg; 0.01 mg to 0.5 mg; 0.01 mg to 1 mg; 0.01 mg to 5 mg; 0.01 mg to 10 mg; 0.01 mg to 25 mg; 0.01 mg to 50 mg; 01 mg to 75 mg; 0.01 mg to 100 mg; 0.01 mg to 125 mg; 0.01 mg to 150 mg; 0.01 mg to 175 mg; 0.01 mg to 200 mg; 0.01 mg to 225 mg; 0.01 mg to 250 mg; 0.01 mg to 275 mg; 0.01 mg to 300 mg; 0.01 mg to 225 mg; 0.01 mg to 350 mg; 0.01 mg to 375 mg; 0.01 mg to 400 mg; 0.01 mg to 425 mg; 0.01 mg to 450 mg; 0.01 mg to 475 mg; 0.01 mg to 500 mg; 0.01 mg to 525 mg; 0.01 mg to 550 mg; 0.01 mg to 575 mg; 0.01 mg to 600 mg; 0.01 mg to 625 mg; 0.01 mg to 650 mg; 0.01 mg to 675 mg; 0.01 mg to 700 mg; 0.01 mg to 725 mg; 0.01 mg to 750 mg; 0.01 mg to 775 mg; 0.01 mg to 800 mg; 0.01 mg to 825 mg; 0.01 mg to 950 mg; 0.01 mg to 875 mg; 0.01 mg to 900 mg; 0.01 mg to 925 mg; 0.01 mg to 950 mg; 0.01 mg to 975 mg; 0.01 mg to 1000 mg; 0.01 mg to 2000 mg; 0.01 mg to 3000 mg; 0.01 mg to 4000 mg; 01 mg to 5000 mg; 0.01 mg to 0.1 mg/kg.; 0.01 mg to 0.5 mg/kg; 01 mg to 1 mg/kg; 0.01 mg to 5 mg/kg; 0.01 mg to 10 mg/kg; 0.01 mg to 25 mg/kg; 0.01 mg to 50 mg/kg; 0.01 mg to 75 mg/kg; and 0.01 mg to 100 mg/kg.
The modified terpene compounds of the present invention are useful for a variety of therapeutic applications. For example, the compounds are useful for treating or alleviating symptoms of diseases and disorders involving CB1 and CB2 receptors, as well as xanthine dehydrogenase and xanthine oxidase, including appetite loss, nausea and vomiting, pain, multiple sclerosis and epilepsy. For example, they may be used to treat pain (i.e. as analgesics) in a variety of applications including but not limited to pain management. In additional embodiments, such modified terpene compounds may be used as an appetite suppressant. Additional embodiments may include administering the modified terpene compounds.
By “treating” the present inventors mean that the compound is administered in order to alleviate symptoms of the disease or disorder being treated. Those of skill in the art will recognize that the symptoms of the disease or disorder that is treated may be completely eliminated, or may simply be lessened. Further, the compounds may be administered in combination with other drugs or treatment modalities, such as with chemotherapy or other cancer-fighting drugs. Implementation may generally involve identifying patients suffering from the indicated disorders and administering the compounds of the present invention in an acceptable form by an appropriate route. The exact dosage to be administered may vary depending on the age, gender, weight and overall health status of the individual patient, as well as the precise etiology of the disease. However, in general, for administration in mammals (e.g. humans), dosages in the range of from about 0.01 to about 300 mg of compound per kg of body weight per 24 hr., and more preferably about 0.01 to about 100 mg of compound per kg of body weight per 24 hr., are effective. Administration may be oral or parenteral, including intravenously, intramuscularly, subcutaneously, intradermal injection, intraperitoneal injection, etc., or by other routes (e.g. transdermal, sublingual, oral, rectal and buccal delivery, inhalation of an aerosol, etc.). In a preferred embodiment of the invention, the water-soluble terpene analogs are provided orally or intravenously. In particular, the phenolic esters of the invention are preferentially administered systemically in order to afford an opportunity for metabolic activation via in vivo cleavage of the ester. In addition, the water soluble compounds with azole moieties at the pentyl side chain do not require in vivo activation and may be suitable for direct administration (e.g. site specific injection).
The compounds may be administered in a pure form or in a pharmaceutically acceptable formulation including suitable elixirs, binders, and the like (generally referred to a “carriers”) or as pharmaceutically acceptable salts (e.g. alkali metal salts such as sodium, potassium, calcium or lithium salts, ammonium, etc.) or other complexes. It should be understood that the pharmaceutically acceptable formulations include liquid and solid materials conventionally utilized to prepare both injectable dosage forms and solid dosage forms such as tablets and capsules and aerosolized dosage forms. In addition, the compounds may be formulated with aqueous or oil based vehicles. Water may be used as the carrier for the preparation of compositions (e.g. injectable compositions), which may also include conventional buffers and agents to render the composition isotonic. Other potential additives and other materials (preferably those which are generally regarded as safe [GRAS]) include: colorants; flavorings; surfactants (TWEEN, oleic acid, etc.); solvents, stabilizers, elixirs, and binders or encapsulants (lactose, liposomes, etc). Solid diluents and excipients include lactose, starch, conventional disintergrating agents, coatings and the like. Preservatives such as methyl paraben or benzalkium chloride may also be used. Depending on the formulation, it is expected that the active composition will consist of about 1% to about 99% of the composition and the vehicular “carrier” will constitute about 1% to about 99% of the composition. The pharmaceutical compositions of the present invention may include any suitable pharmaceutically acceptable additives or adjuncts to the extent that they do not hinder or interfere with the therapeutic effect of the active compound.
The administration of the compounds of the present invention may be intermittent, bolus dose, or at a gradual or continuous, constant or controlled rate to a patient. In addition, the time of day and the number of times per day that the pharmaceutical formulation is administered may vary are and best determined by a skilled practitioner such as a physician. Further, the effective dose can vary depending upon factors such as the mode of delivery, gender, age, and other conditions of the patient, as well as the extent or progression of the disease. The compounds may be provided alone, in a mixture containing two or more of the compounds, or in combination with other medications or treatment modalities. The compounds may also be added to blood ex vivo and then be provided to the patient.
The term “glycosyltransferase” or “UGT” as generally used herein means an enzyme that has glycosylation and/or glycosylation and acetylation activity toward one or more terpenes, cannabinoids or both.
The term “terpene” refers to the large and diverse class of organic compounds produced by a variety of plants, including Cannabis plants. When terpenes are modified chemically, such as by oxidation or rearrangement of the carbon skeleton, the resulting compounds are generally referred to as “terpenoids.” The structure of terpenes are built with isoprenes, which are 5 carbon structures. Flavonoids are generally considered to be 15 carbon structures with two phenyl rings and a heterocyclic ring. So, there could be an overlap in which a flavonoid could be considered a terpene. However, not all terpenes could be considered flavonoids.
As used herein, the terms “terpene” and “terpenoid” are used interchangeably.
Within the context of the inventive technology, the term terpene includes: Flemiterpenes, Monoterpenols, Terpene esters, Diterpenes, Monoterpenes, Polyterpenes, Tetraterpenes, Terpenoid oxides, Sesterterpenes, Sesquiterpenes, Norisoprenoids, or their derivatives. Derivatives of terpenes include Terpenoids in their forms of hemiterpenoids, monoterpenoids, sesquiterpenoids, sesterterpenoid, sesquarterpenoids, tetraterpenoids, Triterpenoids, tetraterpenoids, Polyterpenoids, isoprenoids, and steroids. They may be forms: α-, β-, γ-, oχo-, isomers, or combinations thereof.
Examples of terpenes within the context of the inventive technology include: 7,8-dihydroionone, Acetanisole, Acetic Acid, Acetyl Cedrene, Anethole, Anisole, Benzaldehyde, Bergamotene (α-cis-Bergamotene) (α-trans-Bergamotene), Bisabolol (β-Bisabolol), Borneol, Bornyl Acetate, Butanoic/Butyric Acid, Cadinene (α-Cadinene) (γ-Cadinene), Cafestol, Caffeic acid, Camphene, Camphor, Capsaicin, Carene (Δ-3-Carene), Carotene, Carvacrol, Carvone, Dextro-Carvone, Laevo-Carvone, Caryophyllene (β-Caryophyllene), Caryophyllene oxide, Castoreum Absolute, Cedrene (α-Cedrene) (β-Cedrene), Cedrene Epoxide (a-Cedrene Epoxide), Cedrol, Cembrene, Chlorogenic Acid, Cinnamaldehyde (α-amyl-Cinnamaldehyde) (a-hexyl-Cinnamaldehyde), Cinnamic Acid, Cinnamyl Alcohol, Citronellal, Citronellol, Cryptone, Curcumene (α-Curcumene) (γ-Curcumene), Decanal, Dehydrovomifoliol, Diallyl Disulfide, Dihydroactinidiolide, Dimethyl Disulfide, Eicosane/lcosane, Elemene (β-Elemene), Estragole, Ethyl acetate, Ethyl Cinnamate, Ethyl maltol, Eucalyptol/1,8-Cineole, Eudesmol (a-Eudesmol) (β-Eudesmol) (γ-Eudesmol), Eugenol, Euphol, Farnesene, Farnesol, Fenchol (β-Fenchol), Fenchone, Geraniol, Geranyl acetate, Germacrenes, Germacrene B, Guaia-1 (10),11-diene, Guaiacol, Guaiene (a-Guaiene), Gurjunene (α-Gurjunene), Herniarin, Flexanaldehyde, Flexanoic Acid, Humulene (a-Humulene) (β-Humulene), lonol (3-oxo-a-ionol) (β-IoηoI), lonone (a-lonone) (β-lonone), Ipsdienol, Isoamyl acetate, Isoamyl Alcohol, Isoamyl Formate, Isoborneol, Isomyrcenol, Isopulegol, Isovaleric Acid, Isoprene, Kahweol, Lavandulol, Limonene, γ-Linolenic Acid, Linalool, Longifolene, α-Longipinene, Lycopene, Menthol, Methyl butyrate, 3-Mercapto-2-Methylpentanal, Mercaptan/Thiols, β-Mercaptoethanol, Mercaptoacetic Acid, Allyl Mercaptan, Benzyl Mercaptan, Butyl Mercaptan, Ethyl Mercaptan, Methyl Mercaptan, Furfuryl Mercaptan, Ethylene Mercaptan, Propyl Mercaptan, Thenyl Mercaptan, Methyl Salicylate, Methylbutenol, Methyl-2-Methylvalerate, Methyl Thiobutyrate, Myrcene (β-Myrcene), γ-Muurolene, Nepetalactone, Nerol, Nerolidol, Neryl acetate, Nonanaldehyde, Nonanoic Acid, Ocimene, Octanal, Octanoic Acid, P-cymene, Pentyl butyrate, Phellandrene, Phenylacetaldehyde, Phenylethanethiol, Phenylacetic Acid, Phytol, Pinene, β-Pinene, Propanethiol, Pristimerin, Pulegone, Quercetin, Retinol, Rutin, Sabinene, Sabinene Hydrate, cis-Sabinene Hydrate, trans-Sabinene Hydrate, Safranal, α-Selinene, a-Sinensal, β-Sinensal, β-Sitosterol, Squalene, Taxadiene, Terpin hydrate, Terpineol, Terpine-4-ol, α-Terpinene, y-Terpinene, Terpinolene, Thiophenol, Thujone, Thymol, a-Tocopherol, Tonka Undecanone, Undecanal, Valeraldehyde/Pentanal, Verdoxan, a-Ylangene, Umbelliferone, or Vanillin. Diterpenes: oridonin, phytol, and isophytol. Triterpenes: ursolic acid, oleanolic acid, among those list elsewhere.
To “xylosylated” means to attach a xylosyl moiety to a molecule.
The term “glycosidase inhibitor” and as used in the present invention is used to mean a compound, which can inhibit glycosidase enzymes which catalyze the hydrolysis of glycosidic bonds.
As used herein, the term “homologous” with regard to a contiguous nucleic acid sequence, refers to contiguous nucleotide sequences that hybridize under appropriate conditions to the reference nucleic acid sequence. For example, homologous sequences may have from about 70%-100, or more generally 80% to 100% sequence identity, such as about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; and about 100%. The property of substantial homology is closely related to specific hybridization. For example, a nucleic acid molecule is specifically hybridizable when there is a sufficient degree of complementarity to avoid nonspecific binding of the nucleic acid to non-target sequences under conditions where specific binding is desired, for example, under stringent hybridization conditions. The term “homolog” as used herein means a homologous protein having a similar enzymatic action. For example, a UTG homolog would be any similar homologous enzyme having glycosylation activity towards one or more terpenes or cannabinoids.
The term, “operably linked,” when used in reference to a regulatory sequence and a coding sequence, means that the regulatory sequence affects the expression of the linked coding sequence. “Regulatory sequences,” or “control elements,” refer to nucleotide sequences that influence the timing and level/amount of transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters; translation leader sequences; introns; enhancers; stem-loop structures; repressor binding sequences; termination sequences; polyadenylation recognition sequences; etc. Particular regulatory sequences may be located upstream and/or downstream of a coding sequence operably linked thereto. Also, particular regulatory sequences operably linked to a coding sequence may be located on the associated complementary strand of a double-stranded nucleic acid molecule.
As used herein, the term “promoter” refers to a region of DNA that may be upstream from the start of transcription, and that may be involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A promoter may be operably linked to a coding sequence for expression in a cell, or a promoter may be operably linked to a nucleotide sequence encoding a signal sequence which may be operably linked to a coding sequence for expression in a cell.
As used herein, a “cannabinoid” is a chemical compound (such as cannabinol, THC or cannabidiol) that is found in the plant species Cannabis among others like Echinacea; Acmella oleracea; Helichrysum umbraculigerum; Radula marginata (Liverwort) and Theobroma cacao, and metabolites and synthetic analogues thereof that may or may not have psychoactive properties. Cannabinoids therefore include (without limitation) compounds (such as THC) that have high affinity for the cannabinoid receptor (for example Ki<250 nM), and compounds that do not have significant affinity for the cannabinoid receptor (such as cannabidiol, CBD). Cannabinoids also include compounds that have a characteristic dibenzopyran ring structure (of the type seen in THC) and cannabinoids which do not possess a pyran ring (such as cannabidiol). Hence a partial list of cannabinoids includes THC, CBD, dimethyl heptylpentyl cannabidiol (DMHP-CBD), 6, l2-dihydro-6-hydroxy-cannabidiol (described in U.S. Pat. No. 5,227,537, incorporated by reference); (3S,4R)-7-hydroxy-A6-tetrahydrocannabinol homologs and derivatives described in U.S. Pat. No. 4,876,276, incorporated by reference; (+)-4-[4-DMH-2,6-diacetoxy-phenyl]-2-carboxy-6,6-dimethylbicyclo[3. l. l]hept-2-en, and other 4-phenylpinene derivatives disclosed in U.S. Pat. No. 5,434,295, which is incorporated by reference; and cannabidiol (−)(CBD) analogs such as (−)CBD-monomethylether, (−)CBD dimethyl ether; (−)CBD diacetate; (−)3′-acetyl-CBD monoacetate; and ±AFl l, all of which are disclosed in Consroe et al., J. Clin. Phannacol. 2l:428S-436S, 1981, which is also incorporated by reference. Many other cannabinoids are similarly disclosed in Agurell et al., Pharmacol. Rev. 38:31-43, 1986, which is also incorporated by reference.
As claimed herein, the term “cannabinoid” may also include different modified forms of a cannabinoid such as a hydroxylated cannabinoid or cannabinoid carboxylic acid, an acetylated glycoside cannabinoid, a methylated cannabinoid. For example, if a glycosyltransferase were to be capable of glycosylating a cannabinoid, a resulting cannabinoid glycoside would be included in the term cannabinoid as defined elsewhere, as well as the aforementioned modified forms. It may further include multiple glycosylation moieties.
Examples of cannabinoids are tetrahydrocannabinol, cannabidiol, cannabigerol, cannabichromene, cannabicyclol, cannabivarin, cannabielsoin, cannabicitran, cannabigerolic acid, cannabigerolic acid monomethylether, cannabigerol monomethylether, cannabigerovarinic acid, cannabigerovarin, cannabichromenic acid, cannabichromevarinic acid, cannabichromevarin, cannabidolic acid, cannabidiol monomethylether, cannabidiol-C4, cannabidivarinic acid, cannabidiorcol, delta-9-tetrahydrocannabinolic acid A, delta-9-tetrahydrocannabinolic acid B, delta-9-tetrahydrocannabinolic acid-C4, delta-9-tetrahydrocannabivarinic acid, delta-9-tetrahydrocannabivarin, delta-9-tetrahydrocannabiorcolic acid, delta-9-tetrahydrocannabiorcol, delta-7-cis-iso-tetrahydrocannabivarin, delta-8-tetrahydrocannabiniolic acid, delta-8-tetrahydrocannabinol, cannabicyclolic acid, cannabicylovarin, cannabielsoic acid A, cannabielsoic acid B, cannabinolic acid, cannabinol methylether, cannabinol-C4, cannabinol-C2, cannabiorcol, 10-ethoxy-9-hydroxy-delta-6a-tetrahydrocannabinol, 8,9-dihydroxy-delta-6a-tetrahydrocannabinol, cannabitriolvarin, ethoxy-cannabitriolvarin, dehydrocannabifuran, cannabifuran, cannabichromanon, cannabicitran, 10-oxo-delta-6a-tetrahydrocannabinol, delta-9-cis-tetrahydrocannabinol, 3, 4, 5, 6-tetrahydro-7-hydroxy-alpha-alpha-2-trimethyl-9-n-propyl-2, 6-methano-2H-l-benzoxocin-5-methanol-cannabiripsol, trihydroxy-delta-9-tetrahydrocannabinol, and cannabinol. Examples of cannabinoids within the context of this disclosure include tetrahydrocannabinol and cannabidiol.
A “Cannabis extract” may include one or more compounds extracted from a Cannabis plant. In one embodiment, a “Cannabis extract” may include one or more terpenes extracted from a Cannabis plant. In one embodiment, a “Cannabis extract” may include one or more cannabinoids extracted from a Cannabis plant. In one embodiment, a “Cannabis extract” may include one or more terpenes and one or more cannabinoids extracted from a Cannabis plant. As also used herein, the term Cannabis include hemp.
As used herein, the term “transformation” or “genetically modified” refers to the transfer of one or more nucleic acid molecule(s) into a cell. A plant is “transformed” or “genetically modified” by a nucleic acid molecule transduced into the plant when the nucleic acid molecule becomes stably replicated by the plant. As used herein, the term “transformation” or “genetically modified” encompasses all techniques by which a nucleic acid molecule can be introduced into, such as a plant, or yeast cell and include both stable and transient transformations. Genes encoding by a combination polynucleotide and/or a homologue thereof, may be introduced into a plant, and/or plant cell using several types of transformation approaches developed for the generation of transgenic plants. Standard transformation techniques, such as Ti-plasmid Agrobacterium-mediated transformation, particle bombardment, microinjection, and electroporation may be utilized to construct stably transformed transgenic plants.
An “expression vector” is nucleic acid capable of replicating in a selected host cell or organism. An expression vector can replicate as an autonomous structure, or alternatively can integrate, in whole or in part, into the host cell chromosomes or the nucleic acids of an organelle, or it is used as a shuttle for delivering foreign DNA to cells, and thus replicate along with the host cell genome. Thus, an expression vector are polynucleotides capable of replicating in a selected host cell, organelle, or organism, e.g., a plasmid, virus, artificial chromosome, nucleic acid fragment, and for which certain genes on the expression vector (including genes of interest) are transcribed and translated into a polypeptide or protein within the cell, organelle or organism; or any suitable construct known in the art, which comprises an “expression cassette.”
A polynucleotide sequence is operably linked to an expression control sequence(s) (e.g., a promoter and, optionally, an enhancer) when the expression control sequence controls and regulates the transcription and/or translation of that polynucleotide sequence.
Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), the complementary (or complement) sequence, and the reverse complement sequence, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Because of the degeneracy of nucleic acid codons, one can use various different polynucleotides to encode identical polypeptides. The Table below, contains information about which nucleic acid codons encode which amino acids.
The term “plant” or “plant system” includes whole plants, plant organs, progeny of whole plants or plant organs, embryos, somatic embryos, embryo-like structures, protocorms, protocorm-like bodies (PLBs), and culture and/or suspensions of plant cells. Plant organs comprise, e.g., shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, trichomes and the like). The invention may also include Cannabaceae and other Cannabis strains, such as C. sativa generally.
The term “expression,” as used herein, or “expression of a coding sequence” (for example, a gene or a transgene) refers to the process by which the coded information of a nucleic acid transcriptional unit (including, e.g., genomic DNA or cDNA) is converted into an operational, non-operational, or structural part of a cell, often including the synthesis of a protein. Gene expression can be influenced by external signals; for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).
The term “gene” or “sequence” refers to a coding region operably joined to appropriate regulatory sequences capable of regulating the expression of the gene product (e.g., a polypeptide or a functional RNA) in some manner. A gene includes untranslated regulatory regions of DNA (e.g., promoters, enhancers, repressors, etc.) preceding (up-stream) and following (down-stream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons). The term “structural gene” as used herein is intended to mean a DNA sequence that is transcribed into mRNA which is then translated into a sequence of amino acids characteristic of a specific polypeptide. The term “sequence identity” or “identity,” as used herein in the context of two nucleic acid or polypeptide sequences, refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
The terms “approximately” and “about” refer to a quantity, level, value or amount that varies by as much as 30%, or in another embodiment by as much as 20%, and in a third embodiment by as much as 10% to a reference quantity, level, value or amount. As used herein, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.
As used herein, “heterologous” or “exogenous” in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or is synthetically designed, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention. As used herein, “endogenous” in reference to a nucleic acid is a nucleic acid that originates from a host the same species, or is substantially un-modified from its native form in composition and/or genomic locus by deliberate human intervention.
The term “prodrug” refers to a precursor of a biologically active pharmaceutical agent (drug). Prodrugs must undergo a chemical or a metabolic conversion to become a biologically active pharmaceutical agent. A prodrug can be converted ex vivo to the biologically active pharmaceutical agent by chemical transformative processes. In vivo, a prodrug is converted to the biologically active pharmaceutical agent by the action of a metabolic process, an enzymatic process or a degradative process that removes the prodrug moiety to form the biologically active pharmaceutical agent.
The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. Indeed, while this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
The present inventors generated an in vivo expression system for yeast-based terpene bioconversion, which in one preferred embodiment utilizes Invitrogen Pichiapink™ system. This model system is a eukaryotic expression system based on the methylotrophic yeast Pichia pastoris (Komagataella phaffii). The Pichiapink system includes protease-deficient host strains and allows both intracellular as well as secreted protein production. In addition, the use of the inducible promoter alcohol oxidase (AOX1) uncouples growth from production of desired proteins, so that cells are not stressed by the accumulation of recombinant protein during growth phase. PichiaPink strain 4 (herein referred to as wild-type, WT), a double knockout for proteases prb1, pep4 (to avoid degradation of desired protein) was utilized by the present inventors as the background strain in exemplary yeast transformations. The vector pPINK-HC was used as the backbone for expression of exemplary terpene glycosyltransferases (UGTs) in the yeast system. As shown in
Expression analysis for introduced transgenes was carried out by RT-PCR. For yeast, 2 mL of a 2-day old culture induced by methanol overnight was centrifuged in a microfuge tube. The pellet was ground in a TissueLyser (QIAGEN Inc, USA). RNA was extracted following the EZNA plant RNA extraction kit (Omega Bio-tek Inc, USA). Up to a microgram of total RNA was used to synthesize cDNA using the superscript III cDNA synthesis kit (Thermo Fisher Scientific, USA). The cDNA was used to check for the expression of transgenes by RT-PCR.
The present inventors conduct a 96-well screening procedure to identify the production of terpene glycosides in a novel yeast-based expression system. Cultures listed in Table 2 below were fed 100 μg/mL of linalool or alpha-bisabolol after overnight gene expression induction with 2% methanol. Cultures were harvested 48 h post terpene feeding. As shown in
In another embodiment, the same yeast cultures as identified in Table 2 below were induced with 2% methanol overnight, and then fed with individual terpenes (alpha-bisabolol, nerol, citronellol) at 100 μg/mL final concentration. The cultures were harvested 2 days post-feeding with terpenes. There was no observed negative effect on yeast cell growth rates after treatment with terpenes at this concentration. As summarized in Table 3 below, Nerol was successfully bio-converted to neryl monoglucoside by the tea plant UGT85K11, but not by other enzymes or empty vector control (
The present inventors generated a Cannabis plant based production systems for the bioconversion of terpenes into glycosylated terpene glycosides. As showing in
Overnight cultures of Agrobacterium AGL1 expressing UGT94P1, UGT85K11, and NtGT4 were transferred to a 250 mL flask with 50 mL LB medium supplemented with 50 mg/L of Kanamycin, 50 mg/L of Gentamycin and 10 mg/L of Rifampicin and grown for 4-8 hours until the optical density at 600 nm (OD600) reached approximately between 0.75 and 1. The cells were pelleted in a centrifuge at room temperature and resuspended 45 mL of infiltration medium containing 5 g/L D-glucose, 10 mM MES, 10 mM MgCl2 and 100 μM acetosyringone. Hemp plants (Youngsim 10 genotype), 4-weeks old, were vacuum-infiltrated with Agrobacterium tumefaciens AGL1 cultures expressing UGT94P1, UGT85K11, and NtGT4.
Expression of the transgenes was confirmed 2-4 days after infiltration by RT-PCR. For RT-PCR analysis, 100 mg of leaf tissue were frozen in liquid nitrogen and ground in a TissueLyser (QIAGEN Inc, USA). RNA was extracted following the EZNA plant RNA extraction kit (Omega Bio-tek Inc, USA). Up to a microgram of total RNA was used to synthesize cDNA using the superscript III cDNA synthesis kit (Thermo Fisher Scientific, USA). The cDNA was used to check for the expression of transgenes by RT-PCR.
In one preferred embodiment, the present inventors demonstrated in in vivo production of terpene glycosides in a Cannabis plant. In this embodiment, four weeks old, Cannabis (Hemp) plants (Youngsim 10 genotype), were vacuum-infiltrated with Agrobacterium tumefaciens AGL1 cultures expressing UGT94P1 and UGT85K11 together, or NtGT4 alone. The wild-type concentration of free terpenes present in these samples prior to infiltration was previously determined. Samples were harvested 2-10 days post infiltration, weighted, and extracts analyzed for the formation of terpene glycosides. As shown in
Agrobacterium-infiltrated samples relying on endogenous terpene pools identified above were harvested 5 days post infiltration and analyzed for possible terpene conjugate formation. In one embodiment, While alpha-bisabolyl diglucoside was detected in samples co-expressing UGT85K11 and UGT94P1, this product was not detected in the empty vector (prI201-AN) control samples (
The present in inventors demonstrated a novel non-GM method for the bioconversion of terpenes using wild type tobacco (BY2) cell suspension cultures for in vivo glycosylation of terpenes. In this embodiment, healthy BY2 cell suspension cultures (15 mL) were fed in triplicates with 200 ug/mL of geraniol (1.290 uM). Cultures used as control were fed the equivalent methanol volume used to deliver the terpenes to treated cultures. The cultures were harvested 5 and 7 days-post treatment. As demonstrated in
Detection of Terpene Glycosides
In a preferred embedment, free volatile terpene and terpenyl glycoside content may be analyzed by LC-MS/MS. Endogenous free and bound terpene content may further be screened in both wild type yeast and/or plant cell suspension cultures to establish an expected background profile. Additionally, Cannabis-specific terpenes may be screened by feeding a known concentration of Cannabis terpene standard mixtures to yeast and/or plant cell suspension cultures. Control cultures may be incubated with the delivery solvent (isopropanol). Cultures can then be incubated for 16 hr, followed by extraction for downstream analysis by LC-MS/MS
Free and bound terpenes may be extracted from 0.2-1.0 g starting material or equivalent, in the case of cell suspension cultures, and this material can be homogenized into 10 volumes methanol. For free terpenes, an incubation period of 1 hr may be followed by centrifugation (10,000×g, 5 min) to clear cellular debris. The filtered supernatant can then be spiked with either 0.3% toluene or 5 μM 4-methyl-2-pentanol as an internal standard (ISTD). For terpenyl glycosides, the filtered supernatant can be applied to a pre-conditioned Supleco© XAD-2 solid-phase extraction (SPE) cartridge (Sigma, US), which may then be washed with dichloromethane to remove phenolics and free terpenoids. Bound terpenes may then be eluted with methanol, and concentrated to dryness under nitrogen.
Extracts may then be analyzed by LC-MS/MS using an Acquity M-Series UPLC system coupled with a Synapt G2-Si mass spectrometer (Waters, US). Briefly, a 2 μL aliquot may be injected onto a HALO C18 2.7 μm fused-particle column 150 mm×300 □m (Sciex, US). Mobile phases (A) water with 0.1% formic acid and (B) methanol with 0.1% formic can be used for a gradient separation over 20 min at a flow rate of 0.1 mL min−1, and atmospheric pressure chemical ionization (APCI) may be used to ionize free terpene species. MS and MS/MS data can be collected using multiple reaction monitoring (MRM), with the proposed retention times, collision energies and MRM transitions for free terpenes are listed in table 9 directly below. Gradient separation (retention time) and collision energy may differ between LC-MS/MS systems.
The dominant free terpenoids present in C. sativa are listed above; however, other free terpenoids have been observed within C. sativa, H. lupulus and N. tabacum, and additional MRM parameters may be determined during screening. Additionally, MRM parameters can be established for terpenyl glycosides. Previous reports have also indicated a high degree of promiscuity for certain glycosyltransferases; therefore, the method can be expanded to screen for additional non-target products, such as flavonoids, steroids, phenolic compounds, alcohols, and aldehydes and the like.
Sample Preparation for the Analysis of Water-Soluble Terpenes
From yeast and tobacco cell culture: Yeast and BY2 cell suspension cultures were centrifuged at 4000 rpm, 10 min, and the supernatant were collected in fresh 15 mL falcon tubes. Samples were processed following generally known protocols by those of ordinary skill in the art. Cell pellets were lyophilized overnight, and dry cell weights were collected. Dried cells were extracted with 5 mL methanol containing 0.1 ppm 7-hydroxycoumarin as an internal standard, and homogenized in an ultrasonic bath for 30 min. Supernatant and dried cell extracts were cleaned up by solid-phase extraction (SPE) using 6 mL Resprep bonded reversed-phase (C18) SPE cartridges (Restek). Briefly, cartridges were pre-conditioned with three bed volumes of water, samples were applied, washed with one bed volume of water and one bed volume of 30% methanol, and terpene glycosides were eluted in 5 mL of 100% methanol and free terpenes were eluted in 1 mL of 100% hexane.
From leaf tissue: Hemp leaves were processed following generally known protocols by those of ordinary skill in the art. with the following modifications. Hemp leaves were weighed and then ground in liquid nitrogen with a micropestle in a 2 mL centrifuge tube. Free and glycosylated terpenes were extracted with 1 mL acetone and incubated for 10 min. Extracts were centrifuged (15,000 rpm×2 min×RT) to clear cell debris and then split for analysis by LC-MS and GD-FID. Acetone extracts were dried under a nitrogen stream, and extracts for LC-MS analysis were dissolved in 70% methanol and extracts were dissolved in hexane.
Liquid Chromatography-Mass Spectrometry (LC-MS)
Dried extracts containing terpene glycosides were resolubilized in 1 mL of 70% methanol and 1 μL injections were made onto an HSS T3 C18 column (300 μm×150 mm, particle size 1.8 μm) maintained at 40° C. and equipped to an ACQUITY M-Class UPLC System and a Synapt G2-Si HDMS (Waters). A flow rate of 5.0 μL/min is used with mobile solvents (A) acetonitrile with 0.1% formic acid and (B) water with 0.1% formic acid following a linear gradient: initial conditions 85:15% (A:B %) for 2 minutes, linear ramp to 15:85% in 12 min, hold at 15:85% for 4.5 min, then equilibrate back to initial conditions 85:15% for 5.5 min and a total run time of 22 min. A LockMass solution of 200 μg/mL leucine enkephalin (554.2615 m/z) is infused through an auxiliary pump at a flow rate of 5.0 μL/min to maintain mass accuracy.
Data are acquired in negative ionization mode (ES-) using a data-independent acquisition (MSe) method in continuum mode. Sample and lockspray capillary voltages are set to 2.0 and 3.2 kV, respectively, and sample cone and cone offset are set to 30 and 40 V, respectively. Cone and desolvation gases are set to 20 and 500 L/hr and nebulizer gas is maintained at 6.5 bar. MS acquisition is performed from 0.0-22.0 minutes over a mass range of 100-600 m/z with a 0.486 s scan time with 0.014 s interscan delay. A high energy collision ramp of 5-30 V is applied, and LockSpray measurements are acquired every 30 s.
Data Processing
LC-MS data were processed in UNIFI v 9.3 (Waters) using an MSe analysis method developed to screen for both predicted and known terpene glycosides. Free terpenes were not detected by LC-ESI-MS. A terpene glycoside library was generated in-house using known and predicted structures drawn and exported as (.mol) files in ChemDoodle 2D Sketcher (ChemDoodle). Fragmentation ions produced by cleavage of the glyosidic bond [C6H10O6]− (179.0561 m/z) and [C6H9O5]− (161.0456 m/z), and [C2H4O2]− (59.0142 m/z) produced by fragmentation of glucose, were used to identify terpene glycosides. In addition, transformed products resulting from both phase I and II metabolism were also screened, including acetylation, hydration, dehydration, reduction and desaturation. All LC-MS data are presented as signal intensity—counts per second (CPS)—standardized to dry cell weight.
Mass Spectral Analysis of Water-Soluble Terpenes: Identification of Modified Terpenes by Mass Spectrometry
The present inventors used mass spectroscopy analysis to detect bioconversion products generated from UGTs in transgenic yeast, BY2 cells and hemp. Known and predicted glycosylation reactions for individual terpene glycosides, along with empirical data from chromatographic results, were used to generate both known and predicted structures and physiochemical properties indicating increased water solubility for observed terpene glycosides (
Each of the below embodiments is specifically incorporated into the specification of the current application. Each of the below embodiments may be amended and presented as a formal claim and further represents an independent invention. Also, it should be specifically noted that for each preserved embodiment and supporting description in the specification, the term “glycosylated terpene” encompasses glycosylated-xylosylated terpene, and/or xylosylated terpenes.
1. A composition comprising:
All nucleotide and amino acid sequences listed in U.S. Provisional Patent Application No. 62/746,053, filed Oct. 16, 2018 are specifically incorporated herein by reference. The following sequences are further provided herewith and are hereby incorporated into the specification in their entirety:
Absidia coerulea
Absidia coerulea
Mucor hiemalis
Camellia sinensis
Camellia sinensis
Camellia sinensis
Camellia sinensis
Vitis vinifera
Vitis vinifera
Nicotiana tabacum
Nicotiana tabacum
Nicotiana tabacum
Nicotiana tabacum
Bacillus subtilis
Bacillus subtilis
Vitis vinifera
Vitis vinifera
This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62/746,053, filed Oct. 16, 2018, the entirety of which is hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/56613 | 10/16/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62746053 | Oct 2018 | US |
