PL205945B1

Improved EXTRACTION OF PHARMACEUTICALLY ACTIVE CANNABINOIDS FROM PLANT MATERIALS

Abstract

The invention relates to the extraction of pharmaceutically active components from plant materials, and more particularly to the preparation of a botanical drug substance (BDS) for incorporation into a medicament. It also relates to a BDS of given purity, for use in pharmaceutical formulations. In particular it relates to BDS comprising cannabinoids obtained by extraction from cannabis.

PL205945B1, drawing sheet 1 — Sheet 1 of 3

Term

Term ended

Expired 14 August 2023, 3.1 years ago.

45 claims: 17 independent, 28 dependent

1
Zastrzeżenia patentowe 1. Sposób ekstrakcji kannabinoidów z materiał u roś linnego obejmujący etap dekarboksylacji, ekstrakcję ciekłym ditlenkiem węgla (CO2) i etap zmniejszenia w ekstrakcie udziału materiałów nie będącymi materiałami docelowymi, znamienny tym, że ekstrakcję ciekłym CO2 prowadzi się w warunkach podkrytycznych w temperaturze w zakresie między 5-15°C i przy ciśnieniu w zakresie między 5-7 MPa.
2
Sposób według zastrz. 1, znamienny tym, że etap dekarboksylacji prowadzi się po ekstrakcji ciekłym CO2.
3
Sposób według zastrz. 1, znamienny tym, że etap dekarboksylacji prowadzi się przed ekstrakcją ciekłym CO2.
4
Sposób według jednego z zastrz. 1 do 3, znamienny tym, że temperatura mieści się w zakresie od 8 do 12°C.
5
Sposób według zastrz. 4, znamienny tym, ż e temperatura wynosi 10°C.
6
Sposób według jednego z poprzednich zastrz., znamienny tym, że ciśnienie zawarte jest w zakresie od 5,5 do 6,5 MPa.
7
Sposób według zastrz. 6, znamienny tym, że ciśnienie wynosi 6,0 MPa.
8
Sposób według jednego z poprzednich zastrz., znamienny tym, że CO2 ma masowe natę żenie przepływu w zakresie 1000-1500 Kg/godz.
9
Sposób według zastrz. 8, znamienny tym, ż e CO2 ma masowe natężenie przepływu wynoszące 1250 Kg/godz.
10
Sposób według jednego z poprzednich zastrz., znamienny tym, że ekstrakcję prowadzi się do 10 godzin.
11
Sposób według zastrz. 10, znamienny tym, że ekstrakcję prowadzi się 8 godzin.
12
Sposób według jednego z poprzednich zastrz., znamienny tym, że CO2 usuwa się przez rozszczelnienie, a uzyskany ekstrakt utrzymuje się w temperaturze w zakresie od -15°C do -20°C.
13
Sposób według jednego z poprzednich zastrz., znamienny tym, że etapem zmniejszenia udziału materiałów nie będącymi materiałami docelowymi w ekstrakcie jest strącanie alkoholem C1-C5 i że przed dodaniem alkoholu C1-C5 materiał poddawany obróbce ogrzewa się do temperatury wyższej niż temperatura pokojowa.
14
Sposób według zastrz. 13, znamienny tym, że alkoholem C1-C5 jest etanol.
15
Sposób według zastrz. 13 albo 14, znamienny tym, że ekstrakt ogrzewa się do temperatury od 36 do 44°C.
16
Sposób według zastrz. 15, znamienny tym, że ekstrakt ogrzewa się do 40°C.
17
Sposób według jednego z zastrz. 14 do 16, znamienny tym, że alkohol C1-C5 dodaje się w ilości od 3:1 do 1:1 objętości alkoholu C1-C5 w stosunku masy materiału poddawanego obróbce.
18
Sposób według zastrz. 17, znamienny tym, że alkohol C1-C5 dodaje się w ilości około 2:1 objętości alkoholu C1-C5 w stosunku masy materiału poddawanego obróbce. PL 205 945 B1
19
Sposób według jednego z zastrz. 13 do 18, znamienny tym, że roztwór powstały przez dodanie do materiału poddawanego obróbce alkoholu C1-C5 ochładza się i pozwala się na wytrącenie materiałów nierozpuszczalnych.
20
Sposób według zastrz. 19, znamienny tym, że roztwór powstały przez dodanie do materiału poddawanego obróbce alkoholu C1-C5 ochładza się do temperatury w zakresie od -15 do -25°C.
21
Sposób według zastrz. 19 albo 20, znamienny tym, że roztwór powstały przez dodanie do materiału poddawanego obróbce alkoholu C1-C5 ochładza się w ciągu do 52 godzin.
22
Sposób według jednego z zastrz. 19 do 21, znamienny tym, że strącony osad materiałów nierozpuszczalnych usuwa się przez filtrowanie.
23
Sposób według zastrz. 22, znamienny tym, że filtrowanie przeprowadza się przez membranę 20 μm.
24
Sposób według jednego z zastrz. 13 do 23, znamienny tym, że obejmuje dodatkowo wieloetapowe odparowanie pod obniżonym ciśnieniem.
25
Sposób według zastrz. 24, znamienny tym, że najpierw usuwa się alkohol C1-C5, a następnie usuwa się wodę.
26
Sposób według zastrz. 25, znamienny tym, że alkohol C1-C5 usuwa się przez ogrzewanie do temperatury w zakresie od 58 do 62°C w celu uzyskania temperatury pary w zakresie 38 do 42°C, pod próżnią w zakresie 16,8-17,2 kPa, aż do momentu, gdy kondensat będzie występował w małej ilości lub nie będzie widoczny.
27
Sposób według zastrz. 25 albo 26, znamienny tym, że dodatkowo usuwa się wodę przez wieloetapową redukcję próżni stopniowo do 5,0 kPa.
28
Sposób według jednego z zastrz. 3 do 27, znamienny tym, że etap dekarboksylacji prowadzi się przed ekstrakcją ciekłym CO2, przy czym materiał roślinny ogrzewa się do temperatur i w ciągu okresów czasu zapewniających co najmniej 95% przemianę kwasowych kannabinoidów w ich postać obojętną, oraz termiczną degradację THC do CBN mniejszą niż 10%.
29
Sposób według zastrz. 28, znamienny tym, że prowadzi się wielostopniowy proces ogrzewania, w którym materiał roślinny:i) ogrzewa się do pierwszej temperatury w ciągu pierwszego okresu czasu dla odparowania zaabsorbowanej wody i umożliwienia równomiernego ogrzania materiału roślinnego i ii) temperaturę zwiększa się do drugiej temperatury w ciągu drugiego okresu czasu, aż nastąpi co najmniej 95% przemianę kwasowych kannabinoidów w ich postać obojętną.
30
Sposób według zastrz. 29, znamienny tym, że pierwszy etap prowadzi się w temperaturze w zakresie 100°C do 110°C w ciągu 10-20 minut.
31
Sposób według zastrz. 30, znamienny tym, że pierwsza temperatura wynosi 105°C i pierwszy okres czasu wynosi 15 minut.
32
Sposób według jednego z zastrz. 28 do 31, znamienny tym, że stosuje się materiał roślinny mający dużą zawartość CBD, druga temperatura zawarta jest w zakresie od 115 do 125°C, korzystnie wynosi około 120°C, a drugi okres czasu zawarty jest w zakresie od 45 do 75 minut, korzystnie wynosi około 60 minut.
33
Sposób według jednego z zastrz. 28 do 31, znamienny tym, że stosuje się materiał roślinny mający dużą zawartość CBD, druga temperatura zawarta jest w zakresie od 135 do 145°C, korzystnie wynosi 140°C, a drugi okres czasu zawarty jest w zakresie od 15 do 45 minut, korzystnie wynosi 30 minut.
34
Sposób według jednego z zastrz. 28 do zastrz. 31, znamienny tym, że stosuje się materiał roślinny mający dużą zawartość CBD, druga temperatura zawarta jest w zakresie od 140 do 150°C, korzystnie 145°C, a drugi okres czasu zawarty jest w zakresie od 55 do 90 minut.
35
Sposób według jednego z zastrz. 28 do zastrz. 31, znamienny tym, że stosuje się materiał roślinny mający dużą zawartość THC, druga temperatura zawarta jest w zakresie od 115 do 125°C, korzystnie 120°C, a drugi okres czasu zawarty jest w zakresie od 45 do 120 minut, korzystnie 60 minut.
36
Sposób według jednego z zastrz. 28 do 31, znamienny tym, że stosuje się materiał roślinny mający dużą zawartość THC, druga temperatura zawarta jest w zakresie od 100 do 110°C, korzystnie 105°C, a drugi okres czasu zawarty jest w zakresie od 60 do 120 minut.
37
Sposób według jednego z zastrz. 28 do 31, znamienny tym, że stosuje się materiał roślinny mający dużą zawartość THC, druga temperatura zawarta jest w zakresie od 140 do 150°C, korzystnie 145°C, a drugi okres czasu zawarty jest w zakresie 45 do 55 minut. PL 205 945 B1
38
Sposób według jednego z zastrz. 28 do 37, znamienny tym, że etap dekarboksylacji prowadzi się w temperaturach i w ciągu okresów czasu, które zapewniają co najmniej 97% przemianę kwasowych kannabinoidów w ich postać obojętną i termiczną degradację THC do CBN mniejszą niż 5%.
39
Sposób według jednego z poprzednich zastrz., znamienny tym, że materiał roślinny rozdrabnia się, miele lub poddaje innej obróbce w celu otrzymania wielkości cząstek mniejszej niż 2 mm.
40
Sposób według zastrz. 30, znamienny tym, że wielkość cząstek jest większa niż 1 mm.
41
Sposób według jednego z zastrz. 1 do 40, znamienny tym, że obejmuje ponadto etap obróbki ekstraktu pochodzącego z materiału roślinnego za pomocą węgla aktywnego drzewnego.
42
Sposób według zastrz. 41, znamienny tym, że ekstrakt pochodzący z materiału roślinnego rozpuszcza się w roztworze etanolowym.
43
Sposób według zastrz. 42, znamienny tym, że roztworem alkoholowym jest roztwór etanolowy.
44
Sposób według zastrz. 43, znamienny tym, że obróbka za pomocą węgla aktywnego drzewnego następuje po etapie strącania etanolowego.
45
Sposób wytwarzania kompozycji farmaceutycznej zawierającej jako środek aktywny, botaniczną substancję lekową, która jest ekstraktem z co najmniej jednej rośliny konopnej, znamienny tym, że obejmuje wytwarzanie botanicznej substancji lekowej zawierającej kannabinoidy z co najmniej jednej rośliny konopnej z zastosowaniem sposobu ekstrakcji jak określony w zastrz. 1 do 44 i sporządzanie botanicznej substancji lekowej z jednym lub kilkoma farmaceutycznie akceptowanymi rozcieńczalnikami, nośnikami lub podłożami w celu wytwarzania kompozycji farmaceutycznej.

Independent claims45

474 paragraphs in 20 sections, as filed

Description of the invention

The present invention relates to a method of extracting cannabinoids from plant materials and a method of producing a pharmaceutical composition containing a botanical drug substance (BDS). It also presents a BDS of a given purity for use in pharmaceutical preparations, containing cannabinoids obtained by extraction from hemp.

In PCT / GB02 / 00620 the applicant discloses a method of producing a herbal drug extract (botanical drug substance) from medicinal cannabis. The method comprises the steps of:

1. heating to decarboxylate the acid form of cannabinoids to their neutral form,

2. first extraction determined by volume of liquid carbon dioxide in 6-8 hours and

3. reducing the proportion of non-target materials referred to as de-stearing, in which the waxes are precipitated.

More specifically, PCT / GB02 / 00620 discloses a method in which:

step 1 involves heating cut hemp (2-3 mm) at 100-150 ° C for a period of time sufficient to allow decarboxylation, step 2 involves extracting CO2 using:

a) coarse-grained powder (particles pass through a 3 mm sieve),

b) packing density 0.3 i

c) supercritical conditions of 600 bar at 35 ° C in 4 hours, although it could be confirmed that other combinations of temperature and pressure between 10-35 ° C and 60-600 bar could be used (both supercritical and subcritical conditions ) and step 3 involves performing an ethanol precipitation at -20 ° C for 24 hours and filtering off the wax material.

In the supercritical process disclosed in PCT / GB02 / 00620, the following were produced:

a) extract with a high THC content containing: 60% tetrahydrocannabinol (THC), 1-2% cannabidiol (CBD) 4-5% other trace cannabinoids, including CBN (quantitative yields were 9% w / w in relation to the dry weight of medicinal cannabis) and

b) extract with high CBD content containing 60% CBD, 4% THC and 2% other cannabinoids (quantitative yields were 9% w / w in relation to the dry weight of medicinal cannabis).

Since the obtained BDS is to be used in a pharmaceutical product, it is necessary that the process is safe, that it can be varied according to good manufacturing practice, and that it gives a high degree of product integrity and preferably also good yields.

The principle of supercritical liquid extraction (SFE) has been known since the work of Baron Cagniard de la Tour in 1822, when it was found that the gas-liquid interface disappeared when the temperature of some materials was increased by heating them in a closed glass vessel. In carrying out this initial work, a substance's tipping point was discovered for the first time. The critical point is the temperature above which the gas, liquid or solid phase coexistence of a given substance can occur. It was later found that once the substances were brought to or above critical temperatures and pressures, they could be used as improved solvents for the extraction and fractionation of complex mixtures.

The method is widely used in the treatment of diesel fuel and has been used, for example, for the purification and separation of vegetable oils and fish oils.

An attractive feature of SFE over traditional solvents is that the dissolving power (E °) can be varied by modifying the temperature and pressure above the critical point.

In a typical pressure-temperature diagram for a given substance, there are three lines that represent the equilibrium between the two phases. These lines meet at a triple point. The lines indicate the interface between the gaseous, liquid and solid states, and points along the lines indicate the equilibrium between the pairs of phases. For example, the vapor pressure (boiling point) curve starts at the triple point and ends at the critical point. The critical area begins at this point and the supercritical fluid is any substance that is above its critical temperature (Tc) and critical pressure (Pc). The critical temperature is thus the highest temperature at which a gas can be converted to a liquid by increasing the pressure, and the critical pressure is the highest pressure at which a liquid can be converted to a conventional gas by increasing the temperature. There is only one phase in this so-called critical region and it has some of the properties of both gas and liquid.

PL 205 945 B1

There are many solvents that can be used to extract active substances from plant materials. Table 1 shows the critical temperature and pressure of some of these solvents.

Table 1

Critical conditions of solvents

<td>Solvents</td><td>Critical temperature (° C)</td><td>Critical pressure (bar)</td>

<td>Carbon dioxide</td><td> 31,1</td><td> 73,8</td>

<td>Ethane</td><td> 32,2</td><td> 48,8</td>

<td>Ethylene</td><td> 9,3</td><td> 50,4</td>

<td>Propane</td><td> 96,7</td><td> 42,5</td>

<td>Propylene</td><td> 91,9</td><td> 46,2</td>

<td>Cyclohexane</td><td> 280,3</td><td> 40,7</td>

<td>Isopropanol</td><td> 235,2</td><td> 47,6</td>

<td>Benzene</td><td> 289,0</td><td> 48,9</td>

<td>Toluene</td><td> 318,6</td><td> 41,1</td>

<td>p-xylene</td><td> 343,1</td><td> 35,2</td>

<td>Chlorotrifluoromethane</td><td> 28,9</td><td> 39,2</td>

<td>T richlorofluoromethane</td><td> 198,1</td><td> 44,1</td>

<td>Ammonia</td><td> 132,5</td><td> 112,8</td>

<td>Water</td><td> 374,2</td><td> 220,5</td>

As the preferred solvent, carbon dioxide has been used, having a critical temperature of 31.1 ° C and a critical pressure of 7.38MPa.

Carbon dioxide is particularly preferred because it is available at low cost from many suppliers and can be recycled for reuse if desired. Any losses of CO2 are also neutral to the environment. Moreover, CO2 extraction is a safe production method and very sensitive particles can be extracted with precision.

The decisive point to consider when initially selecting liquid CO2 as a solvent for the production of a standardized extract of high potency cannabis herb was the high degree of selectivity that could be achieved. In the CO2 system, it has been found that the solvating capacity can primarily be considered a function of density and temperature, with the solvent density being a more important factor.

By carefully controlling the temperature and pressure below supercritical temperatures and pressures, specific cannabinoid-rich lipophilic or hydrophilic fractions can be separated along with other ingredients that can be relatively easily isolated to obtain a botanical drug substance (BDS) that contains the desired ingredients in a form that is pharmaceutically acceptable . In this way, components which are known to be active substances can be separated from complex mixtures which are present in the botanical raw material.

Moreover, very good batch-to-batch reproducibility can be obtained, and undesirable components such as heavy metals, which may be present to a varying degree in the botanical raw material, can be left behind in the spent material.

Extraction conditions can also be modified to eliminate pesticide residues that may be present in the starting material.

The benefits of using subcritical conditions include the selective nature of the extraction. In contrast, in the case of SFE, it was found that the solvent, in addition to dissolving the desired cannabinoids, also disadvantageously dissolves other non-target materials that proved difficult to separate in a further purification step.

To explain, the density of subcritical CO2 is low and remains low, even with increasing pressure until the critical point of the system is reached. And so, when the subcritical solvating ability

Since CO 2 is lower, a high degree of selectivity can be achieved since only the most soluble components are efficiently dissolved by CO 2, in this case it is the cannabinoid fraction. The result is a relatively simple extract containing only a limited number of non-target compounds in addition to the cannabinoids, many of which can be removed relatively easily in an uncomplicated manner. In addition, cost savings from operating at relatively low pressures and temperatures are an additional benefit.

In contrast, above the critical temperature of 31 ° C there is a significant increase in CO2 density since it is then in a supercritical fluid state. This results in a large increase in the solvating capacity of the solvent which, while generally advantageous in that more cannabinoids dissolve thus giving good yields, in fact proves to be a disadvantage as the reduced selectivity of the stronger solvent results in increased solubility of a range of non-target compounds. which makes the resulting extract more difficult to clean. In other words, this results in the production of more complex extracts in which the concentration of the target compound may be much weaker (ie the strength of the extract is decreased).

According to the present invention, the method of extracting cannabinoids from plant material comprises a decarboxylation step, liquid carbon dioxide (CO2) extraction and a step of reducing the proportion of non-target materials in the extract, characterized in that the extraction with liquid CO2 is performed under subcritical conditions at a temperature of 5-15 ° C and a pressure of 5-7 MPa.

Preferably, decarboxylation is performed after extraction with liquid CO2 or before extraction with liquid CO2.

Preference is given to using a temperature in the range from 8 to 12 ° C, in particular 10 ° C.

The pressure used is preferably in the range from 5.5 to 6.5 MPa, in particular 6.0 MPa.

Preferably the CO2 has a mass flow rate in the range of 1000-1500 kg / h, especially 1250 Kg / h.

Preferably the extraction is carried out for up to 10 hours, especially 8 hours.

Preferably CO2 is removed by unsealing and the resulting extract is kept at a temperature of -15 ° C to -20 ° C.

Preferably, the step of reducing the proportion of non-target materials in the extract is precipitation with a C1-C5 alcohol, and the treated material is warmed to a temperature greater than room temperature prior to the addition of the C1-C5 alcohol.

Preferably, ethanol is used as the C1-C5 alcohol.

Preferably the extract is heated to a temperature of from 36 ° to 44 ° C, in particular to 40 ° C.

Preferably the C1-C5 alcohol is added in an amount of from 3: 1 to 1: 1, especially 2: 1, by volume of C1-C5 alcohol based on the weight of the material to be treated.

Preferably, the solution formed by adding the C1-C5 alcohol to the material to be treated with the C1-C5 alcohol is cooled and the insoluble materials are allowed to precipitate.

Preferably, the solution formed by adding the C1-C5 alcohol to the material to be treated is cooled to a temperature of -15 ° C to -25 ° C.

Preferably, the solution formed by adding the C1-C5 alcohol to the material to be treated is cooled down within up to 52 hours.

Preferably, the precipitate of insoluble materials is removed by filtration.

Preferably, the filtering is carried out on a 20 µm membrane.

Preferably, the process according to the invention additionally comprises a multi-stage evaporation under reduced pressure.

Preferably, C1-C5 alcohol is removed first, followed by removal of water.

Preferably, the C1-C5 alcohol is removed by heating to a temperature in the range of 58 ° C to 62 ° C to obtain a vapor temperature in the range of 38 ° C to 42 ° C, under a vacuum of 16.8-17.2 kPa, until until there is little or no visible condensate.

Preferably, the water is additionally removed by multi-stage reduction of the vacuum gradually to 5.0 kPa. Preferably, the decarboxylation step is carried out prior to extraction with liquid CO2, the plant material being heated to temperatures and for periods of time that will ensure at least 95% conversion of the acidic cannabinoids to their neutral form and thermal degradation of THC to CBN of less than 10%.

PL 205 945 B1

Preferably, the decarboxylation step is carried out in a multi-stage heating process in which the plant material:

i) is heated to a first temperature during a first time period to evaporate the absorbed water and allow the plant material to be evenly heated; and ii) the temperature is increased to a second temperature over a second period of time until at least 95% of the acidic cannabinoids have converted to their neutral form .

Preferably the first step is carried out at a temperature in the range 100 ° C to 110 ° C for 10-20 minutes.

Preferably, the first temperature is 105 ° C and the first period of time is 15 minutes.

Preferably, plant material having a high CBD content is used in the method of the invention, the second temperature is in the range of 115 ° C to 125 ° C, preferably is about 120 ° C, and the second period of time is in the range of 45 to 75 minutes. preferably 60 minutes.

Preferably, plant material having a high CBD content is used in the process according to the invention, the second temperature is in the range 135 ° C to 145 ° C, preferably is 140 ° C, and the second time period is in the range 15 to 45 minutes, preferably Thirty minutes.

Preferably plant material having a high CBD content is used, the second temperature is in the range of 140 ° C to 150 ° C, preferably 145 ° C, and the second period of time is in the range of 55 to 90 minutes.

Preferably plant material having a high THC content is used, the second temperature is in the range 115 ° C to 125 ° C, preferably 120 ° C, and the second period of time is in the range 45 to 120 minutes, preferably 60 minutes.

Preferably, plant material having a high THC content is used, the second temperature is in the range of 100 ° C to 110 ° C, preferably 105 ° C, and the second period of time is in the range of 60 to 120 minutes.

Preferably plant material is used having a high THC content, the second temperature is in the range 140 ° C to 150 ° C, preferably 145 ° C, and the second period of time is in the range 45 to 55 minutes.

Preferably, the decarboxylation step is performed at temperatures and for periods of time that ensure at least 97% conversion of the acidic cannabinoids to their neutral form and thermal degradation of THC to CBN of less than 5%.

Preferably the plant material is comminuted, milled or otherwise processed to obtain a particle size less than 2 mm but greater than 1 mm.

Preferably, the method according to the invention further comprises the step of treating the extract derived from the plant material with activated charcoal.

Preferably, an extract derived from plant material is dissolved in an alcoholic, especially ethanolic, solution.

Preferably, the treatment with activated charcoal follows the ethanol precipitation step.

The invention also relates to a process for the preparation of a pharmaceutical composition containing, as an active agent, a botanical drug substance which is an extract of at least one hemp plant, characterized by that comprises producing a botanical drug substance containing cannabinoids from at least one cannabis plant using an extraction method as defined above and preparing the botanical drug substance with one or more pharmaceutically acceptable diluents, carriers or excipients to prepare a pharmaceutical composition.

The method of the invention can be used to produce a cannabinoid rich extract from hemp plant material. In a preferred embodiment of the invention, the method can be used to produce a cannabis extract which is a botanical drug substance.

In the context of this use, a 'botanical drug substance' is an extract derived from a hemp plant material to which the definition of 'botanical drug substance' as given in the Draft Guideline on Industrial Botanical Medicinal Products, August 2000, Department of United States of America Health and Social Services, Food and Drug Administration for Drug Evaluation and Scientific Research "A drug substance derived from one or more plants, algae or macroscopic fungi. It is produced from botanical raw materials by one or more of the following methods: pulverization, potting, squeezing, water extraction, ethanol extraction and other similar methods. "

"Plant material" is defined as a plant or part of a plant (e.g., bark, wood, leaves, stems, roots, flowers, fruits, seeds, berries or parts thereof) as well as exudates and includes material falling within the definition of "Botanical raw material" contained in the Guideline the Draft Guideline for Industrial Botanical Medicinal Products, August 2000, U.S. Department of Health and Human Services, Food and Drug Administration for Medicinal Products, Drug Assessment and Research.

The method of the invention can be used to extract cannabinoids from any plant material known to contain such cannabinoids. Most often, but not necessarily, the "plant material" will be "plant material" or "botanical raw material" derived from one or more cannabis plants.

The term "hemp plant (s)" covers wild-type true hemp as well as varieties thereof, including chemical varieties of hemp which naturally contain varying amounts of individual cannabinoids, Indian subspecies of true hemp, including varieties var. indica and var. kafiristanica, cannabis, as well as plants that are the result of genetic crosses, autocrosses or their hybrids. The term "hemp plant material" is therefore to be understood as including plant material derived from one or more hemp plants. For the avoidance of doubt, it is hereby stated that "hemp plant material" contains dry hemp biomass.

Substantially any treatment that results in the selective removal of undesirable components (as opposed to cannabinoids) in the botanical drug substance can be the step to reduce the proportion of non-target materials in the botanical drug substance such that the amount of undesirable components present in the final botanical drug substance is reduced. Non-target materials are any material derived from a plant starting material that is not desired to be present in the final botanical drug substance. In a preferred embodiment of the invention, this step may include a C1-C5 alcohol precipitation, wherein the material treated in the alcohol precipitation step is warmed to a temperature greater than room temperature prior to the addition of the C1-C5 alcohol. Typically, the step to reduce the proportion of non-target materials in the botanical drug substance is carried out after extraction with liquid CO2, in which case the "treated material" in the alcohol precipitation is the extraction product of liquid CO2. This extract is itself a "botanical drug substance" within the definition given above.

Preferably, the C1-C5 alcohol is ethanol. Preferably the extract is heated to a temperature ranging from 36 ° C to 44 ° C, most preferably around 40 ° C. Heating the material to be treated prior to adding the C1-C5 alcohol results in an improved mixing of the material with the C1-C5 alcohol and thus improves the efficiency of the alcohol precipitation step.

The solution formed by adding the C1-C5 alcohol to the treated material is cooled and the insoluble materials are allowed to precipitate. Preferably the solution is cooled to a temperature ranging from -15 ° C to -25 ° C and preferably the solution is cooled over up to 52 hours.

The precipitate of the insoluble materials is then removed, usually by filtration.

Preferably, the filtration is carried out through a 20 µm membrane.

In a further preferred embodiment of the invention, the process may further comprise a multi-stage evaporation under reduced pressure. This may be rotary evaporation or other known techniques.

Typically, the product of the C1-C5 alcohol precipitation step is subjected to a multi-stage evaporation to remove virtually all of the C1-C5 alcohol and water.

Preferably, C1-C5 alcohol is removed first, followed by water.

In a preferred embodiment of the invention, the decarboxylation step is carried out prior to extraction with liquid CO2 and is performed by heating the plant material to temperatures and for periods of time that ensure at least 95% conversion of the acidic cannabinoids to their neutral form and thermal degradation of THC to CBN of less than 10%. .

The decarboxylation of cannabinoid acids is time and temperature dependent. Thus, a shorter period of time will be used to completely decarboxylate a given amount of cannabinoid acid at higher temperatures. In selecting the appropriate decarboxylation conditions, however, consideration should be given to minimizing the thermal degradation of the desired pharmacological cannabinoids to undesirable degradation products, particularly the thermal degradation of THC to cannabinol (CBN).

If the plant material is derived from hemp plants with a high CBD content (defined as> 90% CBD as a percentage of the total cannabinoid content),

Preferably the second temperature is in the range 115 ° C to 125 ° C, preferably 120 ° C, and the second period of time is in the range 45 to 75 minutes, preferably 60 minutes. More preferably, the second temperature is in the range of 135 ° C to 145 ° C, preferably 140 °, and the second period of time is in the range of 15 to 45 minutes, preferably 30 minutes. In another embodiment of the invention most preferred for a weight of the plant material greater than 4 kg, the second temperature is in the range 140 ° C to 150 ° C, preferably 145 ° C, and the second period of time is in the range 55-90 minutes. The latter conditions are favorable for processed amounts of, for example, 4-6 kg of vegetable raw material, and the exact numerical values, especially the time period, may slightly change with increasing weight.

If the plant material is derived from cannabis plants with a high THC content (defined as> 90% THC as a percentage of the total cannabinoid content), preferably the second temperature is between 115 ° C and 125 ° C, usually is 120 ° C and preferably the second period of time is in the range of 45 to 75 minutes, typically 60 minutes. More preferably, the second temperature is in the range of 100 ° C to 110 ° C, preferably is 105 ° C, and the second period of time is in the range of 60 to 120 minutes, preferably 30 minutes. In another embodiment of the invention, most preferred for a weight of the plant material greater than 4 kg, the second temperature ranges from 140 ° C to 150 ° C, preferably 145 ° C, and the second period of time ranges from 45 to 55 minutes.

Most preferably, the decarboxylation step is performed at temperatures and for periods of time that ensure at least 97% conversion of the acidic cannabinoids to their neutral form and thermal degradation of THC to CBN of less than 5%.

Standard conditions for cannabinoid determinations and methods for calculating the cannabinoid content (in%) are given in the attached examples.

Preferably, the plant material used as the starting material in the extraction process is comminuted, ground or otherwise processed to obtain a particle size of less than 2 mm, but preferably greater than 1 mm. Such treatment usually results in a better extraction of cannabinoids from the plant material as the packing density is improved.

In a preferred embodiment of the invention, the method of the invention may further comprise the step of treating an extract (or botanical drug substance material) derived from the plant material with activated charcoal.

Typically, this step will be applied to the C1-C5 alcohol precipitation product, usually immediately after filtering to remove any precipitate. The liquid product of alcoholic precipitation is classified as "botanical drug substance" in accordance with the definition given above. The treatment with activated charcoal may conveniently be carried out by passing the liquid material to be treated down the column of activated charcoal.

As illustrated in the attached examples, the treatment with activated charcoal significantly improves the stability of the botanical drug substances derived from the hemp plant material, significantly improving the resistance to thermal degradation of the active cannabinoids.

In a preferred embodiment of the invention, the method according to the invention will comprise the following steps for treating the hemp plant material, preferably carried out in the order indicated:

i) decarboxylation, ii) extraction with liquid CO2 to produce the crude botanical drug substance, iii) C1-C5 alcohol precipitation to reduce the proportion of non-target materials, iv) filtering to remove precipitate,

v) evaporation to remove C1-C5 alcohol and water to yield the final botanical drug substance (BDS).

An activated charcoal treatment step may be included between step iv) and step v), which improves the stability of the final BDS.

The botanical drug substance, which can be obtained from the botanical raw material of the high-THC hemp plant, has a THC content of at least 90% w / w in relation to the total cannabinoid content, the above-mentioned botanical drug substance being an extract derived from a high-THC hemp plant THC containing at least 50% THC w / w in relation to the extract, not more than 5% CBD w / w in relation to the THC content and not more than 5% of cannabinoids other than THC and CBD in% w / w in relation to the THC content.

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More preferably the THC percentage of the w / w extract is at least 55%, especially at least 60%. Other cannabinoids and quantification methods are given below.

According to the invention, it is also possible to obtain a botanical drug substance from the botanical raw material of a high-CBD hemp plant, having a CBD content of at least 90% w / w in relation to the total cannabinoid content, the above botanical drug substance being an extract derived from a high-CBD hemp plant. CBD content, which extract contains at least 50% CBD w / w in relation to the extract, not more than 7.5% THC w / w in relation to the CBD content and not more than 5% of cannabinoids other than CBD and THC expressed in% w / w in relation to the CBD content.

A person skilled in the art knows that plants with a high THC content, such as, for example, "Skunk", have been grown, although intended to be used as recreational drugs, using traditional cultivation techniques that can also be used to produce plants rich in other cannabinoids, eg CBD, by natural selection or genetic techniques, since the genes for cannabidiolate synthase and THC synthase have been identified, see JP 2001029082 and JP 2000078979. The Turkey CPRO 921018 field breed is an example of a plant with high CBD content.

The botanical drug substances can be obtained from hemp plant material (botanical raw material) using the extraction method of the invention.

The botanical drug substance obtained by the method of the invention contains not more than 4 ppb of aflatoxin, and also not more than 20 ppm of all heavy metals.

The botanical drug substance obtained by the method of the invention contains no more than 15% w / w residual solvents, more specifically no more than 15% w / w ethanol.

The botanical drug substance obtained by the method of the invention contains no more than 105 cfu / g TVC (total indicator bacteria count), not more than 104 cfu / g of mushrooms, no more than 103 cfu / g intestinal bacilli and other organisms non-gram negative and undetectable E. Coli, Salmonella or S. Aureus.

The parameters mentioned above relate to the purity of the botanical drug substance and define a purity level which is advantageous if the botanical drug substance is to be included in a pharmaceutical product. Botanical drug substances having the required level of purity can be obtained using the extraction method of the invention, particularly using the operating conditions and quality control procedures set forth in the accompanying examples. Standard assay techniques are known in the art for determining the levels of alphatoxin, heavy metals, residual solvents, and bacterial contaminants in a botanical drug substance (eg Ph. Eur standard procedures). Additional details are provided in the accompanying examples.

The botanical drug substances obtained from the hemp plant material according to the methods of the invention can be formulated with one or more pharmaceutically acceptable carriers, diluents or excipients or deposited on a pharmaceutically acceptable evaporation surface for the preparation of pharmaceutical preparations containing cannabinoids as pharmaceutically active agents.

Thus, in a further aspect, the invention provides a method for the preparation of a pharmaceutical composition containing, as an active agent, a botanical drug substance which is an extract of at least one variety of hemp plants, which method comprises producing a botanical drug substance containing cannabinoids from at least one cultivar of a cannabis plant using the extraction method of the invention and preparing the botanical drug substance with one or more pharmaceutically acceptable diluents, carriers or vehicles or depositing the botanical drug substance on a pharmaceutically acceptable evaporation surface for the manufacture of a pharmaceutical composition.

Separate botanical drug substances can be made from individual varieties of cannabis plants having different cannabinoid contents (e.g. high THC and high CBD plants) and then mixed or blended together prior to formulation to form the final pharmaceutical composition. This approach is preferred if, for example, it is desired to achieve a specific weight ratio of the individual cannabinoids in the final formulation. Alternatively, the botanical raw material of one or more cannabinoid varieties of a cannabis plant can be mixed together prior to extraction of one botanical drug substance having the desired cannabinoid content, which can then be formulated into the final pharmaceutical composition.

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For the preparation of a pharmaceutical composition, the botanical drug substance can be formulated with any suitable pharmaceutically acceptable diluents, carriers or excipients. The choice of diluents, carriers or excipients will depend on the dosage form desired, which in turn may depend on the intended route of administration to the patient. Preferred dosage forms include, interalia, liquid dosage forms for administration by pump devices or aerosol sprays, tablets, lozenges, gels, capsules, suppositories, powders etc. and vaporizers. Such dosage forms can be prepared according to standard pharmaceutical formulation known to those skilled in the art. Preferred dosage forms and methods for preparing such dosage forms are set out in the applicant's international application pending PCT / GB02 / 00620 (WO 02/064103).

Liquid preparations are particularly preferred. A particularly preferred formulation for the administration of cannabinoids, although not limiting the invention, is a liquid formulation containing the botanical drug substance, ethanol and propylene glycol, and optionally a flavoring agent such as peppermint oil. This formulation may conveniently be administered to the buccal or sublingual mucosa by means of a pump spray. It ensures the effective absorption of active cannabinoids.

Moreover, various aspects of the inventions are illustrated, by way of example only, with the following examples and the accompanying graphs.

Graph 1 shows the loss of THC over time at 40 ° C for the Reference THC-containing botanical drug substance (BDS) and charcoal-treated THC-containing BDS (purified BDS). Y axis: amount of THC (expressed as a percentage of t0 value), x axis: time in months.

Figure 2 shows the loss of CBD over time at 40 ° C for the Reference CBD-containing botanical drug substance (BDS) and the charcoal-treated CBD BDS (purified BDS). Y-axis: amount of CBD (expressed as a percentage of the t0 value), x-axis: time in months.

Graph 3 shows formation of cannabinol (CBN) over time at 40 ° C for Reference THC-containing botanical drug substance (BDS) and charcoal-treated THC-containing BDS (purified BDS). Y axis: amount of CBN (expressed as a percentage of t0 value), x axis: time in months.

Shortcuts

The generally accepted abbreviations for the main cannabinoids are as follows:

Tetrahydrocannabinol (THC), Δ9-tetrahydrocannabinol (THC or Δ'-THC), Δ8-tetrahydrocannabinol O-THC), the propyl equivalent of Δ9-tetrahydrocannabinol (THCV), cannabidiol (CBD), cannabidiol propyl equivalent (CBDV), cannabiol (CBN), cannabichromene (CBC), cannabichromene propyl equivalent (CBCV) and cannabigerol (CBG).

Example 1. Development of a method of extracting cannabinoids from hemp plants

A selection of chemical cannabis varieties

GW Pharma Ltd has developed distinct varieties of cannabis plant hybrids to maximize the yield of specific chemical components, cannabinoids. Two types of plant are used. One cannabis chemical primarily produces THC, and the other chemical hemp primarily produces CBD. However, alternative variations can be obtained - see, for example, common cannabinoid phenotypes in 350 hemp raw materials, Smali and Beckstead, Lloydia, Vol. 36b, 1973, pp. 144-156 and grown using techniques well known to one skilled in the art to maximize the cannabinoid content.

There are chemical and structural similarities between THC and CBD. Due to these similarities and because of the botanical origin of the starting materials, they can be considered interchangeable when developing methods to extract cannabinoids.

Preferably, each cannabis chemical is treated and controlled separately to obtain two different BDS. However, it is possible to mix plant material from two or more chemical varieties, or to use a variety that produces the desired ratio of given cannabinoids prior to extraction, thereby producing one BDS.

Production of botanical raw material

BDS is made from extracts of Cannabis sativa L. (Cannabidaceae family). Cannabis sativa was described in the British Pharmacopoeia 1934. Cannabis is grown under authorization from the UK Home Office, under the control of GW Pharma Ltd in the UK.

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The cultivation facilities are equipped with curtains and an air-conditioning unit with full control (temperature, humidity and high intensity lighting) so that it is possible to obtain several crops per year under almost identical conditions, ensuring continuity of supply.

Cultivation

Hemp plants are propagated from cuttings taken from parent plants obtained from a single hay source. Thus, plants are produced by asexual reproduction in which all plants are female. Propagation with cuttings regulates the consistency of the genotype. The seedlings are planted in pesticide-free compost. During the growth cycle, the plants are watered and sustained-release fertilizer applied. Due to controlled growth conditions, the plants reach maturity in approximately 12 weeks. The plants are irrigated with quality drinking water throughout the entire growth cycle. No synthetic herbicides or pesticides are used in the cultivation of hemp plants.

Compost

Effective cannabis cultivation requires a supply of absolutely homogeneous growth media.

Compost is characterized by a soft structure, high air permeability, easy wetting, low conductivity and a balanced supply of nutrients. The compost consists of peat and added natural minerals, including quicklime (magnesium and calcium carbonates), which ensure that the pH of the compost is regulated during the cannabis plant growth cycle. Compost contains an adequate supply of essential minerals and a minimal amount of minerals with known harmful side effects on plants. Some manganese-containing minerals may be present in the compost in an insoluble form and may be additionally released in an easily soluble form. Controlling the pH of the compost and monitoring irrigation to avoid soil saturation with water will ensure control of soluble manganese levels. The pH of the compost is kept above 5.5. The compost contains no pesticides as no pesticides or herbicides are added.

Artificial fertilizer

Compost contains fertilizer identified in two distinct forms - base fertilizer and slow-release fertilizer. Additional slow release fertilizer is applied to the plants while they are growing.

Pest and disease control

No artificial herbicides or pesticides are used during cultivation. Harsh hygiene conditions will reduce the access of pests and the occurrence of diseases. Controlling growing conditions, environmental stresses such as drought, insufficient light and unfavorable temperatures reduces the risk of disease. Checking the plants regularly during the growth cycle can detect defective plants and pests. Defective male plants may appear, although weeds should be absent due to controlled growing conditions and the environment. Frequent checkups and biological control methods are used to control any pests and diseases that may arise.

Collection of plants

Due to strict control of growing conditions, cannabis plants reach maturity in approximately 12 weeks. Compact resinous flowers develop in the last weeks of growth. By the end of approximately week 11, cannabinoid biosynthesis is noticeably slowing down and the plants are ready for harvest.

The whole plant is cut and dried in a temperature and humidity controlled environment:

- approximately 21 ° C,

- approximately 38-45% RH.

The dry plant is physically assessed at the endpoint.

THC and CBD are the primary bioactive ingredients in BDS.

However, these components are present in BRM as biologically inactive carboxylic acids:

- THCA

- CBDA

The acid forms are slowly decarboxylated during drying over time. Leaves and flowers are removed from larger stems to obtain Botanical Raw Material (BRM).

Storage of BRM

Under storage conditions, the loss on drying reaches an equilibrium of approximately 10%. The conditions for dry storage of BRM will depend on the physical condition of BRM.

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Normal storage conditions for BRM:

- protected against light,

- approximately 15-25 ° C or -20 ° C,

- approx. 38-45% RH. BRM Production at a glance.

Collection of plants

AND

Drying (without light) AND

BRM (contains: THCA + CBDA) AND

Milling to reduce the particle size to less than 2000 µm AND

Decarboxylation of the acid form of cannabinoids (THCA + CBDA) to produce neutral cannabinoids (THC + CBD)

A description of a typical BRM from a high-CBD cultivar is illustrated in Table 2.

Table 2

<td>Research</td><td>Method</td><td>Description</td>

<td>- A</td><td>Visual</td><td>It is correct</td>

<td>- B</td><td>TLC</td><td>Conforms to the standard (for CBD and CBDA) Positive for CBDA</td>

<td>CBDA + CBD designation</td><td>Inside (HPLC / UV)</td><td>Not less than 90% of cannabinoids determined by peak area</td>

<td>Loss on drying</td><td>Ph. Eur.</td><td>Not more than 15%</td>

<td>Aflacosin *</td><td>UKAS method</td><td>Not more than 4 ppb</td>

<td>Microbiological **</td><td>Ph. Eur.</td><td></td>

<td>- Mushrooms</td><td></td><td>No more than 105 cfg / g</td>

<td>Foreign body</td><td>Ph. Eur.</td><td>Not more than 2%</td>

<td>Residual herbicides and pesticides ***</td><td>Ph. Eur.</td><td>It is correct</td>

Analytical methods

Visual identification

The macroscopic properties make it possible to distinguish the characteristics of the cannabis plant from any substances used to adulterate the product and substitutes. It is a visual identification in relation to the photographic pattern.

Identification by TLC

Both the retention time and the characteristic color of the spots are used to successfully identify a cannabis variety by means of TLC. For TLC analysis, laboratory samples are prepared by extracting the dry herb. Aliquots are spotted on a TLC plate alongside reference samples for THC and CBD. When exposed to Fast Blue B reagent, THC and THCA appear as pink patches, while CBD and CBDA are orange in color. Neutral compounds can be distinguished from acids by comparing the Rf values with the value obtained for the standards. Identification is confirmed by comparing the Rf and color of the sample spot with the value obtained for the appropriate standard.

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Identification by HPLC

A comparison of cannabinoid retention times is used to efficiently identify a cannabis strain by HPLC. A specific method for CBD and CBDA is reverse phase HPLC and therefore can be used as an identification test. The biomass samples are extracted and centrifuged. Detection of all analytes is made at 220 nm with additional confirmation of the acid analytes at 310 nm.

Determination (CBD + CBDA)

This assay is used to monitor the CBD and CBDA content of the plant. The assay of CBD and CBDA is determined using the HPLC method. The efficiency of the decarboxylation process is determined by dividing the% w / w CBD content by the total CBD + CBDA content.

Loss on drying

Loss on drying is assessed by the measurement method Ph. Eur.

Aflatoxin

Aflatoxin is analyzed using a method accredited by the UK Accreditation Authority (UKAS).

Microbiological examination

Microbiologically, the plant is assessed by the methodology of Ph. Eur.

Foreign body

The foreign body is assessed using the measurement method Ph. Eur. Flowers, leaves and side stems are spread out as a thin layer on a clean laboratory surface. The foreign body is separated by hand as best as possible and weighed. The results are expressed in% w / w foreign body in the herbal biomass sample. The foreign body may constitute no more than 2% of the biomass.

Residual herbicides and pesticides

Hemp plants grow in a well-controlled environment. No artificial herbicides or pesticides are used, or are not necessary during cultivation. For a high THC-rich variety, a BRM equivalent description is generated (compare table 2) and identical analytical methods are used, except that THC / THCA replaces CBD / CBDA.

Decarboxylation

THC and CBD are the primary bioactive ingredients in cannabis. However, these ingredients are present in cannabis plants as biologically inactive carboxylic acids. In order to extract THC or CBD from hemp plant material, it is necessary to convert the stored THCA and CBDA precursor compounds into easily extractable and pharmacologically active forms. In their natural state, THC and CBD are slowly decarboxylated. The conventional way to increase the rate of decarboxylation is to apply heat. However, THCA is converted not only into THC but also into another cannabinoid, cannabinol (CBN).

THCA or CBDA (C22H30O4) 145° C> THC or CBD (C21H30O2)

The decarboxylation procedure is usually done as part of the preparation of the starting material or botanical raw material (BRM) before starting the extraction process.

Laboratory studies - decarboxylation

Portions of the ground dry plant material were subjected to heat (approximately 0.25 g of particle size 1-2 mm). An experimental pilot-scale system was set up to determine the parameters for the optimal conversion of THCA or CBDA to THC and CBD, respectively, with coexisting minimal loss of these resulting compounds due to thermal degradation to thermal degradation products, in the case of THC this is the formation of CBN.

Brief description of materials and methods

Portions (0.25 g) of ground (approximately 1-2 mm particle size) herbal materials, both THCA and CBDA, were placed in 20 ml glass vials with headspace and the vials sealed with butyl rubber seals covered with Teflon with crimp cap. The closed vials were heated to one of three temperatures for periods of up to 4 hours as follows: 105 ° C, 120 ° C, 140 ° C for 0.5, 1.0, 2.0 and 4.0 hours. Heating was carried out in a forced-air oven. The oven conditions were shown to be accurate to within 0.5-1.0 degrees at the three temperatures used. After the heating process is complete

Representative samples of the decarboxylated herb were assayed using HPLC, GC, and TLC techniques. Standards of THC, CBD and CBN are included in the HPLC and GC sequences.

Results and discussions

HPLC analysis of the solvent extracts showed the disappearance of either CBDA or THCA time dependent at the two lower temperatures. At 140 ° C, the samples at the earlier 0.5 hour time point contained only very small levels of peak elution at the retention times of CBDA or THCA.

Tables 3 and 4 present HPLC data quantifying the conversion of CBDA or THCA to free compounds. Data showing the CBD or THC content and the CBD / CBDA + CBD or THC / THCA + THC ratio are also provided. The conversion of the carboxylic acid form to the corresponding decarboxylated form can be monitored by comparing the ratio of decarboxylated / decarboxylated plus non-decarboxylated compounds with the total amount of decarboxylated compounds. Thus, when the ratio reaches its maximum value (> 0.95), the earlier time / temperature point at which the THC or CBD content is also maximum should be optimal for the conversion process. Thus, for a CBD containing herb, 1 hour at 120 ° C or 0.5 hour at 140 ° C was appropriate. This is confirmed by the examination of the TLC chromatogram for the solvent extracts, CBDA is absent after 1 hour at 120 ° C or at any time point at 140 ° C.

For THC there is a third criterion, CBN formation, where it is desirable to minimize the formation of this compound during the thermal decarboxylation process. Table 5 shows the gas chromatography (GC) data from which a CBN / THC ratio can be obtained. Given both the THC / THCA + THC ratio and the maximum THC content, minimal CBN formation occurs after 0.5 hour or 1.0 hour at 120 ° C. At 140 ° C, even 0.5 hours gives a higher CBN content than either of the lower two time / temperature points.

Therefore, laboratory studies show optimal conditions for decarboxylation:

- a chemical variety that mainly produces CBD - 1 hour at 120 ° C or 0.5 hour at 140 ° C,

- a chemical variety that mainly produces THC to minimize the formation of CBN - 1 to 2 hours at 105 ° C or 1 hour at 120 ° C.

Thin layer chromatography showed that all of the THCA was gone after 4 hours at 105 ° C and after 1 hour at 140 ° C. THCA is not visible at any time point as the herb is heated to 140 ° C. A small amount of residual color at this TLC retention value and the presence at low levels of a peak convergent with THCA in HPLC analysis may indicate less cannabinoid presence than residual THCA.

Table 3

HPLC data from decarboxylation of CBDA herbal material

<td>Temperature</td><td>Time (hours)</td><td>CBD / CBD + CBDA</td><td>CBD peak area / 0.1 g herb</td>

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Table 4

HPLC data from decarboxylation of THCA herbal material

<td>Temperature</td><td>Time (hours)</td><td>THC / THC + THCA</td><td>THC peak area / 0.1 g herb</td>

Table 5

GC data from decarboxylation of THC herbal material

<td>Temperature</td><td>Time (hours)</td><td>CBN / THC (%)</td>

The decarboxylation conditions for a batch scale of about 4 kg of botanical raw material (BRM) are as follows. About 4 kg of milled BRM (THCA or decarboxylated CBDA was preheated to 105 ° C and held at this temperature for about 15 minutes to evaporate absorbed water and allow the BRM to heat evenly. The batch was further heated to 145 ° C and held at this temperature for approximately 45 minutes to allow decarboxylation to be completed with greater than 95% efficiency. The heating time of BRM CBDA was increased to 55 minutes at 145 ° C as CBDA was found to be slightly more resistant to decarboxylation than THCA from the results. In commercial scale batches, the difference between CBD and THC would be even more pronounced. The heating time for BRM THC at 145 ° C was 45 minutes. The conditions used in the pilot scale closely reflect the conditions identified as optimal in the laboratory tests. The differences can be explained by the slower and less efficient heat transfer through the tanks and by the BRM with the larger batch size for the pilot scale.

Tables 6 and 7 present data to demonstrate the efficiency of decarboxylation as measured by the levels of biologically active cannabinoid, THC or CBD.

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Table 6

Decarboxylation efficiency for BRM CBD

<td>CBD batch number</td><td>% Decarboxylation efficiency specification> 95%</td>

An increase in the batch size of BRM CBD from approximately 4 kg to 6 kg necessitated an increase in the decarboxylation time. The decarboxylation time at 145 ° C was increased from 45 minutes to 90 minutes.

Table 7

<td>THC batch number</td><td>% Decarboxylation efficiency specification> 95%</td>

<td>ABOUT</td><td> 100,0</td>

Summary of the extraction method

BDS is extracted from decarboxylated BRM using the liquid carbon dioxide method. It consists in continuously passing liquid carbon dioxide through the chopped biomass, which is contained in a high-pressure tank. The crude extract is dissolved in ethanol, cooled to low temperature, then filtered to remove precipitated components such as waxes. Removal of ethanol and water in vacuo gives BDS containing high concentrations, depending on the biomass used, either CBD or THC.

Technological scheme of a typical extraction method

BRM is decarboxylated by heating to approximately 105 ° C for 15 minutes, then to approximately 145 ° C for a minimum of 55 minutes for THCA and 90 minutes for CBDA and

Extraction with liquid carbon dioxide (CO2) [food grade] for up to 10 hours.

Conditions: pressure of approximately 6 MPa ± 1 MPa and 10 ° C ± 5 ° C and

Removal of CO2 by unsealing to obtain a crude extract and "De-stearing" - dissolving the crude extract in ethanol followed by cooling the solution (-20 ° C ± 5 ° C / up to 52 hours) to precipitate unwanted waxes and

Removal of undesirable wax material by cold filtering (20 μ ^ ι filter) and

Removal of ethanol and water from the filtrate by thin film evaporation under reduced pressure (60 ° C ± 2 ° C, with steam at 40 ° C ± 2 ° C / 172 mbar and 72 mbar ± 4 mbar) and

BDS (Stored at -20 ° C ± 5 ° C)

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Extraction # 1

The first phase of the manufacturing process is extraction with liquid CO2 under subcritical conditions. Experiments have shown that both THC and CBD could be extracted from hemp plant material with high efficiency using subcritical CO2, at a low temperature of approximately 10 ± 5 ° C, using a pressure of approximately 60 bar ± 10 bar. Table 8 below shows the comparative data obtained with THC-rich BDS.

Table 8

<td>No cargo</td><td>Pressure Bar</td><td>Temperature ° C</td><td>% w / w wax removed</td><td>% THC the above-mentioned after de-stearing</td>

<td>Three series</td><td> 60</td><td> 10</td><td> 2,2-4,8</td><td> 59,9-73,7</td>

<td></td><td></td><td></td><td>Wed about 3</td><td>Wed 65%</td>

From the results it can be concluded that there is a loss of selectivity as indicated by the high weight of the wax under supercritical conditions. Although larger amounts of wax can be removed by de-stearing, processing is difficult because, for example, filters are blocked. Similar results were obtained with CBD.

The preferred extraction conditions with liquid CO2 are as follows:

The decarboxylated botanical raw material is packed in a single column and exposed to liquid CO2 under pressure.

- Batch size: approximately 60 kg

- Pressure: 60 bar ± 10 bar

- Temperature: 10 ° C ± 5 ° C

- Time: approximately 8 hours

- CO2 mass flow: 1250 kg / h ± 20%.

The preferred parameters of the BDS production method are as follows: extraction time> 10 hours, CO2 pressure 50-70 bar, extraction temperature 5-15 ° C, CO2 mass 167 kg / kg BRM.

After unsealing and removal of CO2, the crude BDS extract is collected in closed tanks. Primary BRM is reduced to approximately 10% w / w crude BDS extract. The crude BDS extract is stored at -20 ° C ± 5 ° C.

Raw BDS extract contains waxes and long-chain molecules. Their removal is accomplished by a "de-stearinating" procedure (extraction 2) which allows the crude BDS extract to be heated to eg 40 ° C ± 4 ° C in order to liquefy the material. Ethanol is added in a ratio of 2: 1 ethanol volume to the weight of the crude BDS extract. The ethanol solution is then cooled to -20 ° C 5 ° C and held at this temperature for approximately 48 hours. After completion of stearinization, the precipitate formed is removed by cold filtering through a 20 µm filter.

Extraction # 2

The second step in the production method is Extraction 2, referred to as "de-stearing" with ethanol. Raw BDS extract is made from Extraction # 1 and contains ingredients such as waxes. Ethanol efficiently extracts long-chain molecules from the crude extract.

Research

It was found that by heating the crude BDS extract to approximately 40 ° C the miscibility of the crude extract with the solvent was improved. It was preferred to cool the "de-stearin" solution to -20 ° C in about 48 hours. The preferred parameters for the BDS production process are as follows: extraction temperature 36-44 ° C, ethanol: product ratio approximately 2: 1, freezer temperature -25 ° C to -15 ° C, time 48-54 hours.

Filtration

The ethanolic solution produced in the second extraction phase requires filtration to remove the precipitate formed. Preferably, the size of the filter is 20 µm. The preferred parameters of the BDS production method are as follows: total filter time> 6 hours.

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Evaporation

The final step of the manufacturing process is to remove any ethanol and water that may be present. Preferably it is carried out by heating at 60 ° C 2 ° C to obtain a vapor temperature of 40 ° C 2 ° C under a vacuum of 172 mbar 4 mbar. The distillation is continued under these conditions until there is little or no visible condensate. By lowering the vacuum further, the removal of water is completed gradually to approximately 50 mbar. Upon completion of BDS, it is transferred to closed stainless steel containers and stored in a freezer at -20 ° C ± 5 ° C. The preferred parameters of the BDS production method are as follows: temperature of the evaporating steam 38-42 ° C, removal of ethanol under vacuum 167-177 mbar, removal of water under vacuum 7-7.5 kPa, 6.2-5.8 kPa 5.2-4, 8 kPa, time <8 hours.

Characteristics of BDS

BDS containing THC is a brown, sticky, semi-solid extract containing at least 60% cannabinoid ingredients. The cannabinoid ingredients contain at least 90% THC, approximately 1.5% CBD with the remainder consisting of other secondary cannabinoids.

The chemical composition of cannabis has been studied extensively with the identification of more than 400 compounds (Hendricks et al., 1975, Turner et al., 1980). More than 60 cannabinoids have been identified, with CBDA and THCA (CBD and THC precursors) being the most abundant. Typically, the non-cannabinoid constituents make up up to 50% of the extracts, depending on the extraction method. The identified chemical categories include alkanes (chains with 25-30 carbon atoms), nitrogen compounds, amino acids, sugars, aldehydes, alcohols and ketones, flavanoids, glycosides, vitamins, pigments and terpenes. About 95 mono- and sesquiterpenes have been identified in cannabis and are responsible for the characteristic smell. Considerable work has been done to completely elucidate the structure of both CBD and THC (summarized in the materials above) and both have been made synthetically. Pure THC was successfully isolated in sufficient quantity from BDS to be used as reference material for identification and quantification.

pollution

BDS is a selective extract from the dry, decarboxylated leaves and flowering crowns of certain chemical hemp varieties. A range of over 400 compounds have been found in cannabis plants, including over 60 cannabinoids (Turner 1980). As they occur in nature, it is not considered necessary to treat any of these compounds as contaminants. The main contaminants therefore emerge in four areas: pesticides introduced during the growth process, alphatoxins, any new products formed by decarboxylation, and non-cannabinoid materials that constitute BDS.

The growing process is strictly controlled using the guidelines of Good Agricultural Practice and takes place in a growing environment in a closed, climate controlled room. No pesticides are applied to the plants during growth, and all pest control is carried out with biological agents. No pesticides are introduced into the growing environment. To ensure that no pesticide residues are present in the product, the growth environment is periodically tested for pesticides known to be used by the supplier of the growth medium. After the plant material has been harvested and dried, additional samples are periodically tested using a common pesticide screen to ensure that no contamination of the plant has occurred. Possible pesticides are adequately controlled in the BRM phase.

The raw material may be microbiologically contaminated, resulting in alfatoxins in the product, although growth conditions are carefully controlled to prevent this. Therefore, the BRM and BDS are tested periodically for alphatoxin content. The naturally occurring form of THC in a freshly grown plant is acidic THCA, although small amounts of neutral THC are present. Prior to extraction, the THCA is decarboxylated by heating to obtain neutral THC. The method is effective, but a small amount of THCA remains and this is monitored during the final BDS study. Thermal degradation of THCA and THC during the decarboxylation process is possible and gives CBNA and CBN. They are monitored in the BDS. The non-cannabinoid ingredients that are ballast of the BDS include hydrocarbon and triglyceride waxes, plant pigments, and terpenes. They are common to many other medicinal plant extracts and are considered to be of little toxicological and pharmacological importance. The variety of other ingredients present is wide, but they are usually only present in small amounts.

The amount of ballast is reduced by the de-stearing process in which the waxes are precipitated. Ballast materials are considered to be a solvent for the active ingredients and are not marked or controlled.

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Table 9

Specification for the control of BDS high in CBD

<td>Research</td><td>Measurement method</td><td>Limit values</td>

<td>Look</td><td>Inside</td><td>Brown, sticky, semi-solid</td>

<td>Identification - A - B</td><td>TLC HPLC / UV</td><td>The stains have a distinctive Rf and color compared to the CBD pattern Positive for CBD</td>

<td>CBD content</td><td>Internal (HPLC-UV)</td><td>Not less than 55% w / w extract</td>

<td>Related cannabinoids - THC content - other (total)</td><td>Internal (HPLC-UV)</td><td>Not more than 7.5% CBD content Not more than 5% CBD content</td>

<td>Aflacosin *</td><td>TBA</td><td>Not more than 4 ppb</td>

<td>Total heavy metal content</td><td>Ph. Eur.</td><td>Not more than 20 ppm</td>

<td>Residual solvents: - Ethanol</td><td>Inside</td><td>Not more than 5% w / w</td>

<td>Microbiological: *** - TVC - Mushrooms - Other intestinal sticks and certain other non-grammatical organisms - E. Coli - Salmonella - S. aureus</td><td>Ph. Eur.</td><td>No more than 105 cfg / g No more than 104 cfg / g No more than 103 cfg / g Absent in 1 h Absent in 10 g Absent in 1 h</td>

Analytical procedures

Identification, labeling, and related cannabinoids

The content of THC, CBD and cannabinol (CBN) in BRM and BDS was quantified by extraction with methanol or methanol / chloroform (9: 1). The method of quantification is reversed-phase high-performance thin-layer chromatography (HPLC) with UV detection - 220 nm. Any analysis should be performed under amber light as the compounds of interest are known to be photosensitive.

Chromatographic equipment and conditions

Equipment: Agilent (HP) 1100 HPLC system with variable wavelength UV detector or Diode-Array detector HPLC Discovery C8 column 5 μτπ 15 cm x 0.46 cm;

Pre-column: Kingsorb C18 5 µm 3cm x 0.46 cm;

Mobile phase: Acetonitrile: Methanol: 0.25% w / v acetic acid (16: 7: 6 by volume);

Column temperature: 25 ° C;

Flow rate: 1.0 ml min-1;

Detection: 220 nm 600 mA maximum scale excursion - second wavelength 310 nm;

Injection Volume: 10 µ ^ ι;

Transition time: 20-25 minutes (may extend to low-volume samples with peaks later eluting)

The order of elution: BD, CBDA, Δ9 THCV, CBN, Δ9 THC, CBC, Δ9 THCA.

Generation of the pattern

CBD, CBN, Δ stock standard solutions9 THC in methanol approximately 1 mg 1 m-1 stored at -20 ° C.

Diluted working standards (0.1 mg / ml for Δ9 THC and CBD and 0.01 mg / ml for CBN) are prepared in methanol from stock standards and stored at -20 ° C (maximum period of twelve months after initial production).

After preparation, stock solutions should be divided into vials of equal volumes to reduce the amount of standard exposed to room temperature. Prior to sample analysis by HPLC, the required number of standard vials are removed and allowed to equilibrate at room temperature.

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Production of samples

Alternate weights and volumes can be used in all preparations to achieve the same final dilutions.

Botanical raw material

- Accurately weigh approximately 100 mg of cut homogeneous dry material and place in a 10 ml volumetric flask.

- Diffuse material in methanol / chloroform (9: 1 v / v) and make up to volume with the same solvent.

- Extract the sample in an ultrasonic bath within 15 minutes.

- Centrifuge samples containing equal volumes of 3000 rpm for approximately 2 minutes.

- Dilute 100 µm of the supernatant to 1 ml with methanol in a suitable HPLC sample vial. (Further dilution may be required if the main cannabinoid concentration is outside the linear operating range).

Decarboxylated Botanical Raw Material: Same as Botanical Raw Material

Botanical drug substance

- Accurately weigh approximately 80 mg of BDS and place in a 50 ml volumetric flask.

- Dissolve BDS and make up to volume with methanol.

- Dilute 100 µm of the prepared supernatant to 1 ml with methanol in a suitable HPLC autosampler vial.

Chromatographic procedure

The samples are placed on the autosampler rack for listing at the Agilent chemstation. Standard solutions are used to obtain quantitative data and retention times. They can typically be injected two or three times before injecting the sample solutions, and then singly at appropriate intervals throughout the run, with a maximum of 10 test samples between standards.

Chromatographic eligibility criteria

Table 10

Retention times and relative retention times (RRT) values versus Δ9 THC for each analyte

<td>Cannabinoid</td><td>Retention time (minutes)</td><td>RRT (THC)</td>

Table 11

Peak shape (symmetry coefficient according to the British Pharmacopoeia method)

<td>Cannabinoid</td><td>Symmetry Factor</td>

Calculation

Botanical raw material

The following equation is used to obtain a result on the purity of the main cannabinoid as% of the cannabinoids currently measured (CBD, CBDA, CBN, Δ9 THC and Δ9 THCA) in a lot.

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For materials with a high Δ content9 THC:

sum of THC and THCA peak areas% THC = - · h · kP.-TT-. · dx 100 sum of peak areas determined by cannabinoids

For material with high CBD content, CBD and CBDA replace THC and THCA in the top line of the equation.

Decarboxylated botanical raw material

The following equation is used to calculate the efficiency of the decarboxylation process.

For materials with a high Δ content9 THC:

% decarboxylation yield THC peak area sum of THC and THCA peak areas

For material with high CBD content, CBD and CBDA replace THC and THCA in the equation.

Botanical drug substance

The following equations are used to calculate the concentration of a sample of the drug substance, the concentration of cannabinoids in an individual sample, the percentage of the assayed cannabinoids in the drug substance, the amount of the main cannabinoid as% of the currently assayed cannabinoids, and the amount of the main cannabinoid in the total mass of the extracted drug substance.

For material with high Δ content9 THC:

Sample concentration of drug substance = sample weight dilution factor where dilution factor = 50 x 10 = 500

THC concentration of the sample = concentration of the THC standard x average area of the THC sample average area of the THC standard% THC content of the drug substance = concentration of the THC sample concentration of the drug substance sample x100

CBD and CBN can be substituted for all of these equations in place of Δ9 THC to obtain quantitative results for both substances. Δ9 THCA and CBDA are also calculated using the standard concentrations for Δ9 THC or CBD in the absence of its own specific reference standards.

The substances concerned are defined as the sum of the mean% w / w CBN values, Δ9 THC and CBDA.

% w / w THC content

THC as% of the body of the assayed cannabinoids = -—-——-—-———— * 100 sum of the% of all assayed cannabinoids

The total amount Δ is obtained9 THC present in the entire drug substance extract.

Example 2: Analysis of the stabilization of the botanical drug substance (BDS) by partial purification with activated charcoal

The results from the stability studies of THC preparations indicate that THC in the form of

BDS is unstable even at storage temperatures as low as 5 ° C. This contrasts with the behavior of purified THC (Dronabinol USP) in Marinol soft gel capsules, which are assumed to have a shelf life of 2 years at a fairly low ambient temperature. It should also be noted that the shelf life of THC standard solutions in methanol supplied by Sigma-Aldrich is 4 years when stored chilled and protected from light. This apparent discrepancy between BDS (THC) stability and purified THC led to speculation that a certain component of BDS destabilized the main cannabinoid. The solution to this problem would be to purify the BDS (THC) to obtain high purity, preferably crystalline cannabinoid.

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However, the additional processing costs associated with converting BDS into pure cannabinoid would significantly increase the cost of finished cannabinoid-containing pharmaceutical products. Accordingly, it has been attempted to develop a simple purification step that would produce BDS with greater stability, but which would not unduly increase processing costs. It has been found that the activated charcoal purification step can conveniently be carried out in close conjunction with the "de-stearing" process by passing the stearinized ethanol solution in one step through a filter pad to remove any precipitated waxes and then directly through the charcoal column, and that the use of activated charcoal significantly improves storage.

Experimental details

Solutions of either BDS (THC) or BDS (CBD) at a concentration of 100 mg / ml in absolute ethanol BP were passed through a column packed with activated charcoal and the eluate was collected. They were then diluted with additional absolute ethanol to achieve a concentration of approximately 25 mg / ml of cannabinoid. The solution was then transferred to a 10 ml AX1 type vial (ie amber glass) and the clamp was closed. These samples were designated charcoal purified BDS. Samples of the BDS (THC) and BDS (CBD) solutions that had not been passed through the charcoal column were similarly diluted to provide a cannabinoid concentration of 25 mg / ml and then sealed in an amber glass vial of the same type. These samples were designated the "Reference BDS" and served as stability controls. Vials containing the reference BDS and charcoal purified BDS of each type were stored in a stability incubator at 40 ° C and samples were then periodically removed over 1-12 months for cannabinoid analysis by HPLC and determination by TLC profile.

The following conditions were used in the normal phase TLC analysis:

Stationary phase: silica gel G

Mobile phase: 80:20 hexane / acetone

Unfolding: 2 x 8 cm ie double unrolling

Visualization: bath in 0.1% w / v Fast Blue B (water)

The following conditions were used in the reversed-phase TLC analysis:

Stationary phase: C18 coated silica gel

Mobile phase: 6: 7: 16 0.25% v / v acetic acid (aq) / methanol / acetonitrile

Unfolding: 2 x 8 cm ie double unrolling

Visualization: bath in 0.1% w / v Fast Blue B (water)

For each sample on the TLC plate, a volume of solution containing approximately 5 µg of total cannabinoid was used.

Results and discussion

The ethanol solutions of the reference BDS (THC) and the reference BDS (CBD) are quite intense yellow. The passage of the BDS solutions through the activated charcoal effectively discolored the solutions, presumably by adsorption of the plant pigments co-extracted with the cannabinoids during the preparation of BDS from the hemp herb by liquid CO2 extraction.

The HPLC analysis results for the various BDS solutions are given in Table 12 below. They are also shown graphically (Graphs 1-3). All data are given in% of the tO determination. CBN values are given for BDS (THC) solutions as this compound was identified as an indicator of thermal degradation of THC in previous stability studies.

Table 12

Cannabinoid determination values for standard and purified BDS solutions within 1-12 months at 40 ° C

<td>Months</td><td></td><td> 1</td><td> 4</td><td> 6</td><td> 12</td>

<td>Exemplary BDS</td><td>THC</td><td> 97,3%</td><td> 92,4%</td><td> 85,3%</td><td> 74,0%</td>

<td>BDS Cleaned</td><td>THC</td><td> 102,9%</td><td> 107,4%</td><td> 96,0%</td><td> 88,6%</td>

<td>Reference BDS (CBD)</td><td>CBD</td><td> 100,3%</td><td> 103,6%</td><td> 93,3%</td><td> 91,0%</td>

<td>BDS cleaned up</td><td>CBD</td><td> 101,0%</td><td> 100,7%</td><td> 97,2%</td><td> 96,9%</td>

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It is quite clear from the above data that for both BDS (THC) and BDS (CBD), there is a certain component of ballast that can be removed with activated charcoal that destabilizes the cannabinoids.

A comparison of the degradation levels achieved after 12 months at 40 ° C for the reference BDS and the corresponding charcoal-purified BDS shows that for both THC extracts and CBD extracts, charcoal purification increases resistance to thermal degradation by more than 50 %.

Regarding BDS (THC), an increase in CBN levels was found as a function of major cannabinoid depletion (Figure 3). As has been observed with other THC-containing formulations, it is reaffirmed that CBN levels are indicative of thermal degradation.

A comparison between the cannabinoid regions of the HPLC chromatograms of the BDS (CBD) reference samples and the BDS (CBD) purified after 12 months at 40 ° C (data not shown) did not reveal any significant information. However, a similar comparison of the HPLC BDS (THC) chromatograms of the standard and purified degradation provided information.

CBN was more abundant in the more degraded crude standard BDS, but a second significant degradation product was also observed which is again present in both samples but which is more abundant in the more degraded sample. Again, the spectrum of this degradation product was essentially identical to and based on the CBN spectrum, and the retention time appeared to be one of the retention times of the CBN equivalents.

Proposal

By simple treatment with activated charcoal, a significant improvement in resistance to thermal degradation is achieved.

Example 3. Effect of adding modifying agent in CO2 extraction of hemp plant material

The example below presents the study of the effect of adding a polar cosolvent on the properties of an extract made from hemp plant material (chemical variety G5) using liquid CO2 extraction and illustrates the selectivity differences obtained using subcritical CO2 extraction versus supercritical extraction.

Experimental details

Extraction experiments were performed using a 1 liter CO2 extraction device. Food grade CO2 and BP grade absolute ethanol were used as solvents .

A batch of G5 hemp (a high-CBD chemical variety) was used. The CBD content was 7.3% w / w after decarboxylation. The cannabinoid content of the extracts was analyzed by HPLC.

Results and discussion

The composition data of the final extract obtained after a 4-hour extraction period under specified conditions are presented below in Table 13.

Table 13

Composition and yield data for extracts produced under different extraction conditions

<td>A sample</td><td>Extraction conditions</td><td>% w / w extract</td><td>% CBD (w / w)</td><td>% CBD yield</td>

<td>AC472</td><td>40 ° C / 10 MPa + 2% ethanol</td><td> 10,3%</td><td> 64,6%</td><td> 91,0%</td>

The yield efficiency is based on the CBD available in the decarboxylated plant material loaded into the tank for each extraction.

The results illustrate that changing the extraction conditions from subcritical to supercritical increases the solvating capacity of CO2 and results in a higher yield of available CBD. However, critical CO2 can now dissolve a wider variety of compounds, and the extraction of this additional compound reduces the concentration of CBD in the extract to such an extent that it is now less than the concentration obtained in the subcritical extraction. Thus, the marginal additional yield of available CBD from raw material would not compensate for this disadvantage and is evidence that the use of supercritical conditions is not advisable.

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The addition of 2% w / w absolute ethanol to supercritical CO2 modifying agent increases the yield of available CBD to> 90%. Presumably the relatively polar cannabinoid is more soluble in the polarity increased extract.

Interestingly, the addition of a polar modifying agent slightly increases the concentration of CBD in the extract. This would seem to indicate that the coextractable non-cannabinoid material present in the plant material is less polar than the target cannabinoid and thus extraction of this material ("ballast") is discarded as polarity increases. Thus, the extraction of the hemp plant material with supercritical CO2 + 2% w / w ethanol increases the yield of the target active compound without the concomitant drawback of loss of selectivity.

Summary

1. The transition from subcritical to supercritical conditions has little benefit in terms of the total cannabinoid yield from the raw material, but has the disadvantage of reducing the active extract content.

2. The addition of a modifying agent to supercritical CO 2 - 2% absolute ethanol - results in a significant improvement in the yield of cannabinoid from the feed, without the drawback of diluting the active content by the co-extracted material.

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Improved EXTRACTION OF PHARMACEUTICALLY ACTIVE CANNABINOIDS FROM PLANT MATERIALS

Abstract

Term

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- THCA

- CBDA

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AND

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