Donald F. Stanford’s research while affiliated with University of Mississippi and other places

In recent years, cannabis has been proposed and promoted not only as a medicine for the treatment of a variety of illnesses, but also as an industrial crop for different purposes. Being an agricultural product, cannabis inflorescences may be contaminated by environmental pathogens at high concentrations, which might cause health problems if not controlled. Therefore, limits have to be placed on the levels of aerobic bacteria as well as yeast and mold. To ensure the safety of cannabis plant material and related products, a remediation process has to be put in place. Gamma irradiation is a sterilization process mainly used for pharmaceuticals, foods, cosmetics, agricultural, and herbal products including cannabis plant material. This study was designed to determine the effect of irradiation on the microbial count as well as on the chemical and physical profiles of the cannabis biomass, particularly cannabinoids, terpenes, and moisture content. The full cannabinoid profile was measured by GC/FID and HPLC analysis, while terpene profile and moisture content were determined using GC/MS and Loss on Drying (LoD) methods, respectively. Analyses were conducted on the samples before and after gamma irradiation. The results showed that the minimum and maximum doses were 15 and 20.8 KiloGray (KGY), respectively. Total Aerobic Microbial Count (TAMC) and Total Yeast and Mold Count (TYMC) were determined. The study showed that irradiation has no effect on the cannabinoids and little effect on terpenes and moisture content, but it did result in the virtual sterilization of the plant material, as evidenced by the low levels of bacterial and fungal colony-forming units (CFUs) < 10 after gamma irradiation.

Strategies for the quality control of complex mixture drugs are illustrated using plant-derived complex drug products and are comprised of unique challenges in the management of raw materials supply chains as well as the control of processing into drug product. This chapter is focused on quality control approaches from collection of medicinal plants to formulation of a botanical drug and includes cultivation, harvest, drying, processing, storage, extraction of biomass, and formulation of a botanical drug product. Naturally derived complex mixtures carry inherent biological source variabilities, but these can be exaggerated by seasonal or climatological or geographic changes as well as varying agricultural and collection practices. When the large-scale production to that satisfy national and global markets is considered, control strategies for raw material production must consider even such basic criteria as ensuring authentication of batches of plant materials as well as prime sources of variability within the range of sources. A raw materials control program must thus be tailored for each supply chain to ensure quality and consistency across the whole production platform. Many “downstream” issues can be eliminated or mitigated by rigorous control and consistency of raw material inputs, and their comprehensive documentation. The processing and manufacturing controls employed will generally be more focused—e.g., fewer processing/manufacturing sites as compared to sourcing inputs. However, process control strategies will be increasingly refined and technically complex, to include parameters of finished product and formulation consistency, safety and stability. These usually require orthogonal approaches that utilize a number of independent technologies.

Primaquine (PQ), a racemic drug, is the only treatment available for radical cure of relapsing vivax malaria and blocking transmission of falciparum malaria. Recently studies by us have shown differential pharmacologic and toxicologic profiles of individual PQ enantiomers. A study was conducted in six healthy adult human volunteers to determine plasma pharmacokinetic profile of enantiomers of PQ and carboxyprimaquine (cPQ), the major plasma metabolite. The individuals were orally administered with PQ diphosphate, equivalent to 45 mg base, 30 min after a normal breakfast. Blood samples were collected at different time intervals and plasma samples were analyzed for enantiomers of PQ & cPQ. Plasma PQ concentrations were low and variable for both parent enantiomers and peaked around 2-4 hrs. Peak (-)-PQ concentrations ranged from 121-221 ng/ml and peak (+)-PQ concentrations ranged from 168-299 ng/ml. cPQ concentrations were much higher and surprisingly consistent from subject to subject. Essentially all of the cPQ detected in plasma was (-)-cPQ. The peak concentrations of (-)-cPQ were observed at 8 hr (range 1104-1756 ng/ml); however, very high concentrations were sustained through 24 hr. (+)-cPQ was two orders of magnitude lower than (-)-cPQ, and in a few subjects it was only detected under the limit of quantification. The results suggest more rapid metabolism of (-)-PQ to (-) cPQ compared to (+)-PQ. Alternatively, (+)-PQ or (+)-cPQ could be rapidly converted to another metabolite(s) or distributed to tissues. This is the first clinical report on enantioselective pharmacokinetic profiles of PQ and cPQ and supports further clinical evaluation of individual PQ enantiomers. The American Society for Pharmacology and Experimental Therapeutics.

The chemical composition of the volatile oil of cannabis as well as other constituents has been the subject of several investigations [1,2]. Some of these investigations were directed toward establishing a chemical profile for plant materials of different genetic make up or different geographic origin [3,4]. In forensic investigations, the question of where a specific seizure of marijuana was produced is commonly asked. We have previously described a procedure for determining the geographical origin of marijuana samples based on complete chemical profile of the samples' extracts using GC/MS [3]. Broad classification was easily achievable, e.g. domestic vs. foreign, indoor vs. outdoor, and defining the country of origin of foreign samples was possible with a high degree of accuracy (> 80%). The method, however, was lengthy and labor intensive. In an attempt to simplify the chemical profiling method and in recognition of the increased potency of illicit marijuana samples we embarked on a procedure that focuses on the chemical composition of the volatile oil. This avoids the high levels of cannabinoids and simplifies the chemical analysis. This investigation is focused on establishing a protocol for the preparation of the volatile oil from different samples with consistency and establishing the best chromatographic conditions for the separation of the volatile oil constituents for GC/FID analysis. The protocol was used for the analysis of the volatile oil of marijuana samples of the same genetic make-up but produced by either indoor or outdoor cultivation. Data will be presented showing sufficient differences to distinguish indoor vs. outdoor produced materials even if the plants were of the same genetic make-up (all produced from cuttings from the same mother plant). This suggests that perhaps a procedure based on the analysis of the volatile oil of cannabis could be developed for establishing geographical origin. Acknowledgements: This work is supported by the National Institute on Drug Abuse (contract # N01DA-5-7746). References: [1] ElSohly MA (2006) Marijuana and the Cannabinoids, Humana Press, New Jersey. [2] ElSohly MA, Slade D. (2005) Life Sciences 78: 539–548. [3] Brenneisen R, ElSohly MA (1988) Journal of Forensic Science 33: 1385–1404. [4] Hood LVS, Barry GT (1978) Journal of Chromatography 166: 499–506.

The stability of various Cannabinoids in different Cannabis products (Marijuana plant material, Marijuana cigarettes and Cannabis extract) stored at The University of Mississippi - National Center for Natural Products Research was studied under different storage conditions. These included room temperature (17 ⁰C ± 4 ⁰C), refrigerator (4 ⁰C ± 2 ⁰C) and freezer (-20 ⁰C ± 2 ⁰C). The content of each individual cannabinoid was then determined periodically for up to 60 months for Cannabis biomass and Cannabis extract, and for 36 months for Cannabis cigarettes. The Cannabinoids monitored were ∆9-THC, CBD, CBC, CBG, THCV, and CBN. Marijuana plant material of three different potencies was monitored: low potency (approx. 1.5 % THC), medium potency (approx. 2.5 % THC), and high potency (approx. 4.0 % THC), while marijuana cigarettes of approx. 8% of THC were used for the study. The potency of Cannabis extract was approx. 26 % THC.

Delta-9-Tetrahydrocannabinol has been formulated in sesame oil soft gelatin capsules and marketed as Marinol, which is approved by the USFDA as antiemetic for cancer patients receiving chemotherapy and as an appetite stimulant for AIDS patients. The lack of consistent bioavailability and the problems associated with the first-pass effect from this formulation necessitated the search for alternative formulations. One such formulation with the possibility for overcoming the difficulties associated with the oral preparation is suppositories. However, Delta-9-THC has been reported to luck bioavailability from suppositories ( from either lipophilic or hydrophilic bases ). Therefore, a prodrug had to be developed to effect rectal bioavailability. Delta-9THC-Hemisuccinate ( THC-HS ) was found to be ideal prodrug.

The analysis of meconium specimens for metabolites of substances of abuse is a relatively accurate method for the detection of fetal exposure to drugs. Most of the methods reported in the literature before the early 1990s relied on radioimmunoassays. The purpose of this study was to develop and validate methods for meconium sample preparation for the screening and gas chromatography-mass spectrometry (GC-MS) confirmation of meconium extracts for cannabinoids, cocaine, opiates, amphetamines, and phencyclidine. EMIT and TDx immunoassays were evaluated as screening methods. The sample preparation method developed for screening included extraction and purification prior to analysis. Cutoff levels were administratively set at 20 ng/g for 11-nor-delta9-THC-9-COOH (THCCOOH) and phencyclidine and at 200 ng/g for benzoylecgonine, morphine, and amphetamines, although lower levels could be detected in meconium using the EMIT-ETS system. Ninety-five meconium specimens were subjected to the screening procedure with GC-MS confirmation of presumptive positives. In addition, 30 (40 for cocaine) meconium specimens were subjected to GC-MS analysis for all analytes regardless of the screening results to determine the false-negative rate, if any, of the immunoassay. Although there were no false negatives detected, the GC-MS confirmation rate for the immunoassay-positive specimens was generally low, ranging from 0% for amphetamines to 75% for opiates. The lowest rate of confirmed positives was found with the cannabinoids, suggesting that tetrahydrocannabinol (THC) metabolites other than free 11-nor-9-carboxy-delta9-THC may be major contributors to the immunoassay response in meconium.

GC/MS analysis of biological specimens is believed to be the most forensically accepted method for confirming the presence of abused drugs. 11-Nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (delta 9-THC-COOH) is the major metabolite of delta 9-tetrahydrocannabinol (delta 9-THC) for which testing (including GC/MS) is directed as an indication of marijuana use. The currently available internal standard for delta 9-THC-COOH is d3-delta 9-THC-COOH, which has the deuterium atoms located on the side chain. In addition to the high cost of this compound, it suffers from a limited dynamic range of analysis, especially when the methyl derivative is used. This is because of a contribution to one of the internal standard ions (m/z 316) from a fragmentation of the natural drug which involves loss of the side chain. The new internal standard, d6-11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (d6-delta 9-THC-COOH), avoids these disadvantages. The six deuterium atoms are located on the two methyl groups of Carbon 6 in the dibenzopyran structure. The dynamic range of analysis with the new internal standard was tested between 6.25 to 1,000 ng/mL with a correlation coefficient of 0.998. Analysis of several urine specimens for delta 9-THC metabolite using both d3- and d6-internal standards showed a correlation coefficient of 0.9987.

The bioavailability of delta-9-tetrahydrocannabinol (delta 9-THC) from suppository formulations containing several polar esters was studied. The esters tested were the hemisuccinate, N-formyl alaninate, N-methyl carbamate, and methoxy acetate. These esters were administered to monkeys in both lipophilic and hydrophilic suppository bases, namely, Witepsol H15 and polyethylene glycol, respectively. Each suppository contained a dose equivalent to 10 mg delta 9-THC. Blood samples were analyzed for both delta 9-THC and its carboxylic acid metabolite (ll-nor-delta 9-THC-9-COOH) using gas chromatography/mass spectrometry. The data showed that, with the exception of the hemisuccinate, no delta 9-THC or its metabolite was detected in the blood samples using the Witepsol H15. Using polyethylene glycol, low levels of delta 9-THC and its metabolite were detected in blood for all esters tested. The levels, however, were lower than those observed with delta 9-THC hemisuccinate using Witepsol H15. Subsequent studies in the conscious dog using the hemisuccinate in Witepsol H15 showed 67% bioavailability of delta 9-THC with a linear response in the dose range equivalent to 5-20 mg of delta 9-THC. No significant bioavailability differences were found when delta 9-THC hemisuccinate ester was administered in various lipophilic bases (Hydrokote 25, Kaomel, Suppocire AIML, and Witepsol H15).