The metamorphosis of the „joint“ to a medicine
The history of cannabis begins already in antiquity and its therapeutic use has been documented for 2700 years . In the year 1850, cannabis was included in the United States Pharmacopoeia (USP) before it was deleted in 1942, after a new legislation in 1937 classified cannabis as a narcotic and was subsequently prohibited . Since that time, the story of cannabis has been told by baby boomer generations as “the mind-expanding, peace-making smoke” that can land you in jail.
But beside the psychotropic effects, many patients and even doctors swore by the effectiveness of cannabinoids in important therapeutic areas like chronic pain, terminal cancer, and multiple sclerosis. Due to the illegal status these applications remained anecdotal until its legalization. Within a short period thereafter scientific interest took off at a rocket speed triggering a flood of clinical studies with parts of the cannabis plant, its resin or extracts investigating different indications. Until today only a few products were approved by regulatory authorities using either synthetic Δ9-tetrahydrocannabinol (THC) (Marinol ®, Syndros®, Cesamed®) or plant derived standardized cannabidiol (CBD) or CBD/THC extracts (Epididex®, Sativex®). Overconfidence in cannabis per se was soon challenged by many failed endpoints in clinical trials. These setbacks led to more basic research on the different cannabis varieties, the active components, their pharmacology and metabolomics, structure-activity relationships, synergistic effects of compounds (entourage effect) and targeted receptors [3-5]. The more these research progresses the more we understand the complex nature of cannabis as well as the multitude of chemical compounds and structures with the potential of novel therapeutic application.
The cannabis plants
Cannabis sativa is a polymorphic and complex plant growing under different environmental conditions, altitudes, and soils leading to a variety of genotypes and chemovars. For therapeutic applications more than 600 different varieties are available with substantial different cannabinoid and terpenoid compositions as well as other pharmacologic active compounds . These differences lead to various and variable physiological and pharmacological responses . Cannabis sativa L. contains about 565 chemical compounds from 23 different chemical classes of which around 100 are attributed to cannabinoids . Yet, the chemical characterization of the plants and their extracts require specific analytical procedures to capture the multitude of components present. One of the challenges of cannabis analytics is the impact of the processing and extraction conditions, which can alter the chemical structures of the active compounds as well as the composition original present in the plant [8, 9].
The variety of different cannabinoids in the plant and their extracts display multiple often contrasting receptor interactions which might counteract desired effect. It is well established that an excessive central nervous system effect by some components is responsible for serious side effects including dizziness, dry mouth, nausea, fatigue, somnolence, euphoria, vomiting, disorientation, drowsiness, confusion, loss of balance, and even hallucination . Therefore, purification technologies of cannabis are progressing for medical applications . Identifying the chemical structures and compounds contributing to the desired but also undesired pharmacological effects provide a rich source of new active ingredients being it plant based compounds, semi-synthetic derivatives or newly created synthetic structures .
Cannabis analytical characterization
Characterizing botanical plants and their products can be very challenging as they are complex and dynamic systems. Metastable components in organs or cells of the plant can easily degrade or form artefacts based on the processing and analytical procedures. Some of the active chemical compounds might only be present in quantities which are close to the limit of detection. Suitable analytical procedures, which are required for medicinal use, are validated with regard to effective extraction and separation technology coupled with sensitive, selective as well as reproducible detection systems. Over the past years different approaches have been proposed coupling HPLC, RP-HPLC, GC, LC with HRMS/MS, MS/MS, FID or VUV [8, 13-15]. Most recent work also suggested that MIR spectroscopy can be used as a Process Analytical Technology (PAT) for the quantitative determination and monitoring of the major cannabinoids during product manufacturing . In addition, the natural origin of cannabis makes it susceptible to microbiological, pesticide and heavy metal contamination which are subject to strict limitation in medical products . The microbiology contamination can be determined for example by ICP-MS, MALDI-MS, APC or qPCR .
Cannabis regulations for medicinal use
The development of cannabis based medical products follow the established principles of evidence for efficacy, safety and quality as pharmaceuticals. The FDA considers Cannabis based products to be developed according to their guidance for botanical drug development  and the draft guidance on the quality considerations for clinical research of cannabis and cannabis-derived compounds . One of the major concern specific to cannabis derived products remains the addiction potential of THC for which reason the FDA has set a maximum THC concentration of no more than 0.3 % by dry weight. Otherwise, it will be classified as Schedule I controlled substances under the Controlled Substance Act. Due to the multicomponent nature of cannabis, it is essential for new drug products to either standardize the cannabis-based product (e.g. extract) to specific quantities of the active components or extract and purify the active components into a single compound system or into fixed dose combination.
Drug development challenges of cannabis products
The physicochemical properties that are decisive for the formulation strategy are very different between the cannabis and cannabis derived actives. They depend on the provenance of the plants, the processing method and the properties of the individual components. The challenges related to cannabinoids like THC and CBD is their high lipophilicity (clogP > 6), low aqueous solubility (2 – 10µg/mL), low melting point (< 70°C), low oral bioavailability (< 20%), highly variable orally inhaled bioavailability, poor taste as well as their limited stability under standard storage conditions (< 6 months). After oral intake, cannabinoids are extensively metabolized by the cytochrome P450 system. Despite this high first pass metabolism, due to their lipophilicity they distribute substantially into lipid tissue leading to individual elimination half-lifes of > 24 h and in steady state of 2 – 50 days .
Pulmonary, nasal, topical, oral, mucosal and parenteral dosage forms have been investigated whereby each of the dosage forms has its own merits for specific clinical targets . The highest bioavailability has been achieved through the pulmonary route with high inter- and intrasubject variability due to the individual inhalation performance. The bioavailability of oral delivery is dependent of the formulation. Lipophilic solutions or surfactant-based emulsion systems have demonstrated to achieve therapeutic plasma levels even though the bioavailability was low. Transmucosal delivery forms applied in the mouth or nasal cavity were able to achieve bioavailability of around 40 % as they circumvent the first pass effect, but might be limited in the achievable dose that can be delivered. Transdermal applications demonstrated their benefit based on their long term, constant delivery of small doses of cannabinoids for pain management. In the recent years engineered particles provided an interesting formulation approach that can be used for oral and parenteral delivery. Recently lipid, polymeric carriers or nanocarriers have been developed to achieve targeted delivery of cannabis derived products [22, 23].
Determination of cannabinoids and its metabolites in biological fluids
Bioanalysis of cannabis in biologic fluids and tissues has been of interest for many decades for forensic purposes. For medicinal purposes, bioanalytics are key for product development and regulatory clearance. Especially in the case of cannabis based medicinal products with multiple pharmacological active components, the existing lack of sufficient pharmacokinetic and pharmacodynamic understanding has to be seen as an opportunity rather than a threat . Even though the determination of the pharmacokinetic profile of cannabinoids in blood and plasma remains a challenge  creative scientists have been successful in developing bioanalytical methods to support medicinal cannabis-based product development.
Within the development of the dosage forms bioanalysis is essential to quantify the desired concentration at the targeted site of action. This includes solid procedures to measure absorption, bioavailability and distribution of the drug and its metabolites in blood or serum. This is especially true for cannabinoids due to the high lipophilicity, instability at low pH and extensive hepatic first pass metabolism which require a targeted formulation approach. Chemical optimization of the cannabinoids to achieve better drug-like properties through structure-activity analysis can also be explored to improve the pharmacokinetic and pharmacodynamic properties [3, 26]. Consequently, the pharmacokinetic profiling of the different cannabinoids is a critical aspect to understand the complex kinetics of cannabinoids and their metabolites to improve efficacy and reduce adverse drug reactions.
The opportunity space for cannabis derived medicinal products
Cannabis and their derived products are being investigated for multiple clinical conditions . A major challenge remains the complexity and variability of the cannabis plants and plant derived products that can explain a lot of disappointing results in clinical trials [28, 29]. This opportunity can be successfully seized if a systematic approach is followed. This approach includes a sequence of steps in purification, analytical, formulation, manufacturing, clinical study design, bioanalysis, and regulatory processes for which specific expertise is required.
Serving several medicinal cannabis projects successfully, some of the critical issues, their solutions and opportunities provided by Ardena will be explained with the help of the following examples.
As for any new drug development program, the cannabis derived product is considered as the active pharmaceutical ingredient (API), requiring an exhaustive characterization of its composition. An accurate analytical method must be able to separate all components qualitatively and capture them quantitatively. A method based on liquid chromatography (LC) coupled with ultraviolet spectra (UV) determination was used to qualify and quantify the major components (CBD and THC) in the cannabis derived product. The method was validated with regard to accuracy, precision, repeatability, intermediate precision, specificity detection limit, quantitation limit, linearity and range. The comparison of different batches of the API revealed sufficient compositional reproducibility so that purification and further standardization of the extract via column chromatography was not necessary for the formulation development.
Dependent on the intended therapeutic target the API has to be formulated into a dosage form which is capable to release the API at its site of absorption. To achieve fast and sufficiently high plasma concentration of purified CBD for pediatric use, a sublingual dosage form was considered. Due to its high lipophilicity, three different crystal engineering approaches were developed for CBD: nanocrystals, spray dried particles and amorphous solid dispersion by freeze drying. Different tablet formulations were screened and evaluated for stability and in-vitro dissolution. The lead formulation was than scaled up to assure processability as well as tablet quality and performance targets. While the tablet achieved all the targeted quality criteria, the initial palatability assessment raised concerns about acceptability by children. Based on data, literature and internal reviews, the potential root cause could be identified and the taste issue was resolved by formulation optimization.
Another case was the urgent request for the development and clinical supply of a stable oily oral formulation of THC and CBD at a defined ratio. Based on prior experience, the number of formulations in the screening could be kept at a minimum which also reduced the number of technical batches to be manufactured to serve initial stability testing. In parallel the analytical methods for CBD and THC in the oil formulation were developed and validated according to GMP standards. Clinical batches of the selected oily CBD & THC formulation as well as the respective placebo were manufactured and released for the clinical trials on time. To secure the program and its continuation, Ardena also qualified the supplier as well as put samples of the clinical batch in passive stability.
Measuring plasma concentrations of cannabis components and their metabolites is another specific challenge due to the very low plasma concentrations and required sample handling procedures.
During the course of many preclinical and clinical studies supported by Ardena, a set of fully validated cannabinoid bioanalytical methods based on LC-MS/MS analysis have been developed and validated according to the latest FDA and EMA guidelines for bioanalytical method validation.
For cannabinol (CBN), a sophisticated sample processing method has been designed and optimized. After addition of an internal standard (a stable isotope labeled variant of CBN) and sample derivatization combined with an extensive sample clean-up, a sensitive and quantitative bioanalytical assay has been validated in the range as low as 1.00 to 100 pg/mL. This method has been successfully applied in numerous regulated preclinical studies and in phase I and II clinical programs to provide detailed PK data in plasma and in tissue samples.
Another valuable example can be found in the combined assay for tetrahydrocannabinol (THC), 11-hydroxy tetrahydrocannabinol (11OH-THC) and cannabidiol (CBD). After liquid-liquid extraction of the samples, the compounds and their respective internal standards are derivatized and purified using solid phase extraction. The subsequent LC-MS/MS analysis then allows the accurate and precise determination of plasma levels as low as 0.1 ng/mL for each of these analytes. If necessary, the 11-nor-9 carboxy-THC metabolite of THC (i.e. THC-COOH) can be analysed by our laboratory as a separate assay. Due to the different physicochemical properties of THC and THC-COOH and the fact that THC-COOH levels in plasma remain relatively high over time, a combined assay is not optimal
Chemistry, Manufacturing and Controls (CMC) is yet another, often underestimated, requirement for the successful development of medicinal cannabis products. CMC is a mandatory procedure for the comprehensive documentation of the entire product development according to the respective guidelines to support any clinical trial and product approval.
After the legalization of cannabis, a dedicated science has emerged on the variety of therapeutic components and possible uses, from which new medicines are now emerging. The botanical origin of cannabis derived products poses special challenges to its development into a medical product and approved drug. Despite all the enthusiasm, it is therefore important from the outset to develop and document the product systematically in terms of quality, clinical efficacy and safety. In order to keep risks to a minimum during the course of development and to take advantage of new opportunities as they arise, cooperation with experienced experts has proven its worth. Especially in a highly competitive environment, they can be decisive for the market entry and success.
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