John Smith, MD1; Jane Doe, PhD2; Frederick Jones, MD, PhD1,2. 1University of Affiliation, 2Medical Center of Affiliation. We appreciate the financial support ...
® Development of a cannabinoid-based Cell-in-a-Box
therapeutic system targeted toward malignant tumors
R.M. Hyslop1, C.E. Brown1, A. Magiotta1, B. Morgan1, M. Brown, T. Sherman2, D. Petty, S. Desa3, J. O’Neil1, S. Flora, K. Kellogg, C. Hansen1, S. Bydalek1, T. Cale, C. Laster1, J. Folsom, A. Hawkinson2 1Department of Chemistry and Biochemistry, UNC;; 2School of Biological Sciences, UNC, 3Department of Biology, Universiti Pendidikan Sultan Idris
Abstract
Methods and Materials
Cannabis sativa represents a sustainable, “green chemistry” source of potentially therapeutically-‐active compounds. Several Cannabis-‐derived phytocannabinoids have been reported to have a variety of medicinal benefits including anticancer activity. We are currently investigating the feasibility of a patented cell-‐encapsulation system in which cells producing enzymes capable of converting an inactive phytocannabinoid prodrug into an active anti-‐cancer drug are encapsulated in a cellulose-‐based porous polymer, which can be injected immediately upstream from a tumor. An administered phytocannabinoid prodrug can be activated by the encapsulated cells at the site of the tumor. Using both specific phytocannabinoids and model compounds, a variety of cell lines were screened for the appropriate enzymatic activity to convert an inactive cannabinoid prodrug into an active drug. Five cell lines were observed that were actively producing the desired enzyme. These cell lines are currently being assessed with a specific phytocannabinoid prodrug. Initial results are discussed.
Cell culture. Cells were c ultured in 500-‐700 mL of appropriate growth medium and incubated at room 1 2 1,2 temperature or 37 °C according to species. Once c ell cultures had been propagated, the cells were 1 to model compounds in order to induce 2expression of the target gene, and thus production of exposed the target enzyme (3). Cells were induced with model c ompounds incubating for varying periods ranging from 1-‐24 hours. Following induction, cells w ere centrifuged at 4000g for 10 minutes to collect all cells. Wet cell mass of the final cell pellet was obtained in mg/mL. Cells were resuspended in 50 mL of c ulture medium and split between two 50 mL conical tubes. After a period of induction, c ells are processed in two groups; one sample was left intact while the other was sonified to lyse the cells.
Introduction A 2009 mini-‐review of 51 published scientific articles concluded that cannabinoids could be useful in the treatment of cancer due to their ability to regulate cellular signaling pathways critical for cell growth and survival (1). The primary aim of this research is to develop a human clinical trial-‐ready phytocannabinoid-‐based therapy for the targeted treatment of pancreatic, brain, and other cancers utilizing a novel human cell line that has been encapsulated via the Cell-‐in-‐a-‐Box® live cell encapsulation technology and engineered to activate a marijuana-‐derived prodrug in situ. The Cell-‐in-‐a-‐Box® treatment platform is a cellulose-‐based live cell encapsulation technology that encloses living cells into bio-‐inert protective porous capsules about the size of the head of a pin. Capsules contain pores providing for the exchange of essential nutrients/waste products and allowing cells inside the capsules to live and function for long periods of time (2+ years). Encapsulated cells may be stored at -‐80 °C or lower and successfully thawed for use. Various cell types, both prokaryote and eukaryote, have been reported to possess enzymes capable of catalyzing a specific chemical reaction equivalent to the reaction necessary for conversion of several individual cannabinoid prodrugs into their corresponding active antineoplastic forms. Thus, our goal is to identify an organism with the appropriate enzyme, isolate the gene for the enzyme, incorporate the gene into a human embryonic kidney (HEK) cell, and encapsulate the HEK cell. Preliminary studies have focused on screening these cell types using non-‐cannabinoid model compounds. Conditions for separation of model compound prodrugs and their corresponding active drug were optimized and a standard curve for each compound was constructed. A method to extract the model compounds and their corresponding products from the cell incubation medium was developed (2). The cells were then screened using the cannabinoid prodrug.
Inactive Prodrug
Capsules Containing Prodrug Activating Cells
John Smith, MD ; Jane Doe, PhD ; Frederick Jones, MD, PhD University of Affiliation, Medical Center of Affiliation
Analytical methods. To separate the pro-‐ and active drugs, high pressure liquid chromatography (HPLC) was performed using a Shimadzu L C-‐10AT HPLC equipped with a Luna Omega 5µm Polar C18 (150 mm x 4.6 mm) reverse-‐phase column, SPD-‐10A UV detector, and 10 µL injector loop. Conditions:A linear gradient consisting of 20 mM ammonium formate:acetonitrile atarting at 40:60 was increased to 95:5 over a period of 9 min with a flow rate of at 1.2 mL/min; detector wavelength 220 nm; run time 10 min. Enzymatic activity. Assessment of enzymatic activity with model compounds has been reported. Enzymatic activity for cannabinoids was assessed as follows: Incubation media contained 13 mL of cell suspension and 0.1 mL of purified cannabinoid pro-‐drug (20 mg/mL in ethanol). The medium w as incubated at 37 ̊C with constant shaking. Two-‐mL aliquots were taken at 0, 0.5, 1, 2, 3, and 4 hr intervals. The aliquots were immediately added to a solution containing 5 mL n-‐pentane and 0.5 mL ethanol, vortexed for 5 sec, and centrifuged. The organic layer was transferred, evaporated to dryness, and reconstituted in 0.1 mL ethanol followed by analysis using HPLC.
Results
Enzymatic activity was observed in five cell lines for two model compounds (Table 1). These three cell lines w ere assessed using a specific cannabinoid pro-‐drug. Three c ell lines were observed to have detectable activity converting the pro-‐drug into the active drug following 3 hr incubation (Table 1). Table 1. Cell lines screened for activity.
Tumor Cells are Destroyed
Figure 1: Using Cell-‐in-‐a-‐Box® to Treat Cancer
1200000 1000000 800000 600000 400000
Intact
Lysed
Induced
Substrate
Lactobacillus casei
No
No
No
Aerobacter aerogenes
Yes
Yes
Yes
Model compound only
Klebsiella pneumoniae
Yes
Yes
Yes
Model compound only
Escherichia coli
No
No
No
Pseudomonas putida
No
Yes
Lactobaccilus plantarum
No
No
No
Trichosporon monilliforme
No
No
No
Aspergillus niger
No
No
No
Lactobaccilus brevis
No
No
NA
Conclusions
Pseudomonas chrysogenum
No
No
No
Penicillium chrysogenium
No
Yes
Yes
Current work involves the identification of the gene coding from the enzyme responsible for activating the prodrug. We have attempted to transfect HEK cells with a specific gene potentially capable of encoding for the desired enzymatic activity. Future work will involve identification of the respective genes from the other lines and attempts to transfect HEK.
Asperigillus clavatus
No
Yes
0 0
Yes
Cannabinoid
0.1
0.2
0.3
0.4
0.5
0.6
Figure 2: Standard curve for cannabinoid active drug (220nm)
Discussion Currently, three cell lines have shown marginal activity for the conversion of the cannabinoid pro-‐drug into an active antineoplastic drug. The activity requires induction using a model compound. Lysis of the cells prior to incubation was required, which suggests that the pro-‐drug cannot be absorbed through the cell surface of the intact cell. Screening of additional cell lines will be continued. In addition, an attempt will be made to isolate the gene for the enzyme from the cell line that demonstrated activity.
Cannabinoid Cannabinoid
Yes
References
Acknowledgments We appreciate the financial support provided by PharmaCyte Biotech (to RMH), and the support from UNC School of Biological Sciences, and Department of Chemistry and Biochemistry.
R² = 0.9771
200000
Activated Drug
Cancer Prodrug is “Activated” by the Encapsulated Cells
1600000 1400000
The HPLC method for separating the specific cannabinoid pro-‐drug from the active drug gave base-‐line separation (Figure 1). The extraction method gave reproducible quantitative recovery (greater than 90%) for both pro-‐ and active drug. The analysis was linear over the range from 60 µg/mL to 500 µg/mL (Figure 2) with a limits detection of 30 µg/mL.
Cell Type
Figure 1. Separation of the cannabinoid active drug (7.2 min) and prodrug drug (7.9 min)
1. Alexander A, Smith PF, Rosengren RJ. Cannabinoids in the treatment of cancer. Cancer Letters. 2009; 285(1):6-‐12 2. Brown CE, Rabe ML, Crabtree G, Hyslop RM. Sustainable, green chemistry and medicine: Targeted cannabinoid-‐based chemotherapy utilizing Cell-‐in-‐a-‐Box® cellular encapsulation technology” 2014; 248th ACS National Meeting, San Francisco, CA. 3. Jimenez N, Curiel JA, Reveron I, De las Rivas B, Munoz R. Uncovering the Lactobacillus plantarum WCFS1 Gallate Decarboxylase Involved in Tannin Degradation. Applied and Environmental Microbiology. 2013; 79(14):4253-‐63. .
