Spanish Cannabis Tests for Cancer Tumours

I found it very difficult to find lots of information initially about the ‘famous’ tests done here in Spain that many of us allude to when it comes to discussing cannabis and  cancer; in layman’s terms at least, my chemistry and biology qualifications didn’t prepare me for such depth. I spoke with Manual Guzman at the university and he was quite candid at the time but half of it was over my head…

I am going to edit and repost some of the actual procedures (and bold some of the more important points ) that were used and why many people jumped on the bandwagon if they were not already on it. If you do find it difficult to follow, simply jump to the conclusion at the end of the page…. Basically a factual statement explaining that cannabis can kill cancer. I think its worth a battle through tho, to see what kind of depth it has and how science comes to these types of conclusions.

Ethics statement animal work

This study was carried out in strict accordance with the Spanish regulation for the care and use of laboratory animals. The protocol was approved by the committee on animal experimentation of Computense University.  All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.


Δ9-tetrahidrocannabinol (THC) and cannabidiol (CBD) were from THC Pharm GmbH (Frankfurt, Germany). All chemicals and reagents were used as received. In order to avoid cannabinoid binding to labware, materials were pre-treated with Sigmacote®.

Cannabinoid solution

For in vivo administration to mice, cannabinoid solutions were prepared at 1% (v/v) DMSO in 100 µL of PBS supplemented with 5 mg/mL of bovine serum albumin. No significant influence of the vehicle was observed on any of the variables determined in this study.

Microparticles preparation

Biodegradable polymeric microparticles (MPs) were prepared by the oil-in-water emulsion solvent evaporation technique.

Analytical method

High performance liquid chromatography was used to quantify the cannabinoid loaded in the microspheres and the amount of cannabinoid released at different time-points.

Drug content and encapsulation efficiency

Briefly, 10 mg of MPs were dissolved with 1 mL of methylene chloride. Subsequently, mobile phase was added to the solution in order to precipitate the polymer and extract the cannabinoid. Samples were filtered prior to analysis by HPLC.

The encapsulation efficiency was obtained by calculating the percent of total cannabinoid loaded in the microspheres, divided by the initial cannabinoid added during the preparation of the microspheres.

In vitro release of CBD and THC from PCL microspheres

For the in vitro release studies, microspheres were incubated in PBS pH 7.4-Tween®80 0.1% (v/v) and maintained in a shaking incubator at 37°C (n = 3). At predetermined time intervals supernatants were withdrawn and media was replaced. The concentration of CBD or THC in the release medium was quantified by HPLC. The percentage of drug released was presented as a cumulative curve.

Cell culture

U87MG human glioma cells were obtained from ATCC. Cells were cultured in DMEM containing 10% FBS and maintained at 37°C in a humidified atmosphere with 5% CO2.

Nude Mouse Xenograft Model of Human Glioma

Tumors were generated in athymic nude mice (Harlan Laboratories). The animals were injected subcutaneously on the right flank with 5*106 U87 human glioma cells in 0.1 ml of PBS supplemented with 0.1% glucose. Tumors were measured using an external caliper, every day of treatment, and volume was calculated by the formula: 4π/3 *(length/2) *(width/2)2. When tumors reached a volume of 200 mm3, mice were randomly distributed into 8 experimental groups and treated daily with vehicle of the corresponding cannabinoid in solution or with blank or cannabinoid-loaded MPs at a dose of 75 mg MPs every 5 days. Mice were monitored daily for health status and for tumor volumes. After 22 days of treatment mice were scarificed and tumors were removed, measured and weighed.

Preparation and characterization of cannabinoid-loaded microparticles

In order to evaluate the potential anticancer efficacy of microencapsulated cannabinoids, we prepared biodegradable polymeric poly-ε-caprolactone (PCL) microparticles (MPs) containing THC or CBD by using the oil-in-water emulsion solvent evaporation technique.

Evaluation of the anticancer activity of cannabinoid-loaded microparticles

To investigate the potential anticancer activity of the above-described cannabinoid-loaded MPs, we generated tumor xenografts by injecting subcutaneously U87MG cells (a well-established cellular model of glioma, that has been widely used to investigate the anticancer action of cannabinoids in this type of tumors [8], [10]) into the right flank of immunodeficient mice.

Once the tumours reached a 200–250 mm3 volume, animals were treated every 5 days with blank MPs (prepared in the absence of cannabinoids) or with microparticles loaded with THC or CBD. In addition, as the combined administration of submaximal doses of THC and CBD (1∶1) has been shown to reduce the growth of glioma xenografts [18], animal were also treated with a mixture of THC and CBD MPs.(1∶1 w:w) In the same experiment, another set of tumours was treated daily with a single peritumoral injection of a solution containing vehicle, THC, CBD or a mixture of THC and CBD (1∶1).

As shown in Figure 2 administration every five days of cannabinoid-loaded microparticlesFigure 2 (THC, CBD or THC + CBD) reduced tumor growth at the same extent than daily treatment with THC, CBD or THC + CBD in solution (Figure 2A–D). A similar effect was observed when the weight of the tumors on the last day of the treatment was analyzed (Figure 3A and 3B).

To note, animals treated with cannabinoids in solution and with cannabinoid-loaded MPs received approximately the same amount of cannabinoids along the treatment (Table 2). Thus, we found that 59 % of the initial amount of CBD present in CBD-loaded MPs (5.3±0.22 mg CBD/100 mg of MPs of the initial 8.93±0.13 mg CBD/100 mg MPs) and 58% of THC present in THC-loaded MPs (4.73±0.13 mg THC/100 mg MPs of the initial 8.21±0.07 mg THC/100 mg MPs) were still present in the MPs remaining at the site of injection at the end of the experiment (Table 2).

Table 2

Taken together, these observations support that administration of cannabinoid-loaded MPs every five days reduces tumor growth with the same efficiency than a daily injection of cannabinoids in solution and suggest that effective concentrations of cannabinoids could be reached at the tumour site using a lower frequency of MPs administration.

Treatment with cannabinoid-loaded microparticles activates apoptosis and inhibits tumor angiogensis

Figure 4The mechanism of cannabinoid anticancer action relies on the ability of these compounds to promote cancer cell death – via stimulation of apoptosis – and inhibit cancer cell proliferation and tumour angiogenesis [6]. Therefore, we analyzed whether these mechanisms were activated in the tumour xenografts that had been treated with cannabinoid-loaded MPs. Unlike tumors that have been treated with blank MPs, treatment of U87-derived xenografts with THC- or CBD-loaded MPs or with a mixture of THC and CBD MPs reduced cancer cell proliferation (as determined by Ki67 immunostaing, Figure 4A), enhanced apoptosis (as determined by TUNEL; Figure 4B) and decreased tumour vascularization (as determined by immunostaining with the endothelial cell marker CD31, Figure 4C). These observations confirm that cannabinoid microencapsulation does not interfere with the mechanism by which these agents inhibit tumor growth.


One of the strategies that are currently under investigation to improve the efficacy of anticancer treatments is the utilization of drug carrier systems facilitating the local delivery of antineoplasic agents.

THC and CBD – two phytocannabinoids with potent anti-cancer activity – can be efficiently encapsulated into biodegradable PCL microspheres. Our data show that PCL micro spheres permit continuous release of these drugs and that its administration every 5 days to tumour-bearing mice reduces the growth of glioma xenografts with similar efficacy than a daily local administration of these cannabinoids in solution. Furthermore, results show that using this frequency of administration a significant fraction of the two cannabinoids is still present in the MPs at the end of the treatment. These observations suggest that effective concentrations of THC and CBD could be reached at the tumour site using a higher dosing interval.

Of note, different observations suggest that the doses of THC required to produce its cell death-promoting effect in cancer cells (IC 50 of around 1.5 to 6 μM in vitro depending on the type of cancer cell and the conditions of cell culture) are higher than the ones required for other actions of this agent or other CB1 receptor agonists in non-transformed cells. Thus, reaching effective concentrations of THC at the tumour site using a systemic route of administration may require increasing the doses of THC administered to humans, which would enhance the risk of undergoing the undesired side effects of THC derived from its binding to CB1 receptors present in different brain regions.

Local administration of cannabinoid-loaded MPs can help to circumvent this problem as their administration in the proximity of the tumour would ensure that effective concentrations of THC are reached at the therapeutically relevant site without enhancing acutely the levels of this agent in the brain regions responsible for its pyschoactivity.

In addition, in this study we also found that the anticancer efficacy of the individual treatments with THC-loaded MP (containing approximately 6.15 mg of THC per administration) or CBD-loaded MP (containing approximately 6.7 mg of CBD per administration) is similar to that produced by co-administration of a mixture (1∶1 w:w) of THC- and CBD-loaded MPs (containing approximately 3.075 mg of THC and 3.75 mg of CBD per administration).

These results are in line with previous observations by our laboratory , and suggest that rather than producing a synergistic effect, the combined administration of sub-maximal doses of THC and CBD could help to reduce the doses of these compounds required to produce their inhibitory effects on tumour growth.

Cannabinoids have been shown to produce a potent anticancer action in different types of tumour xenografts including some of the ones that exhibit a higher resistance to standard chemotherapies such as gliomas,  tumour types that are susceptible of being treated with drug-loaded MPs.  

This anticancer action of cannabinoids is based on the ability of these compounds to enhance apoptosis, inhibit proliferation of cancer cells and inhibit tumour angiogenesis.

Although additional research should clarify whether a similar effect can be produced in other types of tumour xenografts, and whether MPs loaded with THC, CBD or its combination are equally efficacious in different tumour types and sub-types, these observations strongly support that micro-encapsulation could be a promising strategy to optimize the utilization of cannabinoids as anticancer agents.

Of interest, we have recently found that the combined administration of THC or THC + CBD with temozolomide synergistic ally reduces the growth of glioma xenografts. The findings presented here now provide a rational for the design of novel anticancer strategies based on the use of cannabinoid-loaded MPs in combinational therapies.


Data presented in this manuscript show for the first time that in vivo administration of micro-encapsulated cannabinoids efficiently reduces tumor growth thus providing a proof of concept for the utilization of this formulation in cannabinoid-based anti-cancer therapies.

Synergies or as I have spoke about for years ‘entourage effect’ is real. THC AND CBD work better together than when isolated.

Semi conclusions

Cannabinoids stay in your system active for several days, it seems that we might not be needed to medicate every day, but rather every 5.  Tho some of this will undoubtedly be related to the method of production and ingestion.

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