Elsevier

Biochemical Pharmacology

Volume 62, Issue 6, 15 September 2001, Pages 755-763
Biochemical Pharmacology

Antitumor effects of ajulemic acid (CT3), a synthetic non-psychoactive cannabinoid4

https://doi.org/10.1016/S0006-2952(01)00700-6Get rights and content

Abstract

One of the endogenous transformation products of tetrahydrocannabinol (THC) is THC-11-oic acid, and ajulemic acid (AJA; dimethylheptyl-THC-11-oic acid) is a side-chain synthetic analog of THC-11-oic acid. In preclinical studies, AJA has been found to be a potent anti-inflammatory agent without psychoactive properties. Based on recent reports suggesting antitumor effects of cannabinoids (CBs), we assessed the potential of AJA as an antitumor agent. AJA proved to be approximately one-half as potent as THC in inhibiting tumor growth in vitro against a variety of neoplastic cell lines. However, its in vitro effects lasted longer. The antitumor effect was stereospecific, suggesting receptor mediation. Unlike THC, however, whose effect was blocked by both CB1 and CB2 receptor antagonists, the effect of AJA was inhibited by only the CB2 antagonist. Additionally, incubation of C6 glioma cells with AJA resulted in the formation of lipid droplets, the number of which increased over time; this effect was noted to a much greater extent after AJA than after THC and was not seen in WI-38 cells, a human normal fibroblast cell line. Analysis of incorporation of radiolabeled fatty acids revealed a marked accumulation of triglycerides in AJA-treated cells at concentrations that produced tumor growth inhibition. Finally, AJA, administered p.o. to nude mice at a dosage several orders of magnitude below that which produces toxicity, inhibited the growth of subcutaneously implanted U87 human glioma cells modestly but significantly. We conclude that AJA acts to produce significant antitumor activity and effects its actions primarily via CB2 receptors. Its very favorable toxicity profile, including lack of psychoactivity, makes it suitable for chronic usage. Further studies are warranted to determine its optimal role as an antitumor agent.

Introduction

Several years ago, a number of reports noted in vitro and in vivo antineoplastic activity of cannabinoids but concluded that their efficacy was limited by both the development of tolerance and psychoactivity [1], [2], [3], [4]. Recently, however, there has been renewed interest in cannabinoids as anticancer agents. For example, recent studies demonstrated an increased rate of apoptosis of C6 glioma cells exposed to THC [5] and inhibition of human breast and prostate cancer cell proliferation by the endogenous cannabinoid agonist anandamide [6], [7]. More intriguing is another study that demonstrated in vivo efficacy of THC and similar agonists when administered intratumorally to rats harboring intracranial C6 gliomas [8]. The mechanisms underlying the antineoplastic actions of cannabinoids remain unclear, however; possible etiologies include blockade of growth factor stimulation and sustained ceramide accumulation [5], [6], [8].

Because the psychoactivity of THC limits its medicinal usage, great effort has been directed toward finding an agent that separates these activities. One such strategy is based on the properties of the acid metabolites of THC, which show little or no psychoactivity and bind less avidly to cannabinoid receptors [9], [10], [11]. Several recent studies indicated efficacy for these acids and suggest that they might contribute to the analgesic properties of THC and attenuate its psychotropic effects [12], [13]. A more potent synthetic analog, AJA (dimethylheptyl-THC-11-oic acid), was therefore synthesized by modifying the pentyl side chain (Fig. 1); this compound is both analgesic [14], [15] and anti-inflammatory [16] and has no detectable psychoactive properties.

We hypothesized that since AJA proved equipotent or more potent than THC in several anti-inflammatory bioassays, that it would also have significant antitumor effects. This property would then make AJA an attractive substitute for THC based upon its lack of known psychoactive effects and favorable toxicity profile.1 To test this hypothesis, we compared AJA with THC as an antineoplastic agent and found that it was nearly equipotent to THC in vitro and superior in vivo. Furthermore, we determined that its antineoplastic effect was medated primarily through actions on CB2 receptors and was associated with a marked increase in cell DG levels.

Section snippets

Reagents

Δ8-THC and THC-11-oic acid were obtained from NIDA. AJA was obtained from Organix. SR141716a [N-(piperidin-1-yl)-5-(4-chlorophenyl-1H-pyrazole-3-carboximide] and SR144528 [N-[(1S)-endo-1, 3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl) ([C3H]4-methylbenzyl)-pyrazole3-carboxamide] were obtained from Dr. Francis Barth (Sanofi Research). All drugs were stored as concentrated solutions in DMSO at 4°. The concentrations were such that 10 μL added to 1.0 mL of medium gave the

Results

After 48 hr, C6 glioma cell growth was inhibited in a concentration-dependent fashion by THC (ic50 5.8 μM). By comparison, the ic50 of THC-11-oic acid was almost an order of magnitude higher (49.5 μM). The inhibitory effect of AJA was greater than that of THC-11-oic acid, but after 48 hr was approximately one-half as potent as THC (ic50 16.0 μM) (Fig. 2). The character of the inhibition curves suggested that AJA and THC were nearly equipotent and that both inhibited these cells more potently

Discussion

Prompted by reports that marihuana smoke alters DNA synthesis in lung cell cultures [4] and inhibits both bone marrow leukopoiesis [20] and lymphocyte activity [21], a number of reports in the late 1970s examined the potential of the active principle of Cannabis, THC, as an antineoplastic agent. These reports demonstrated that THC could inhibit both cancer cell DNA synthesis and certain murine neoplasms in vivo[1], [2], [3], although tolerance tended to develop [2]. Recently, there has been

Acknowledgments

The authors would like to thank Dr. Greg Hendricks for his help with the transmission electron microscopy and Dr. R. Hulspas for assistance with performing flow cytometry. This work was supported by grants from the NIDA (DA12178) and the NCI (CA68426).

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    Abbreviations: AJA, ajulemic acid, dimethylheptyl-THC-11-oic acid; DG, diglyceride; FBS, fetal bovine serum; MEM, Minimum Essential Medium; MTT, microculture methyl-thiazolyl tetrazolium; PKC, protein kinase C; TEM, transmission electron microscopy; TG, triglyceride; and THC, tetrahydrocannabinol.

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