Beta-Caryophyllene, a CB2-Receptor-Selective Phytocannabinoid, Suppresses Mechanical Allodynia in a Mouse Model of Antiretroviral-Induced Neuropathic Pain
Esraa Aly, Maitham A. Khajah and Willias Masocha
Molecules, 2020, 25, 106;
Neuropathic pain associated with nucleoside reverse transcriptase inhibitors (NRTIs), therapeutic agents for human immunodeficiency virus (HIV), responds poorly to available drugs. Smoked cannabis was reported to relieve HIV-associated neuropathic pain in clinical trials. Some constituents of cannabis (Cannabis sativa) activate cannabinoid type 1 (CB1) and cannabinoid type 2 (CB2) receptors. However, activation of the CB1 receptor is associated with side eects such as psychosis and physical dependence. Therefore, we investigated the effect of B-caryophyllene (BCP), a CB2-selective phytocannabinoid, in a model of NRTI-induced neuropathic pain. Female BALB/c mice treated with 20-30-dideoxycytidine (ddC, zalcitabine), a NRTI, for 5 days developed mechanical allodynia, which was prevented by co treatment with BCP, minocycline or pentoxifylline. A CB2 receptor antagonist (AM 630), but not a CB1 receptor antagonist (AM 251), antagonized BCP attenuation of established ddC-induced mechanical allodynia. B-Caryophyllene prevented the ddC-induced increase in cytokine (interleukin 1 beta, tumor necrosis factor alpha and interferon gamma) transcripts in the paw skin and brain, as well as the phosphorylation level of Erk1/2 in the brain. In conclusion, BCP prevents NRTI-induced mechanical allodynia, possibly via reducing the inflammatory response, and attenuates mechanical allodynia through CB2 receptor activation. Therefore, BCP could be useful for prevention and treatment of anti retroviral-induced neuropathic pain.
Keywords : neuropathic pain; nucleoside reverse transcriptase inhibitor; cytokines; ddC; phytocannabinoid; B-caryophyllene; antiretroviral;mechanical allodynia; CB2 receptor
Antiretroviral combination therapy is used to treat human immunodeficiency virus (HIV) infection and has resulted in the lowering of viral load, minimized viral transmission and made HIV a chronic disease rather than a fatal one [1,2]. The introduction of highly-active antiretroviral therapy (HAART) dramatically increased HIV patients’ life expectancies. Unfortunately, this increase in life expectancy came with a price. All antiretroviral therapies possess side eects and toxicities, which range from mild to life-threatening toxicities . The initial antiretroviral therapy (ART) regimen for a treatment of naïve patient generally consists of two nucleoside reverse transcriptase inhibitors (NRTIs) plus a drug from one of three drug classes: an integrase strand transfer inhibitor (INSTI), a non-nucleoside reverse transcriptase inhibitor (NNRTI) or a boosted protease inhibitor (PI) .
Zidovudine, a NRTI, was the first drug approved to treat HIV [3,5]. Since then, NRTIs have been the cornerstone for ART. However, some of the NRTIs cause a dose-limiting neuropathy which has a huge impact on compliance, adherence and patient quality of life. These neuropathies were mainly associated with the dideoxynucleoside reverse transcriptase inhibitors (D drugs), which include zalcitabine (20-30-dideoxycytidine, ddC), didanosine (ddI) and stavudine (d4T) [6–8]. Moreover, the HIV virus produces distal symmetric polyneuropathy (HIV-DSP) which cannot be clinically distinguished from the anti-retroviral toxic neuropathy (ATN) [7,9].
The clinical picture for both neuropathies involves a combination of negative (e.g., loss of sensation, hypoesthesia, hypoalgesia) and positive sensory symptoms (e.g., spontaneous pain, evoked pain, allodynia, hyperalgesia). Pain arises gradually, typically described as aching or numbness, and is characterized by a classical distal “glove and stocking” distribution as symptoms occur mainly in feet and lower extremities followed by hands [8,10]. The most prominent pathological features include Wallerian distal axonal degeneration, neuronal loss in dorsal root ganglia (DRG) of affected nerves, inflammatory cell infiltration (especially macrophages), reduced epidermal nerve fiber (ENF) density and a ‘dying back’ sensory neuropathy [9,10].
Over the years several animal models using ddC as a representative NRTI have been established in an attempt to understand the pathophysiology or to find treatment options for ATN. The ddC-induced neuropathy is accompanied by mechanical allodynia , hyperalgesia , decreased conduction velocity in C fiber aerents  and upregulation of inflammatory molecules such as tumor necrosis factor alpha (TNF-), stromal cell-derived factor (SDF1-) , and chemokine receptor type 4 (CXCR4) , caspases , interleukin 1 beta (IL-1) and Wnt5a ; ddC-induced neuropathy also is accompanied by mitochondrial dysfunction [18–20].
Despite several clinical trials, there is no Food and Drug Administration (FDA) approved medication for either prevention or treatment of ATN . The regular pharmacological treatments for other forms of neuropathic pain are widely used, although not proven effective inpatients withHIV-associated neuropathic pain.These include antidepressants [22,23], anticonvulsants [24,25], topical agents, non steroidal anti-inflammatory drugs (NSAIDs) and opioids [6,26,27]. Smoked cannabis [28,29] and the capsaicin 8% transdermal patch  have proven to be eective against HIV-associated neuropathic pain in randomized clinical trials . Despite these findings, it is well known that cannabis (Cannabis sativa) produces psychosis as a side eect; its use is prohibited in most countries and smoking carry significant health risks [31,32].
Recently, Munawar et al., reported the involvement of the endocannabinoid system in ddC-induced neuropathy and that the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have anti-hyperalgesic activity via both cannabinoid type 1 (CB1) and cannabinoid type 2 (CB2) receptors . The facts that cannabis reduced HIV sensory neuropathy (HIV-SN) and that endocannabinoids reduced ATN all lead to the supposition that a CB2 receptor agonist that does not produce psychosis as a side effect would be a good candidate to prevent or treat ATN.
Beta-Caryophyllene (BCP) is a phytocannabinoid found in essential oils of various plants including black pepper (Piper nigrum), cinnamon (Cinnamomum spp.), lemon balm (Melissa ocinalis), cloves (Syzygium aromaticum), oregano (Origanum vulgare), hops (Humulus lupulus) and cannabis (Cannabis sativa) [33,34]. It is approved to be used as a natural flavoring agent by the FDA . Beta-Caryophyllene is a CB2-receptor-selective agonist  that produces antinociception but lack the psychotic side eects produced through CB1 receptors . It has pleiotropic activities including antioxidant , anti-inflammatory , neuroprotective , anxiolytic , anti-cancer and analgesic effects . In addition, it attenuated neuropathic pain in several models including models of chemotherapy-induced neuropathy , diabetic neuropathy , sciatic nerve partial ligation  and chronic constriction injury of the sciatic nerve [44,45].
In this study, we investigated whether concomitant treatment with BCP and ddC can prevent the development of ddC-induced neuropathic pain and inflammation. In addition, we compared the anti-allodynic eects of BCP to minocycline and pentoxifylline, which have been shown to have anti-inflammatory and antiallodynic eects against various types of neuropathic pain [46–49].