HEPATITIS & Cannabis studies completed

Myeloid-derived suppressor cells (MDSCs) are getting increased attention as one of the main regulatory cells of the immune system. They are induced at sites of inflammation and can potently suppress T cell functions. In the current study, we demonstrate how activation of TRPV1 vanilloid receptors can trigger MDSCs, which in turn, can inhibit inflammation and hepatitis.

Methodology/Principal Findings

Polyclonal activation of T cells, following injection of concanavalin A (ConA), in C57BL/6 mice caused acute hepatitis, characterized by significant increase in aspartate transaminase (AST), induction of inflammatory cytokines, and infiltration of mononuclear cells in the liver, leading to severe liver injury.

Administration of cannabidiol (CBD), a natural non-psychoactive cannabinoid, after ConA challenge, inhibited hepatitis in a dose-dependent manner, along with all of the associated inflammation markers. Phenotypic analysis of liver infiltrating cells showed that CBD-mediated suppression of hepatitis was associated with increased induction of arginase-expressing CD11b⁺Gr-1⁺ MDSCs. Purified CBD-induced MDSCs could effectively suppress T cell proliferation in vitro in arginase-dependent manner. Furthermore, adoptive transfer of purified MDSCs into naïve mice conferred significant protection from ConA-induced hepatitis.

CBD failed to induce MDSCs and suppress hepatitis in the livers of vanilloid receptor-deficient mice (TRPV1^−/−) thereby suggesting that CBD primarily acted via this receptor to induce MDSCs and suppress hepatitis. While MDSCs induced by CBD in liver consisted of granulocytic and monocytic subsets at a ratio of ∼2 [ratio] 1, the monocytic MDSCs were more immunosuppressive compared to granulocytic MDSCs. The ability of CBD to induce MDSCs and suppress hepatitis was also demonstrable in Staphylococcal enterotoxin B-induced liver injury.

Conclusions/Significance

This study demonstrates for the first time that MDSCs play a critical role in attenuating acute inflammation in the liver, and that agents such as CBD, which trigger MDSCs through activation of TRPV1 vanilloid receptors may constitute a novel therapeutic modality to treat inflammatory diseases.

Introduction

Cannabidiol (CBD) is a major non-psychoactive cannabinoid component of marijuana (Cannabis sativa). CBD has been shown to have potent immunosuppressive and anti-inflammatory properties and is currently approved for clinical use in some countries for the treatment of pain in multiple sclerosis (MS) patients. In addition to MS, CBD has shown promise in several rodent models of inflammation. A single dose of CBD has been shown to suppress serum TNF-α production induced by lipopolysaccharide (LPS) in mice and has been found to be beneficial in murine collagen-induced arthritis by inhibiting IFN-γ production and T cell proliferation.

Hepatitis is the inflammation of the liver that can be caused by various agents such as viruses, chemicals, drugs, alcohol, genetic factors, or the patient's own immune system. The inflammation can be acute, flaring up and then resolving within a few weeks to months, or chronic, enduring over many years. Chronic hepatitis may simmer for 20 years or more before causing significant symptoms related to advanced liver damage such as cirrhosis (scarring and liver failure), liver cancer, or death. American liver foundation estimates that one in every 10 people in North America is afflicted with a liver, biliary or gallbladder disease.

Hepatitis represents a worldwide health problem in humans for which pharmacological treatments currently available are not adequate. Development of new drugs, however, requires proper animal models relevant to human hepatitis. Majority of the liver diseases such as viral hepatitis, autoimmune hepatitis (AIH), primary biliary cirrhosis, primary sclerosing cholangitis, and liver allograft rejection are caused by activated T lymphocytes that infiltrate and destroy liver parenchyma leading to liver injury.

Injection of mice with the T-cell mitogenic plant lectin Concanavalin A (ConA), results in polyclonal activation of T lymphocytes leading to liver selective inflammatory response, which mimics activated T-cell mediated hepatitis. ConA-induced hepatitis has been well established as an ideal animal model to study T-cell mediated hepatic injury and has been used extensively to elucidate various aspects of human T cell-mediated liver diseases, such as AIH and viral hepatitis. It is characterized by elevated levels of aspartate transaminase (AST) and alanine transaminase (ALT) enzyme activities, and inflammatory cytokines in blood and liver.

Histologically, ConA injection induces dramatic inflammatory infiltrates in the liver, particularly T cells. In this model, liver injury occurs without sensitization or priming as compared to other models of liver inflammation such as galactosamine-lipopolysaccharide (LPS) induced hepatitis.

Myeloid-derived suppressor cells (MDSCs), are a newly identified suppressor cells of myeloid lineage which co-express CD11b and Gr-1 antigens. These cells, originally identified in tumor bearing hosts, have been shown to possess potent suppressive functions and regulate inflammatory responses. Although, cannabidiol is known to be highly immunosuppressive and anti-inflammatory, its effect on this important suppressive cell population has not been investigated.

Autoimmune hepatitis is generally treated with medications that suppress the immune system, such as prednisone and azathioprine, although these treatments are not universally effective and long term side effects exist. New treatments, vaccines, and prevention strategies for hepatitis continue to emerge.

Here, we describe our finding that a single dose of CBD is effective in significantly suppressing ConA-induced T cell-mediated hepatic inflammation in mice. Importantly, we have identified a novel pathway through which CBD suppresses hepatitis involving the induction of MDSCs in liver following activation of vanilloid receptor, TRPV1. These observations will help in developing CBD as a potential drug to treat inflammatory liver diseases.

Results

Cannabidiol suppresses ConA-induced hepatitis

A single injection of concanavalin-A (ConA) has been shown to induce hepatitis in mice mimicking the symptoms of human autoimmune hepatitis. Acute liver inflammation occurs within 8–24 h of injecting ConA, with clinical and histological evidence of hepatitis, elevation of transaminase activities in the plasma and hepatic inflammatory lesions characterized by massive leukocyte accumulation and hepatic necrosis. In this study, we investigated if CBD can be used to treat hepatitis using this model.

WT mice were injected with PBS (vehicle), or ConA to induce hepatitis. ConA injected mice were administered (i.p.) with vehicle (ConA+veh group) or different doses of CBD, ranging from 5 mg/kg to 50 mg/kg body wt (ConA+CBD groups), 5 minutes after ConA injection. Some mice received CBD alone at the maximum dose of 50 mg/kg (CBD group). Next, blood was collected at 6, 12, 24 and 48 h and plasma AST (aspartate transaminase) was determined by spectrophotometry using AST assay kit, as described.

As shown in Fig. 1A, intravenous injection of ConA resulted in dramatic increase in plasma AST levels over vehicle control, indicative of acute hepatitis. Increased AST levels were seen as early as 6 h after ConA injection, reaching a peak around 12 h and declining thereafter. At 48 h, the plasma AST reached normal levels. Mice which received both ConA and cannabidiol (ConA+CBD) showed significantly less plasma AST activity compared to ConA-injected (ConA+veh) group demonstrating reduced liver injury upon CBD treatment. Cannabidiol alone injected at the maximum dose showed AST levels similar to that of vehicle control at all-time points tested thereby suggesting that CBD did not mediate any direct hepatotoxic effects.

Figure 1

Cannabidiol (CBD) attenuates ConA-induced hepatitis in mice.

Histological examination of paraformaldehyde fixed liver sections was performed. Vehicle and CBD alone injected groups showed normal tissue morphology and did not show any signs of liver inflammation. Significant leukocyte infiltration and tissue necrosis was observed 24 h after ConA-injection (Fig. 1B). Although, CBD treated groups (ConA+CBD) still had significant cellular infiltrates, CBD treatment resulted in marked decrease in liver tissue injury with a significant decrease in necrotic lesions (Fig.1B, C), thereby corroborating that CBD was very effective in protecting against ConA-induced autoimmune liver injury.

Cannabidiol suppresses pro-inflammatory cytokines

Several cytokines and chemokines were analyzed in the serum of mice 12 h after ConA-challenge using multiplex cytokine array system. This time point was selected because AST levels peak at around 12 h (Fig. 1A). ConA+veh injection resulted in significant increase in the levels of proinflammatory cytokines, predominantly IL-2, TNF-α, IFN-γ, IL-6, IL-12(p-40), IL-17, MCP-1 and eotaxin-1 (CCL11) (Fig. 2A) compared to vehicle control. The levels of these pro-inflammatory cytokines were significantly decreased in ConA+CBD mice, demonstrating that CBD treatment led to effective suppression of multiple inflammatory cytokines which may afford protection against hepatocellular damage.

Figure 2

Analysis of inflammatory cytokines, SOCS1 and SOCS3.

We also determined the levels of mRNA for suppressor of cytokine signaling 1 and 3 (SOCS-1 and SOCS-3) at an early time point (2 h) in livers by semi-quantitative RTPCR. Although, there was no significant difference in SOCS-1 mRNA levels, SOCS-3 was significantly induced in ConA+CBD injected mice when compared to ConA+veh injected mice (Fig. 2B &C), suggesting a role for SOCS-3 mediated mechanism in the suppression of cytokines by CBD during hepatitis.

Analysis of liver infiltrating cells

To understand the cellular mechanisms involved, we isolated the liver infiltrating cells and subjected them to phenotypic characterization. While performing these studies, we noted that the number of T cells or mature macrophages (F4/80^high) did not show any significant change following CBD treatment (data not shown). However, we noticed a dramatic increase in the percentage and absolute numbers of cells expressing CD11b and Gr-1.

In-depth analysis revealed that vehicle-treated mice had significant (∼10%) CD11b⁺Gr-1⁺ in the liver and CBD treatment alone did not affect this percentage or the absolute number (Fig. 3A, B). Administration of ConA caused an increase in both the percentage and absolute number of CD11b⁺Gr-1⁺ cells. ConA+CBD treatment caused a further robust induction of CD11b⁺Gr-1⁺ cells when compared to ConA+veh treatment group. We also noted that ConA+CBD treatment caused a relatively modest increase in CD4⁺Foxp3⁺ Tregs (Fig. 3C, D) in liver when compared to ConA+veh group indicating that CBD acts by predominantly inducing CD11b⁺Gr-1⁺ cells in liver.

Figure 3

Phenotypic characterization of liver infiltrating cells: CBD treatment of hepatitis increases predominantly CD11b⁺Gr-1⁺ cell numbers in liver.

Identifying CD11b⁺Gr-1⁺ in the liver as MDSCs

Recently, myeloid-derived suppressor cells (MDSCs) that express CD11b and Gr-1 antigens have been shown to be induced at sites of inflammation that help down-regulate immune responses. The increased presence of CD11b⁺Gr-1⁺ cells in mice with hepatitis suggested that such cells may represent immunosuppressive MDSCs and that they may play a critical role in suppressing the acute inflammation and liver injury as evidenced by the AST levels reaching normal levels by 48 h (Fig. 1A). Furthermore, CBD may promote the induction of such cells thereby further protecting the host from acute hepatitis.

In order to characterize the CD11b⁺Gr-1⁺ cells found in the liver as MDSCs, we triple-stained the isolated infiltrating cells for CD11b, Gr-1, and for the expression of intracellular arginase 1 (Arg1), which is one of the characteristic features of MDSCs (Fig. 4A). We found that CD11b⁺Gr-1⁺ cells in all groups were Arg-1⁺.

However, CD11b⁺Gr-1⁺ cells from ConA+CBD group showed increased Arg-1 expression as indicated by higher mean fluorescence intensity (MFI). There was also a significant increase in arginase functional activity in infiltrating cells isolated from livers of ConA+CBD group as compared to ConA+veh group (Fig. 4B).

Immunohistochemistry for Arg-1 expression in liver sections revealed large number of positively stained cells in ConA+CBD injected mice (Fig. 4C). Wright-Giemsa staining of cytospin preparations of Percoll isolated infiltrating liver cells (Fig. 4D) showed granulocytic type cells with circular nuclei as well as monocytic type cells in both ConA+veh and ConA+CBD groups, although the frequency of such cells was higher in ConA+CBD group.

Figure 4

Characterization of MDSCs in liver.

CBD-induced liver CD11b⁺Gr-1⁺ MDSCs are immunosuppressive in vitro and in vivo

To assess the immunosuppressive activity of CD11b⁺Gr-1⁺ MDSCs induced by CBD in livers, we analyzed them for their ability to suppress T cell proliferation. To this end, we sorted CD11b⁺Gr-1⁺ cells (>90% purity) from the livers of mice injected with ConA+CBD, irradiated them (2000 rad) and co-cultured at different ratios with purified syngeneic lymph node T cells in the presence of mitogen ConA (4 µg/mL) for 48 h. T cell proliferation was determined by thymidine incorporation during the last 8 h of culture. CBD-induced CD11b⁺Gr-1⁺ cells from liver significantly suppressed T cell proliferation at 100 [ratio] 1 and 10 [ratio] 1 ratios of T cell: MDSC (Fig. 5A). In some wells with T cell: MDSC ratio of 10 [ratio] 1, arginase-1 inhibitor (nor-NOHA) was added at the start of the culture. As can be seen, co-incubation with arginase inhibitor significantly reversed the suppression of T cell proliferation induced by MDSCs. Together, these data conclusively demonstrated that the CD11b⁺Gr-1⁺ cells found in the livers were actually immunsuppressive MDSCs.

Figure 5

CBD-induced MDSCs in liver are immunosuppressive in vitro and in vivo.

To further test if CBD-induced MDSCs can suppress liver injury, we adoptively transferred purified MDSCs into naïve mice before challenging them with ConA. Five million CD11b⁺Gr-1⁺ cells isolated from livers of ConA+CBD group were injected into naïve mice, followed by ConA 12 h later. The transferred MDSCs were able to significantly protect against liver injury as indicated by decreased AST levels (Fig. 5B). These data showed that the CBD-induced MDSCs exhibit immunosuppressive functions in vivo and that they can prevent acute liver injury.

Role of vanilloid receptors (TRPV1) in CBD-mediated suppression of liver injury

Next, we addressed the mechanism of action of CBD. CBD has been shown to primarily function through vanilloid receptors (transient receptor potential vanilloid1, TRPV1). To test the role of TRPV1 in this model, we used vanilloid receptor knockout (TRPV1^−/− or VR1-KO) mice. VR1-KO mice developed hepatitis in response to ConA as indicated by increase in AST levels, which was similar to that seen in ConA-injected wild-type (WT) mice (Fig. 6A).

However, unlike in WT mice, CBD was not able to suppress AST levels in VR1-KO mice, suggesting a critical role for TRPV1 in mediating the anti-inflammatory activity of CBD. Moreover, when we enumerated the number of MDSCs in the liver in this experiment, CBD was able to induce MDSCs in ConA-injected WT but not VR1-KO mice (Fig. 7B & C), thereby suggesting that induction of MDSCs by CBD in the livers of hepatitis mice was dependent on TRPV1 receptors. It was interesting to note that administration of ConA alone also induced lower levels of MDSCs in the liver which was similar in both WT and VR1-KO mice, suggesting that this response was independent of TRPV1 receptor.

Figure 6

Role of TRPV1 (vanilloid) receptors.

Figure 7

Identification of MDSC subsets induced by CBD in liver and their suppressive activity.

Analysis of MDSC subsets

The CD11b⁺Gr-1⁺ MDSCs are known to contain heterogeneous mixture of myeloid cells with suppressive function. Recently, two major subsets of MDSCs have been identified based on the expression of CD11b, Ly6-G and Ly6-C antigens. Granulocytic subsets (Gr-MDSC) express both Ly6-G and Ly6-C along with CD11b (CD11b⁺Ly6-G⁺Ly6-C^+(int)) while monocytic subsets (Mo-MDSC) express only Ly6-C and CD11b (CD11b⁺Ly6-G^–Ly6-C⁺).

To identify these subsets, we used mAbs specific to Ly6-G (clone: 1A8) and Ly6-C (clone: HK1.4). Infiltrating cells from the liver of CBD-treated hepatitis mice showed significant frequency of CD11b⁺Ly6-G⁺Ly6C^+(int) granulocytic and CD11b⁺Ly6-G^–Ly6C⁺ monocytic MDSCs in close to 2 [ratio] 1 ratio (Fig. 7A). These subsets were purified by sorting and used in T cell proliferation assay to determine their relative suppressive potential. While both the subsets significantly suppressed T cell proliferation in vitro (Fig. 7B & C), Mo-MDSCs were highly immunosuppressive compared to Gr-MDSCs.

CBD attenuates SEB-induced acute liver injury

We sought to see if the suppressive effect of CBD was specific to ConA-induced liver inflammation or would it work in any other acute liver inflammation model. To this end, we used Staphylococcal Enterotoxin B (SEB)-induced acute liver inflammation. Injection of SEB into GalN-sensitized mice led to increased AST levels at 12 h, indicative of acute hepatitis (Fig. 8A). CBD was able to decrease the AST levels significantly in a dose dependent manner, showing that CBD was effective in suppressing liver inflammation in this model. Moreover, in this model as well, CBD treatment of hepatitis was associated with significant increase in the frequency and number of CD11b⁺Gr-1⁺ MDSCs in liver (Fig. 8B & C).

Figure 8

CBD attenuates SEB-induced acute liver injury by inducing MDSCs in liver.

Discussion

ConA-induced hepatitis is a well-established model for hepatitis caused as a consequence of T and NKT cell activation. In the current study we demonstrate for the first time that CBD, a non-psychoactive cannabinoid, can significantly reduce ConA-induced inflammation and protect the mice from acute liver injury, as indicated by marked decrease in plasma AST levels and necrotic lesions. We observed that a single dose of CBD as low as 20 mg/kg body weight was effective in this model. CBD is already approved for clinical use in Canada, in combination with THC under the trade name Sativex (GW Pharmaceuticals) to alleviate neuropathic pain, spasticity and overactive bladder in multiple sclerosis and also prescribed for cancer patients to reduce nausea and improve appetite. CBD is also in clinical trials to reduce schizophrenic symptoms. The daily recommended dose of Sativex is 5 oral sprays per day which is equivalent to 12.5 mg CBD per day as a long term treatment.

In one of the earliest double-blind studies on CBD, normal volunteers received 3 mg/kg daily CBD for 30 days and epileptic patients received 200–300 mg per day for up to 4 1/2 months without any signs of toxicity or serious side effects. The study concluded that CBD was effective as an anti-epileptic drug or as a potentiating agent for other epileptic drugs compared to placebo. In another randomized double-blind controlled study of Huntington disease patients, CBD was given orally at an average daily dose of 700 mg/day for six weeks, where it was found neither symptomatically effective nor toxic relative to placebo.

In the current study, we used a single dose of CBD at 20–50 mg/kg body weight in mice, which showed significant efficacy in an acute inflammation model. This dose converts to 1.6–4.1 mg/kg of human equivalent dose (HUD) based on body surface area normalization (BSA) method, and translates to a single dose of 96–246 mg in an average individual of 60 kg, which seems to be safe and acceptable dose based on several previous studies in humans mentioned above.

ConA-induced hepatitis is primarily mediated by activated T cells and NKT cells, and the induction of hepatitis is associated with the surge in the production of various pro-inflammatory cytokines. TNF-α and IFN-γ are the first cytokines produced after ConA injection, and are the most critical in inducing hepatitis as anti-TNF and anti-IFN-γ antibodies confer protection against the disease. We found that CBD treatment resulted in suppression of various pro-inflammatory cytokines including TNFα and IFN-γ induced by ConA.

The protective effect of SOCS3 in liver inflammation is known. Replenishing the intracellular stores of SOCS3 with cell penetrating forms of SOCS3 has been shown to effectively suppress the devastating effects of acute inflammation in ConA, LPS or SEB-induced hepatitis models. Attenuated liver injury in STAT1^–/– and IFN-γ^–/– mice in response to ConA was associated with enhanced SOCS3 activation Whereas, decreased SOCS3 activation in IL-6^–/– mice seem to result in enhanced hepatitis in response to ConA. Furthermore, forced expression of SOCS3 in T cells has been shown to protect against the development of ConA-induced hepatitis. In the current study, we observed that CBD induced SOCS-3 in the liver after ConA challenge which may contribute to suppression of inflammatory cytokines observed, and decreased liver injury.

CBD treatment caused a dramatic decrease in inflammatory loci or necrotic lesions in livers of ConA-treated mice. Our phenotypic analysis and detection of CD11b⁺Gr-1⁺ cells in livers showed that majority of the infiltrating cells in the CBD treated group consisted of MDSCs. MDSCs express arginase which can metabolize and deplete L-Arginine, an essential amino acid required for the proliferation and function of T cells.

This seems to be the primary mechanism by which MDSCs suppress activated T cells. In the current study, using arginase enzyme activity assay based on the conversion of L-arginine to L-ornithine, we demonstrated that CBD-induced MDSCs in liver expressed functionally active arginase. Purified MDSCs from CBD treated mice were able to suppress ConA-stimulated T cell proliferation in vitro in Arg-1 dependent manner. In vivo, the suppressive activity of adoptively transferred MDSCs could be demonstrated in models of inflammation and cancer. In the current model, we showed that the adoptively transferred MDSCs induced by CBD were able to significantly protect mice from ConA-hepatitis, thereby conclusively demonstrating that CBD-induced MDSCs were indeed functional and can suppress hepatitis.

It should be noted that ConA-induced hepatitis by itself showed a small increase in the number of MDSCs in livers. This may be a natural mechanism following inflammatory stimuli to regulate inflammation. Such MDSCs may play a crucial role in helping the host recover from inflammation as evidenced by the restoration of AST levels to basal levels by 48 h.

Nevertheless, CBD treatment further triggered the induction of MDSCs which also expressed higher density of Arg-1. It is interesting to note that CBD alone did not induce any MDSCs in liver of naïve mice. We speculate that CBD triggers the induction of MDSCs only when there is an insult or inflammation in the liver.

Cannabidiol has been shown to bind and function primarily through TRPV1 or vanilloid receptors. Vanilloid receptors mediate anti-hyperalgesic effect of CBD, in a rat model of acute inflammation. TRPV1 ion channels also mediate the response to painful heat, extracellular acidosis, and capsaicin, the pungent compound from plants which upon prolonged use decreases TRPV1 activity by a phenomenon called desensitization. CBD possesses no, or very weak affinity for the central and peripheral canabbinoid receptors (CB1 and CB2) and is not psychoactive.

Use of vanilloid receptor knock-out mice in our study clearly showed that CBD induced suppression of inflammation in ConA-hepatitis was dependent on TRPV1, so was the induction of MDSCs by CBD in the livers of ConA-injected mice. Recently, we demonstrated that activation of cannabinoid receptors can trigger massive induction of immunosuppressive MDSCs. We noted this was dependent on the production of G-CSF. In the current study, we tested if some specific chemokine or cytokine may be involved in TRPV1-mediated induction or accumulation of MDSCs by CBD in liver. Our attempt to identify such factor, particularly looking at GM-CSF, G-CSF, and KC by ELISA was not conclusive (data not shown).

Even though there was a significant increase in G-CSF 24 h after CBD treatment of hepatitis corresponding with increase in number of MDSCs in liver as well as decrease in liver enzymes (inflammatory marker), blocking studies with anti-G-CSF Ab failed to reverse the CBD effect.

Finally, we showed that CBD-induced suppression of acute liver inflammation is not specific to ConA-induced hepatitis, but it is also equally effective in other acute hepatitis models such as sensitization with GalN followed by induction of liver inflammation by sub lethal doses of SEB. Overall, the current study demonstrates that MDSCs may play a critical role in protecting the liver from acute inflammation.

The most interesting observation in this study was robust induction of CD11b⁺Gr-1⁺ MDSCs by CBD in the livers of ConA-induced mice that were immunosuppressive, which protected mice from hepatitis upon adoptive transfer. Moreover, CBD was found to induce MDSCs following activation of TRPV1 inasmuch as, CBD failed to trigger MDSCs in the livers of TRPV1 deficient mice and failed to protect them from hepatitis.

Together, these studies not only demonstrate that CBD can protect the host from acute liver injury but also provide evidence for the first time that MDSCs may play a critical role in protecting the liver from acute inflammation. Non-psychoactive cannabinoids such as CBD possess great therapeutic potential in treating various inflammatory liver diseases, including autoimmune hepatitis.

Materials and Methods

Mice

Female C57BL/6 mice (8–12 weeks) were obtained from National Cancer Institute (Frederick, MD). Female vanilloid receptor knockout (TRPV1) mice on BL/6 background were purchased from the Jackson labs (Bar Harbor, ME, USA).

Ethics Statement

Mice were housed and maintained under specific pathogen-free conditions in the Animal Resource Facility of University of South Carolina and all experiments were pre-approved by the Institutional Animal Care and Use Committee (ICAUC approval number AUP # 1620).

Reagents

Cell culture grade concanavalin A was from Sigma-Aldrich (St. Louis, MO). The monoclonal antibodies (mAb), FITC-conjugated anti-CD11b and anti-Ly6-C (clone: HK1.4), PE-conjugated anti-CD3, anti-Gr-1 and anti-F4/80, AlexaFlour647-conjugated CD11b, and purified anti-CD16/CD32 were from eBioscience. PE-conjugated anti-Ly6-G (clone: IA-8) was from BD Bioscience. Cannabidiol was provided by NIDA, NIH. N^ω-hydroxy-nor-Arginine (nor-NOHA) was obtained from Cayman Chemical Company. Cell culture reagents including RPMI 1640 medium were from Invitrogen Corp. All other reagents and chemicals used were from Sigma-Aldrich.

Induction of hepatitis and treatment with cannabidiol

Concanavalin A was dissolved in pyrogen-free PBS at a concentration of 2.5 mg/ml and injected intravenously at a dose of 12.5 mg/kg body weight to induce hepatitis as described. Mice were administered intraperitoneally with indicated doses of cannabidiol (DMSO stock diluted in PBS + a drop of Tween-80) or vehicle (DMSO similarly diluted in PBS) 5 min after ConA injection. One group received cannabidiol alone. Blood was collected after 6, 12, 24 and 48 h by retro-orbital bleeding and sera were separated and stored below −20°C until further use.

Aspartate transaminase (AST) activity

Liver enzyme aspartate transaminase activity in sera from individual mice obtained at various time points after ConA injection was measured at 340 nm by spectrophotometric method using a commercially available AST assay kit (Pointe Sci.), as described previously.

Liver histology

After 48 h of post ConA-injection, livers were harvested, carefully rinsed with PBS and fixed in 4% paraformaldehyde. Fixed liver tissue was embedded in paraffin, cut into 5 µm thick sections, deparaffinized in xylene, and serially dehydrated in decreasing concentrations of ethanol. Sections were stained with hematoxylin-eosin (H&E) and examined under light microscopy to evaluate and liver damage. Area of necrotic lesions as a percentage of total liver parenchyma was quantified using NIH ImageJ software. At least five fields per section were analyzed from each liver sample.

Cytokine analysis

Serum obtained 12 h after ConA-injection was used for analyzing 23 different cytokines and chemokines including IL-2, TNF-α, IFN-γ, IL-1α, IL-1β, IL-3, IL-4, IL5, IL-6, IL-10, IL-12p40, IL-12p70, IL-17, GM-CSF, G-CSF, KC, MIP-1α, RANTES and eotaxin-1 by Bioplex cytokine array system (BioRad).

Reverse Transcription-PCR (RT-PCR)

RT-PCR was performed by standard protocol. The total RNA was prepared using RNeasy kit (QIAGEN), and cDNA was prepared using random hexamer primers (Invitrogen). Gene-specific primers used for amplification were: SOCS-1 (forward 5′ GAG GTC TCC AGC CAG AAG TG 3′ and reverse 5′ CTT AAC CCG GTA CTC CGT GA 3′), SOCS-2 (forward 5′ AAG ACA TCA GCC GGG CCG ACT A 3′ and reverse 5′ GTC TTG TTG GTA AAG GTA GTC 3′), 18S (forward 5′ GCC CGA GCC GCC TGG ATA C 3′ and reverse 5′ CCG GCG GGT CAT GGG AAT AAC 3′). 18S served as the internal control. The PCR products were electrophoresed on 1.5% agarose gel in the presence of ethidium bromide and visualized in a gel imaging system (BioRad). The densities of bands were analyzed with NIH Image J software and normalized to internal control.

Cell preparation and flow cytometric analysis

Liver infiltrating cells were isolated using Percoll density separation. Briefly, single cell suspensions from livers were prepared by using a tissue homogenizer and passing the homogenate through sterile nylon mesh (70 µM).

Cell suspension was washed once with PBS and pellet suspended in 33% Percoll (Sigma-Aldrich) diluted in sterile PBS and centrifuged at 2000 rpm for 15 min at 25°C. Leukocyte cell pellet was washed twice with PBS. Contaminating RBCs were lysed using RBC-lysis solution.

For FACS analysis, cells were blocked using mouse Fc-block (anti-CD16/CD32) and stained for various cell surface markers using fluorescently labeled mAb (10 µg/mL, in PBS containing 2% FBS). After washing, stained cells were analyzed in a flow cytometer (FC500, Beckman Coulter). Only live cells were counted by setting gates on forward and side scatters to exclude cell debris and dead cells. Cells stained similarly with isotype antibodies served as staining controls to set the voltage. Data obtained were analyzed in Cytomics CXP software (Beckman Coulter).

Arginase Activity Assay

Cell lysates were obtained by suspending the cell pellet in lysis buffer (50 mM HEPES, 150 mM NaCl, 5 mM EDTA, 1 mM NaVO₄, and 0.5% Triton X-100) containing 50 µg/ml aprotinin, 50 µg/ml leupeptin, 100 µg/ml trypsin-chymotrypsin inhibitor, and 2 mM PMSF.

Lysates were centrifuged at 3000×g for 10 min at 4°C. Protein content was determined by BCA method (Sigma-Aldrich) and cell lysates (5 µg) were tested for arginase activity by measuring the production of L-ornithine. Briefly, cell lysates were added to 25 µL of Tris-HCl (50 mM; pH 7.5) containing 10 mM MnCl₂. This mixture was heated at 55–60°C for 10 min to activate arginase.

Then, a solution containing 150 µL carbonate buffer (100 mM) and 50 µL L-arginine (100 mM) was added and incubated at 37°C for 20 min. The hydrolysis reaction from L-arginine to L-ornithine was identified by a colorimetric assay after the addition of ninhydrin solution and incubation at 95 °C for 1 h.

Wright-Giemsa staining

Cells were collectedby cytospin onto glass slides and dried completely for 30 min. Slides were then stained with Wright-Giemsa stain (Fisher Sci.) according manufacturer's instructions and analyzed by light microscopy.

Cell sorting

Mice were injected with ConA (12.5 mg/kg) + CBD (25 mg/kg). Liver infiltrating cells were isolated after 12–16 h by Percoll separation. Cells were blocked using Fc-block and stained using appropriate mAb. CD11b⁺Gr-1⁺ cells (MDSCs), CD11b⁺Ly6-G⁺Ly-6C⁺ granulocytic (Gr-MDSC) and CD11b⁺Ly6-G⁻Ly-6C⁺ monocytic (Mo-MDSC) subsets were sorted to >90% purity using FACS Aria (BD Biosci.) after labeling with appropriate fluorescently conjugated mAbs.

T cell proliferation assay

Purified MDSCs were irradiated at approximately 2000 rads and cultured at different ratios with purified syngenic T cells (2×10⁵) stimulated with ConA (4 µg/ml) in a 96-well round bottom plates, in complete RPMI 1640 medium (Invitrogen) supplemented with 10% FBS, 10 mM HEPES, 1 mM penicillin-streptomycin, and 50 µM β-mercaptoethanol. T cell proliferation was determined after 48 h culture by pulsing with [3H]thymidine (2 µCi/well) during the final 8 h of culture. Cultures were harvested using a cell harvester and thymidine incorporation was measured in a beta counter (Perkin Elmer).

Adoptive transfer experiments

ConA was injected into naïve C57BL/6 (WT) mice to induce hepatitis as described before. For adoptive transfer, purified CD11b⁺Gr-1⁺ MDSCs from the livers of ConA+CBD injected mice were transferred to a group of naïve mice (5×10⁶ purified MDSCs/mouse, i.v) 12 h before injecting ConA. Blood was collected 12 h after ConA challenge. Hepatitis was assessed by measuring liver enzyme aspartate transaminase (AST) in sera.

Statistical Analysis

Data are expressed as mean ± S.E.M. Student's t-test was used for comparison and P≤0.05 was considered statistically significant. Experiments were repeated at least twice unless otherwise mentioned.

Acknowledgments

The authors acknowledge Dr. Udai Singh, Instruments Resource Facility, University of South Carolina School of Medicine for his help in sorting MDSCs and technical assistance by Mrs. Shweta Hegde.

Footnotes

Competing Interests: The authors have declared that no competing interests exist.

Funding: The research was funded by National Institutes of Health, USA grant awards, R01-DA016545 and P01-AT003961 to MN and PSN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Cannabidiol causes activated hepatic stellate cell death through a mechanism of endoplasmic reticulum stress-induced apoptosis

source: Cell death & disease

The major cellular event in the development and progression of liver fibrosis is the activation of hepatic stellate cells (HSCs). Activated HSCs proliferate and produce excess collagen, leading to accumulation of scar matrix and fibrotic liver. As such, the induction of activated HSC death has been proposed as a means to achieve resolution of liver fibrosis.

Here we demonstrate that cannabidiol (CBD), a major non-psychoactive component of the plant Cannabis sativa, induces apoptosis in activated HSCs through a cannabinoid receptor-independent mechanism. CBD elicits an endoplasmic reticulum (ER) stress response, characterized by changes in ER morphology and the initiation of RNA-dependent protein kinase-like ER kinase-, activating transcription factor-6-, and inositol-requiring ER-to-nucleus signal kinase-1 (IRE1)-mediated signaling cascades.

Furthermore, CBD induces downstream activation of the pro-apoptotic IRE1/ASK1/c-Jun N-terminal kinase pathway, leading to HSC death. Importantly, we show that this mechanism of CBD-induced ER stress-mediated apoptosis is specific to activated HSCs, as it occurs in activated human and rat HSC lines, and in primary in vivo-activated mouse HSCs, but not in quiescent HSCs or primary hepatocytes from rat.

Finally, we provide evidence that the elevated basal level of ER stress in activated HSCs has a role in their susceptibility to the pro-apoptotic effect of CBD. We propose that CBD, by selectively inducing death of activated HSCs, represents a potential therapeutic agent for the treatment of liver fibrosis. Cell Death and Disease (2011) 2, e170; doi:10.1038/cddis.2011.52; published online 9 June 2011
Subject Category: Experimental Medicine...read full publication (pdf)

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A Novel Synthetic Cannabinoid Derivative Inhibits Inflammatory Liver Damage via Negative Cytokine Regulation

source: Molecular Pharmacology

Dr. Iris Lavon, Pharmos LTD, Kiryat Weizmann, Bld #13b, Rehovot 76326, Israel. E-mail: lavoni@netvision.net.il

Abstract

The therapeutic potential of cannabinoids has been described previously for several inflammatory diseases, but the molecular mechanisms underlying the anti-inflammatory properties of cannabinoids are not well understood. In this study, we investigated the mechanism of action of a novel synthetic cannabinoid, [(+)(6aS,10aS)-6,6-Dimethyl-3-(1,1-dimethylheptyl)-1-hydroxy-9-(1H-imidazol-2-ylsulfanylmethyl]-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran (PRS-211,092) that has no psychotropic effects but exhibits immunomodulatory properties.

Treatment with PRS-211,092 significantly decreased Concanavalin A-induced liver injury in mice that was accompanied by: 1) promotion of early gene expression of interleukin (IL)-6 and IL-10 that play a protective role in this model; 2) induction of early gene expression of the suppressors of cytokine signaling (SOCS-1 and 3), followed by 3) inhibition of several pro-inflammatory mediators, including IL-2, monocyte chemoattractant protein-1 (MCP-1), IL-1β, interferon-γ, and tumor necrosis factor α.

Based on these results, we propose a mechanism by which PRS-211,092 stimulates the expression of IL-6, IL-10 and the SOCS proteins that, in turn, negatively regulates the expression of pro-inflammatory cytokines. Negative regulation by PRS-211,092 was further demonstrated in cultured T cells, where it inhibited IL-2 production and nuclear factor of activated T cells activity. These findings suggest that this cannabinoid derivative is an immunomodulator that could be developed as a potential drug for hepatitis as well as for other short- or long-term inflammatory diseases.... Read full publication

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Cannabis Compound Induces Death Of Cells Associated With Liver Fibrosis

New York, NY: The administration of the non-psychotropic cannabinoid CBD (cannabidiol) induces selective apoptosis in hepatic stellate cells (HSCs), according to preclinical findings reported in the journal Cell Death and Disease. The activation of HSCs is considered to be a key cellular event underlying hepatic fibrogenesis (excessive tissue build up), a condition that can result in liver failure.

Authors reported: "In this study, we find that CBD selectively kills activated HSCs. ... We provide a molecular basis of action for CBD and identify CBD as a novel potential therapeutic agent for liver fibrosis."

They concluded, "These promising findings warrant future investigation evaluating the anti-fibrotic effect of CBD in vivo. The prospect of CBD as a new anti-fibrotic compound is rendered more appealing by the fact that CBD is a non-psychoactive small drug-like molecule already approved for clinical use in many countries."

Liver fibrosis is the tenth leading cause of death in the United States.

Previous studies have consistently reported that cannabinoids can selectively promote cell suicide in various malignant cell lines, including breast cancer, lung cancer, and glioma.

For more information, please contact Paul Armentano, NORML Deputy Director, at: paul@norml.org.

Full text of the study, "Cannabidiol causes activated hepatic stellate cell death through a mechanism of endoplasmic reticulum stress-induced apoptosis," appears in Cell Death and Disease.

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Prevention of Fibrosis Progression in CCl4-Treated Rats: Role of the Hepatic Endocannabinoid and Apelin Systems.

This work was previously presented in part at the following conferences: Reichenbach V, Ros J, Fernández-Varo G, Muñoz-Luque J, Morales-Ruiz M, Makriyannis A, and Jiménez W (2008) Chronic stimulation of cannabinoid CB2 receptor represses fibrosis progression in CCl₄-treated rats.

59th Annual Meeting of the American Association for the Study of Liver Diseases; 2008 Oct 31–Nov 4; San Francisco, CA. American Association for the Study of Liver Diseases, Alexandria, VA. Reichenbach V, Ros J, Fernández-Varo G, Casals G, Melgar-Lesmes P, Pauta M, Morales-Ruiz M, and Jiménez W (2010) Activation of the hepatic apelin system is of major relevance in early stages of liver fibrosis. International Liver Conference 2010; 2010 Apr 14–18; Vienna, Austria. European Association for the Study of the Liver, Geneva, Switzerland.

Abstract

Endocannabinoids behave as antifibrogenic agents by interacting with cannabinoid CB2 receptors, whereas the apelin (AP) system acts as a proangiogenic and profibrogenic mediator in the liver.

This study assessed the effect of long-term stimulation of CB2 receptors or AP receptor (APJ) blockade on fibrosis progression in rats under a non-discontinued fibrosis induction program.

The study was performed in control and CCl₄-treated rats for 13 weeks. Fibrosis-induced rats received a CB2 receptor agonist (R,S)-3-(2-iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole (AM1241) (1 mg/kg b.wt.), an APJ antagonist [Ala¹³]-apelin-13 sequence: Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Ala (F13A) (75 μg/kg b.wt.), or vehicle daily during the last 5 weeks of the CCl₄ inhalation program. Mean arterial pressure (MAP), portal pressure (PP), hepatic collagen content, angiogenesis, cell infiltrate, and mRNA expression of a panel of fibrosis-related genes were measured in all animals.

Fibrosis-induced rats showed increased hepatic collagen content, reduced MAP, portal hypertension, and increased expression of the assessed messengers in comparison with control rats. However, fibrotic rats treated with either AM1241 or F13A had reduced hepatic collagen content, improved MAP and PP, ameliorated cell viability, and reduced angiogenesis and cell infiltrate compared with untreated fibrotic rats.

These results were associated with attenuated induction of platelet-derived growth factor receptor β, α-smooth muscle actin, matrix metalloproteinases, and tissue inhibitors of matrix metalloproteinase. CB2 receptor stimulation or APJ blockade prevents fibrosis progression in CCl₄-treated rats. The mechanisms underlying these phenomena are coincident despite the marked dissimilarities between the CB2 and APJ signaling pathways, thus opening new avenues for preventing fibrosis progression in liver diseases.

Footnotes

This work was supported by the Dirección General de Investigación Científica y Técnica [Grants SAF09-08839, SAF07-63069] (to W.J. and M.M.-R., respectively); Agència de Gestió d'Ajuts Universitaris i de Recerca [Grant SGR 2009/1496]; Dirección General de Investigación Científica y Tecnológica [Grant BES-2004-5186] (to P.M.-L.); and Instituto de Salud Carlos III [“Contrato Post Formación Sanitaria Especializada” FIS CM07/00043] (to G.C.). Centro de Investigación Biomédica en Red-Enfermedades Hepáticas y Digestivas was founded by the Instituto de Salud Carlos III (Spain).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

http://dx.doi.org/10.1124/jpet.111.188078.

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Cannabis use improves retention and virological outcomes in patients treated for hepatitis C

Eur J Gastroenterol Hepatol Oct 2006 18:1057-1063
Diana L. Sylvestrea,b, Barry J. Clementsb and Yvonne Malibub
Department of Medicine, University of California, San Francisco, California, USA
and bOrganization to Achieve Solutions in Substance-Abuse (OASIS), Oakland, California, USA

Note from Jules Levin: this study was first presented at a conference 2 years ago or longer.

"...The results of this observational study suggest that the use of cannabis during HCV treatment can improve adherence by increasing the duration of time that patients remain on therapy; this translates to reduced rates of post-treatment virological relapse and improved SVR....."

As shown in Fig. 3, cannabis users were no more likely than non-users to take at least 80% of the prescribed interferon, 91 versus 76% (P=0.2), nor were they more likely to take at least 80% of the prescribed ribavirin, 91 versus 84% (P>0.5). However, cannabis users were significantly more likely than non-users to remain on HCV treatment for at least 80% of the projected treatment course, 95 versus 67% (P=0.01). The average duration of HCV treatment in cannabis users was 38 weeks compared with 33 weeks for the non-users.

ABSTRACT
Objectives: Despite the widespread use of polypharmacy, the management of hepatitis C virus (HCV) treatment-related side-effects is often incomplete, and many patients turn to cannabis for symptom relief. Unfortunately, there are few data about cannabis use on treatment outcomes, leaving clinicians without the data needed to inform recommendations.

Methods: To define the impact of cannabis use during HCV treatment, we conducted a prospective observational study of standard interferon and ribavirin treatment in 71 recovering substance users, of whom 22 (31%) used cannabis and 49 (69%) did not.

Results:
Seventeen of the 71 study patients (24%) discontinued therapy early, one cannabis user (5%) and 16 non-users (33%) (P = 0.01).

Overall, 37 patients (52%) were end-of-treatment responders, 14 (64%) cannabis users and 23 (47%) non-users (P =0.21).

A total of 21 out of 71 (30%) had a sustained virological response: 12 of the 22 cannabis users (54%) and nine of the 49 non-users (18%) (P =0.009), corresponding to a post-treatment virological relapse rate of 14% in the cannabis users and 61% in the non-users (P = 0.009).

Overall, 48 (68%) were adherent, 29 (59%) non-users and 19 (86%) cannabis users (P =0.03).

Although cannabis users were no more likely than non-users to take at least 80% of the prescribed interferon or ribavirin, they were significantly more likely to remain on HCV treatment for at least 80% of the projected treatment duration, 95 versus 67% (P =0.01).

Conclusions: Our results suggest that modest cannabis use may offer symptomatic and virological benefit to some patients undergoing HCV treatment by helping them maintain adherence to the challenging medication regimen.

Introduction
Although hepatitis C virus (HCV) treatment outcomes have improved dramatically over the past decade, the intolerability of interferon/ribavirin combination therapy remains a barrier to treatment success.

The majority of patients develop significant treatment-related side effects, with almost 80% experiencing an initial 'flulike syndrome that includes fevers, chills, and muscle and joint aches. Although the acute effects of treatment tend to modulate over time, many will experience debilitating fatigue (70-72%), headaches (66-67%), nausea (35-46%), anorexia (19-27%), depression (21-44%), and insomnia (30-39%) among others.

Many patients require the use of adjunctive pharmacological agents for side-effect management [5,8]. These include a spectrum of medications including antiemetics, anti-inflammatory agents, antihistamines, sleeping pills, antidepressants, anxiolytics, stimulants, and antipsychotics. Unfortunately, symptom relief is often incomplete despite the widespread use of polypharmacy, and patients so affected may compensate by reducing their interferon or ribavirin doses or by discontinuing treatment altogether.

Maximizing HCV treatment outcomes thus requires a thorough familiarity with an array of successful side-effect management strategies.

Faced with intolerable treatment-related side-effects that respond inadequately to conventional medications, some patients turn to Cannabis sativa (marijuana) for symptom relief. Cannabis sativa contains over 400 chemical entities, but delta-9-tetrahydrocannabinol (THC) is the major psychoactive component.

Although the majority of studies of cannabis are observational in nature, there is anecdotal evidence that it may have benefits in modulating some of the common side-effects associated with HCV treatment, including nausea, anorexia, weight loss, musculoskeletal pain, insomnia, anxiety, and mood instability. However, the benefits of cannabis during HCV treatment remain unconfirmed and concerns about its safety remain.

Cannabinoid receptors appear to be upregulated in hepatic myofibroblasts of human cirrhotic liver samples, and smoking daily cannabis has been reported to accelerate the progression of hepatic fibrosis in patients with chronic HCV.

Cannabinoid receptors are also present on immune cells, and cannabis use may suppress a variety of immune functions, including antibody production, cell proliferation, natural killer cell activity, and macrophage function, and also alter the production of such cytokines as interferon gamma and tumor necrosis factor.

In addition, there is a potential drug-drug interaction between ribavirin and marijuana, as both are metabolized by the cytochrome P450 system.

Obviously, the overall benefit of cannabis in terms of side-effect management may be outweighed by worsening histology and impairments in virological outcomes; therefore, its use as a potential therapeutic agent must be more clearly defined in the setting of HCV treatment.

Although widespread restrictions limit the ease with which these questions can be formally studied, the pervasive use of cannabis during HCV treatment provides a means for an observational study of its potential risks and benefits. I

n the context of a prospective study of HCV treatment in recovering heroin users maintained on methadone we have conducted such a study, by measuring the impact of intercurrent cannabis use on treatment adherence, retention rates, and virological outcomes.

Discussion
The results of this observational study suggest that the use of cannabis during HCV treatment can improve adherence by increasing the duration of time that patients remain on therapy; this translates to reduced rates of post-treatment virological relapse and improved SVR.

Although other potential mechanisms may contribute to its enhancement of treatment outcomes, such as altered immunological function and improved nutritional status, it appears that the moderate use of cannabis

Although its availability in the United States has been restricted since 1937 and its benefit unconfirmed, cannabis is frequently obtained illicitly for self-medication. It has been used recreationally for millennia, and is the third most commonly used drug after tobacco and alcohol. In the United States, 6.2% of individuals aged 12 years or older have used cannabis in the past month, with 4.8 million individuals using it on 20 or more days.

THC can produce alterations in mood, perception,cognition, and memory, and studies have shown that THC has anticonvulsive, analgesic, anti-anxiety, and anti-emetic properties.

Clinical trials have demonstrated that cannabinoids reduce nausea and improve appetite in humans, and cannabis has shown benefit in modulating the nausea of cancer chemotherapy, multiple sclerosis-related spasticity, and the wasting syndrome of HIV.

Progress has been made in understanding the pharmacology of cannabinoids in humans. Of the two known cannabinoid receptors, CB1 is responsible for the neurological and behavioral effects of marijuana. CB1 was the first cannabinoid receptor identified, and is the most abundant G-protein-coupled receptor in the central nervous system.

It is also expressed on peripheral neurons and is found abundantly in the basal ganglia, cerebellum, and hippocampus, accounting for its effects on motor coordination and short-term memory.

It is also expressed at high concentrations on primary afferent nociceptors of the dorsal spinal cord, which are responsible for the ability of cannabinoids to inhibit pain.

Although CB1 cannabinoid receptors mediate the central nervous system effects of cannabinoids , an additional subset of cannabinoid receptors, the CB2 receptors, is present on immune cells. The presence of these receptors on B lymphocytes and natural killer cells suggests that cannabinoids may impact upon the immune response.

Some studies have shown that THC can be immunosuppressive and can impair cell-mediated immunity, humoral immunity, and cellular defences against a variety of infectious agents in experimental animals. There is an increased recurrence of herpes simplex viral lesions in marijuana smokers and an altered responsiveness of human papilloma virus to IFN-a 2a treatment.

Although uncontrolled studies suggested an association between marijuana use and the progression of HIV disease, a recent prospective study demonstrated no evidence of detrimental effects of cannabinoids on immune parameters in patients with HIV.

The majority of studies on the effects of cannabis have been conducted in cell culture or on animal models with supraphysiological doses of the compound, and their clinical relevance is unclear.

Although their potential contribution to liver disease is not understood, both the CB1 and CB2 receptors have also been reported to be expressed on hepatic myofibroblasts in cirrhotic livers.

Activation of these receptors can lead to cellular apoptosis, and a recent study demonstrated that the use of cannabis on a daily basis may enhance the progression of hepatitis fibrosis in patients with HCV. By implicating these receptors as mediators of the fibrotic process, these results raise concerns about the safety of cannabis use in patients with HCV.

In spite of this, our results suggest that moderate cannabis use during HCV treatment may offer significant benefit to certain patients.

Although the lack of a direct dose response suggests that its principal contribution is related to a non-specific improvement in the tolerability of the challenging medication regimen, we cannot rule out additional biological effects.

We did not measure relevant immune parameters in our patients, nor did we assess potential differences in nutritional status. P450-mediated drug-drug interactions between cannabinoids and ribavirin may have led to additional benefit, but these were not assessed.

However, the lack of dose response in our study argues against specific receptor or metabolismrelated effects, and suggests instead that cannabis exerted its benefit by non-specific improvements in symptom management. Interestingly, because the bene fits of heavy cannabis use were less apparent, we cannot rule out the possibility that detrimental biological or immunological mechanisms may be relevant at higher levels of consumption. Obviously, further study is needed.

Our study has a number of additional limitations that warrant caution in its interpretation. First, we confined our study to methadone-maintained patients, a population with relatively high rates of medical and psychiatric co-morbidity.

Second, the use of additional illicit substances was not uncommon, and although not differing between the two cohorts, the impact of these substances or even of methadone on study outcomes cannot be excluded.

Third, the use of marijuana was quantified by self-report and may have introduced bias as a result of underreporting or even overreporting.

Fourth, illicitly obtained marijuana, even that obtained through 'cannabis clubs', may be highly variable in its content of bioactive compound, leaving in question a true quantitation of the amount of cannabis that may or not be beneficial.

And finally, significant limitations are introduced by our observational study design; however, with legal proscriptions against cannabis use limiting its study, the design and conduct of randomized, prospective research studies is virtually impossible at this time.

Despite its shortcomings, this study begins to answer some of the key questions that arise about the use of cannabis during HCV treatment.

Our results suggest that the modest use of cannabis does not appear to impact negatively upon HCV treatment outcomes and need not elicit undue alarm.

The widespread use of illicit cannabis during HCV therapy highlights the inadequacies of our current side-effect management strategies; our study suggests that cannabis use may offer benefit for some patients undergoing HCV treatment by helping them maintain adherence to the frequently debilitating medication regimen.

However, the mechanisms through which cannabis exerts its benefit are unclear, and controlled studies may further elucidate the mechanisms through which cannabis may impact upon clinical outcomes during HCV treatment.

Results
Baseline characteristics
Seventy-one patients were enrolled; 22 (31%) smoked cannabis while undergoing HCV treatment and 49 (69%) did not. The demographic characteristics of the study patients are shown in Table

1. The median age was 50 years, and 43 (61%) were male, 53 (75%) Caucasian, 10 (14%) African-American, and eight (11%) Latino; there were no differences in the demographic characteristics between the cannabis users and non-users.

The median estimated duration of HCV exposure was 30±9 years. Forty patients (56%) had genotype 1, 29 (41%) had genotypes 2 or 3, one patient had genotype 8a, and one patient's genotype was untypable. There was no difference in the frequencies of genotypes between the cannabis users and non-users; 30 of the non-users (61%) and 10 of the cannabis users (48%) had genotype 1 (P=0.31). Thirty patients underwent liver biopsy.

Among these, the mean METAVIR inflammation grade was 2.4 (1.5-3.5) and the mean fibrosis stage was 2.6 (0-4). There was no significant difference in liver fibrosis between the two groups; the mean fibrosis stage was 2.5±0.4 for the cannabis users and 2.7±0.2 for the nonusers (P=0.36). The 20 patients (28%) who had platelet counts of less than 100 000 cells/ml were also equally divided between the groups, comprising 29% (n=14) of the non-users and 27% (n=6) of the group that used cannabis.

Forty-two patients (59%) reported a previous psychiatric diagnosis; the majority had depression (n=33) or depression/anxiety (n=6). Cannabis users were no more likely to report a psychiatric diagnosis than non-users (P>0.5), and there were no differences in the rates of antidepressant use between users and non-users during HCV treatment (P>0.5).

Similarly, a total of 25 (35%) used other illicit substances during HCV treatment, including heroin, cocaine, and methamphetamine, but this did not differ between the two groups (37% in the cannabis non-users and 32% in the users; P>0.5), nor were there differences in rates of alcohol consumption (24% in the non-users and 14% in the users; P=0.36).

Treatment outcomes
The majority of patients, 93% (n=66), reported at least one treatment-related side-effect, most commonly 'flulike symptoms, nausea, or headache, but there was no difference in reported symptoms between the cannabis users and non-users (P>0.5).

The association of cannabis use with HCV treatment outcomes is shown in Fig. 1. Seventeen of the 71 study patients (24%) discontinued therapy before completing the full course. Of these, 16 did not use cannabis and one was a cannabis user. The discontinuation rate of the 49 cannabis nonusers was 33%; it was 5% in the cannabis users (P=0.01). Of the 16 non-users who terminated treatment early, eight discontinued as a result of intolerable side-effects and four discontinued because of depression.

Three of the 16 were terminated at the discretion of the medical provider: one because of excessive alcohol intake, one because of worsening liver disease, and one because of intractable anemia. The remaining patient in this cohort relocated and was unable to obtain medications. The single cannabis user who discontinued treatment developed worsening liver disease and was unable to continue.

Overall, 37 of the 71 patients (52%) were end-of-treatment responders and 21 (30%) had an SVR. The association of cannabis use with response rates is shown in Fig. 1. Fourteen of the cannabis users (64%) and 23 of the non-users (47%) were end-of-treatment responders (P=0.21).

Twelve of the 22 cannabis users (54%) and nine of the 49 non-users (18%) had an SVR, corresponding to a post-treatment relapse rate of 14% (n=2) with the cannabis users and 61% (n=14) with the non-users. Multivariate logistic regression analysis taking sex, race, genotype, and the use of other illicit substances into account, revealed that this finding was statistically significant (P=0.009).

The association of the estimated quantity of cannabis used with virological outcomes is shown in Fig. 2. Ten of the 16 occasional cannabis users (62%) had an end-oftreatment virological response compared with four of the six regular users (67%, P>0.5). SVR were also not statistically different between the occasional and regular users of cannabis, seen in two of six of the regular users (33%) and 10 of the 16 (62%) occasional users (P=0.35).

Adherence
The association of cannabis use with the components of treatment adherence is shown in Fig. 3. Overall, 48 of the 71 study patients (68%) took at least 80% of the prescribed interferon and ribavirin for at least 80% of the projected duration of treatment, and were therefore considered adherent. Of those, 29 did not use cannabis and 19 were cannabis users.

The corresponding adherence rates were 59% in the non-cannabis group and 86% in the cannabis group (P=0.03); there was no difference in adherence between occasional users (87%) and regular users (83%) (P>0.5).

As shown in Fig. 3, cannabis users were no more likely than non-users to take at least 80% of the prescribed interferon, 91 versus 76% (P=0.2), nor were they more likely to take at least 80% of the prescribed ribavirin, 91 versus 84% (P>0.5). However, cannabis users were significantly more likely than non-users to remain on HCV treatment for at least 80% of the projected treatment course, 95 versus 67% (P=0.01). The average duration of HCV treatment in cannabis users was 38 weeks compared with 33 weeks for the non-users.

Methods
Study setting and eligibility Recruitment and treatment took place at OASIS (Organization to Achieve Solutions in Substance-Abuse), a community-based non-profit clinic providing medical and psychiatric treatment to substance users in Oakland, CA.

Although the clinic does not provide methadone treatment, comprehensive primary medical and psychiatric care services are provided on-site. All experimental procedures were followed in accordance with the Helsinki Declaration of 1975, as revised in 1983, and were approved by the Ethical Review Committee (Kansas City, Missouri, USA).

Men and women aged 18 years and older were considered eligible if they had been maintained on methadone for a period of 3 months or more and had a positive HCV polymerase chain reaction (PCR). Patients with non-HCV-related liver disease or decompensated liver disease were excluded. Those with untreated depression were excluded until stabilized on antidepressant treatment. Drug use was assessed by self-report as well as by random monthly urine toxicology testing, as per standard protocol at the methadone clinics.

Medications
HCV treatment consisted of IFN-a 2b, 3 x 106 units administered subcutaneously three times a week and ribavirin capsules, 1000 mg taken orally daily in two divided doses for patients weighing less than 165 lb, or 1200 mg daily for those weighing 165 lb or more.

Patients were initially treated for 48 weeks regardless of genotype; however, subsequent data supporting the efficacy of 24 weeks of treatment for genotypes 2 and 3 led to a protocol amendment that shortened the treatment course for patients with these genotypes. Medications were selfadministered unless patients specifically requested otherwise.

Cannabis use
The use of cannabis during study was neither endorsed nor prohibited by study staff, and all patients obtained their cannabis outside the construct of the study protocol. However, because marijuana use was legalized for medical use in the state of California, it was often obtained with outside medical approval through local 'cannabis clubs'. Cannabis use was quantified by self-report, with 'regular' use defined as the use of cannabis every day or every other day for a minimum duration of 4 weeks; 'occasional' reflected the use of less than daily quantities.

Procedures
After providing informed consent, participants completed a questionnaire that elicited baseline demographic, psychosocial, psychiatric, and substance use characteristics. The duration of HCV infection was estimated as one less than the number of years since injection drug use was initiated. Liver biopsy was suggested but not required, and was scored on the METAVIR scale of 0-4, with 0, none; 1, minimal-mild; 2, mild-moderate; 3, moderate-severe; and 4, cirrhosis.

Patients were monitored for treatment-related neutropenia, thrombocytopenia, and hemolytic anemia using standard published algorithms, and medication doses were adjusted accordingly .

Drug and alcohol consumption were assessed by monthly self-report questionnaires, and monthly random urine drug test results were obtained from the subject's methadone treatment program.

An HCV-RNA PCR was performed at baseline, at 6 months, at the end of treatment, and 6 months after the completion of therapy. Substance use during HCV therapy was actively discouraged, but did not result in treatment discontinuation unless the patient became unreliable in attending appointments or the clinician felt it represented a safety risk.

HCV treatment was discontinued if requested by the patient, or for severe cytopenias, uncontrolled or worsening psychiatric conditions, or decompensating liver disease. The protocol was evaluated and approved by the Ethical Review Committee, Kansas City, Missouri, USA.

Outcome measures
The primary study endpoint was sustained virological response (SVR), as determined by undetectable levels of HCV RNA on analysis 6 months after the completion of therapy using the Bayer HCV-RNA branched DNA 3.0 assay, with a lower limit of detection of 550 IU/ml.

Patients were classified as sustained virological responders at this time point if they had no detectable virus, or as non-responders if the PCR was positive. End-oftreatment response was defined as undetectable levels of HCV RNA at the completion of therapy. All analyses were performed on an intent-to-treat basis.

Adherence
Adherence to interferon was assessed by the timing of returned empty interferon vials and by a monthly questionnaire that detailed the number of missed doses of medication. Adherence to ribavirin therapy was assessed by pill counting and by query during a monthly questionnaire. Using adherence criteria developed by others, patients were considered adherent to the HCV treatment regimen if they took 80% or more of the prescribed interferon and 80% or more of the prescribed ribavirin for at least 80% of the projected treatment course [9].

Statistical analysis
All data were compiled in and analysed using SPSS version 11.5.0 for Windows (SPSS Inc., Chicago, Illinois, USA). Associations between outcome measures and cannabis use were determined using the Student's t or Wilcoxon signed rank test for categorical variables.

Bivariate analysis of categorical data was performed using the chi-square and Fisher's exact tests. P values less than 0.05 in two-tailed comparisons were considered statistically significant. Logistic regression was used to assess for statistical independence among variables that showed a univariate association with a P value of 0.20 or less.

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Hepatitis C - The Silent Killer Can Medical Cannabis Help?

Jay R. Cavanaugh, PhD

Hepatitis C (HCV) is a blood borne pathogen that presently infects some four to eight million Americans making it the leading blood borne virus in America.

HCV is the primary cause of liver cancer and cirrhosis and kills over 10,000 Americans each year. Hepatitis C is the leading factor in patients who require liver transplant. Some 80% of those who contract HCV will go on to develop chronic infection and 20% of these will develop cirrhosis, liver cancer, or liver failure. A slim 20% of those infected will eliminate the virus from their body on their own.

Patients can contract Hepatitis C from using shared needles, accidental needle stick injuries, blood transfusions (prior to 1990), and to a minor degree from unprotected sex. HCV can be transmitted from an infected tattoo needle, dental instruments, or tools used in commercial nail care. Anything that assists the transfer of HCV infected blood from one person to another can be a vector. The blood of a patient with HCV can be highly contagious and precautions should be taken to not come in contact with it.

Hepatitis C usually produces no early symptoms. The disease can go unrecognized for decades. This is why HCV is termed a "silent killer". During the decades of quiescence the virus can continue to slowly destroy liver cells without the patient having any idea this is happening. The following groups are considered "high risk" and should be tested for the virus:

IV drug users who have shared needles
Sexual partners of HCV patients
Family members of HCV patients
Individuals receiving a blood transfusion prior to 1990
Patients who undergo dialysis
Individuals with tattoos or who have their nails frequently done
Individuals who have suffered a needle stick incident
Patients who have been diagnosed with any liver disease

Symptoms:

While patients are generally unaware of HCV infection for many years, some 80% may eventually develop symptoms which can include:
Fatigue and malaise
Loss of appetite
Weight loss
Jaundice
Joint pain and headache
Fluid retention in the abdomen (ascites)
Nausea and vomiting
Itching

Diagnosis:

Diagnosis begins with a simple history and blood test. The history looks at risk factors and symptoms. The blood test is generally to measure both liver enzymes (produced when the liver is damaged) and to detect antibodies to the Hepatitis C virus and/or to quantify the amount of virus present in the blood stream. The amount of virus is called the "titer" and is determined using the polymerase chain reaction (PCR) with HCV "primers".

Additional blood tests can determine the specific strain of virus present (some strains are more pathogenic than others). A physical examination will be conducted that includes probing the liver for enlargement and looking for other possible factors such as swelling in the legs and feet and jaundice.

Prior to the initiation of treatment, it is common practice to conduct additional tests to more closely ascertain the status of the liver and the need for treatment. A liver scan using radioactive isotopes and X-ray can highlight liver structures and blood flow.

A CAT scan may be conducted to look for tumors or blockage. The definitive diagnostic procedure is a liver biopsy where small samples of the liver are extracted through an office surgical procedure and examined for pathology. A liver biopsy taken from different quadrants of the liver can reveal hepatocarcinoma (liver cancer), fatty deposits, and cirrhosis (scarring).

Why is the liver important?

The liver is the largest internal organ. It is within the liver that medications and toxins are neutralized, metabolized, and eliminated (with the help of the kidneys). The liver is the site of sugar storage (glycogen) and plays a vital role in maintaining normal blood sugar along with the pancreas. The body’s fluid balance and blood clotting are largely controlled by the liver as is the processing of proteins.

Bile produced in the liver aides in the digestion of food. Most noticeable is the role of the liver in energy production which is why fatigue is so common in liver disease.

Treatment for Hepatitis C:

Current therapy focuses on the subcutaneous administration of a combination of Interferon alpha (an immunomodulator) and the anti-viral drug Ribavirin. Depending upon the type of Interferon used, dosing can be one to three times a week for six to 18 months.

Approximately 50% of those treated respond although it is not yet known how long the response might last. Combination drug therapy is usually not attempted when there is no sign of liver damage as determined by histopathology following biopsy.

Drug therapy is also contraindicated when patients have long standing problems with depression or heart disease. The major side effects of the therapy include flu like symptoms, joint pain, nausea, anemia and depression.

The decision to undergo combination therapy is a very serious one and should be done only under the supervision of a qualified and experienced physician. HCV patients with liver impairment must avoid hepatotoxins, particularly alcohol and acetaminophen (Tylenol).

Alcohol is a key toxin that damages the liver. Normal healthy adults are advised to drink no more than two drinks a day for men and one for women. With liver disease that recommendation drops to zero. Yet, many in the general population and the HCV population are heavy drinkers. Cannabis can be an effective harm reduction agent for those with alcohol problems along with therapy and self help.

Long term Complications of Hepatitis C:

Cirrhosis - As liver cells are destroyed by virally induced inflammation, they can be replaced by scar tissue (hence the name) which does not function to conduct normal liver functions. Cirrhosis is chronic and progressive.

Cirrhosis occurs in approximately 20% of all HCV cases and may lead to cancer. The course of cirrhosis is variable but usually includes fluid build up in the abdomen (ascites), portal hypertension, and esophageal varices (swollen blood vessels).

Treatment is limited to alleviating symptoms. Fluid may be periodically drained and medicines provided to reduce hypertension and fluid imbalances. Cirrhosis can cause uncontrolled bleeding, coma, and death.

Hepatocellular carcinoma (liver cancer) - Constant cell death and division caused by HCV can lead to tumors in 1-5% of all patients. Liver cancer is curable only in its earliest stages if it is contained within the liver in an area approachable by surgery. In other cases various treatments can be used including cryotherapy (freezing) and ethanol ablation.

Chemotherapy, at present, is not usually effective with liver tumor. Liver cancer is generally terminal with treatment limited to symptomatic relief and improving the quality and length of life.

Liver failure and transplantation - As HCV destroys liver tissue; liver function can be increasingly compromised leading to failure. As the liver fails toxins can circulate that harm other organs and effect perception and behavior.

Medications are not metabolized normally and have an increased risk of side effects and adverse reaction. Essential clotting factors may not be produced leading to uncontrolled internal bleeding. Complete failure results in coma and death.

Patients with cirrhosis, cancer, and/or liver failure can sometimes be helped by a liver transplant. Hard to come by, transplants are usually restricted to those cases where they may materially help.

Patients who continue to abuse alcohol or drugs are often excluded from transplant waiting lists as are those whose cancer has spread beyond the liver. Others excluded are surgical risks (usually those with cardiac disease) and those with compromised immune systems. Patients survive transplantation in nearly 80% of trials although continued use of immunosuppressants is needed.

Can medical cannabis help?

The short answer is yes. The primary role of cannabis is to stimulate appetite, reduce nausea and vomiting, and treat joint pain. This role is applicable to HCV patients undergoing chemotherapy, those with cancer or cirrhosis, and those with joint pain and headache. Cannabis is far less toxic than other medications that might be prescribed for these conditions and where liver impairment is concerned, it is vital to avoid toxicity.

Cannabis may help alleviate the depression often produced by chronic illness and by combination drug therapy. Additionally, cannabis based food products may provide needed extra nutrition without taxing the liver. Using cannabis in place of alcohol is an established harm reduction technique particularly important when liver disease is present.

Perhaps more important but still unknown is the possibility that some of the chemical components of cannabis (the Cannabinoids) may actually reduce liver inflammation and slow the progression of both cirrhosis and Hepatocellular carcinoma.

The cannabinoids have been shown to be powerful anti-inflammatories and anti-oxidants. They have also been shown to have anti-neoplastic activity, at least in gliomas (a form of brain cancer). Cannabinoids both slow programmed cell death (apoptosis) in normal cells while accelerating apoptosis in cancer cells.

Since cannabis is nontoxic it might as well be tried, particularly in patients who have chronic progressive disease that is likely to result in death. It is important to point out that whole cannabis (whether smoked, vaporized, elixir, or in food products) is preferable to Marinol. The prescription drug Marinol contains only one cannabinoid (THC) and lacks the other healing properties of the whole herb and its extracts.

Dosing is up to the physician and patient. Usually patients "self-titrate" or use only what they feel they need for symptomatic relief. This may be a mistake as the protective effects of cannabis are best achieved with a steady state minimal blood level of Cannabinoids. It is recommended that a base line level of Cannabinoids be maintained with regular doses of oral cannabis products and the smoked or vaporized form of cannabis used for acute symptomatic relief.

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Study: Pot Helps Hepatitis Treatment

Josh Richman, The Oakland Tribune, 14th September 2006

Medical marijuana users are more likely to finish Hepatitis C treatment and so are more likely to be cured, according to a newly published study conducted in San Francisco and Oakland.

Other studies have shown marijuana relieves symptoms, but medical marijuana advocates said this could be the first to show improved cure rates for a life-threatening illness.

The study authored by researchers at the University of California, San Francisco and the Oakland-based Organization to Achieve Solutions in Substance Abuse (OASIS), and published in the European Journal of Gastroenterology and Hepatology found marijuana users being treated for HCV were three times more likely to have a "sustained virological response," meaning the virus can't be detected six months after treatment ends.

HCV treatment with ribavirin and interferon causes severe side effects such as nausea, vomiting, weight loss, sleeplessness and depression, causing many patients to quit the long regimen too early. Of 71 HCV patients studied, 21 finished with a sustained virological response: 12 of the 22 cannabis users and nine of the 49 non-users.

"Modest cannabis use may offer symptomatic and virological benefit to some patients...by helping them maintain adherence to the challenging medication regimen," the study concluded.

Rob Kampia, executive director of the Marijuana Policy Project in Washington, D.C., issued a news release touting this as "a landmark study, showing that medical marijuana can literally save lives.

Every day that our government continues punishing the sick for using this medicine is literally a crime against humanity."

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Attenuation of Experimental Autoimmune Hepatitis by Exogenous and Endogenous Cannabinoids

Author Affiliations

Department of Pathology, Microbiology, and Immunology, School of Medicine, University of South Carolina, Columbia, South Carolina (V.L.H., S.H., M.N., P.S.N.); The Skaggs Institute for Chemical Biology and Departments of Cell Biology and Chemistry, The Scripps Research Institute, La Jolla, California (B.F.C.); and Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina (L.J.H.)

Address correspondence to:
Dr. Prakash S. Nagarkatti, Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, 6439 Garners Ferry Rd., Columbia, SC 29209. E-mail: pnagark@gw.med.sc.edu

Abstract

Immune-mediated liver diseases including autoimmune and viral hepatitis are a major health problem worldwide.

Natural cannabinoids such as Δ⁹-tetrahydrocannabinol (THC) effectively modulate immune cell function, and they have shown therapeutic potential in treating inflammatory diseases.

We investigated the effects of THC in a murine model of concanavalin A (ConA)-induced hepatitis. Intraperitoneal administration of THC after ConA challenge inhibited hepatitis as shown by significant decrease in liver enzymes and reduced liver tissue injury.

Furthermore, THC treatment resulted in significant suppression of crucial inflammatory cytokines in ConA-induced hepatitis. It is noteworthy that THC treatment in ConA-injected mice led to significant increase in absolute number of Forkhead helix transcription factor p3⁺ T regulatory cells in liver. We were surprised to find that select cannabinoid receptor (CB1 or CB2) agonists were not able to block hepatitis either independently or in combination. However, CB1/CB2 mixed agonists were able to efficiently attenuate hepatitis similar to THC.

The modulatory effect of THC in ConA-induced hepatitis was reversed by both CB1 and CB2 antagonists. We also observed that endogenous cannabinoid anandamide was able to reduce hepatitis by suppressing cytokine levels.

In addition, deficiency or inhibition of endocannabinoid hydrolyzing enzyme fatty acid amide hydrolase (FAAH), which leads to increased levels of endogenous cannabinoids, resulted in decreased liver injury upon ConA challenge. Our data demonstrate that targeting cannabinoid receptors using exogenous or endogenous cannabinoids and use of FAAH inhibitors may constitute novel therapeutic modalities to treat immune-mediated liver inflammation.

Footnotes

This study was funded by National Institutes of Health grants R01-DA016545, R01-ES09098, R01-AI053703, R01-AI058300, R01-HL058641, and P01-AT003961 (to P.S.N. and M.N.).
ABBREVIATIONS: AIH, autoimmune hepatitis; NKT, natural killer T; TNF, tumor necrosis factor; IFN, interferon; mAb, monoclonal antibody; THC, δ-9-tetrahydrocannabinol; CB, cannabinoid; AEA, arachidonoylethanolamide (anandamide); ConA, concanavalin A; JWH-133, 1,1-dimethylbutyl-1-deoxy-Δ⁹-tetrahydrocannabinol; foxp3, Forkhead helix transcription factor p3; MAFP, methylarachidonyl fluorophosphate; ACEA, arachidonyl-2′-chloroethylamide; CP55,940, (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol; WIN55212, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone; SR144528, N-[(1S)-endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide); URB532, 4-benzyloxyphenyl-n-butylcarbamate; FAAH, fatty acid amide hydrolase; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; AST, aspartate transaminase; ALT, alanine transaminase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; H&E, hematoxylin and eosin; IL, interleukin; GM-CSF, granulocyte macrophagecolony-stimulating factor; G-CSF, granulocytecolony-stimulating factor; KC, CXC-chemokine; MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell expressed and secreted; MNC, mononuclear cell; PKC, protein kinase C; CS, ConA-activated splenocyte; Treg, regulatory T cell; KO, knockout; WT, wild type; AM251, N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AM630, iodopravadoline.
- Received March 6, 2008.
- Accepted April 2, 2008.

The American Society for Pharmacology and Experimental Therapeutics

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Evaluation of oral cannabinoid-containing medications for the management of interferon and ribavirin-induced anorexia, nausea and weight loss in patients treated for chronic hepatitis C virus

Title
Author(s)	Costiniuk CT, Mills E, Cooper CL
Journal, Volume, Issue	Can J Gastroenterol. 2008 Apr;22(4):376-80.
Major outcome(s)	Improvement of appetite and reduction of nausea and vomiting by nabilone and dronabinol (THC)

Indication	Nausea/vomiting;Appetite loss/weight loss	Abstract
Medication		OBJECTIVES: The systemic and cognitive side effects of hepatitis C virus (HCV) therapy may be incapacitating, necessitating dose reductions or abandonment of therapy. Oral cannabinoid-containing medications (OCs) ameliorate chemotherapy-induced nausea and vomiting, as well as AIDS wasting syndrome. The efficacy of OCs in managing HCV treatment-related side effects is unknown. METHODS: All patients who initiated interferon-ribavirin therapy at The Ottawa Hospital Viral Hepatitis Clinic (Ottawa, Ontario) between August 2003 and January 2007 were identified using a computerized clinical database. The baseline characteristics of OC recipients were compared with those of nonrecipients. The treatment-related side effect response to OC was assessed by c2 analysis. The key therapeutic outcomes related to weight, interferon dose reduction and treatment outcomes were assessed by Student's t test and c2 analysis. RESULTS: Twenty-five of 191 patients (13%) initiated OC use. Recipients had similar characteristics to nonrecipients, aside from prior marijuana smoking history (24% versus 10%, respectively; P=0.04). The median time to OC initiation was seven weeks. The most common indications for initiation of OC were anorexia (72%) and nausea (32%). Sixty-four per cent of all patients who received OC experienced subjective improvement in symptoms. The median weight loss before OC initiation was 4.5 kg. A trend toward greater median weight loss was noted at week 4 in patients eventually initiating OC use (-1.4 kg), compared with those who did not (-1.0 kg). Weight loss stabilized one month after OC initiation (median 0.5 kg additional loss). Interferon dose reductions were rare and did not differ by OC use (8% of OC recipients versus 5% of nonrecipients). The proportions of patients completing a full course of HCV therapy and achieving a sustained virological response were greater in OC recipients. CONCLUSIONS: The present retrospective cohort analysis found that OC use is often effective in managing HCV treatment-related symptoms that contribute to weight loss, and may stabilize weight decline once initiated. All Conditions Benefited by Medical Marijuana Top Home
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Moderate Cannabis Use Associated with Improved Treatment Response in Hepatitis C Patients on Methadone

By Liz Highleyman

Interferon-based therapy for chronic hepatitis C virus (HCV) infection is often limited by side effects including flu-like symptoms, fatigue, insomnia, loss of appetite, nausea, muscle and joint pain, and depression, which can lead to poor adherence, dose reduction, or treatment discontinuation.

Medicinal cannabis may relieve such side effects and help patients stay on treatment, according to a study published in the October 2006 European Journal of Gastroenterology and Hepatology.

Several studies - as well as ample anecdotal evidence - have demonstrated that medical marijuana can reduce nausea, increase appetite, and improve wasting in people with HIV.

Diana Sylvestre, MD, of the University of California at San Francisco and colleagues conducted a study to define the impact of cannabis use during HCV treatment.

The prospective observational study included 71 patients at OASIS (Organization to Achieve Solutions in Substance Abuse), a community-based clinic providing medical and psychiatric treatment to substance users in Oakland, California.

Patient Demographics

Eligible participants were recovering substance users with HCV who had been on methadone maintenance therapy for at least 3 months.

Patients with non-HCV-related liver disease or decompensated cirrhosis were excluded.

Among the 30 patients with liver biopsy results, the mean Metavir inflammation grade was 2.4 and the mean fibrosis stage was 2.6. Subjects with untreated depression were first stabilized on antidepressants.

Use of cannabis during the study was "neither endorsed nor prohibited." About one-third of participants used marijuana during hepatitis C treatment. "Regular" marijuana use was defined as every day or every other day for at least 4 weeks.

Drug and alcohol use were assessed by self-report and random monthly urine testing.

22 patients (31%) reported cannabis use during ant-HCV treatment, while 49 (69%) did not.

Baseline characteristics were generally similar between marijuana users and non-users.

The median age was about 50 years in both groups.

Compared with non-users, cannabis users were somewhat more likely to be male (68% vs 57%) and Caucasian (86% vs 69%), but less likely to have genotype 1 HCV (48% vs 61%).

About 60% of participants reported a previous psychiatric diagnosis (usually depression); cannabis users and non-users had similar rates of psychiatric diagnosis and antidepressant use.

32% of cannabis users and 37% of non-users reported use of other illicit substances during HCV treatment (including heroin, cocaine, and methamphetamine), while 14% and 24%, respectively, reported alcohol consumption; these differences were not statistically significant.

Participants were treated with conventional interferon alfa-2b (3 million units 3 times weekly) plus 1000-1200 mg daily ribavirin. Patients were initially treated for 48 weeks regardless of genotype, but the protocol was later amended to allow 24-week therapy for those with genotypes 2 or 3.

Adherence to therapy was assessed by self-report, ribavirin pill counts, and returned empty interferon vials. Participants were considered adherent if they took 80% or more of prescribed interferon and ribavirin for at least 80% of the projected treatment course.

Results

In an intent-to-treat analysis, 37 patients (52%) achieved an end-of-treatment response (undetectable HCV RNA at the end of 24 or 48 weeks of therapy):

- 14 cannabis users (64%);
- 23 non-users (47%) (P = 0.21).

Overall, 21 out of 71 participants (30%) achieved sustained virological response (SVR), or continued undetectable HCV RNA 6 months after the end of therapy:

- 12 of 22 cannabis users (54%);
- 9 of 49 non-users (18%) (P = 0.009).

Post-treatment virological relapse rates were 14% for cannabis users and 61% for non-users (P = 0.009).

End-of-treatment response rates were similar among occasional cannabis users (10 of 16; 62%) and regular users (4 of 6; 67%).

10 of 16 occasional users (62%) went on to achieve SVR, compared with 2 of 6 regular users (33%), but the difference was not statistically significant.

Most patients (93%) reported at least one treatment-related side-effect, with similar rates among cannabis users and non-users.

Overall, 17 of 71 patients (24%) discontinued therapy early:

- 1 cannabis user (5%);
- 16 cannabis non-users (33%) (P = 0.01).

Overall, 48 patients were adherent (68%):

- 19 cannabis users (86%);
- 29 non-users (59%) (P = 0.03).

There was no significant difference in adherence between occasional and regular cannabis users (87% vs 83%)

91% of cannabis users took at least 80% of prescribed interferon, compared with 76% of non-users. For ribavirin, the corresponding rates were 91% and 84%; these differences were not statistically significant.

However, cannabis users were significantly more likely than non-users to remain on therapy for at least 80% of the projected treatment duration (95% vs 67%; P = 0.01).

The average duration of HCV treatment was 38 weeks for cannabis users, compared with 33 weeks for non-users.

Conclusion

In conclusion, the authors wrote, "Our results suggest that modest cannabis use may offer symptomatic and virological benefit to some patients undergoing HCV treatment by helping them maintain adherence to the challenging medication regimen."

Discussion

In their discussion, the authors wrote that their results "suggest that the use of cannabis during HCV treatment can improve adherence by increasing the duration of time that patients remain on therapy; this translates to reduced rates of post-treatment virological relapse and improved SVR."

"Although other potential mechanisms may contribute to its enhancement of treatment outcomes, such as altered immunological function and improved nutritional status," they added, "it appears that the moderate use of cannabis during HCV treatment does not lead to deleterious consequences."

In this study, it appears that the treatment response benefit was primarily due to improved ability to stay on adequate doses of interferon and/or ribavirin. Sylvestre told HIV and Hepatitis.com that the researchers could not judge whether there was a direct antiviral effect. "It was probably more of a side-effect management effect than an antiviral effect, but we can't rule out the latter," she said.

There remain concerns about the safety of marijuana use by individuals with chronic hepatitis C. Cannabinoid receptors are present on immune cells, and use of the drug may suppress immune function. In addition, there is some evidence that frequent marijuana use may contribute to liver fibrosis. As reported in the July 2005 issue of Hepatology, French researchers found that HCV positive individuals who smoked cannabis daily were more likely to have severe fibrosis and were at higher risk for rapid fibrosis progression than those who used marijuana only occasionally or not at all. However, the participants in that study were not receiving treatment for hepatitis C.

Notably, in the current study, there was no direct dose-response relationship between the amount of cannabis consumed and the likelihood of sustained virological response. In fact, the patients who used the largest amounts of cannabis did not show as much benefit from hepatitis C therapy. The researchers did not perform pre- and post-treatment histological assessments using paired liver biopsies, and did not measure immune parameters.

"The lack of dose response in our study argues against specific receptor or metabolism-related effects, and suggests instead that cannabis exerted its benefit by non-specific improvements in symptom management," the authors stated. "Interestingly, because the benefits of heavy cannabis use were less apparent, we cannot rule out the possibility that detrimental biological or immunological mechanisms may be relevant at higher levels of consumption. Obviously, further study is needed."

Unfortunately, because cannabis is strictly controlled in the U.S. and the federal government considers the drug illegal even in states with medical marijuana laws, it is difficult to conduct randomized, controlled trials.

Editorial

In an accompanying editorial, a group of hepatitis C experts from Canada and Germany noted that people who use illicit drugs are the main risk group for new hepatitis C infections, and "will form the largest HCV treatment population for years to come."

While past treatment guidelines advised against hepatitis C treatment for active substance users and those with a recent history of active use, this categorical recommendation is no longer in effect in the U.S. and Europe, since recent studies have shown that such patients can achieve good treatment outcomes as long as they are able to maintain adequate adherence. Treatment remains a challenge for this population, however, in part because substance users have a higher prevalence of depression and other psychiatric conditions, which are associated with an increased likelihood of neuropsychological side effects during interferon therapy.

Sylvestre's study, the editorial authors wrote, "suggests that cannabis use may benefit treatment retention and outcomes in illicit drug users undergoing HCV treatment" and that "there is substantial evidence that cannabis use may help address key challenges faced by drug users in HCV treatment." Several recent studies have demonstrated the benefits of combining anti-HCV therapy with methadone maintenance, in effect offering "one-stop shopping."

The authors suggested that the therapeutic effects of cannabis "may be of principal importance and benefit for the distinct needs of illicit drug users" on methadone maintenance, because methadone itself is associated with some of the same side effects as interferon (bone aches, loss of energy, depression).

"Overall, cannabis use may thus even offer dual benefits, in facilitating adherence to both methadone maintenance therapy and HCV treatment in the HCV-infected drug user, and thus contribute to public health benefits related to both these interventions," they noted.

"While further research is required on the biological and clinical aspects of the benefits of cannabis use for HCV treatment, and the effectiveness of cannabis use for HCV treatment needs to be explored in larger study populations," they concluded, "we advocate that in the interim existing barriers to cannabis use are removed for drug users undergoing HCV treatment until the conclusive empirical basis for evidence-based guidance is available."

In particular, they suggested that medical marijuana laws and programs that specify its use for patients with specific conditions such as AIDS and cancer should also include people with hepatitis C.

9/15/06

References

D L Sylvestre, B J Clements, Y Malibu. Cannabis use improves retention and virological outcomes in patients treated for hepatitis C. European Journal of Gastroenterology and Hepatology 18(10): 1057-1063. October 2006.

B Fischer, J Reimer, M Firestone, and others. Treatment for hepatitis C virus and cannabis use in illicit drug user patients: implications and questions. European Journal of Gastroenterology and Hepatology 18(10): 1039-1042. October 2006.

C Hezode, F Roudot-Thoraval, S Nguyen, and others. Daily cannabis smoking as a risk factor for progression of fibrosis in chronic hepatitis C. Hepatology 42(1): 63-71. July 2005.

Source

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