Ajulemic acid: potential treatment for chronic inflammation

Abstract Ajulemic acid (AJA, CT‐3, IP‐751, JBT‐101, anabasum) is a first‐in‐class, synthetic, orally active, cannabinoid‐derived drug that preferentially binds to the CB2 receptor and is nonpsychoactive. In preclinical studies, and in Phase 1 and 2 clinical trials, AJA showed a favorable safety, tolerability, and pharmacokinetic profile. It also demonstrated significant efficacy in preclinical models of inflammation and fibrosis. It suppresses tissue scarring and stimulates endogenous eicosanoids that resolve chronic inflammation and fibrosis without causing immunosuppression. AJA is currently being developed for use in 4 separate but related indications including systemic sclerosis (SSc), cystic fibrosis, dermatomyositis (DM), and systemic lupus erythematosus. Phase 2 clinical trials in the first 3 targets demonstrated that it is safe, is a potential treatment for these orphan diseases and appears to be a potent inflammation‐resolving drug with a unique mechanism of action, distinct from the nonsteroidal anti‐inflammatory drug (NSAID), and will be useful for treating a wide range of chronic inflammatory diseases. It may be considered to be a disease‐modifying drug unlike most NSAIDs that only provide symptomatic relief. AJA is currently being evaluated in 24‐month open‐label extension studies in SSc and in skin‐predominant DM. A Phase 3 multicenter trial to demonstrate safety and efficacy in SSc has recently been initiated.

several of its potentially important preclinical actions are now reviewed in some detail.

| BACKGROUND AND HISTORY
The preclinical literature on AJA has been briefly reviewed, 2 however, in this presentation, several selected, as well as newly reported areas, are discussed in more detail. These have been chosen because they suggest therapeutic targets where AJA may be efficacious in the treatment of human disease. Specifically, they are arthritis, fibrosis and metastatic disease. Each of the 3 represent a major under met medical need.

| Rheumatoid arthritis
Joint tissue injury in patients with rheumatoid arthritis (RA) is due in part to activation of T lymphocytes in the synovia; T lymphocytes in synovia of RA patients are resistant to apoptosis.
Oral administration of AJA prevents joint cartilage and bone damage in an experimental model of arthritis in rats. 3 Thus, a potential mechanism whereby AJA prevents joint tissue injury in this animal model might be an enhanced apoptosis of T lymphocytes. Apoptosis of human T cells in vitro was assessed by annexin V expression, caspase-3 activity, DNA fragmentation, and microscopy. 4,5 AJA induced apoptosis of T cells in a dose-and time-dependent manner. Apoptosis preceded loss of cell viability by trypan blue dye exclusion, confirming that cell loss was due to programmed death rather than necrosis. This suggests that a nontoxic compound such as AJA may be a useful therapeutic agent for patients with diseases such as RA that are characterized by T-cell-driven chronic inflammation and tissue injury.
Reduced T-cell proliferation was promoted by the addition of AJA to cells 60 minutes before stimulation with anti-CD3/CD4 monoclonal antibodies. AJA suppressed 5-bromo-2-deoxyuridine (BrdU) incorporation in a concentration-dependent manner; halfmaximal inhibition was achieved with concentrations lower than 1 lmol/L.
Because activation of osteoclasts is essential in the pathogenesis of bone erosion in patients with RA, the influence of AJA on osteoclast differentiation and survival was studied. Osteoclast cultures were established by stimulation of RAW264.7 (RAW) cells, and primary mouse bone marrow cultures, with the receptor activator of NF-kappa B ligand (RANKL). 5 Simultaneous addition of AJA (15 or 30 lmol/L) and RANKL to both culture systems significantly suppressed development of multinucleated osteoclasts (osteoclastogenesis) in a dose-dependent manner, as determined by quantification of multinuclear, tartrate-resistant, acid phosphatase-positive cells. AJA impaired growth of RAW264.7 monocytes and prevented further osteoclast formation in cultures in which osteoclastogenesis had already begun. Reduction by AJA of both monocyte growth and osteoclast formation was associated with apoptosis, assayed by annexin V, propidium iodide staining, and caspase activity. The antiosteoclastogenic effects of AJA did not require the continuous presence of AJA in the cell cultures. Based on these findings, it seems likely that AJA may be a useful drug for diseases such as RA and osteoporosis in which bone resorption is a central feature.

| Multiple sclerosis
In multiple sclerosis (MS), damage to myelin in the central nervous system (CNS) and to the nerve fibers themselves interferes with the transmission of nerve signals between the brain, the spinal cord, and other parts of the body. The loss of myelin promotes several inflammatory processes, which results in the release of soluble factors like cytokines and antibodies that further perpetuates nerve damage. Data were obtained in a mouse model of experimental autoimmune encephalomyelitis (EAE) at doses of 0.1 and 1.0 mg/kg AJA. It was identified as a substance that is able to reduce MS-induced spasticity presumably by its inflammation resolving action on peripheral nerves. 6

| Antifibrotic effects
Bleomycin (BLM)-induced experimental fibrosis in mice was used to assess the antifibrotic effects of AJA in vivo. 7,8 Initially, acute inflammation characterized by neutrophil and macrophage accumulation were the main changes present in lung parenchyma. 8 Between 8 and 14 days after BLM administration, the response progresses from inflammation to fibrosis and involves the bronchi and vasculature.
The fibrotic response in lung tissue at day 21 after BLM treatment was significantly reduced in mice receiving either AJA in the inflammatory or in the early fibrogenic phase. A marked change in the expression pattern of substances implicated in fibrogenesis, such as TGF-b1, pSMAD2/3, CTGF, and a-SMA was observed.

| Cancer and antimetastatic effects
Early observations on inhibition of cell proliferation by AJA (Burstein SH and Zurier RB, unpublished) prompted a study of its possible anticancer actions. 9 AJA was compared with THC as an antineoplastic agent and was found to be nearly equipotent in vitro and more F I G U R E 1 The structure of ajulemic acid. (6aR,10aR)-1-hydroxy-6,6-dimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10a-tetrahydrobenzo[c] chromene-9-carboxylic acid. Molecular formula: C 25 H 36 O 4 . Exact mass: 400.261 g/mol. It is asynthetically modified analog of THC-11oic acid, the principal metabolite of THC. THC, tetrahydrocannabinol effective than THC in vivo. For example, at 14 days following inoculation of glioma cells, tumor diameter was 50% less in AJA-treated mice when compared to vehicle or THC-treated animals. Data obtained with the CB2 receptor antagonist SR144528 suggested that CB2 was involved in the mechanism of the antitumor action.
It has been proposed that inflammation can be promoted by endogenous mediators through Toll-like receptors (TLRs) to enhance tumor progression and metastasis. 10 Many studies have shown a correlation between TLR signaling and tumor progression and metastasis; endogenous mediators, including HMGB1 (high mobility group box 1), have been implicated in the triggering of tumor-associated inflammation. A gene array study showed that AJA significantly reduced the expression of TLR 3, 5, 7, 8, and 9 (Skulas A and Zurier RB, personal communication) suggesting a role for these receptors in the antimetastatic effects of AJA.
Inflammation in the tumor microenvironment that is mediated by IL-1b is believed to have an important role in cancer invasiveness, progression, and metastases. 11 Thus, further support for the anticancer action of AJA comes from the report that it can inhibit the production of IL-1b. 12 An example of such a mechanism comes from a randomized, double-blind, placebo-controlled trial of the IL-1b inhibitor, canakinumab. 13 Thus, it is quite reasonable to suggest that IL-1b inhibitors, such as AJA, could produce a similar therapeutic action without side effects. mice. This may be partly due to the anti-inflammatory properties of AJA and to its effect on several matrix metalloproteinases. 14 Also, the appearance of the primary tumors was different in the drug-treated mice vs. the vehicle-treated mice. The AJA-treated mice had discrete well-defined tumors, whereas the vehicle-treated control mice produced diffuse, poorly defined tumors; the latter is typical of the human clinical situation and is a major problem in the treatment of glial cell brain cancer. This suggests that AJA, with its good safety profile, would be a useful follow up long-term treatment to suppress the development of metastatic disease.  16 In the first series of studies, wild-type (WT) C57BL/6J animals were utilized to evaluate oral dosing, safety and toxicity of AJA. In the second series of studies, a limited number of both WT and CF mice were evaluated for safety, toxicity, and efficacy upon oral dosing AJA. As controls, PA infected WT and CF mice were given the vehicle. The mice were followed daily for clinical score and weights for 10 days. At day 10, animals were euthanized and evaluated for bacterial load (colony forming units [CFUs]), total and differential bronchoalveolar lavage (BAL) white blood cell counts (WBCs). In the first study in WT mice, AJA was well tolerated and more efficient at resolving both inflammation and infection than  The primary efficacy outcome of the Phase 3 study will be the change from baseline at week 52 in modified Rodnan skin scores

| Cystic fibrosis
Cystic fibrosis (CF) is a genetic disorder that affects not only the lungs, but also the pancreas, liver, kidneys, and intestine. CF is inher- Recently a Phase 2, double-blinded, randomized, placebo-controlled multicenter study to evaluate safety, tolerability, and pharmacokinetics of AJA in CF was completed (ClinicalTrials.gov Identifier: NCT02465450) and the topline results have been reported. 18,5,6 This trial tested the safety, tolerability, and pharmacokinetics (PK) of AJA in 85 subjects between 18 and 65 years of age with documented CF. An earlier trial in healthy humans measured PK values and is dis-  Chronic bacterial infection in CF lungs is associated with an excessive inflammatory response, characterized by elevated cytokine levels.

| Dermatomyositis
Dermatomyositis (DM) is a connective tissue disease that is characterized by inflammation of the muscles and the skin. It is a systemic disorder that may also affect the joints, the esophagus, the lungs, and the heart. In the United States, the incidence of DM is estimated at 5.5 cases per million people.
An in vitro study using peripheral blood mononuclear cells (PBMCs) from dermatomyositis patients showed that AJA treatment BURSTEIN | 5 of 10 significantly reduced the production of pathogenic cytokines in a dose-related manner compared with vehicle controls. 22 Specifically, moderate and high AJA concentrations significantly reduced TNF-a secretion; all doses significantly decreased the levels of IFN-a. Cell viability was always greater than 95% following treatment with AJA or vehicle control. These encouraging data supported the initiation of a phase 2 clinical trial.
An NIH supported phase 2 trial of AJA entitled "double-blind, randomized, placebo-controlled study to investigate the safety, tolerability, and efficacy in subjects with dermatomyositis" is currently

| Lack of ulcerogenicity
The most significant unwanted side effect of many anti-inflammatory agents is the formation of gastrointestinal ulcers. For this reason, AJA was carefully examined to detect any possible ulcerogenicity. When given acutely to rats in doses up to 1000 mg/kg, no evidence for ulcer formation was seen. Chronic intragastric administration of up to 30 mg/kg likewise resulted in no ulcerogenicity, whereas the positive control indomethacin-treated rats showed extensive ulcer formation. 23 AJA has a very favorable therapeutic index for ulcerogenicity vs inflammation when compared to several NSAIDs (Table 1).

| Toxicity
Because of its structural similarity to THC, AJA was studied for possible induction of opiate like physical dependence in a 14-day rat study. 24 None of the typical opiate withdrawal effects such as writhing, diarrhea, wet dog shakes, etc., were observed indicating that AJA has a low dependence liability. There were no effects on renal, cardiovascular, or gastrointestinal function, and no signs of respiratory depression as well. Lethal doses were estimated following single doses in mice (600 mg/kg) and in rats (400 mg/kg). AJA was well tolerated at doses up to 50 mg/kg. Three different standard tests for mutagenic potential gave negative findings.

| Cannabinoid receptors
In recent years, the discovery of CB2-specific agonists has been an important goal in the area of therapeutics. CB2 is a member of the G-protein-coupled receptor (GPCR) superfamily and its pharmacology has been reviewed in some detail. 25 CB2 receptors are primarily expressed in the periphery, including cells involved in immune system activities. Although CB2 has been detected in the CNS, its actions do not include mood alteration or behavioral responses.
Receptor binding studies using highly purified preparations of AJA showed~12-fold selectivity for CB2 over CB1 receptors, whereas preparations described in previous reports showed much higher activity at CB1 relative to CB2 ( Table 2). The highly purified preparation of AJA stimulated release of PGJ in a CB2-dependent manner and produced minimal catalepsy or hypothermia, effects primarily mediated by CB1 receptors. 2 In contrast, AJA preparations described in earlier reports had higher CB1 binding activity and caused concomitantly greater catalepsy and hypothermia, 2 suggesting possible contamination by impurities such as 11-hydroxy-DMH-THC, an intermediate in the synthesis of AJA.

| Drug distribution
The structure of AJA suggests an inability to cross the blood-brain barrier in humans due to its polarity and molecular weight. Evidence from distribution studies in rats is in agreement with this possibility. 26 Therefore, in addition to its selectivity for CB2 over CB1, the poor brain uptake of AJA will further favor its actions toward activating CB2 receptors on immune cells in the periphery, as opposed to stimulating CB1 receptors in the CNS.

| Preclinical mechanism studies
The major precursor for all of the eicosanoids is free arachidonic acid (AA) whose release from phospholipid storage sites is robustly stimulated by AJA ( Figure 3). 27  The ability of AJA to increase the production of the proresolving eicosanoid PGJ has been confirmed experimentally; application of AJA to HL60 cells produced a robust stimulation of PGJ. 27 The AJAinduced increase in PGJ was effectively inhibited by the CB2

Resolution of inflammation
Unregulated CB2 F I G U R E 4 In vitro CB2-initiated pathways for the synthesis of the proresolving eicosanoids PGJ 2 and LXA 4 . All of the steps are tightly regulated except for the conversion of PGD 2 to PGJ 2 , which proceeds in the absence of enzymic mediation. PGD 2 , prostaglandin D 2 ; LXA 4 , lipoxin A 4 antagonist SR144528 (1 lmol/L), whereas the CB1 antagonist SR141716 (10 lmol/L) had only a small effect, suggesting a CB2 receptor-mediated mechanism of action. 1,27 A second eicosanoid, lipoxin A 4 (LXA 4 ), has also been reported to contribute to the inflammation resolving action of AJA. 29 In a different branch of the arachidonic acid cascade, LXA 4 production results from the sequential actions of 5-and 15-lipoxygenases (LOX) following the release of free arachidonic acid (Figure 4). 29 It was further shown that the 12/15 LOX inhibitor baicalein reduces this action of AJA. LXA 4 is an important member of the emerging class of molecules known as specialized proresolving mediators (SPMs) that promote the resolution of chronic inflammation. 30 Following trauma or tissue injury, SPMs play pivotal roles in vascular responses and leukocyte trafficking, from initiation to resolution.
Organ fibrosis can be an ultimate result of unresolved chronic inflammation.

| Mechanism studies in humans
To further explore its mechanism of action, and establish a pharma- AJA was studied in the above model and preliminary data were reported by Gilroy et al 9 Subjects were divided into 3 groups of 5, each receiving 5 mg or 20 mg BID AJA, or placebo, all of which were given before the procedure. Both doses of AJA caused a 70% reduction in inflammation based on a decrease in neutrophil infiltration and decreased blood flow around the site of inflammation. In addition, the treatment resulted in a progressive increase in blood flow around the site of inflammation during the early phases of resolution indicative of an efficient inflammatory response followed by a timely resolution 9 .

| SUMMARY AND CONCLUSION S
Thus far, the available preclinical (Table 3) and clinical data ( Table 4) suggest that AJA could provide a safe and effective treatment for diseases that are characterized by chronic inflammation and eventually result in fibrosis, loss of function, etc. Data from in vitro experiments suggest that the CB2 receptor is an important mediator and that AJA can act as an inflammation-resolving agent through its stimulatory action on the production of the proresolving agents PGJ and LXA 4 . Its ability to lower IL-1b inhibitor a factor in some of its actions. Based on its structural overlap with AA, it has been suggested that AJA should be referred to as an arachidomimetic agent. 1 Therapeutic targets for AJA such as scleroderma, CF, dermatomyositis, lupus, arthritis, MS, and metastatic cancer are possibilities based on the current body of data. Its lack of adverse side effects makes it especially suitable for chronic administration. Finally, a long-standing goal in the cannabinoid field has been the separation of therapeutic actions of THC from its psychoactivity. AJA appears to represent, in a large measure, such an achievement of this goal.

Observed action
Model Reference