Cancer, Inflammation, and Therapy: Effects on ...

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Cancer, Inflammation, and Therapy: Effects on Cytochrome P450–Mediated Drug Metabolism and Implications for Novel Immunotherapeutic Agents R Donald Harvey1 and ET Morgan2 Immune system activation through innate and adaptive systemic mechanisms is critical for protection from pathogens and other antigens. However, uncontrolled systemic inflammation may occur as a consequence of acute and chronic conditions and has multiple clinically relevant effects. Inflammation and cancer are fundamentally linked during development, invasion, and metastasis, yet, paradoxically, many cancers evade immune system detection. Components of cancer inflammation include chemokines, prostaglandins, and cytokines, and these have been shown to downregulate cytochrome P450 (CYP) enzyme activity. Recently, promising novel anticancer agents that upregulate immune responses have entered into clinical practice and have shown high response rates. These agents, either alone or in combinations, may cause systemic immune-related adverse events, with potential clinical implications for use of concurrent agents metabolized by CYP and other pathways. In this article, the authors focus on what is known about inflammation, cancer, and CYP-mediated drug metabolism; discuss clinical and pharmacologic data regarding novel immunomodulators; and consider their potential interactions with concurrent agents. BACKGROUND

Inflammation and immune responses are critical endogenous mechanisms of prevention and treatment of a variety of insults and diseases, including cancer. Interactions among the immune system, the invading pathogen or antigen, and the host are complex communication networks intended to protect through specific, layered, and adaptable responses. The innate immune system is nonspecific and designed to immediately respond to acute challenges, such as bacteria or other invaders, whereas the adaptive system is focused on individualized and refined long-term recognition and response. Systemic activation of the innate immune system involves a number of critical components, including mast cells, complement, neutrophils, natural killer cells, and monocytes (circulation)/macrophages (tissuebound). Upon activation, these and other immune cells produce cytokines, chemokines, eicosanoids, and growth factors, all designed to augment local and systemic responses to pathogens and antigens through vasodilation, additional cell recruitment, and inducing fever. As the immediate stimulus is controlled or

abates, these responses are quickly diminished or downregulated through regulation of monocyte/macrophage cell fate.1 Importantly, the innate system serves as a critical bridge to the adaptive immune system rather than as a stand-alone response to pathogens, as was previously believed.2 The adaptive immune system theoretically has a limitless ability to catalog antigen responses through memory. Cells of the adaptive system include dendritic cells and T- and B-lymphocytes, with multiple subsets within each group. T cells control T- and B-lymphocyte function, thus regulating cell-mediated immunity, primarily following antigen-presenting cell (including dendritic cells and monocytes/macrophages) initiation. Categorization of T cells may be done based on CD domains by flow cytometry (e.g., CD3, CD4, CD8) and/or based on their roles (e.g., helper, cytotoxic, and regulatory). The innate and adaptive systems have multiple interfaces and enhance the functions of one another, with one example being pattern recognition receptors. These receptors recognize and are activated by molecular profiles generated from invading

1Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, Georgia, USA; 2Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, USA. Correspondence: ET Morgan ([email protected])

Received 29 May 2014; accepted 25 June 2014; advance online publication 13 August 2014. doi:10.1038/clpt.2014.143 CLINICAL PHARMACOLOGY & THERAPEUTICS

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pathogens and subsequent tissue damage; they are teleologically focused on microorganisms and carbohydrate components. The group of transmembrane pattern recognition receptors called the Toll-like receptor family is found on cells associated with both innate (monocytes/macrophages, neutrophils) and adaptive (dendritic cells) responses.3–5 These receptors are one example of the complex interplay between acute and chronic immune responses, and offer additional potential targets for therapies for a variety of immune-mediated diseases. Regulation and homeostasis of the innate and adaptive immune systems are dependent on pro- and anti-inflammatory signals released from constitutive components and, in particular, cytokines. Cytokines are small glycoproteins produced primarily by macrophages, B- and T-lymphocytes, and mast cells, but fibroblasts and endothelial cells may also generate them (Table 1). They are categorized as interleukins (ILs), interferons (INFs), and those in the tumor necrosis factor (TNF) family, as well as by specific functional names (e.g., colony-stimulating factors and vascular endothelial growth factor). Once released, cytokines may act locally or systemically, depending on adjacent effector cells and receptors and proximity to vascular beds. Paracrine functions are also observed, as T helper (Th) cell subtype differentiation may be influenced by a predominance of local cytokine(s); for example, IL-4 and IL-33 in high concentrations preferentially drive the production of Th2 cell subsets.6 Cytokines initially amplify an immune response (e.g., IL-6), through recruitment of other cells in a positive feedback loop, then accelerate a dampening effect through other cytokines (e.g., transforming growth factor-β, IL-10, IL-35), leading to deactivation of selected immune cells.7,8

The complexity of immune system regulation leads to both challenges and opportunities in understanding and treating disease. Acute and chronic conditions associated with immune system upregulation may have off-target systemic complications and effects as a result of inflammation. The inability to balance measured inflammatory response and surveillance with more sustained inflammation creates a dysregulated environment for cancer and other diseases associated with chronic immune dysregulation, such as atherogenesis, rheumatoid arthritis, psoriasis, and systemic lupus erythematosus, to develop. This background of immune system dysfunction, its causes and systemic effects, and methods for intervention have led to recent advances in the understanding of cancer biology and treatment, with implications for clinical pharmacology. CANCER AND INFLAMMATION

Cancer has been linked with inflammation at all stages of the disease—risk of development, initiation, invasion, metastasis, and mortality (Figure 1).9 There are clear links between certain immune-mediated diseases and cancer; for example, inflammatory bowel disease is associated with increased risk of colorectal cancer, and chronic inflammation due to infectious agents such as Helicobacter pylori is associated with increased cancer risk. A recently published epidemiologic study showed an association between chronic psoriasis and multiple cancers, regardless of antipsoriatic therapy, with the highest rates seen in lymphomas and skin cancers.10 Cancer development in the setting of increased inflammation has been linked to other chronic conditions, such as obesity and hepatocellular carcinoma, with proinflammatory signals originating from adipose tissue and mediated by macrophages and cytokines.11,12 The interaction

Table 1 Origin and effects of cytokines that may affect drug metabolism Cytokine IL-1

Cell(s) of origin Macrophages

Effect in inflammation and cancer

Effect on CYP metabolism

Proinflammatory; increases angiogenesis signaling

Downregulates CYP2C8 and 3A4; no effect on CYP2B6, CYP2C9, CYP2C18, or CYP2C19

Dendritic cells Keratinocytes IL-2

T cells (Th1, some CD8+)

T-cell expansion, growth

Decreased activity of CYP1A2, CYP2C, CYP2E1, and CYP3A4

IL-3

Th1 and Th2 cells, CTLs

Growth factor for hematopoietic progenitor cells

None reported

IL-4

T cells

Promotes T-cell growth; epithelial transition and metastasis

Downregulates CYP1A2 and CYP2C; upregulates CYP2E1

IL-6

Macrophages

STAT3 activation; stimulates myeloma growth; induces acute-phase protein secretion

Downregulates CYP1A2, CYP2C19, and CYP3A4

Dendritic cells IL-10

Th2, Tregs

Inhibits cytokine release, Th1 cells

Decreased activity of CYP3A; no changes in CYP1A2, CYP2C9, or CYP2D6

IL-23

Macrophages

STAT3 and NF-κB activation

None reported

INF-γ

Th1, CTLs

Inhibits Th2 cells; activates macrophages

Downregulates CYP1A2, CYP2A6, and CYP3A4

TNF-α

Macrophages

Induces changes in vascular endothelium

Decreased activity of CYP2C19

Inhibits macrophage activation and T-cell growth

Downregulates CYP1A1 and CYP1A2

Dendritic cells TGF-β

CD4+ T cells

CD, cluster domain; CTL, cytotoxic T lymphocyte; CYP, cytochrome P450; IL, interleukin; INF, interferon; NF-κB, nuclear factor-κ light-chain enhancer of activated B cells; STAT, signal transducer and activator of transcription; TGF, transforming growth factor; Th, T helper; TNF, tumor necrosis factor; Treg, T regulatory cell. Adapted from refs. 23,71,72.

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Therapy-induced inflammation Tumor reemergence Resistance to therapy Antigen presentation Cancer cell killing

Inflammation caused by environmental and dietary exposure

Mutations Genomic instability Tumor promotion Angiogenesis

Tumor development

Mutations Genomic instability Tumor promotion Angiogenesis

Chronic inflammation Infection Autoimmunity

Tumor growth/survival Genomic instability Angiogenesis Immunosuppression Metastasis Cancer cell killing

Tumor-associated inflammation

Figure 1 Cancer and inflammation. Reprinted with permission from ref. 9.

between immune system constituents and cancer growth is most relevant in the tumor microenvironment, where cells, including macrophages, mast cells, neutrophils, and lymphocytes, have been observed to be in tumor stroma since the original observation by Rudolf Virchow in 1863.13 The tumor microenvironment is critical to cancer expansion and invasion, and the immune system is in a local–systemic flux of activation and suppression as a response to the presence of mutated cells. An initial procancer inflammatory response consisting of cytokines produced by tumor cells acts to increase transcription factors, including nuclear factor-κB light-chain enhancer of activated B cells and signal transducer and activator of transcription 3, as well as to induce epigenetic changes and initiate angiogenesis.9 These and other procancer immune signals are opposed by antigenpresenting cell–mediated anticancer immune responses, including cytotoxic T lymphocytes, natural killer cells, and Th1 cells. Greater intratumoral numbers of these cells are associated with improved outcomes in a number of solid and hematological cancers.14,15 Some T-cell subsets, however, are also associated with tumor promotion, including interferon-γ-producing Th1, Th2, and Th17 populations. Adding to the heterogeneity is the differing immune cell populations and proportions across cancer types, making some more susceptible to immune intervention (e.g., melanoma, renal cell carcinoma) as compared with others (e.g., sarcomas).9 In the end, cancer cell survival depends on the immune balance tipping in favor of angiogenesis, survival signaling, and evasion from immune-directed surveillance. Markers of inflammation have been shown to be prognostic in patients with cancer, and prior data showing a reduced risk of cancer in patients receiving nonsteroidal anti-inflammatory drugs suggests a pivotal role of inflammation in the proliferation CLINICAL PHARMACOLOGY & THERAPEUTICS

of malignant cells.16,17 Chemoprevention trials in patients with high risk for developing colorectal cancer utilizing nonsteroidal anti-inflammatory drugs, including sulindac, aspirin, and others, have yielded mixed results, however, leading the US Preventive Task Force to recommend against their routine use in the population (http://www.uspreventiveservicestaskforce. org/uspstf/uspsasco.htm).18,19 In an epidemiologic study of more than 2,400 elderly (70–79 years of age at entry) people, circulating levels of IL-6, C-reactive protein (CRP), and TNF-α were assessed for their relationship with cancer development and outcomes.20 Elevations of a 1-unit increase on a log scale in each marker were associated with a higher risk of death from cancer, but not consistently with cancer development, with the notable exceptions of lung cancer (elevations in all three predicted higher rates) and colorectal cancer (elevations in only IL-6 and CRP predicted higher rates). The use of nonsteroidal anti-inflammatory drugs in the group was also unequally associated with reductions in each of the three measures, suggesting additional interventions are necessary to reduce cancer risks. INFLAMMATION AND DRUG METABOLISM

Systemic inflammation due to acute and chronic disease is known to have effects on hepatic cytochrome P450 (CYP)mediated drug clearance and extrahepatic metabolism.21 Bacterial sepsis as an acute model of inflammation has offered insight into the complex mechanisms of immune-mediated CYP suppression.22,23 Reduced hepatic CYP activity and expression are mainly the result of transcriptional suppression but may also involve, in part, posttranslational protein modification, caused by mediators produced as a consequence of inflammation and infection. These mediators include multiple proinflammatory 3

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Inhibition of CYP3A4 mRNA by IL-6

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CYP3A4 mRNA (fold change from control)

cytokines (IL-6, INF-γ, TNF-α, and IL-1β), eicosanoids, and histamine, which also regulate acute-phase proteins (e.g., CRP). The proinflammatory cytokines IL-6, INF-γ, TNF-α, and IL-1β are generally recognized as the most potent mediators of reduced CYP activity and expression. CYP-derived eicosanoids are modulated in inflammation in cancer24,25; however, the roles of eicosanoids in alterations of drug metabolism are not known. In general, CYP expression and activity are impaired in the setting of high concentrations of proinflammatory cytokines.22 Animal and preclinical human hepatocyte models of cytokine-induced changes in CYP activity have been described since the 1980s.26,27 The proinflammatory cytokines IL-6, TNFα, and interferon-γ have consistently shown suppression of CYP expression and activity in human hepatocyte cultures.28–30 The translation to patients has been challenging, and the ability to predict changes following immune stimulation has been limited, possibly due to high cytokine concentrations used and an unclear physiologically relevant concentration range. Likewise, the overall effect of multiple pro- and anti-inflammatory cytokine changes in models has not been fully assessed, which is a more likely physiologic scenario than alterations in just one cytokine in the complex setting of acute and chronic inflammation. Of the proinflammatory cytokines and CYP isozymes investigated, the concentration–inhibition relationship between IL-6 and CYP3A4 appears to be the strongest (Figure 2), and the link between increased IL-6 and reduced CYP3A-family proteins (CYP3A4 in humans) has been demonstrated in animal models and humans.31–34 Proof of principle for clinical relevance of the link between IL-6 and CYP3A4 activity can be found in drug interaction studies of tocilizumab, a monoclonal antibody directed against the IL-6 receptor and approved for use in rheumatoid arthritis.26,35 Simvastatin exposures were used as a marker of CYP3A4 alteration and were compared after a single 40-mg p.o. dose prior to and 1 and 5 weeks following a single dose of tocilizumab (10 mg/kg) in 12 patients with rheumatoid arthritis. A doubling of simvastatin clearance with a 400% decrease in area under the curve was seen at 1 week, but these trended toward pretreatment values at 5 weeks, showing that IL-6 inhibition improves CYP3A4 activity. A concordant reduction in mean CRP from 4–5 to ~0.3 mg/dl was also observed, and the baseline mean IL-6 concentration was 50 pg/ml across the population, increasing fivefold 1 day following tocilizumab, consistent with its mechanism of action. By contrast, no pharmacokinetic changes were seen with tocilizumab and omeprazole (a CYP2C19 substrate) or dextromethorphan (a CYP2D6 substrate). These data suggest a critical relationship among an inflammation-driven disease, IL-6, and CYP3A4 activity. Relationships between other cytokines and CYP activity in humans is less clear. Infusion of high doses of the therapeutic proinflammatory cytokine interferon-α-2b reduced CYP1A2 and CYP2C19 by only 60 and 40%, respectively.36 Approved monoclonal antibodies targeting TNF-α include etanercept, infliximab, adalimumab, certolizumab, and golimumab. None of these agents has been formally investigated in patients for potential interactions with CYP substrates, although a case report has been published suggesting that adalimumab

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Figure 2 CYP3A4 activity as a function of IL-6 concentration. Representative data from an evaluation of suppression of human hepatocyte CYP3A4 expression and activity by IL-6 in a single pharmaceutical company laboratory by assessment of testosterone conversion to 6-β-hydroxytestosterone. CYP, cytochrome P450; IL, interleukin. Reprinted with permission from ref. 26.

reduced duloxetine clearance.37 The effect of therapeutic doses of TNF-α active agents on CYP-mediated clearance in patients with inflammatory diseases remains unanswered. However, infliximab was shown to reverse CYP3A downregulation in a rat model of arthritis.38 A recent review of cytokine agents used in psoriasis concluded that, due to few reports of clinically relevant interactions in the literature, biologics have low potential for drug interactions; however, the sole victim agent in the trials reviewed was methotrexate, which is a substrate for multiple transporters, but its clearance is not mediated by CYP.39 The cytokines targeted by ustekinumab, IL-12 and IL-23, did not affect CYP expression in cultured hepatocytes, suggesting that it has little potential for affecting CYP–mediated drug clearance.40 DRUG CLEARANCE IN CANCER PATIENTS

Cancer-related inflammation has gained attention in recent years for its biologic and therapeutic implications. Since initial observations of spontaneous remissions in renal cell cancer and melanoma, understanding and modulating the immune system for anticancer benefit has driven the development and www.nature.com/cpt

STATE approval of improved therapies such as ipilimumab, allogeneic stem cell transplantation, and the therapeutic cancer vaccine sipuleucel-T.41,42 With these approaches comes a need to understand implications of systemic effects of inflammation, both as a result of a diagnosis of cancer and from iatrogenic causes. The differential effect of cancer on drug metabolism has been studied for two specific isozymes, CYP2C9 and CYP3A4.32,43 In a small study, as compared with those without cancer, CYP2C9 activity measured by tolbutamide clearance was not different in cancer patients; however, mean IL-6 and TNF-α values were 4.8- and 17.4-fold higher, respectively, in those with cancer.43 CYP3A4 is responsible for the clearance of multiple oral and intravenous anticancer agents, such as docetaxel, vinorelbine, vemurafenib, and erlotinib. Reduced CYP3A4 activity in cancer is linked to inflammation-induced changes in CYP3A4 gene expression, with a concurrent rise in IL-6 concentrations.44 Cancer patients with reduced CYP3A4 activity have elevations in acute-phase reactants, including α-1 acid glycoprotein and CRP, and this reduced CYP3A4 activity is associated with relatively modest increases (≥10 mg/l) in CRP, potentially providing a biomarker to select patients at risk for impaired clearance and adverse events.45,46 Impaired CYP activity has clear importance when considering concurrent anticancer agents but likewise has implications for other comedications with narrow therapeutic indexes, for example, warfarin and amiodarone, and that are cleared by these enzymes. As alluded to previously, a potential biomarker or set of biomarkers for patients at risk for inflammation-related changes in CYP activity remains undefined. Candidates include CRP, serum IL-6, and other cytokines, and potentially, ratios in endogenous metabolic markers of CYP activity.47 It is clear that preclinical models by themselves are ineffective at predicting the likelihood of an interaction among disease, intervention, and substrate. A call for additional measurement and interpretation of biomarkers in phase II and III studies of therapeutic proteins known to affect cytokine concentrations has been made.26 Any prospective model would need to account for the effect of the intervention on inflammation measures as well as potential for harm, taking into consideration both perpetrator (disease, immunotherapeutic agent) and victim (CYP substrate and therapeutic index). NOVEL IMMUNOMODULATORS IN CANCER TREATMENT

The treatment of cancer has evolved rapidly over the past decade. Greater understanding of the origins of cancer cells, proliferative signals, self-sufficiency mechanisms, and intrinsic and acquired resistance to therapies has led to the greatest number of first-in-class approvals for cancer in the history of drug development. Targeting the so-called hallmarks of cancer, including tissue invasion and metastasis, sustained angiogenesis, evasion of apoptosis, and limitless replicative potential, has produced new targets and agents.48 However, the most promising hallmark to target for treatment is arguably the ability of tumors to evade immune surveillance. Numerous preclinical evaluations with existing and potential therapies have demonstrated deep and sustained anticancer responses when the immune system is utilized to induce and maintain a response.49–52 Immunotherapy CLINICAL PHARMACOLOGY & THERAPEUTICS

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has been an integral part of cancer therapy since the development and introduction of IL-2 for renal cell cancer and melanoma in the early 1980s. The effect of IL-2 on CYP-mediated drug metabolism provides an example of immune-based therapies and subsequent CYP inhibition. In human hepatocytes cocultured with Kupffer cells (macrophages within the liver-based reticuloendothelial system), a 50–75% concentration-dependent reduction of CYP3A activity is seen at 72 h, a clinically relevant observation for cancer treatment.53 In addition, these mechanistic studies showed circulating mononuclear cells that are taken up and that infiltrate liver parenchyma are, in part, responsible, adding complexity to the understanding of how immune therapies cause reductions in CYP3A activity. Patients receiving high-dose IL-2 therapy show marked reduction in CYP1A2, CYP2C, CYP2E1, and CYP3A4 activity in phenotypic probe studies, with clear implications for concurrent anticancer and other coadministered medications.54 Therapeutically, although durable responses are seen with IL-2, only 10–15% of advanced melanoma patients benefit, and toxicities are substantial. Newer agents offer more directed therapy to enhance T-cell function for the immediate and long-term regression and surveillance of cancer cells. Immune checkpoint inhibitors are a new class of agents designed to remove negative regulatory steps in the recognition of antigen-presenting cells by T cells and their receptors. In the presence of these checkpoints, T cells remain quiescent. The two checkpoints recently exploited for drug development are the cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) and the programmed cell death protein-1 (PD-1). A number of immune checkpoint inhibitors are currently in development across multiple cancer types (Table 2). CTLA-4 is a glycoprotein on T cells and secretory granules that, when activated, downregulates adaptive immune responses. 55 Ipilimumab, approved in 2011 for the treatment of advanced melanoma, is a monoclonal antibody directed against CTLA4, which, upon binding, blocks the interaction of CTLA-4 with its ligands, CD80 and CD86, resulting in a disinhibition of T-cell activation and proliferation. In metastatic melanoma, ipilimumab showed unprecedented response rates, with more than 33% of patients experiencing improvement in overall survival, and 5-year survival rates of up to 25% across clinical trials.56,57 A consequence of upregulated T-cell activity is, unfortunately, unregulated T-cell activity. Adverse events with ipilimumab include autoimmune, inflammatory damage to organs historically seen with donor T cells in the setting of graft-vs.-host disease in allogeneic stem cell transplantation. These immune-related adverse events (irAEs) most commonly include enterocolitis, endocrinopathies, dermatitis, and hepatitis. Of these, one might consider hepatitis to be a potential indicator of likelihood of immune-mediated changes in CYP activity. At doses of 3 and 10 mg/kg, grade 3 or 4 elevations in hepatic transaminases and/or bilirubin occurred in 0–0.8% and 2–9% of patients treated, respectively, suggesting a dose-dependent relationship.56,58,59 Tremelimumab, another CTLA-4 monoclonal antibody, also caused grade 3 or 4 hepatotoxicity in 1% of patients in a phase III trial. However, it 5

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Table 2 Immune checkpoint inhibitors in clinical development in solid-tumor malignancies Immune target CTLA-4

PD-1

Agent

Type of antibody

Ipilimumab

Humanized IgG1

Phase III prostate cancer, phase II multiple (pancreatic, ovarian, gastric, and cervical)

Stage of development

Tremelimumab

Fully human IgG2

Phase I and II multiple (melanoma, NSCLC, and renal cell cancer)

Nivolumab

Fully human IgG4

Phase III multiple (NSCLC, melanoma, renal cell cancer, and head and neck cancer)

Pembrolizumab

Humanized IgG4

Phase III melanoma, NSCLC Phase I and II multiple (renal cell cancer, breast cancer)

PD-L1

Pidilizumab

Humanized IgG1

Phase II multiple (pancreatic cancer, colorectal cancer, melanoma, breast cancer, renal cell cancer, and prostate cancer), Phase I and II glioma

BMS-936559

Fully human IgG4

Phase I

MEDI4736

Engineered human IgG1

Phase III NSCLC

MPDL3280A

Engineered human IgG1

Phase III NSCLC, phase II multiple tumors (bladder and renal cell cancers)

CTL, cytotoxic T lymphocyte; IgG, immunoglobulin G; NSCLC, non–small cell lung cancer; PD-1, programmed cell death protein-1; PD-L1, programmed cell death protein ligand-1.

failed to show improvements in overall survival as compared with conventional chemotherapy.60 When severe irAEs occur, prompt initiation of corticosteroids is generally effective at reversing toxicities and preventing morbidity. Patients who experience irAEs with ipilimumab may be more likely to have durable responses,61 making mechanistic investigations in drug metabolism important for clinical management. Inhibition of the interaction between the PD-1 and its ligands, PD-L1 and PD-L2, has also shown benefit in a number of cancers. PD-1 functions as a critical inhibitory checkpoint for activated T cells and resides in peripheral tissues to prevent damage by dampening immune responses.62 It is expressed by innate B and T cells, natural killer cells, dendritic cells, and activated monocytes, and is induced by multiple cell types as a response to inflammation. Cancer cells can upregulate the primary ligand, PD-L1, to effectively silence T cells that would otherwise attack the tumor. To date, the promise of PD-1 antagonism is greatest in non–small cell lung cancer and melanoma, and phase II/ III trials of multiple agents are ongoing.62,63 Nivolumab, pembrolizumab, and pidilizumab all target either the PD-1/CD80 or PD-1/PD-L1 interaction, whereas BMS-936559, MEDI4736, and MPDL3280A block binding of the ligand PD-L1 to PD-1 and CD80. Production of TNF-α, IL-6, and INF-γ by immune cells is increased following anti-CTLA-4 and anti-PD-1 treatment.64,65 These cytokines are potentially responsible for additional T-cell recruitment, leading to disease response and irAEs. Baseline reductions in CYP function due to cancer have substantial clinical implications independently; however, the use of agents such as ipilimumab and anti-PD-1 antibodies has potential to further impair CYP-mediated metabolism in the setting of cancer-related inflammation. Because improved response rates are seen following treatment with novel immune-modifying agents, the potential for patients to return to a normal CYP phenotype has implications for dosing of concurrent and subsequent therapies. 6

IMMUNE STIMULATION WITH NOVEL IMMUNOTHERAPEUTICS AND DRUG–DRUG INTERACTIONS

The addition of immunotherapy to conventional cytotoxic chemotherapy (e.g., carboplatin and paclitaxel) and smallmolecule inhibitors (e.g., vemurafenib) is a rational approach to improve tumor regression rates and maintain response through differing mechanisms. When combined, an integral part of therapeutic success is continuing to optimize exposure to each agent without untoward toxicity. Initial data with combinations of new immunomodulating agents with small-molecule drugs and/or conventional cytotoxic chemotherapy have yielded either mixed or incomplete clinical results when considering the potential effect of adverse events due to CYP-mediated drug interactions. Data combining ipilimumab with conventional chemotherapy, including dacarbazine (a CYP1A2 substrate) and carboplatin (renally cleared) and paclitaxel (a substrate of both CYP2C8 and CYP3A4), have been published.66–70 A phase I, randomized pharmacokinetic trial comparing ipilimumab 10 mg/ kg alone or with either dacarbazine or carboplatin and paclitaxel in advanced melanoma demonstrated no difference in dacarbazine or paclitaxel exposure alone or in combination with ipilimumab.70 Rates of increased aspartate aminotransferase and alanine aminotransferase were higher with combination dacarbazine (52.6 and 47.4%, respectively) and carboplatin/ paclitaxel (both 25%) than with ipilimumab alone (15%), and response rates were either equivalent to single-agent ipilimumab (29.4%, dacarbazine: 27.8%) or, interestingly, inferior (carboplatin/paclitaxel: 11%). In a separate trial, immune-mediated hepatitis with ipilimumab 10 mg/kg and dacarbazine occurred in 31.6% of patients as compared with 6% of those treated with dacarbazine alone and was increased by more than threefold as compared with treatment with ipilimumab alone.58,69 Similarly, grade 3 or 4 transaminase elevations were observed in 10% of patients in a single-arm study in advanced melanoma of ipilimumab 10 mg/kg with fotemustine, with grade 3 or 4 neutropenia and thrombocytopenia seen in 19 and 24% of patients, www.nature.com/cpt

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Healthy patient Disease

Cancer patient proinflammatory cytokines P450 Drug clearance

Plasma concentration

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Time

T cells Proinflammatory cytokines P450 Drug clearance

Plasma concentration

Immunotherapy

Time Reduced tumor burden

Proinflammatory cytokines P450 Drug clearance

Plasma concentration

respectively, rates significantly higher than those seen with fotemustine with dacarbazine (4 and 5%, respectively) in a similar population.71,72 These data suggest an additive effect on hepatotoxicity and cytopenias when ipilimumab is combined with dacarbazine or fotemustine. The combination of ipilimumab 10 mg/kg with carboplatin and paclitaxel has shown mixed results in hepatotoxicity and hematologic toxicity. In a randomized phase II trial in non– small cell lung cancer, no difference was observed in hepatotoxicity or hematologic adverse events with chemotherapy alone as compared with the combination.67 However, in a separate trial in small-cell lung cancer, grade 3 or 4 elevations in transaminases were seen in 13% of those treated with the combination vs. none in those treated with chemotherapy alone.68 Grade 3 or 4 neutropenia rates were 8 and 2%, respectively, but thrombocytopenia was not different between treatments. The rates of grade 3 or 4 irAEs in each of the carboplatin/paclitaxel and concurrent ipilimumab studies were 20 and 21%, respectively. These observations, overall, do not support an immune-mediated CYP interaction with ipilimumab and CYP substrates; however, the differences in hematologic and hepatotoxicity adverse events warrant additional mechanistic investigation. Moreover, the question of whether CYP-mediated clearance begins to normalize in parallel with tumor regression has not been addressed. The effects of agents targeting PD-1/PD-L1 are also unknown. A phase I investigation of the combination of ipilimumab with the V600E mutation–directed BRAF inhibitor vemurafenib was stopped early due to dose-limiting hepatotoxicity at the initial dose level.73 Both agents have hepatotoxicity as an adverse event; however, rarely does it limit therapy with either. Interestingly, vemurafenib is primarily metabolized by CYP3A4 and inhibits a number of CYP isozymes; however, no pharmacokinetic data are available from the combination trial to assess potential alterations in exposure following ipilimumab dosing. Combinations of novel immunotherapeutics have also been performed, due to preclinical synergy and nonredundancy seen with CTLA-4 and PD-1 antagonists. A phase I trial of ipilimumab and nivolumab in patients with advanced melanoma provided initial information on efficacy and adverse events associated with the combination.74 A total of 53 patients with advanced melanoma were enrolled across dose levels of ipilimumab ranging from 1 to 3 mg/kg and of nivolumab ranging from 0.3 to 3 mg/kg. The maximum tolerated doses were ipilimumab 3 mg/kg with nivolumab 1 mg/kg, with dose-limiting toxicities occurring in 6 of 28 (21%) patients and 53% of patients experiencing grade 3 or 4 toxicities while on treatment. Elevations in lipase, aspartate aminotransferase, and alanine aminotransferase were seen in 13, 13, and 11% of patients, respectively. Response rates paralleled toxicities, with 53% of patients experiencing objective reduction in tumor volumes, an unprecedented response in the disease. At 12 weeks, 16 of 53 patients (30%) had reductions of 80% or more in their tumor volumes. Although no correlative studies of systemic inflammation (e.g., cytokine concentrations and CRP levels) were reported, the increased rates of disease response and irAEs suggest a profound increase in immune activation, much more

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Figure 3 Hypothetical pattern of changing drug clearance in the course of cancer development and successful treatment. The healthy patient is stabilized on a regular regimen of a small-molecule comedication (e.g., a statin). Upon development of cancer, cytochrome P450 (CYP)-mediated clearance of the drug is reduced due to the inflammatory cytokines, resulting in higher steady-state drug concentrations. Treatment with an immunotherapeutic agent stimulates T-cell activity, initially resulting in further CYP suppression and higher drug concentrations. As the tumor is successfully treated, however, the attendant inflammation is also reduced, resulting in a return of drug clearance and plasma levels toward the values in the healthy patient. This scenario assumes that no adjustments are made to the dosage of the small-molecule drug.

than with either class of agents alone, with potential implications for CYP metabolism. Further studies of immune correlates and potential drug–disease–drug interaction studies are anticipated. For novel agents early in development, the 2012 regulatory guidance from the US Food and Drug Administration for assessment of potential drug–drug interactions includes information on therapeutic proteins (http://www.fda.gov/downloads/Drugs/ GuidanceComplianceRegulatoryInformation/Guidances/ UCM292362.pdf). Cytokines or agents known to modulate cytokines should be evaluated in in vivo studies with relevant CYP substrates, particularly when used in combination with small molecules, and most importantly, with small molecules with narrow therapeutic indexes. FUTURE IMPLICATIONS OF NOVEL IMMUNOTHERAPEUTICS AND CYP-MEDIATED METABOLISM

As clinical trial data evolve with new immune strategies, a number of scenarios with direct consequences for patients and drug metabolism emerge. Therapies that expand T-cell numbers 7

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and function, including additional vaccines, chimeric antigen receptors, and bispecific T-cell engager antibodies, and continued development of T cell–active antibodies (e.g., natural killer cell activators) are in various stages of development. As noted above, facilitated T-cell function may initially tend to downregulate drug metabolism. Because cancer patients have reduced CYP activity as a result of the disease, the potential exists for reversion to a normal metabolism state if and when cancer burden is substantially reduced (partial remission) or eradicated (complete remission). This potential invokes the need for investigation of CYP activity in patients across the treatment time course (Figure 3). Tools to define those at risk for alterations in CYP function may help to identify and refine investigations, and may include biomarkers such as changes in circulating lymphocyte count, CRP, or cytokines of interest (e.g., IL-6) before, during, and following therapy with novel immunotherapeutics. Likewise, if a cancer response is obtained, maintenance treatment strategies with immune therapies and/or other anticancer agents may be used, necessitating an understanding of potential exposure changes to concurrently administered medications. Cancer patients frequently have multiple comorbidities, such as diabetes, hyperlipidemia, arrhythmias, hypertension, and depression, that require treatment with agents that are hepatically metabolized and may have narrow therapeutic indexes, requiring vigilance and caution in their clinical management as we incorporate agents and approaches that may lead to alterations in disposition of both anticancer and chronically administered oral drugs for comorbidities. The main drawback to mechanistic studies in individual patients or groups is the difficulty in performing metabolism studies of CYP activity. A personalized approach, however, may be possible, particularly if further applications of endogenous markers of CYP metabolism continue to be refined.47,75 ACKNOWLEDGMENT The support of a Georgia Research Alliance Distinguished Cancer Scientist award to E.T.M. is gratefully acknowledged.

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CONFLICT OF INTEREST The authors declared no conflict of interest.

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