REVIEW

Understanding Disease–Drug Interactions in Cancer Patients: Implications for Dosing Within the Therapeutic Window DE Coutant1, P Kulanthaivel1, PK Turner2, RL Bell2, J Baldwin2, SR Wijayawardana3, C Pitou2 and SD Hall1 The human inflammatory response can result in the alteration of drug clearance through effects on metabolizing enzymes or transporters. In this article we briefly review the theory of how cancer can lead to indirect changes in drug metabolism, review acute phase proteins and cytokines as markers of changes in cytochrome P450 (CYP) activity in cancer patients, and provide clinical case examples of how the inflammation in advanced cancer patients can lead to altered CYP-mediated drug clearance.

Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? þ There is widespread and growing understanding that disease state (e.g., inflammation) can lead to altered drug enzyme and drug transporter activity. However, the clinical magnitude of the effect of cancer on CYP-mediated drug clearance is not well established to this point. Further, clinical pharmacokinetic and inflammatory biomarker data are lacking as to how to translate this knowledge into clinical practice, in particular for cancer patients  WHAT QUESTIONS HAS THIS STUDY ADDRESSED? þ What does new clinical data teach us about the magnitude of the effect of advanced cancer on drug enzyme activity? Are there current reliable surrogate markers for inflammatory downregulation of drug clearance in cancer patients?  WHAT HAS THIS STUDY ADDED TO OUR KNOWLEDGE? þ This article illustrates the clinical effect of advanced cancer on drug exposure in several case examples.  HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY AND THERAPEUTICS? þ Clinicians will better understand when monitoring of patient response is advised.

It is established that certain inflammatory conditions can increase cytokine production leading to downregulation of cytochrome P450 (CYP) enzymes, conjugative enzymes, or transporters.1,2 Disease-induced changes in drug clearance can result in increased variability in drug exposure and increase the challenge to the prescribing physician to predictably dose patients within the therapeutic window. Dose adjustment or drug exposure monitoring may be justified in patients with pronounced inflammatory response,2,3 particularly in patients taking narrow therapeutic index (NTI) drugs. Therefore, a broader understanding of the mechanisms by which inflammatory disease can affect drug exposure is needed. Recently, Harvey and Morgan published a review of the effects of cancer on CYP-mediated drug metabolism.2 This article continues on that theme by providing supplemental investigation into the putative use of inflammatory markers as markers of altered drug clearance, and by providing new clinical data on the effects of cancer on CYPmediated drug clearance.

THEORETICAL BASIS FOR HOW CYTOKINES MODULATE ENZYME- AND TRANSPORTER-MEDIATED CLEARANCE

Decreased hepatic expression of drug-metabolizing enzymes and transporters due to inflammation and infection is a combination of transcriptional regulation involving various cytokines, enzyme destabilization by nitric oxide, oxidative stress, and phosphorylation.1,2 The most established effects of proinflammatory cytokines are the downregulation of CYPs by interferon-g (IFN-g), interleukin-1b (IL-1b), IL-6, and tumor necrosis factor-a (TNFa).2 There are also non- or antiinflammatory cytokines such as IL-10 and transforming growth factor-b that are responsible for the downregulation of the immune response, and that can decrease levels of multiple cytokines. Due to the complexities of transcriptional regulation and the interplay of various cytokines, the subsets of CYP enzymes modulated by each particular cytokine are different. For instance, TNF-a downregulates CYP2C19, whereas IL-6 downregulates CYP3A4, CYP2C19, and CYP1A2.2

1

Department of Drug Disposition, Eli Lilly and Company, Indianapolis, Indiana, USA; 2Pharmacokinetics and Pharmacodynamics, Eli Lilly and Company, Indianapolis, Indiana, USA; 3Department of Statistics-Oncology, Eli Lilly and Company, Indianapolis, Indiana, USA. Correspondence: DE Coutant (cout@lilly. com) Received 7 January 2015; accepted 19 March 2015; advance online publication 25 March 2015. doi:10.1002/cpt.128

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VOLUME 98 NUMBER 1 | JULY 2015 | www.wileyonlinelibrary/cpt

REVIEW Effects of inflammation on CYP expression and activity

To date, inflammatory disease state effects on CYPs through cytokine modulation have been of greater clinical magnitude and have been more clearly elucidated than inflammatory effects on either transporters or UDP-glucuronyltransferases (UGTs),1,4 In cancer patients, it appears that IL-6, TNF-a, IL-1b, and IFN-g are the key proinflammatory cytokines that mediate the inflammatory response.2,5,6 A more detailed discussion on the mechanisms by which inflammation alters hepatic CYP-mediated drug clearance can be found in reviews by Morgan et al.1,2,7 We wish to add, however, that intestinal CYPs are also downregulated in response to inflammation.8–10 In one murine model of infection (due to toxoplasma infection), total CYP and CYP3A contents fell in the liver (239% and 249%, respectively) and intestine (243% and 248%), as did the CYP3A towards the marker substrate erythromycin (236% in liver and 258% in intestine).8 In another mouse study, in which IFN-g was injected intravenously, western blot analysis of microsomal CYP3A showed a 22% reduction in the liver, a 29% reduction in upper intestine, and 55% reduction in lower intestine.9 Currently, the degree of intestinal downregulation is difficult to predict, as the pathways of CYP3A4 gene regulation appear to have some differences in the intestine compared to the liver.11 Effects of inflammation on conjugative enzyme expression and activity

There is little information available concerning the effects of inflammation on sulfation, acetylation, methylation, amino acid conjugation, and glucuronidation.12 Investigations into the effects of inflammation on the gene expression of certain UGTs in primary rat hepatocytes as well as in vitro experiments in human cancer cells that have suggested that certain mRNA and protein levels (such as UGT1A1, UGT1A6, UGT2B3, and UGT2B17) are downregulated by increased cytokine levels (such as IL-1a and IL-6).13–15 Therefore, for narrow therapeutic index drugs that are primary substrates for UGTs (such as morphine and zidovudine) and some glucuronidated anticancer agents (such as irinotecan and etoposide) it is reasonable to expect that clinically meaningful changes in therapeutic drug levels could occur, due to downregulation of UGT expression that in turn might cause parent drug and/or active drug metabolite exposures to drift outside the therapeutic window. There are limited data of cancer-induced UGT enzyme downregulation. In one clinical study, it was reported that in normal large bowel mucosa the UGT proteins were always present, whereas their expression was reduced substantially in low-grade adenomas and absent in high-grade adenomas and colon cancer.16 These data suggest that a disease state such as cancer can downregulate intestinal UGT expression and that the magnitude of UGT downregulation is influenced by the severity of the inflammation. Effects of inflammation on transporter expression and activity

In a recent review article, Cressman et al. stated that multiple proinflammatory cytokines (such as IL-1b, IL-6, TNF-a, and IFN-g) modulate transporter expression in inflammatory disease CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 98 NUMBER 1 | JULY 2015

states, and that the modulation of transporter expression and activity can lead to changes in the pharmacokinetics and pharmacodynamics of clinically important therapeutics.4 Changes in transporter expression have been clinically observed in patients with inflammatory bowel disease (IBD), such as ulcerative colitis or Crohn’s disease.4 When compared to normal controls, patients with IBD showed significant decreases in levels of P-glycoprotein and breast cancer-resistant protein transporters in the actively inflamed jejunum and ileum that negatively correlated with circulating levels of IL-1b, IL-6, and CRP.17 Significant changes in the mRNA expression of organic anion transporting polypeptides and organic cation transporters were observed in resected terminal ileum and colon samples from IBD patients.18 In this same study, disease state (Crohn’s disease vs. ulcerative colitis) and the degree of inflammation appeared to be a key determinant of the magnitude of change of mRNA expression of these transporters.18 Although clinical data are lacking with regard to examining the effects of cancer on transporter expression, there is preclinical data that showed in the livers of tumor-bearing mice4 significant downregulation of multiple transporters was seen in correlation with an increase in IL-6. The authors have concluded that the suppression of hepatic drug transporters “. . .could contribute to variable drug responses and potentially cytotoxicity in cancer patients.” MARKERS OF DECREASED DRUG CLEARANCE (I.E., ACUTE PHASE REACTANTS AND CYTOKINES) IN CANCER AND OTHER INFLAMMATORY DISEASES

Several markers (acute phase reactants and cytokines) have been studied as measures of inflammation, including the acute phase proteins C-reactive protein (CRP) and alpha-1-acid glycoprotein (AAG). Albumin is not an acute phase protein, but its circulating concentrations can change in response to inflammation. Other surrogate markers of inflammation and/or prognostic markers of disease outcome include the cytokines IL-6 and TNF-a. CRP as surrogate marker of decreased CYP-mediated drug clearance

While there have been several publications that have studied CRP as a prognostic measure of disease outcome,6,19,20 such as in cancer or in certain cardiovascular diseases, there have been far fewer investigations as to whether increases in CRP (as a result of inflammation, infection, or surgery) can be used as an indirect surrogate marker for decreased CYP enzyme activity. In gastric cancer patients,19 CRP levels (median 6 standard deviation) were 0.8 mg/L in nonmetastatic cancer patients (n 5 109), but increased to 4.1 mg/L (n 5 6) in metastatic cancer patients, and CRP increases were positively correlated with increases in serum IL-6. When cancer patients in another study (n 5 40) were divided into those with (CRP > 10 mg/L) and without an acute phase response (CRP < 10 mg/L), cancer patients with an acute phase response had an average 30% reduction in CYP3A drug metabolism as measured by the erythromycin breath test.21 Among the cytokines, CRP levels appear to be modulated predominantly by IL-6.22 77

REVIEW AAG as surrogate marker of decreased CYP-mediated drug clearance

It is important to note that increases in AAG can serve as a marker for altered drug clearance, insofar as inflammation can increase AAG in concert with increases in various cytokines, which in turn lower CYP gene expression and activity. Lowered CYP activity can in turn lead to higher drug exposure of sensitive CYP substrates and an increased risk of toxicity with narrow therapeutic index (NTI) drugs (such as for cyclosporine or warfarin). In the past, it was mistakenly thought that increased toxicity would occur through altered drug free fraction via the displacement of one drug from plasma proteins by another drug or by reduced protein concentration. However, Benet et al. and Smith et al. have established that clinically meaningful changes in unbound drug exposure do not occur except in very rare cases, such as in cases of NTI drugs that have a high extraction ratio and are given parentally (such as intravenous lidocaine), or in NTI oral drugs that are eliminated by nonhepatic high extraction ratio routes.23,24 For these reasons, there is no clinical relevance to increases in protein binding due to higher total AAG in cancer patients. Total (bound and unbound) concentrations of drugs increase as plasma protein binding increases and unbound drug fraction decreases. However, because unbound drug concentrations are driven by intrinsic clearance, increases in AAG due to cancer or inflammatory disease will not alter unbound drug exposure, and are not expected to influence clinical outcome.23 It is also important to remember that changes in total drug exposure (total AUC) are dependent on the acid–base properties of the drug. For a basic drug that binds preferentially to AAG, the total AUC may increase as the drug unbound fraction decreases. However, a more acidic drug that binds predominantly to AAG may not show a resultant change in total AUC, as it may be that decreased binding to albumin is offset by an increase in binding to AAG. In some instances, therefore, changes in protein binding may confound the overall assessment of whether enzyme activity is decreased in an inflammatory disease state. In cancer patients, increases in AAG have coincided with indirect changes in CYP-mediated drug clearance that have led to increased incidence of drug-related toxicity. In 2006, Charles et al. examined the toxicity of 40 mg/m2 docetaxel administered intravenously once-weekly in a mixed population of advanced cancer patients.25 Docetaxel is an NTI anticancer agent whose clearance is predominately CYP3A4-mediated. Charles et al. found that the odds of severe hematological toxicity were  9fold higher for patients with reduced docetaxel clearance, and the odds of severe nonhematological toxicity were about 3-fold higher for patients with elevated levels of inflammatory markers AAG or CRP (AAG > 1.5 g/L, CRP > 10 mg/L).25 The higher risk of nonhematological toxicity associated with elevated AAG was also observed in cancer patients treated thrice-weekly with docetaxel.26 We postulate that in these studies increases in AAG levels reflected a greater inflammatory response (e.g. greater severity of disease) in certain cancer patients, which in turn led to greater suppression of CYP activity, higher docetaxel concentrations, and increased toxicity. 78

Figure 1 Peak circulating levels (mean 6 SEM) of IL-6 or TNF-a in response to fever or various moderate-to-severe inflammatory states.6,27,32–51 The IL-6 and TNF-a concentrations in cancer patients and in healthy subjects were obtained by compiling data from multiple references and calculating pooled standard deviations taking into account the different number of subjects, n, between trials.

Albumin as surrogate marker of decreased CYP-mediated drug clearance

Decreases in serum albumin levels are a sign of a bad prognosis in many inflammatory diseases. There is evidence that the presence of advanced cancer decreases albumin levels to  37 to 38 g/L and to  35 g/L in cancer patients with cachexia.25,27 This is in contrast to healthy subjects whose albumin levels are typically in the 40–50 g/L range.25 Monitoring of albumin in conjunction with CRP has recently shown to be a good prediction for survival and outcome in cancer patients.28 In particular, the modified Glasgow Prognostic Score estimates systemic inflammation response through a selective combination of CRP and albumin. The modified Glasgow Prognostic Score then “bins” the degree of inflammation as to whether an individual CRP score is greater or less than 10 mg/L and whether albumin is greater or less than 35 g/L. Despite the recent interest in albumin as a prognostic tool, there is inconclusive clinical evidence to date to determine whether albumin levels might be a good surrogate marker of decreased drug clearance in cases of acute inflammation. Cytokines as surrogate markers of decreased CYP-mediated drug clearance

Proinflammatory cytokines such as IL-6, IL-1b, and TNF-a have been shown to be primary inhibitors of hepatic CYP expression in a variety of inflammatory disease states, leading to reduced drug metabolism and clearance.1,2,7,29–31 Figure 1 illustrates the mean (6 standard error of the mean [SEM]) peak circulating IL6 and TNF-a concentration of several inflammatory disease states compared to levels in healthy subjects. The mean 6 standard deviation (SD) values for circulating IL-6 was 4.5 6 10.8 pg/mL in healthy subjects and 28.8 6 60.6 pg/mL in VOLUME 98 NUMBER 1 | JULY 2015 | www.wileyonlinelibrary/cpt

REVIEW advanced cancer patients, while circulating TNF-a levels were 13.5 6 10.9 pg/mL in healthy subjects and 16.7 6 31.8 pg/mL in cancer patients. The IL-6 and TNF-a levels in other disease states are less definitively determined due to scarcity of data. In some disease states, the magnitude of cytokine upregulation correlated with the severity of disease. For instance, in one study mean TNF-a concentrations were much higher in patients classified with severe coronary heart failure than in patients with moderate coronary heart failure.48 In Figure 1, the IL-6 to TNF-a ratio is seen to vary dramatically among inflammatory disease states. CLINICAL LITERATURE EXAMPLES ILLUSTRATING HOW CANCER CAN ALTER EXPRESSION AND ACTIVITY OF CYP DRUG METABOLIZING ENZYMES

“. . .Much of the inter-patient variability in the clearance of anticancer drugs can be attributed to differences in the levels of drugmetabolizing enzymes, especially CYP3A4. . .it is important to elucidate the impact of one other source of variability in hepatic drug metabolism, namely cancer-induced inflammation, as it is present in at least 60% of patients with advanced cancer. . .”.1 This statement was one of the conclusions made by symposium members brought together in 2007 to discuss the clinical consequences and underlying mechanisms of cytochrome P450 and drug transporter regulation brought about by inflammation and infection.1 The symposium members concluded it would be beneficial to have more clinical data wherein investigators measured CYP activity in addition to collecting concentration data from a wide array of acute phase reactants. There are data from one study where the erythromycin breath test (EBT) was used to measure CYP3A activity in an in-patient setting in 32 healthy volunteers and 29 patients with cancer.29 Increases in circulating CRP were highly correlated (P < 0.0001) with decreases in CYP3A activity as measured by EBT. Further, a significant (P < 0.001) correlation was observed between decreases in CYP3A activity and the severity of toxicity observed in cancer patients who received the CYP3A substrates docetaxel or vinorelbine.29 In related work, plasma inflammatory proteins CRP, AAG, and IL-6 levels were inversely correlated with reduced hepatic CYP3A4-mediated drug clearance, as assessed by the EBT.21,25 In patients with metastatic and nonmetastatic prostate cancer, mean 6 SEM circulating IL-6 concentrations were high in patients with prostate cancer metastatic to bone (47.4 6 21.3 pg/ mL, n 5 20), but IL-6 concentrations in the nonmetastatic prostate cancer patients (2.5 6 1.6 pg/mL, n 5 19) did not separate from healthy control values (1.9 6 0.6 pg/mL, n 5 44).46 This is an example of how within a particular disease state, there may be significant variability in the degree of inflammation observed. In 6 of 10 terminally ill cancer patients with cachexia, sharp increases in circulating IL-6 levels (>100 pg/mL) were observed in the week prior to death.52 The changes in circulating IL-6 levels in these cancer patients appeared to be affected by the degree of inflammation.52 In contrast to CYP3A4 activity, CYP2C9 activity has not been shown to appreciably decrease in cancer patients.53 In a CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 98 NUMBER 1 | JULY 2015

Figure 2 NONMEM code and compartmental model.

comparison of cancer patients (n 5 10) to healthy subjects (n 5 10) who were given tolbutamide (a CYP2C9 substrate), the mean apparent oral clearance in cancer patients was 19.5 6 10.5 mL/min vs. 15.8 6 5.0 mL/min in healthy subjects. In summary, there is a growing body of literature that indicates that cancer-mediated inflammation can lead to altered drug disposition in cancer patients. We now present additional case examples of altered drug clearance in cancer patients. PART II: CASE EXAMPLES OF EFFECTS OF CANCER ON CYP-MEDIATED DRUG CLEARANCE

In all of the following case studies, the studies were designed and executed under the direction of Eli Lilly and Co (Indianapolis, IN). The Institutional Review Boards for the study sites approved the protocols, which were developed in accordance with the ethical guidelines of Good Clinical Practice and the Declaration of Helsinki. CASE STUDY I (META-ANALYSIS OF MIDAZOLAM CLEARANCE IN CANCER PATIENTS AND IN HEALTHY SUBJECTS)

Although cancer is associated with changes in inflammatory markers (i.e., CRP, albumin, AAG) and certain cytokines, the effects of cancer on the pharmacokinetics (PK) of midazolam, a model CYP3A substrate, are not completely understood. Unanticipated reductions in CYP3A activity caused by inflammatory disease states such as cancer can lead to increased PK variability in drugs metabolized through the CYP3A pathway. A population PK model (Figure 2), which was derived from the model described by Chien et al.,54 was developed using NONMEM 7 (ICON Development Solutions, Ellicott City, MD) to describe midazolam PK in cancer patients and healthy subjects, with the primary objective of quantifying the disease state effect on intrinsic midazolam clearance, particularly when other potentially explanatory covariates were accounted for in the model. A midazolam PK database was assembled using baseline data from drug–drug interaction (DDI) studies conducted in cancer 79

REVIEW Table 1 Case Study I: baseline demographics of cancer and healthy patients utilized in building of NONMEM model Baseline demographics (unit)

N

Mean

Median

Range

Healthy

205

35.6

33

18 – 64

Cancer

54

62.5

62

33 – 84

Healthy

205

78.7

77.1

47.7 – 129.8

Cancer

54

81.0

77.8

48.5 – 123

Healthy

185

43.6

43

36 – 52

Cancer

54

35.1

36

23 – 44

Healthy

185

0.93

0.93

0.59 – 1.50

Cancer

54

0.89

0.90

0.38 – 1.53

Healthy

185

121

118

65.2 – 222

Cancer

54

97.4

90.1

47.7 – 234

C-reactive protein (mg/L)

Cancer

18

44.5

19.3

0.79 – 179

a-1 acid glycoprotein (mg/dL)

Cancer

21

134

123

65 – 307

Gender

Healthy

Male 5 167; Female 5 38

Cancer

Male 5 27; Female 5 27

Healthy

Caucasian 5 96; Black 5 41; Asian 5 56; Hispanic 5 1; Other 5 3; Unknown 5 8

Cancer

Caucasian 5 50; Black 5 4

Healthy

0.04mg 5 8; 0.2mg 5 99; 1.2mg 5 8; 2.5mg5 12; 5mg 5 76; 7.5mg 5 20; 15mg 5 34

Cancer

0.2mg 5 5; 1.2mg 5 10; 2.5mg 518; 5mg 5 21

Age (years)

Total body weight (kg)

Albumin (g/L)

Serum creatinine (mg/dL)

Estimated creatinine clearance (mL/min)

Race

Doses administered

Of the 54 cancer patients in whom midazolam PK was obtained, 11 patients had colorectal/anal cancer, 6 had lung cancer, 6 had pancreatic/gastrointestinal cancer, 5 had renal/genitourinary cancer, 5 had breast cancer, 5 had acute myeloid leukemia, 5 had prostate cancer, and 4 had skin cancer/melanomas. There were 9 patients with other types of advanced cancer.

patients (n 5 4 Eli Lilly and Co. (Lilly) trials, n 5 54 cancer patients) and from healthy subjects (n 5 11 Lilly trials, n 5 205 healthy subjects). Demographic and clinical laboratory data evaluated in this modeling exercise are summarized in Table 1. Ideally, the distribution of covariates across the healthy and cancer subpopulations would be balanced in order to clearly assess the disease state effect. Two potentially important covariates, age and albumin concentration, were not well balanced and therefore contribute to collinearity (correlation r 5 0.61) in the overall population. Despite this relatively high correlation, age and albumin levels were not considered redundant and were evaluated together as separate covariates on intrinsic midazolam clearance (CLint) and peripheral compartment apparent volume (Vp). Additional covariates assessed include body weight to describe allometric relationships with compartmental parameters and a categorical fed/fasted covariate was added to absorption rate (ka) due to midazolam being administered during a fed state in one of the healthy volunteer drug–drug interaction studies. Serum creatinine was evaluated on CLint due to possible association with disease state and gender was evaluated on Vp due to prior midazolam modeling findings.54 Population estimates for all the structural, covariate, and random variance parameters during PK model development are summarized in Table 2. Disease state (i.e., the presence of cancer as a 80

covariate) was the main predictor of midazolam exposure even with other potentially explanatory covariates present in the model. Across the range of cancer types studied, the presence of cancer would be expected to decrease intrinsic midazolam clearance by an average of 28% (90% confidence interval (CI), 17 to 39%). When assuming a midazolam-free fraction in plasma of 0.024 and a blood to plasma partitioning ratio of 0.86,55,56 the PK population estimates of intrinsic clearance correspond to an apparent plasma clearance of 65.5 L/h in a 70 kg healthy subject vs. 46.4 L/h in a 70 kg cancer patient. In other words, the apparent midazolam plasma clearance is 1.4-fold higher in healthy subjects than in cancer patients. Figure 3 illustrates the difference in midazolam concentration–time profiles between healthy subjects and cancer patients observed in Lilly’s drug–drug interaction trial database. CRP data were available from a single cohort of 18 cancer patients and AAG data from a separate cohort of 21 cancer patients. Due to the limited data available, CRP and AAG were not included as covariates. Secondary exploratory analyses were performed to relate CRP and AAG serum concentration to the remaining unexplained variability in CLint. This analysis did not reveal any conclusive evidence that either CRP or AAG independently predict midazolam intrinsic clearance (data not shown). However, we conclude that it would be beneficial to measure CRP and AAG in future VOLUME 98 NUMBER 1 | JULY 2015 | www.wileyonlinelibrary/cpt

REVIEW Table 2 Case Study I: NONMEM model parameter summary Parameter estimate (%SEE) Pharmacokinetic parameter (unit)

Base model

Full model

95% CI

Absorption rate constant – solution/syrup ka (h-1)

2.36 (5.85)

2.15 (5.81)

1.91 – 2.40

—

20.740 (4.34)

20.803 – 20.679

1.46 (29.7)

0.591 – 2.30

0.211 (40.9)

0.249 (40.2)

0.053 – 0.444

0.192 (17.0)

0.178 (21.9)

0.104 – 0.253

926 (3.81)

1060 (3.04)

993 – 1120

Effect of cancer (categorical)

—

20.275 (23.5)

20.405 – 20.149

Effect of age (linear)

—

20.00578 (38.4)

20.00156 – 20.0100

Effect of serum creatinine (power)

—

0.284 (45.1)

0.0321 – 0.537

Effect of albumin (linear)

—

0.00431 (187)

20.0118 – 0.0200

59.1 (3.10)

60.9 (2.89)

57.4 – 64.3

—

0.617 (25.1)

0.319 – 0.931

63.0 (4.08)

57.8 (3.34)

54.1 – 61.4

Effect of body weight (power)

—

2.13 (9.91)

1.71 – 2.54

Effect of age (power)

—

0.0111 (20.4)

0.00668 – 0.0155

0.0116 (53.9)

20.000556 – 0.0235

—

0.709 (16.1)

0.486 – 0.934

16.1 (4.60)

16.8 (4.60)

15.3 – 18.3

—

0.946 (28.8)

0.408 – 1.47

Effect of food (categorical) Effect of cancer (categorical) Duration of zero-order absorption –tablet D2 (h) Absorption lag time – tablet Lag (h) Intrinsic metabolic clearance Clint (L/hr)

Central volume of distribution Vc (L) Effect of body weight (power) Peripheral volume of distribution Vp (L)

Effect of albumin (linear) Effect of female gender (categorical) Intercompartmental clearance Q (L/h) Effect of body weight (power) Intersubject variability x2 – ka

80.2% CV (14.6)

54.5% CV (20.6)

0.155 – 0.362

2

38.2% CV (14.9)

30.3% CV (15.9)

0.0607 – 0.115

2

29.9% CV (22.8)

25.6% CV (27.6)

0.0296 – 0.0977

2

47.2% CV (19.6)

22.2% CV (25.9)

0.0240 – 0.0717

2

27.7% CV (37.8)

25.4% CV (37.4)

0.0173 – 0.108

11.9% CV (35.2)

12.3% CV (34.9)

0.00509 – 0.0253

50.3% CV (2.56)

50.3% CV (2.56)

0.241 – 0.266

x – Clint x – Vc x – Vp x –Q Within-subject variability x2 – Clint Residual error r2 – Proportional

CYP3A drug–drug interaction studies to better assess their potential to improve the prediction capability of the population PK model. CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 98 NUMBER 1 | JULY 2015

The population PK modeling results of midazolam PK in cancer patients and healthy subjects provide the most direct clinical evidence to date that CYP3A metabolism is impacted in cancer 81

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Figure 3 Case Study 1. Observed dose-normalized midazolam blood concentration data in healthy subjects (D) and in cancer patients (䊊) compared to NONMEM-model predicted midazolam concentrations. Concentration–time course data were analyzed with nonlinear mixed effects modeling software, NONMEM 7.2 (ICON Development Solutions, Ellicott City, MD) using the first-order conditional estimation method with interaction (FOCE-INT). A wellstirred liver model with allometric-scaled hepatic blood flow was used to develop expressions for first-pass hepatic extraction and systemic clearance. Gut and hepatic availability were expressed as functions of free intrinsic clearance. All interindividual and interoccasion variability terms were described by an exponential error model, while the unexplained residual variability was described by a proportional error model.

patients. There were no obvious relationships observed between population PK parameters such as intrinsic midazolam clearance and cancer subtype in our dataset (data not shown). Interestingly, others have come to opposing conclusions as to the effect of cancer on CYP-mediated drug clearance. For instance, Cheeti et al. built an extensive SimCYP model of an oncology patient and concluded CYP3A activity was not altered in patients with cancer.57 However, cytokine-mediated changes in CYP protein content were not included as part of that SimCYP model, as the authors did not have access to an internal, robust dataset of clinical midazolam PK data in cancer patients and healthy subjects.57 It is therefore not surprising that a change in midazolam PK was not predicted. In contrast, our results are consistent with the report by Rivory et al.,21 in which the use of the erythromycin breath test showed an average 30% reduction in drug metabolism when comparing healthy control subjects to cancer patients. CASE STUDY 2 (ENZASTAURIN PHARMACOKINETICS IN CANCER PATIENTS COMPARED TO HEALTHY SUBJECTS)

Enzastaurin is an oral PKCb inhibitor that is a CYP3A substrate (fm 5 0.74, with fm 5 fraction of systemic clearance dependent on CYP3A) with pharmacokinetic data available in both healthy subjects and cancer patients.58–62 An enzastaurin population pharmacokinetic model was developed in NONMEM 7 based on PK data from 24 studies in healthy subjects and cancer patients. Enzastaurin PK data were available from 121 healthy subjects in six studies and 1,065 cancer patients in 19 studies. The final model consisted of two compartments for enzastaurin with first82

order oral absorption and two compartments for the metabolite LSN326020, which is formed by CYP3A. About 97% of enzastaurin and 95% of LSN326020 are bound to plasma proteins in vitro. Both enzastaurin, which is an acidic compound, and LSN326020 show higher binding to human serum albumin than to AAG or immunoglobulin G. Overall, the mean enzastaurin systemic clearance was lower in cancer patients (11 L/h) than in healthy subjects (16 L/h). The difference in enzastaurin systemic clearance of 30% between healthy subjects and cancer patients is similar to the difference observed for midazolam intrinsic clearance. Albumin was a significant covariate for enzastaurin systemic clearance, such that individuals with lower albumin had lower clearance. Specifically, for every g/L unit below the population mean of 39 g/L, enzastaurin clearance drops 2.37%. Among patients with diffuse large B-cell lymphoma, there was an approximately 2-fold range in enzastaurin systemic clearance (12 to 23 L/h). This variability within the cancer patient population may be due to variation in CYP activity or expression and may be reflected in part by levels of serum albumin. The difference between healthy subjects and cancer patients in clearance of enzastaurin may be attributable to lower CYP3A expression in cancer patients and could be linked to an inflammatory response in cancer. CASE STUDY 3 (P-38 MITOGEN-ACTIVATED PROTEIN KINASE-DRIVEN CHANGES IN PHARMACOKINETICS DUE TO REVERSAL OF DISEASE STATE)

The potential of p38 mitogen-activated protein kinase (MAPK) inhibitors to block the synthesis and release of proinflammatory VOLUME 98 NUMBER 1 | JULY 2015 | www.wileyonlinelibrary/cpt

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Figure 4 The effect of LY2228820 on the pharmacokinetics of midazolam and its metabolite, 1’-hydroxymidazolam. Data are presented as the geometric mean AUC, Cmax, Cl/F, Vz/F, t1/2 and MR ratios postLY2228820 treatment vs. baseline values with 90% CI (n 5 18). Patients received an oral dose of midazolam (2.5 mg) 2 days before the first dose of LY2228820 and again on Day 8 after the morning dose of LY2228820. LY2228820 was administered on Days 1 through 14 of a 28-day cycle at the maximum tolerated dose of 420 mg every 12 hours. MR 5 metabolite ratio 5 AUC of 1’-hydroxymidazlam/AUC of midazolam.

cytokines63 formed the basis for their development as therapeutics for treating chronic inflammatory disorders64 and cancer.65 LY2228820 dimesylate is a small-molecule ATP-competitive inhibitor of p38 MAPK. An in vitro study in human liver microsomes indicated that LY2228820 may inhibit the catalytic activity of CYP3A4 in vivo in humans at clinically relevant concentrations. However, results from an exploratory DDI study arm as part of a phase I study in advanced cancer patients indicated that LY2228820 is not an inhibitor of CYP3A4 in vivo in humans. The geometric mean midazolam AUC ratio (LY2228820-treated/nontreated baseline) was 0.84 with 90% CIs of 0.67, 1.06 and the geometric mean Cmax ratio was 1.01 with 90% CIs 0.77, 1.33 (Figure 4). However, as indicated by the midazolam AUC ratio, treatment with LY2228820 may have resulted in a slight increase in CYP3A4 activity (P-value 0.2). For instance, the midazolam mean apparent clearance (Cl/F) increased by  19% when compared to the baseline value. The apparent increase in CYP3A4 activity following LY2228820 treatment is further suggested by the pharmacokinetics of 1’-hydroxymidazolam, the primary metabolite of midazolam formed via the CYP3A4 pathway. The geometric mean metabolite ratio (MR 5 1’-hydroxymidazolam to midazolam AUC ratio when midazolam administered with LY2228820 vs. alone) was 1.8 (90% CIs 1.45, 2.29) and is statistically significant (P-value 30 lg/mL. A maximum increase of 1.8- and 2-fold in midazolam clearance was observed in two advanced cancer patients. As shown in Figure 6, inspection of serum protein levels in these patients revealed that of the 32 proteins for which data are available, 16 of the proteins (50%) showed reduced levels (ratio 1.0). The authors note that certain proinflammatory cytokines such as IL-6 and IFN-g were downregulated and certain antiinflammatory cytokines such as IL-10 and TNFR2 were upregulated following p38 MAPK kinase inhibitor treatment. Altogether, Figure 6 demonstrates that there is great variability in serum protein levels, and it is difficult to clearly draw correlations between select serum protein levels and cancer disease state. This is not altogether surprising, 83

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Figure 6 Down- or upregulation of plasma proteins following LY2228820 treatment for two patients who showed the maximum fold difference in midazolam clearance. A1A 5 alpha-1-antitrypsin; A2M 5 alpha-2-macroglobulin; BDNF 5 brain-derived neurotropic factor; C3 5 complement component 3; ET1 5 eotaxin-1; FaVII 5 factor VII; FRTN 5 ferritin; FGN 5 fibrinogen; ICAM-1 5 intercellular adhesion molecule 1; IFN-g 5 interferon gamma; IL6 5 interleukin-6; IL-8 5 interleukin-8; IL-10 5 interleukin-10, IL-18 5 interleukin-18; MIP-1a 5 macrophage inflammatory protein-1 alpha; MMP-3 5 matrix metalloproteinase-3; MMP-9 5 matrix metalloproteinase-9; RANTES 5 T-cell specific protein RANTES; TIMP-1 5 tissue inhibitor of metalloproteinase-1; TNFR2 5 tumor necrosis factor receptor 2; VCAM-1 5 vascular cell adhesion molecule-1; VDBP 5 vitamin D binding protein; VEGF 5 vascular endothelial growth factor; vWF 5 von Willebrand factor.

due to the complex interplay of serum protein homeostasis, and the great variability in the subtype and severity of cancer in the patients studied. CONCLUSION

Both biologics and small-molecule drugs that treat moderate-tosevere inflammatory disease states (e.g., advanced cancer, rheumatoid arthritis, influenza, and sepsis) have the potential to lead to indirect drug–drug interactions. Specifically, in this article we have reviewed the theoretical understanding of how advanced cancer status can lead to changes in cytochrome P450-mediated drug metabolism, we explored acute phase proteins and cytokines as markers of changes in CYP activity, and we provided clinical evidence of how the inflammatory response in advanced cancer patients can lead to altered CYP-mediated drug clearance. Based on our literature review and findings in our case studies, physicians are encouraged to understand under what circumstances meaningful changes in drug clearance might occur, and be prepared to more closely monitor patient response in those circumstances. Pharmaceutical companies (i.e., “sponsors”) that are developing drugs for inflammatory disease states should consider that an inflammatory disease state can be an important covariate in drug exposure variability. Changes in the level of inflammation due to cancer (rapid onset or full remission) appear to result in only modest changes in CYP-mediated drug clearance. In our pharmacokinetic metaanalysis of midazolam clearance, intrinsic midazolam clearance was on average 28% lower in advanced cancer patients compared to that in healthy subjects. For most small-molecule drugs this change in drug clearance would not be clinically meaningful. To date, there is only evidence of changes in CYP3A4 expression 84

due to cancer; meaningful clinical changes due to cancer have not been demonstrated in the expression of drug metabolizing enzymes CYP1A2, CYP2C9, or CYP2C19, for instance. Patients of possible greater need for careful monitoring of patient response may include those that are taking narrow therapeutic index medicines and whose drug clearance is predominantly CYP-mediated. Monitoring of drug concentration (e.g., cyclosporine) in these instances is suggested and the individual dose of the medicinal product adjusted as needed. Certain at-risk populations, such as neonates with differing CYP protein levels than adults, may also be of greater concern. Patients who are concomitantly taking drugs that are strong time-dependent inhibitors of CYP enzymes, such as clarithromycin, may be at increased risk of drug–drug interaction (or drug–disease interaction) due to the ability of the time-dependent inhibitor drugs to work in concert with inflammatory disease-mediated effects to produce even lower CYP enzyme activity in patients. Further, the enzyme CYP3A4 is predominantly responsible for a number of oral or intravenous anticancer agents, including docetaxel, vinorelbine, vemurafenib, and erlotinib, so changes in cancer status (partial remission vs. full remission) could result in important changes in drug exposure.2 From the literature, CRP, IL-6, AAG, and TNF-a appear to be the inflammatory markers that have the greatest correlation between increased inflammatory response and decreased drugmetabolizing capacity in advanced cancer patients.3,25,29 As increases in IL-6 and TNF-a levels are not as dramatic in cancer patients as in other inflammatory diseases, a better correlation with IL-6 and/or TNF-a and CYP-mediated clearance would be expected in disease states with a greater degree of inflammation (i.e., rheumatoid arthritis). Monitoring of other cytokines or VOLUME 98 NUMBER 1 | JULY 2015 | www.wileyonlinelibrary/cpt

REVIEW acute-phase proteins may ultimately prove to be predictive of altered drug metabolism in response to inflammation, but data are currently lacking. While the authors believe that the use of such inflammatory markers might be of use in understanding and controlling drug PK/PD variability in patients with inflammation, it is acknowledged that there should be caveats or cautions in their use. For instance, CRP levels are highly variable from day-to-day, even within a specific patient in a specific disease state. It is also unrealistic to expect practicing physicians to collect and quantitate cytokine levels and dose-adjust based on cytokine levels (such as IL-6). Also, it should be acknowledged that many disease states will result not in increased circulating levels of just a single cytokine, but rather will cause the modulation of multiple cytokines. Precise understanding and modeling of the complex interplay of cytokines is currently beyond our reach.

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ACKNOWLEDGMENTS The authors thank the anonymous reviewers whose comments improved the manuscript. The authors would like to acknowledge Rod Decker and Jenny Chien for their input and guidance in the building of various pharmacokinetic models.

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CONFLICT OF INTEREST All authors were employees and shareholders of Eli Lilly and Company at the time of manuscript preparation. AUTHOR CONTRIBUTIONS D.E.C., P.K., P.K.T. and S.D.H. wrote manuscript; P.K. and P.K.T. designed the research; D.E.C., P.K., R.L.B., S.R.W., C.P. and J.B. performed the research; D.E.C., R.L.B., P.K., S.D.H., J.B., S.R.W. and C.P. analyzed the data.

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