British Journal of Clinical Pharmacology

DOI:10.1111/bcp.12834

Pharmacokinetics and pharmacodynamics of everolimus in patients with renal angiomyolipoma and tuberous sclerosis complex or lymphangioleiomyomatosis Klemens Budde,1 Bernard A. Zonnenberg,2 Michael Frost,3 Wing Cheung,4 Shweta Urva,4 Thomas Brechenmacher,5 Karen Stein,4 David Chen,4 John Christopher Kingswood6 & John J. Bissler7 1

Correspondence Dr John J. Bissler MD, FedEx Chair of Excellence, University of Tennessee Health Science Center, LeBonheur Children’s Hospital and St Jude Children’s Research Hospital, 51 N. Dunlap St. Memphis Tennessee 38103, USA. Tel.: +1 901 287 5336 Fax: +1 901 287 8420 E-mail: [email protected]; john.bissler@ stjude.org ----------------------------------------------------

Keywords angiomyolipoma, biomarkers, collagenIV, lymphangioleiomyomatosis, tuberous sclerosis complex, VEGF-D ----------------------------------------------------

Received 26 March 2015

Accepted 15 November 2015

Accepted Article Published Online 18 November 2015

Charité Universitätsmedizin, Berlin, Germany, 2Universitair Medisch Centrum, Utrecht, The

Netherlands, 3Minnesota Epilepsy Group, St Paul, Minnesota, 4Novartis Pharmaceuticals Corporation, East Hanover, New Jersey, USA, 5Novartis Pharma Corporation, S.A.S., RueilMalmaison, France, 6Royal Sussex County Hospital, Brighton, UK, 7Lebonheur Children’s Hospital and the University of Tennessee, Memphis, Tennessee, USA

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Everolimus has been shown to effectively reduce renal angiomyolipoma volume on imaging in patients with tuberous sclerosis complex (TSC) or sporadic lymphangioleiomyomatosis (sLAM) • Identification of blood tests to monitor efficacy of medication in patients at increased risk from the effects of imaging assessments (such as anaesthetic side effects) might be of clinical relevance

WHAT THIS STUDY ADDS

• Trough concentrations (Cmin) of everolimus correlated with the percentage change in angiomyolipoma volume from baseline • Everolimus modulated plasma levels of multiple angiogenesis-related molecules, including collagen-IV, VEGF-A, and VEGF-D. The measurement of plasma collagen-IV and VEGF-D levels may offer an alternative method for disease monitoring, thus reducing the need for imaging

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AIMS The purpose was to determine the exposure response relationship of everolimus in patients with angiomyolipoma from the EXIST-2 trial and to analyze the correlation between exposure and plasma concentrations of angiogenic biomarkers in these patients.

METHODS One hundred and eighteen patients with angiomyolipoma associated with tuberous sclerosis complex (TSC) or sporadic lymphangioleiomyomatosis (sLAM) were randomly assigned 2 : 1 to receive everolimus 10 mg (n = 79) or placebo (n = 39) once daily. Blood samples for determining everolimus concentration were collected at weeks 2, 4, 12, 24 and 48 during double-blind treatment. Plasma samples for biomarker analysis were collected at baseline and weeks 4, 12, 24, 36, 48 and at the end of treatment. Concentrations of eight angiogenic biomarkers associated with tumour growth were determined by enzyme-linked immunosorbent assay (ELISA).

RESULTS Peak and trough concentrations of everolimus in blood remained stable over time and similar to those reported in other indications. Substantial pharmacodynamic effects were observed in the everolimus, but not placebo, arm for three biomarkers: After 24 weeks of treatment, reduction of vascular endothelial growth factor D (VEGF-D) and collagen type IV (COL-IV) (mean foldchanges with 95% confidence intervals [CI] were 0.36 [0.33, 0.40], and 0.54 [0.51, 0.57], respectively, P < 0.001 for both), along with increased VEGF-A (mean foldchange of 1.59 [1.39, 1.80], P < 0.001), were seen. Furthermore, baseline VEGF-D and COL-IV levels were associated with angiomyolipoma size at baseline and with angiomyolipoma response to everolimus.

CONCLUSIONS

These findings suggest that plasma angiogenic markers may provide an objective measure of patient response to everolimus. © 2015 The British Pharmacological Society

PK and PD of everolimus in patients with renal angiomyolipoma and TSC

Introduction Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in the TSC1 (encodes hamartin) or TSC2 (encodes tuberin) tumour suppressor genes that normally inhibit the mTOR complex-1 (mTORC1), a central controller of cell growth and proliferation [1, 2]. Possessing an inactivating mutation in either gene leads to a loss of a functional hamartin-tuberin protein heterodimer, which in turn leads to constitutive mTORC1 activation, aberrant downstream signalling and growth of non-cancerous hamartomas in the kidney (renal angiomyolipomas), brain (subependymal giant cell astrocytomas [SEGA], cortical tubers), heart (cardiac rhabdomyomas), liver, eyes and skin (angiofibromas) [1, 3–5]. Renal angiomyolipomas occur in up to 80% of patients with TSC and cause the largest proportion of deaths in adults with TSC [5, 6]. Angiomyolipomas are highly vascularized tumours that appear to arise from a vascular pericyte lineage [7]. As the tumours enlarge, there is an increased risk that blood vessels will develop aneurysms that subsequently rupture and haemorrhage [5, 8–10]. Lymphangioleiomyomatosis (LAM), a disorder of the lung, occurs in 30% to 80% of women with TSC and increases in prevalence with increasing age [5, 11]. It is characterized by infiltration of the lungs by smooth muscle-like cells and pulmonary remodelling that results in diffuse pulmonary cysts. Following neurologic and renal disease, LAM is the third most common cause of death in patients with TSC [5]. Sporadic LAM (sLAM) is a rare form of LAM that occurs in patients without TSC as a result of somatic mosaicism at the TSC2 gene locus [5, 12–15]. LAM and sLAM are associated with angiomyolipomas in 93% and 50% of cases, respectively [5, 16]. Loss of the TSC1 or TSC2 genes has been associated with increased production of vascular endothelial growth factor (VEGF)-A [17]. The VEGF family of growth factors are essential mediators of tumour angiogenesis [18]. Elevated VEGF-D levels in blood have been shown in patients with renal angiomyolipomas or LAM and have been correlated with angiomyolipoma size and LAM disease severity [19–22]. Sirolimus (also known as rapamycin), an oral mammalian target of rapamycin (mTOR) inhibitor, has been shown to inhibit production of VEGF-A, tumour growth and angiogenesis, both in vitro in TSC1- or TSC2-null fibroblasts and in vivo in a mouse model of TSC [17]. Historically, therapeutic options for patients with TSC-related angiomyolipomas were limited to watchful waiting until the need for invasive interventions such as embolization or nephrectomy [5, 6]. The efficacy and safety of the mTOR inhibitor everolimus for treating non-life threatening angiomyolipomas was demonstrated in EXIST-2 (NCT00790400), the first large, randomized phase 3 trial to evaluate a systemic treatment for renal angiomyolipomas (not requiring

immediate surgery) in adult patients with either TSC or sLAM [20]. Everolimus, at a dose of 10 mg daily, significantly reduced renal angiomyolipoma volume compared with placebo. The renal angiomyolipoma response rate (primary analysis, database cutoff 30 June 2011) was 42% for everolimus vs. 0% for placebo (P < 0.0001), where response was defined as 50% reduction of angiomyolipoma volume from baseline. During the study, the patients reported mostly mild to moderate adverse events, which were managed through everolimus dose reduction and/or interruption of therapy [20]. The long term extension phase of the EXIST-2 study (median duration of everolimus exposure of 28.9 months, database cutoff 01 May 2013) reported sustained reductions in angiomyolipoma volumes, with an overall angiomyolipoma response rate of 54% (95% confidence interval [CI], 43.9%, 63.0%) [23]. The current analyses characterized the pre-dose trough everolimus concentration (Cmin) and 2 h postdose everolimus concentration (C2 h), and examined the relationship between everolimus Cmin, the pharmacodynamic changes of various circulating proteins and treatment efficacy in the EXIST-2 patient population. The goal of these analyses was to better discern the variability of everolimus exposure and the pharmacodynamic effect of everolimus on a broad panel of angiogenic growth factors. Patients with TSC often may require sedation or general anaesthesia for imaging to follow their renal disease. We therefore also explored whether changes in the pharmacodynamics could be used as an alternative to multiple, repeated imaging to monitor disease progression.

Methods The details of EXIST-2 have been previously published [20]. In brief, the trial enrolled 118 patients with angiomyolipoma associated with TSC or sLAM who were randomized 2 : 1 to receive 10 mg of everolimus (n = 79) or placebo (n = 39) once daily, stratified by enzymeinducing anti-epileptic drug (EIAED) use or no use. EIAEDs are strong inducers of CYP3A4 activity and included phenytoin, mephenytoin, carbamazepine, phenobarbital, pentobarbital, primidone and oxcarbazepine. The everolimus dose could be reduced to 5 mg daily or further to 5 mg every other day if necessary to manage any unacceptable toxicity. Patients were eligible for the study if they were aged ≥ 18 years, had at least one renal angiomyolipoma ≥ 3 cm in its longest diameter and had either a definite diagnosis of TSC (per consensus criteria) or sLAM (per biopsy or chest computed tomography [CT] scan). Independent ethics committees and/or local ethics review boards approved the protocol and all patients or their guardians provided written informed consent. A list of all institutional review boards is Br J Clin Pharmacol

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provided in Supplementary Table 1. The primary endpoint of the study was the angiomyolipoma response rate, defined as the proportion of patients with renal angiomyolipoma with a confirmed response of ≥ 50% reduction from baseline in the sum of volumes of all target lesions, in the absence of new lesions ≥ 1 cm in longest diameter, kidney volume increase of > 20% from nadir and angiomyolipoma-related bleeding of grade ≥ 2 (CTCAE, version 3.0).

Pharmacokinetic analysis Pre-dose blood samples (2 ml) for determination of everolimus Cmin were collected immediately prior to everolimus dosing at weeks 2, 4, 12, 24 and 48 during the double-blind treatment period 20 to 28 h after the patient’s last dose of study drug. To estimate peak everolimus concentrations (Cmax), blood samples were collected 2 h post-dose (C2 h) as a surrogate for Cmax reflecting that a daily dose of everolimus 10 mg is rapidly absorbed with a median tmax of 1 to 2 h [24]. The everolimus concentration in whole blood was determined by a validated liquid chromatography-tandem mass spectroscopy (WuXi App Tec, Shanghai, China) method [25] as detailed previously [26]. The lower limit of quantification was 0.3 ng ml 1. The method consists of protein precipitation followed by solid phase extraction. The extract was analyzed by reversed phase HPLC with C18 column (CAPCELL PAK MG, Shiseido) by gradient elution with aqueous ammonium acetate buffer and acetonitrile. The detection was made by API4000 (Applied Biosystems) with selected reaction monitoring 975.6 m/z to 908.6 m/z for everolimus and 980.8 m/z to 913.8 m/z for the internal standard of everolimus. The mobile phase consisted of gradient with water and 95% of acetonitrile with ammonium acetate. The coefficient of variation of the assay ranged from 4.7% to 7.8%.

Biomarker analysis Plasma samples for biomarker analysis were prepared from 3 ml peripheral blood collected from all trial participants prior to everolimus dosing, at baseline and day 1 of treatment weeks 4, 12, 24, 36 and 48 and at the end of treatment. The concentrations of eight biomarkers associated with the angiogenesis and tumour growth were determined by enzyme-linked immunosorbent assay (ELISA) for VEGF-D and COL-IV or by electrochemiluminescence using a multiplexed Meso Scale Discovery® (Meso Scale Diagnostics LLC, Rockville, MD, USA) platform formatted as four-plex (placental growth factor [PLGF], VEGF-A, basic fibroblast growth factor [bFGF] and soluble VEGF receptor 1 [sVEGFR-1]) and two-plex (soluble VEGF receptor 2 [sVEGFR-2] and c-Kit) combinations. All assays were validated prior to use according to the manufacturer’s specifications. Coefficients of variation and lower limit of determination are listed in Supplementary Table 2. All assays were performed by a central laboratory. 960

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Statistical analysis Summary statistics were provided by planned dose (10 mg day 1) and by time window for Cmin and C2 h. Because a number of patients treated with everolimus had their dose reduced from 10 mg day 1 to 5 mg day 1, an additional analysis was undertaken by actual dose received. The relationship between everolimus exposure and change from baseline in sum of volumes of target renal angiomyolipoma was investigated by a linear mixed model that included the sum of volumes at baseline, the time-normalized Cmin between the previous and current imaging assessments (CT or MRI) as fixed effect and patient as random effect. A similar linear model was also fitted using log-transformed variables. Descriptive statistics (mean, median, percentiles and outliers) were used to describe the distribution of biomarkers at baseline. Values below the lower limit of quantitation (LLOQ) were imputed as 50% of LLOQ. Pearson correlation was used to investigate the relationship among baseline biomarker levels, and Spearman’s correlation was used to determine the relationship between biomarker level and angiomyolipoma volume (sum of volumes of target angiomyolipoma) at baseline. For the correlation between the baseline biomarker levels and angiomyolipoma response rate, everolimus-treated patients were divided into subgroups defined by ≤ or > overall median value for each biomarker at baseline. Odds ratios for response rate were obtained by logistic regression. The effects of everolimus on biomarker levels over time were evaluated using summary statistics at each time point. Mean fold-change and 95% CI were obtained using a linear mixed model, including terms for baseline value, treatment, time and interaction between time and treatment, P values for test of effect were calculated for each covariate in the model to assess the dynamic change of biomarkers over time. In addition, estimate at week 24 was used to report descriptively fold-change from baseline for each biomarker, because all pharmacodynamic effects had achieved steady-state and the number of patients with evaluable biomarker data was large enough for a meaningful analysis and interpretation of the results. The relationship between percentage change from baseline in biomarkers and everolimus exposure was investigated by a linear mixed model, with log-value of biomarker as dependent variable, log-baseline value of biomarker and time-normalized Cmin as fixed effect independent variables, and patient as random effect independent variable. In this analysis, the time-normalized Cmin was calculated for each patient as area under the concentration curve between the current biomarker assessment and the previous one, divided by the time of this corresponding period. Percentage change in biomarker from baseline for 2-unit Cmin increase was derived from the 1), mixed model estimates as 100 × (exp(2 × Cmin) and 95% CI was calculated in the same way. All provided

PK and PD of everolimus in patients with renal angiomyolipoma and TSC

P values are nominal. Because this was a post hoc exploratory analysis, no multiplicity adjustments were performed. Therefore, statistical interpretation should be made with caution. Statistical analyses were performed using SAS software version 9.2 (SAS Institute, Cary, NC, USA).

Results Patient baseline demographic and disease characteristics The baseline demographic and disease characteristics have been described previously and, in general, were well balanced between the treatment arms [20]. Overall, 78% of patients had bilateral renal angiomyolipomas and 29% of patients had a large (≥ 8 cm in longest diameter) renal angiomyolipoma. Almost 40% of patients had received a previous invasive intervention, including 19% with prior nephrectomy [20].

Everolimus exposure The mean (standard deviation [SD]) relative dose intensity (RDI) for everolimus patients (n = 79) through the double-blind phase of the study from which the PK data were derived was 0.85 (± 0.263) and the median RDI was 1.0 (range 0.2–1.8). A total of 59.5% of patients had an RDI between 0.90 and 1.10, indicating that the doses they received were close to the planned daily dose of 10 mg.

Everolimus pharmacokinetics Median Cmin and C2 h remained relatively stable over time (Table 1). In patients who required a dose reduction to 5 mg day 1 (n ≤ 5), everolimus blood concentrations were correspondingly lower after dose reduction (Table 1). Intersubject variability of Cmin and C2 h was relatively high in the total patient population listed in Table 1, due to inclusion of data from patients receiving both a 5 mg day 1 and 10 mg day 1 dose and data from patients with or without co-administration of a CYP3A4 inducer. In patients who received an actual dose of 10 mg day 1 with or without co-administration of a CYP3A4 inducer, the intersubject variability in Cmin ranged from 56.7% to 105% and in C2 h ranged from 39.5% to 57.6%. In the small number of patients (n ≤ 9) whose daily everolimus dose had been reduced to 5 mg day 1, the intersubject variability in Cmin ranged from 3.6% to 57.6% and in C2 h ranged from 14.6% to 33.6% (Table 1). At randomization, 13 patients in the everolimus arm and seven patients in the placebo arm were using an EIAED. Irrespective of the actual administered dose, Cmin and C2 h values were lower by approximately one-third to one-half in patients using an EIAED at randomization compared with patients who were not (Table 2). In patients using EIAEDs at randomization, the intersubject variability in Cmin ranged from 39.0% to 59.2% and in

C2 h ranged from 44.5% to 66.4%. In patients not using an EIAED at randomization, the intersubject variability in Cmin ranged from 52.4% to 108.2% and in C2 h ranged from 42.4% to 53.4% (Table 2). Analyses using linear mixed models based on the actual dose received also demonstrated lower mean Cmin and C2 h values in patients using EIAEDs at randomization compared with the respective exposure indices in patients who were not using EIAEDs at randomization. For patients receiving an actual dose of 10 mg day 1, the geometric mean (90% CI) for Cmin was 3.79 (2.85, 5.03) ng ml 1 in patients using EIAEDs (n = 11) compared with 8.21 (7.19, 9.37) ng ml 1 for patients not using EIAEDs (n = 49), and for C2 h was 19.74 (16.23, 24.02) ng ml 1 in patients using EIAEDs (n = 11) compared with 35.04 (32.01, 38.34) ng ml 1 for patients not using EIAEDs (n = 51). The difference in the number of patients between Cmin and C2 h for EIAED non-users was due to the number of evaluable samples available. Data for the EIAED users receiving 5 mg daily dose were not interpretable due to the small sample size (n = 0 and n = 1 for Cmin and C2 h, respectively).

Relationship between everolimus concentration and response to treatment Fitting a linear mixed model for the absolute change from baseline in angiomyolipoma lesion volume resulted in a slope of 8.12 cm3 per 1-unit log Cmin increase (95% CI 18.80, 2.55, P = 0.135), which was not statistically significant. An interaction between angiomyolipoma lesion volume at baseline and the absolute volume reduction was noted, suggesting that the exposure response relationship may have varied in patients with different angiomyolipoma lesion volumes at baseline. To assess this, the data were further analyzed by categorizing the angiomyolipoma size at baseline (200 cm3). No significant correlation was found between Cmin and the absolute change of angiomyolipoma lesion volume from baseline in any of the subgroups. An analysis that fitted a linear mixed model for the percentage change from baseline in angiomyolipoma lesion volume did indicate a statistically significant 10.37% reduction in tumour size from baseline (95% CI 15.96%, 4.40%, P = 0.001) for a two-fold increase in Cmin. Thus, data suggest percent change, rather than absolute change, from baseline in angiomyolipoma lesion volume was correlated with everolimus Cmin concentration.

Baseline biomarker analysis VEGF-A, VEGF-D, placental growth factor (PLGF), collagen type IV (COL-IV), and basic fibroblast growth factor (bFGF), soluble VEGF receptor 1 and 2 (sVEGFR1 and 2) and c-Kit/mast/stem cell growth factor receptor/CD117, all related to angiogenesis and cell growth, were selected for the exploratory biomarker analysis of this disease with highly vasculature lesions. The plasma Br J Clin Pharmacol

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Table 1 Everolimus Cmin and C2 h at weeks 2, 4, 12, 24 and 48 of the study irrespective of the actual dose received by the patients or by the actual dose received -1 -1 by the patients (10 mg day or 5 mg day ) for all evaluable everolimus-treated patients during double-blind period

Week 2

Week 4

Week 12

Week 24

Week 48

All patients, irrespective of the dose received -1

Cmin (ng ml ) n

43

44

49

46

15

Mean ± SD

7.63 ± 4.32

7.72 ± 4.35

8.79 ± 6.75

9.37 ± 8.83

11.49 ± 12.01

CV%

56.7%

56.4%

76.8%

94.2%

104.5%

Median (range)

6.6(0.6–19.6)

7.0(1.4–22.2)

7.5(1.0–32.6)

6.7(0.0–52.8)

6.9 (2.5–50.0)

-1

C2 h (ng ml ) n

55

49

56

50

14

Mean ± SD

33.38 ± 15.66

30.89 ± 14.96

34.48 ± 15.10

39.27 ± 22.25

33.20 ± 18.45

CV%

46.9%

48.4%

43.8%

56.7%

55.6%

Median (range)

31.5(4.9–75.8)

28.2(4.3–75.9)

34.4(10.5–77.9)

38.9(5.4–98.6)

29.0(10.0–71.2)

-1

Patients who received 10 mg day dose -1

Cmin (ng ml ) n

43

41

42

39

11

Mean ± SD

7.63 ± 4.32

7.85 ± 4.49

9.40 ± 7.03

10.13 ± 9.34

13.10 ± 13.69

CV%

56.7%

57.2%

74.8%

92.2%

105%

Median (range)

6.6(0.6–19.6)

7.2(1.4–22.2)

7.6(1.0–32.6)

6.9(0.0–52.8)

7.8(2.5–50.0)

-1

C2 h (ng ml ) n

55

46

45

39

10

Mean ± SD

33.38 ± 15.66

31.44 ± 15.28

37.39 ± 14.76

44.18 ± 22.55

36.54 ± 21.03

CV%

46.9%

48.6%

39.5%

51.0%

57.6%

Median (range)

31.5(4.9–75.8)

29.1(4.3–75.9)

36.2(10.5–77.9)

41.5(5.4–98.6)

35.0(10.0–71.2)

-1

Patients who received 5 mg day dose -1

Cmin (ng ml ) n

3

5

5

3

Mean ± SD

0

6.04 ± 0.22

4.89 ± 2.82

4.57 ± 1.92

5.30 ± 0.93

CV%

3.6%

57.6%

41.9%

17.6%

Median (range)

6.0(5.8–6.3)

3.9(2.1–8.0)

5.0(2.5–6.8)

5.7 (4.2–6.0)

-1

C2 h (ng ml ) n

3

9

9

4

Mean ± SD

0

22.47 ± 3.29

19.40 ± 6.26

19.93 ± 6.69

24.88 ± 4.34

CV%

14.6%

32.3%

33.6%

17.5%

Median (range)

21.6(19.7–26.1)

19.7(11.4–30.9)

19.6(12.6–35.7)

24.0(20.8–30.7)

Cmin, predose trough concentration; C2 h, 2 h post-dose concentration; CV, coefficient of variation; SD, standard deviation.

concentrations of these eight biomarkers were determined in triplicate in samples collected at baseline and in up to five on-treatment time points. Evaluable results were obtained from 83% to 94% of the trial population (biomarker population), except those for bFGF. The number of samples with evaluable bFGF data was too small (