Polyphenon E, non-futile at neuroprotection in ... - Semantic Scholar

Journal of the Neurological Sciences 358 (2015) 46–52

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Polyphenon E, non-futile at neuroprotection in multiple sclerosis but unpredictably hepatotoxic: Phase I single group and phase II randomized placebo-controlled studies☆ Jesus Lovera a,⁎, Alexander Ramos a, Deidre Devier a, Virginia Garrison b, Blake Kovner a, Tara Reza a, Dennis Koop e, William Rooney d, Anne Foundas f, Dennis Bourdette c a

Department of Neurology, Louisiana State University Health Sciences Center — New Orleans, New Orleans, LA, United States Clinical Translational Research Center, New Orleans, LA, United States c Department of Neurology, Oregon Health & Science University, Portland, OR, United States d Advanced Imaging Research Center, Oregon Health & Science University, Portland, OR, United States e Bioanalytical Shared Resource/Pharmacokinetics Core, Oregon Health & Science University, Portland, OR, United States f Department of Neurology and Cognitive Neuroscience, University of Missouri-Kansas City School of Medicine, Kansas City, MO, United States b

a r t i c l e

i n f o

Article history: Received 29 June 2015 Received in revised form 31 July 2015 Accepted 4 August 2015 Available online 7 August 2015 Keywords: Multiple sclerosis Magnetic resonance spectroscopy Green tea EGCG Polyphenon E N-acetyl-aspartate Hepatotoxicity Liver toxicity

a b s t r a c t Objectives: Phase I (PhI): assess the safety of Polyphenon E in people with multiple sclerosis (MS) and determine the futility of Polyphenon E as a neuroprotective agent. Correlate plasma levels of EGCG with neuroprotective effects. Phase II (PhII): Further assess safety and confirm the neuroprotective effects of Polyphenon E. Design: PhI: single group futility study. PhII: parallel group randomized double-blind placebo-controlled study. Participants: Recruitment area (both studies): LSU MS Center, New Orleans, LA and general public from surrounding areas. Inclusion criteria (both studies): 1) MS per 2005 McDonald criteria; 2) relapsing remitting or secondary progressive MS; 3) stable for six months prior to enrollment on either no therapy or glatiramer acetate (GA) for the PhI study and on either on GA or Interferon β for the PhII study. Exclusion criteria (both studies): 1) complete bone marrow ablation or alentuzumab use at any time; 2) mitoxantrone, cyclophosphamide, natalizumab or fingolimod use in the prior nine months; 3) liver problems or significant medical problems. Interventions: PhI: Polyphenon E, a green tea extract containing 50% of the antioxidant Epigallocatechin-gallate (EGCG), two capsules twice daily (200 mg of EGCG per capsule; total daily dose 800 mg) for six months. PhII: Polyphenon E or matching placebo capsules, same dose for one year. Only the research pharmacist knew treatment assignment and she randomized participants (one-to-one, stratified by GA or Interferon β, blocks of 4 or 6). Outcome evaluators did not discuss side effects with participants. Outcome measures: PhI: 1) adverse events (AE); 2) futility: decrease in N-acetyl aspartate (NAA) from baseline to six months of 10% or more; 3) association between EGCG plasma levels and change in NAA. PhII: 1) AEs; 2) difference in the rate of change of NAA-levels over twelve months.We measured NAA using a point resolved magnetic resonance spectroscopic imaging sequence (TE30/TR2000) on a 10 cm × 10 cm × 1 cm volume of interest (VOI) located just superior to the lateral ventricles. The field of view was 16 × 16 resulting in 1 cm3 voxels. We quantified NAA and creatine/phosphocreatine (Cr) levels using LCModel for post-processing. Results: PhI: Ten participants enrolled and completed all assessments with no serious AEs. One discontinued therapy due to grade (G) I abnormal liver function tests (LFTs). We included all participants in the analysis. NAA adjusted for creatine increased by 10% [95% CI(3.4%,16.2%), p b 0.01] rejecting the futility endpoint. PhII: Thirteen participants enrolled and twelve started treatment. The DSMB stopped the study because 5/7 participants on Polyphenon E had abnormal LFTs (G I, and 1 G III). Median time to onset of abnormal LFTs was 20 weeks [Inter-Quartile Range (IQR) (10,23)]. Only two participants completed the six-month visit, so we could not analyze the NAA levels. PhI participants took capsules from lot 189I1107 while 6/7 PhII participants took capsules from a new lot

☆ J Lovera conducted the statistical analyses. ⁎ Corresponding author at: Rm 718A Dept of Neurology, 1542 Tulane Ave, New Orleans, LA 70112, United States. E-mail addresses: [email protected] (J. Lovera), [email protected] (A. Ramos), [email protected] (D. Devier), [email protected] (V. Garrison), [email protected] (B. Kovner), [email protected] (T. Reza), [email protected] (D. Koop), [email protected] (W. Rooney), [email protected] (A. Foundas), [email protected] (D. Bourdette).

http://dx.doi.org/10.1016/j.jns.2015.08.006 0022-510X/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

J. Lovera et al. / Journal of the Neurological Sciences 358 (2015) 46–52

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(L0206306). Both lots had similar levels of EGCG but differed in the levels of minor catechins. There were no significant differences between the lots on participants' median free EGCG plasma levels at either 3 h or 8 h as well as conjugated EGCG levels at 3 h (all p N 0.4, Wilcoxon exact test). Free EGCG levels at 8 h correlated with changes in NAA adjusted by water content. A 1 ng/ml higher EGCG plasma concentration correlated with a 0.9% increase in NAA[95% CI(0.5%,1.4%), visit*level interaction F = 14.4, p b 0.001]. However, EGCG plasma concentrations did not correlate with NAA adjusted by creatine (1 ng/ml higher EGCG was associated with 0.02%,[95% CI(−0.27%,0.3%) change in NAA, p N 0.5]). There was a trend towards an increase in creatine levels (referenced to water content) from baseline to exit (1 5% increase, [95% CI(−6%,17%), p = 0.4]). The free EGCG levels at 8 hours correlated significantly with change in creatine levels (1 ng/ml higher EGCG level at 8 h was associated with a 1.1% increase in creatine [95% CI(0.6%,1.6%)]). Thus it is possible that the discrepancy between the correlation of the EGCG 8 h levels with NAA changes referenced to water and the 8 h EGCG levels with NAA changes referenced to creatine was due to a change in creatine among the subjects with higher EGCG levels. Conjugated 3 h and 8 h levels and free 3 h levels did not correlate with NAA changes (all p N 0.5). Conclusions/classification of evidence: Class III evidence: Polyphenon E at a dose of 400 mg of EGCG twice a day is not futile at increasing brain NAA levels. Class I evidence: some lots of Polyphenon E have a high risk of hepatotoxicity. Funding: National Center for Complementary and Alternative Medicine K23AT004433, National Multiple Sclerosis Society RG4816-A-1 and National Institute of General Medical Sciences 1 U54 GM104940. Mitsui Norin provided Polyphenon E and placebo and their representative reviewed the manuscript prior to publication. Mitsui Norin was not involved in other aspects of the study. The decision to submit the manuscript remained with the investigators. Registration: NCT00836719 and NCT01451723. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

2.2. Participants

Polyphenon E (Mitsui Norin Co., Ltd; Shizuoka, Japan) is an extract from leaves of green tea (Camellia sinensis). The main active component in Polyphenon E is Epigallocatechin-gallate (EGCG), an antioxidant that accumulates within the mitochondria of neurons and decreases apoptosis of neurons undergoing oxidative stress [1]. In mice with experimental autoimmune encephalomyelitis (EAE), mitochondrial dysfunction leads to axonal injury that can be reversible [2]. For people with multiple sclerosis (MS), axonal injury is a major determining factor of disability. EGCG effectively treats EAE in SJL/J mice [3,4]. The EAE animal data and the in vitro neuroprotective effects of EGCG led us to hypothesize that Polyphenon E may be neuroprotective in MS. The primary objectives of the pilot study were: 1) to assess the safety of Polyphenon E in people with MS, 2) to perform a futility assessment of the ability of Polyphenon E to be neuroprotective based on its effects on brain levels of N-acetyl aspartate (NAA) and 3) correlate the changes in NAA with plasma levels of EGCG. NAA is made almost exclusively in neurons [5] and its synthesis is closely coupled with energy production by mitochondria [5,6]. NAA is present in oligodendrocyte 2A progenitors mainly during development [7]. However, in the adult brain, NAA is a specific marker of neuronal and axonal pathology [8]. NAA is degraded by oligodendrocytes, but even in conditions of oligodendrocyte loss where degradation of NAA could be impaired, NAA does not increase [9]. Thus, NAA levels measured non-invasively using proton magnetic resonance spectroscopy (MRS) can indicate neuronal metabolic health and may be a good outcome measure when testing potential neuroprotective compounds [10]. Our pilot study was limited to assessing futility comparing our data with a similar study done by Khan et al. [11]. The futility cutoff was set at a 10% decline in NAA over six months. Based on the results of the PhI study we conducted a PhII randomized controlled trial to confirm the PhI results.

2.2.1. Inclusion criteria For both the PhI and PhII studies, we recruited participants ages 18– 60 with MS per the 2005 McDonald criteria (either relapsing remitting or secondary progressive) [12,13] and an Expanded Disability Status Scale (EDSS) [14] score ≤ 7.0. For the PhI study, participants had to have been stable for at least six months prior to starting the study on either GA or no disease-modifying therapy (Fig. 1). For the PhII, participants had to have been stable on either GA or interferon β-1a or 1b. Participants had to have normal leukocyte, neutrophil, and platelet counts as well as normal creatinine and liver function tests. We screened all participants for hepatitis. Women with childbearing potential had to agree to reliable contraception. Participants agreed to refrain from drinking green tea or taking green tea supplements and to drink at most only one cup of black tea or two cups of coffee a day. 2.2.2. Exclusion criteria Participants that had received second-line therapies such as mitoxantrone, cyclophosphamide, T-cell vaccination, natalizumab, or (for the PhII study) fingolimod in the prior nine months were ineligible for the study. Dymethyl-fumarate was not available at the time of the study. We excluded participants who had ever received bone marrow radiation or alentuzumab. Participants could not have consumed green tea or green tea extracts in the thirty days prior to start of the study. They could not have history of liver or kidney diseases, an active clinically relevant illness or predisposition to gastrointestinal bleeding. We did not allow participation in other studies using investigational agents six months prior to the start or during our studies. Participants allergic to compounds in the Polyphenon E capsules or to gadolinium based contrast agents could not participate. All female participants of childbearing potential completed a screening pregnancy test; we excluded pregnant women from the study.

2. Methods

2.3. Intervention

2.1. Standard protocol approvals, registrations, and patient consents

The National Cancer Institute (NCI), Division of Cancer Prevention produced, under contract with Mitsui Norin, the lot of Polyphenon E capsules taken by the participants in our PhI study (lot 189I1107). Three subjects in the PhII study also took capsules from lot 189I1107 with one taking capsules only from this lot. Catalent (formerly Aptuit) produced the Polyphenon E (lot L0206306) and Placebo capsules used

The LSUHSC-New Orleans IRB approved both trials, which we registered with ClinicalTrials.gov (NCT00836719 and NCT01451723). The protocols are available at http://goo.gl/aOh5ja and http://goo.gl/ xaAAVb. All participants signed the informed consent.

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administered EDSS and MSFC assessments at baseline and exit. We instructed participants not to discuss adverse events with the evaluating RA. 2.5. Adverse events In both studies, we provided a calendar at each monthly visit so participants could record the onset and termination of adverse events. The study nurse reviewed the calendars and recorded adverse events on a monthly basis. The study's treating physician further evaluated AEs as he deemed necessary and determined attribution. He also determined the category, name and grading according to the Common Terminology Criteria for AE (v3.0). The study physician also evaluated and treated MS relapses. 2.6. Plasma levels

Fig. 1. CONSORT flow-chart PhI study.

One month after beginning the study, participants fasted overnight and took their morning capsules with a standardized breakfast. Afterwards, participants gave blood to measure free and conjugated plasma levels of EGCG at three hours (peak) and eight hours (trough). After adding a preservative solution (20% ascorbic acid and 0.05% Na2EDTA in 0.4 M phosphate buffer, pH 7.2), we aliquoted and stored the samples at −80 °C until analysis. Staff at OHSU Bioanalytical/Pharmacokinetics Shared Resource measured the EGCG levels using liquid chromatography with tandem mass spectrometry following a modification of Masukawa's method [18]. OHSU staff processed all PhI samples in a single run. When investigating the PhII liver toxicity issues, OHSU staff processed the PhII study samples along with stored aliquots of PhI samples in a single run in order to compare EGCG plasma levels between the studies. 2.7. Magnetic resonance imaging and spectroscopy

by six of the participants in the PhII study. PhI participants took two Polyphenon E capsules, each containing 200 mg of EGCG, twice daily with meals (total dose of EGCG 800 mg/day) for six months. PhII participants took two Polyphenon E capsules, each containing 200 mg of EGCG, twice daily with meals (total dose of EGCG 800 mg/day), or matching placebo capsules for a year. We selected the dose of 400 mg twice a day because healthy volunteers in previous studies tolerated this dose for a month well [15]. Single daily doses of 800 mg are also well tolerated [15] while single doses of 1200 mg cause nausea in 70% of participants when given without food and in 20% when given with food [16]. Although Polyphenon E was safe in beagle dogs and rats when given with food, beagle dogs when given Polyphenon E without food had high mortality rates [17]. Because of the toxicities seen in dogs, at the time we initiated our study, the FDA would not allow a study lasting more than a month with a dose higher than 800 mg a day given with food. 2.4. Study design The PhI study was an open-label, six-month study. Study physicians performed cognitive, EDSS, and Multiple Sclerosis Functional Composite (MSFC) assessments at baseline and exit. The participants had MR spectroscopy at baseline and exit to measure NAA. The PhII study was a randomized double-blind placebo-controlled study. The research pharmacist randomized participants to Polyphenon E or placebo. She was the only person aware of treatment assignments. There were two strata for the randomization: 1) treatment with GA and 2) treatment with interferon β. The randomization sequence used randomly alternating blocks of four and six. The target sample size was forty-eight participants per arm. NAA levels were to be measured at baseline, six and twelve months. A trained research assistant (RA)

We used a GE Signa-LX 3.0 T scanner to acquire MRI and MR spectroscopy data. Using a fast-spoiled-gradient recalled (FSPGR) sequence, we acquired a T 1-weighted anatomical image on an oblique plane parallel to the anterior–posterior commissural (AC– PC) line. All sequences following the SPGR used the same plane as the SPGR. First we obtained a GE Proton Brain Exam using a point resolved spectroscopy (PROBE-P) magnetic resonance spectroscopic imaging (MRSI; TE30/TR2000) sequence to select an approximately 10 cm × 10 cm × 1 cm volume of interest (VOI) located just superior to the lateral ventricles. The field of view for the MRSI acquisition was (16 cm)2 with a 162 matrix to provide isotropic spectroscopy voxels (10 mm)3. After the MRSI, we acquired proton density (PD); T 2-weighted fast spin echo (FSET2) and T 2 -weighted fluidattenuated-inversion-recovery (FLAIR) scans. 2.8. Post-imaging analysis We quantified NAA and creatine/phosphocreatine (Cr) levels using LCModel (http://s-provencher.com/pages/lcmodel.shtml). Using the FSL package (http://www.fmrib.ox.ac.uk/fsl), all MRI images underwent radiofrequency field bias correction and registration to the FSPGR space. We created lesion masks manually from the T2 scans and used SIENAX to segment the brain into gray matter (GM), white matter (WM) and cerebrospinal fluid (CSF). Using the MRS viewer SIVIC (http:// sourceforge.net/projects/sivic), we mapped the MRS VOI onto the FSPGR scans and created a mask representing VOI. We co-registered the baseline and exit FSPGR images to the halfway space between the two time-points using SIENA [19], and used the corresponding matrices to register MRS VOI masks into the halfway space. In order to use the PD scan as a reference for water content, we used baseline and exit MRS VOI masks. We extracted the VOI from the PD scans and then resampled


the VOI using a template created using FSL to match the dimensions of the MRS voxels (10 mm)3.

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Table 1 Participant demographics for the PhI and II studies. PhI

PhII

2.9. Definition of futility Futility was defined as 95% one-sided confidence that Polyphenon E treatment could not increase NAA by at least 5% from baseline to 6month follow-up; based on our sample size this level of confidence would be achieved if NAA declined by 10%. 2.10. Statistical analyses We included only voxels with less than 30% standard deviation error in NAA and Cr determined from the Cramer-Rao bounds in LCModel. The median number of usable voxels per participant/ time-point was 35 [IQR:17–40, Max:70]. The average composition of the spectroscopy region was (31% gray, 52% white, 11% CSF and 0.06% lesions) with a small, not statistically significant difference from baseline and exit (+ 0.4% for gray, − 1.7% white, + 0.2% CSF and − 0.5% lesions; p N 0.6 for all differences); there were no significant differences in the composition between the included and excluded voxels (differences included–excluded: 1% gray, − 0.6% white, 0.9% lesion, 0.6% CSF; p N 0.2 all differences). To account for the correlation between voxels we used a mixed model (proc mixed SAS 9-2, Cary, NC) with an anisotropic power spatial covariance matrix with terms for time and the voxel coordinates in the halfway space. To calculate the degrees of freedom, we used Kenward Rogers' approximation. The model's dependent variable was NAA, and the main effect was visit. The covariates were percent of GM, WM, CSF and lesions in the voxel. To reference NAA to Cr, LCModel's estimate of Cr was included as a covariate; similarly, to reference to the water content the resampled PD was included. NAA and Cr were log transformed so the model would be in a multiplicative scale. The analyses were intention to treat. 3. Results 3.1. PhI Recruitment for the PhI study started 9/2009 and ended 4/2010. Follow-up completed on 11/2010. We screened 11 participants with one screen failure due to abnormal LFTs. Table 1 shows the participant demographics. All participants completed the study. One discontinued therapy because of grade 1 (b 2 × normal) elevation of liver enzymes but still completed the exit assessment. Treatment with EGCG resulted in an NAA increase of 10% [95%, CI(3%–16%), p b 0.01] when referenced to Cr signal intensity and 13% [95%, CI(1%–23%) p b 0.01] when referenced to water content measured from the PD image. No significant changes in brain atrophy, EDSS, MSFC, or cognitive measures occurred. There were no significant correlations between conjugated or free plasma levels at either 3 h or 8 h and change in NAA levels adjusted for creatine. The free plasma levels of EGCG at 8 h correlated to the changes over six months in NAA levels adjusted by water content; an increase in 1 ng/ml in free EGCG levels was associated with in a 0.9% [95% CI(0.5%–1.4%), p b 0.01] increase in NAA between baseline and exit. 3.2. PhII Table 1 also shows the demographics for the PhII study and Fig. 2 the CONSORT flow-chart. Enrollment started on 7/15/2011. The MRS data and other clinical outcomes were uninterpretable because only two participants in the treatment arm completed the six-month follow up point.

PhII Placebo

Polyphenon E

5

8

N

10

13

Age and gender Age years median(range) Gender female N(%)

45 (39–56) 9 (90%)

48 (33–59) 49 (40–56) 47.5 (33–59) 10 (77%) 5 (100%) 5 (62%)

Race Caucasian N(%) African American N(%)

9 (90%) 1 (10%)

13 (100%)

Ethnicity Hispanic Unknown

0 (0%) 1(10%)

0 (0%) 0 (0%)

Type of MS RRMS SPMS

9 (90%) 1 (10%)

MS disability and duration EDSS median(range) Disease duration in years median(range) Disease modifying treatment Glatiramer acetate Interferon β No DMT

5 (100%)

8 (100%)

0 (0%) 0 (0%)

0 (0%) 0 (0%)

13 (100%)

5 (100%)

8 (100%)

4 (1–6.5) 14 (3–34)

2.5 (1–4) 12 (2–29)

3 (1–4) 13 (4–29)

2 (1–4) 9.5 (2–20)

7 (70%)

8 (62%) 5 (38%)

3 (60%) 2 (40%)

5 (63%) 3 (27%)

3 (30%)

3.3. Adverse events 3.3.1. Serious adverse events There were no serious adverse events (SAE) in the PhI study. There were two serious adverse events in the PhII study: one participant on placebo had a basal cell carcinoma and one participant on active treatment had AST and ALT elevated 15 times above normal Grade III and elevated bilirubin (total 1.3 mg/dl and indirect 0.38 mg/dl). The concomitant elevation of transaminases and bilirubin made this a serious adverse event. The participant's liver function tests (LFTs) normalized two months after discontinuing treatment.

3.3.2. Decision to terminate the PhII study The higher frequency of abnormal LFTs in the PhII study compared to the PhI study and the abnormal LFT SAE, led the DSMB to request an unblinded analysis from the DSMB's statistician on 9/12/2012. The analysis included the six participants on active treatment and five participants on placebo who had at least one follow-up visit. Five out of six participants treated with Polyphenon E (Grade I:4 participants, Grade III:1 participant) and one out of five participants treated with placebo had abnormal LFTs (Fisher's exact test p = 0.07, exact conditional mid-pvalue p b 0.05). Furthermore, the participant on the placebo arm only had a single episode of isolated elevated bilirubin; disregarding this event the p-value for the Fisher's exact test was less than 0.05. The LFT elevations did not seem related to concurrent use of interferon treatment as 4/4 participants on glatiramer acetate and 1/2 participants on interferon had abnormal LFTs (Fisher's exact test = 0.16). The DSMB requested that all participants stop treatment on 9/26/2012 and requested a comparison of the results of the two studies. Two out of ten PhI participants vs. 5/6 PhII participants treated with Polyphenon E had liver enzyme elevations. One of the PhI participants had a history of fatty liver that she had forgotten to discuss on enrollment; excluding this participant 1/9 PhI vs. 5/7 PhII participants had liver enzyme elevations (Fisher's exact test = 0.01). The difference in incidence of abnormal LFTs between the PhI and II studies made us review the product lots. All ten PhI participants received Polyphenon E from lot 189I1107. Of the PhII participants, one participant received product

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Fig. 2. Consort flow-chart phase II study.

only from lot 189I1107, and the other two received product initially from lot 189I1107 and then changed to lot L0206306. Two out of twelve participants had abnormal LFTs while using lot 189I1107 and 4/6 participants had abnormal LFTs while using lot L0206306 (p b 0.03 Fisher's exact test). Since there were no alternative lots of Polyphenon E available, the DSMB and the PI decided to terminate the study. 3.3.3. Other adverse events Table E1 of the electronic appendix shows a detailed list of adverse events. Aside from the elevated LFT's, nausea (6/17 Polyphenon E vs. 1/5 placebo) and abdominal pain (5/17 Polyphenon E vs. 1/5 placebo) were the most common adverse events. 3.4. Pharmacokinetics Table 2 shows the pharmacokinetic data. There was a trend towards lower free 3 h EGCG levels for the PhII study participants vs. the PhI but none of the comparisons between studies reached statistical significance.

shipments of 300 to 600 capsules. Thus, no individual shipment problem could account for the problems with the product. 3.5.2. Quality control We repeated the quality control analyses on a sample from the lot used in the PhI study (lot 189I1107) obtained from the NCI repository, on a sample from lot L0206306 from the Catalent (formerly Aptuit) repository, and on a sample from lot L0206306 that remained stored at our research pharmacy. Aptuit analyzed the samples using a validated reversed phase HPLC with 280 nm UV light detection method. The validation only included the EGCG content. The HPLC results also included percent areas referenced to EGCG for minor catechins peaks identified by their expected relative retention times. However, the assay only included spiked EGCG standards. Appearance, moisture content, and levels of EGCG were quite similar for the three sources.

Table 2 Pharmacokinetic data. Time

Study

N

EGCG

Median [IQR] ng/ml

p value1

3h

PhI PhII PhI PhII PhI PhII PhI PhII

10 6 10 6 10 6 10 6

Free Free Conjugated Conjugated Free Free Conjugated Conjugated

160 [132–267] 102 [86–179] 205 [181–483] 325 [156–472] 19 [10–53] 40 [34–44] 78 [43–116] 162 [98–254]

0.2

3.5. Quality control 3.5.1. Shipping and storage Review of the storage records showed stable room temperatures in the pharmacy locker throughout both phases of the study. We instructed participants to store the product at room temperature but we have no documentation of how exactly they stored it. NCI and Catalent (formerly Aptuit) shipped our study drug in multiple overnight

8h

[IQR] Interquartile range. 1 Wilcoxon rank sum two-sample test.

0.7 0.6 0.03


Lot 189I1107 had 93–96% of expected EGCG, our sample from lot L0206306 had 95.1%–94.8%, and the sample from lot L0206306 obtained from the Catalent (formerly Aptuit) repository had 95.8%–96.1%. The two samples from lot L0206306 had nearly identical contents of minor catechins; however, Lot 189I1107 had higher levels of Catechingalleate and Gallo Catechin-galleate than lot L0206306 and had lower levels of Epicatechin, Epigallo-catechin and Catechin. The samples from the three sources released more than 90% of their content by 60 minutes. Table E2 presents details on the results for the quality control analyses. 4. Discussion This is the first study to evaluate the safety of Polyphenon E in patients with MS and assess its futility as a neuroprotective treatment. A futility study does not try to prove efficacy but instead provides a clear decision rule as to whether a treatment should be studied in a larger study. NAA measurement using MRS is a unique tool for assessing neuroprotection. Neurons with unhealthy mitochondria will produce less NAA and after receiving a treatment that improves mitochondrial function neurons should produce more NAA. Polyphenon E contains EGCG, an antioxidant that mitochondria concentrate and thus EGCG could protect them from the free radical injury associated with lowgrade inflammation in MS. Therefore, Polyphenon E could potentially restore mitochondrial function resulting in an increase in NAA levels as the PhI study suggested. NAA increased in the participants in our PhI study rejecting the futility cutoff. This indicated that Polyphenon E warranted further investigation in a larger placebo-controlled trial, which we started. There were no changes made to the MRI instrument, to either its hardware or its software, throughout the study so changes in the scanner were not likely the cause of increased NAA levels. However, with no control group, we cannot exclude drifts in the instrument as the cause of the increase in NAA but the normalization to creatine and to less extent to water content protect against this bias. Unfortunately, our PhII study terminated prematurely due to the unexpected high frequency of elevated liver enzymes in the participants treated with Polyphenon E. The high frequency of elevated liver enzymes appeared related to the use of a new product lot, which the majority of PhII participants used. The cause of the difference in toxicity between the lots remains unclear. The quality control analyses showed identical concentrations of EGCG between the two lots, as expected considering that the capsules are standardized by their EGCG content. Differences in levels of the minor catechins between the lots could account for the differential toxicity. The quality control analyses suggested differences between the lots in several minor catechins relative to the EGCG peak but absolute quantitation was not performed. Another contaminant not measured in the quality control analyses could also be responsible for the toxicity. We did not analyze the samples for concentrations of the extraction solvents (methanol and ethyl acetate), but their concentrations were likely very low since both solvents are highly volatile and the extract is completely dry. Other trials using the same lot of our PhII study have not experienced similar difficulties related to liver toxicity so it is possible that our participants had a unique genetic background that made them more susceptible. Higher plasma EGCG levels are achieved when Polyphenon E is given without food [16]; a dose of 800 mg without food is well tolerated but 1200 mg results in frequent nausea [16]. The FDA does not allow any further human studies with Polyphenon E when given in the fasting state because of high mortality seen in dogs when given a daily dose comparable to 1200 mg of Polyphenon E without food [17]. Due to these results in dogs, at the time of our study initiation, 400 mg twice a day dose given with food was the highest safe dose that the FDA would allow for more than a month. However, more recent studies

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using Polyphenon E to treat chronic lymphocytic leukemia have been able to escalate the doses up to 2000 mg twice a day given with food [20] with reasonable safety for up to six months (16% discontinuation due to elevated LFTs and 5% discontinuation due to other causes) [21]. For breast cancer the maximum tolerated dose is estimated to be 600 mg twice a day (b25% participants experiencing dose limiting toxicities) [22]. Thus, some people may be able to tolerate the higher doses used for cancer but the dose we used of 400 mg twice a day is probably the dose that most participants would be able to consistently tolerate. Although Polyphenon E appeared promising as a neuroprotective agent in the PhI study, this effect remains unconfirmed. Further studies with Polyphenon E are unviable as there are no additional lots of the drug. People with MS should be very cautious about using other green tea extracts available in the supplement market as the evidence so far suggests that they may not be safe. At the very least, people using these supplements should have frequent liver test monitoring. Future studies may want to focus on pure EGCG products whose composition may be easier to monitor. Nonstandard Characters

Code

β

03B2

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jns.2015.08.006.

Acknowledgments This publication was made possible by grant number K23AT004433 from the National Center for Complementary and Alternative Medicine (NCCAM) at the National Institutes of Health. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCCAM. This investigation was supported in part by a grant (RG4816-A-1) from the National Multiple Sclerosis Society (NMSS). NMSS also advertised the study in their newsletter. Supported in part by 1 U54 GM104940 from the National Institute of General Medical Sciences of the National Institutes of Health which funds the Louisiana Clinical and Translational Science Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Thanks to Mitsui Norin for providing Polyphenon E and placebo capsules and supporting CCS Associates, the clinical research organization (CRO) that facilitated regulatory affairs. Thanks to Linda Doody, PhD; Beverly Smolich, PhD; Amy Kelley; Margaret R. Scheitrum, RAC, CIP; Zhdana Khomenko; Margaret Schreinburg, PhD and Kisha Thomas, MPH, CCRA from CCS Associates for their assistance with protocol development and support with regulatory affairs.

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