Phase 1 study of quercetin, a natural antioxidant for children and young adults with Fanconi anemia

Fanconi anemia (FA) is a rare inherited DNA-repair disorder characterized by progressive bone marrow failure (BMF), variable congenital anomalies, and a predisposition hematological malignancy and solid tumors.^1-3 FA is genetically heterogeneous, and 23 different genotypes have been identified.^4-6 BMF occurs in the first and second decades of life in most patients, typically requiring hematopoietic cell transplant (HCT) with its associated potentially life-threatening complications.^3,7-10 Androgens can temporarily improve blood counts in a subset of patients with FA but return of marrow failure and significant liver toxicities are common.^11-14 Novel therapeutic agents with better toxicity profiles are needed to delay or prevent BMF in FA. Previous reports of treatment with resveratrol, direct activators of sirtuin 1, or metformin in FA mouse models, and etanercept or metformin for patients with FA, have shown limited success.^15-19 

Studies in FA-pathway–deficient mice and FA human samples indicate that high levels of systemic reactive oxygen species (ROS) and increased sensitivity of hematopoietic progenitors to ROS play a key role in the pathogenesis of marrow failure associated with FA.^20-29 Additionally, previous reports have shown that FA cells are vulnerable to oxygen-induced chromosomal aberrations, and FA fibroblasts along with primary bone marrow cells grow better under hypoxic conditions than in ambient air.^30-33 Hypersensitivity to ROS has been documented in many studies using primary and immortalized cell lines derived from patients with FA, as well as FA-knockout mouse models.^20-29 ROS is also implicated in leukemogenesis in FA.³² Importantly, our group has shown previously that that lipopolysaccharide/tumor necrosis factor-α–generated hematopoietic suppression in Fancc^–/– mice is reversible by treatment with antioxidants. Additionally, treatment with natural antioxidant quercetin restores insulin resistance signaling and the diabetes-prone phenotype in FA mice.^29,33-35 Together, these data suggest a possible benefit in supporting hematopoiesis with quercetin in patients with FA.

Quercetin (3, 30, 40, 5, 7-pentahydroxyflavone) is a naturally occurring flavonoid with a wide range of biological activities including free-radical scavenging, anti-inflammatory, and antineoplastic activities.^36-42 Quercetin exerts its potent inhibitory effect on oxygen radical generation through chelation of transition metal ions and catalysis of electron transport through the xanthine/xanthine-oxidase pathway together with scavenging of hydrogen peroxide.^43-50 Quercetin reduces spontaneous and diepoxybutane (DEB)-, formaldehyde- and acetaldehyde-induced cell cycle arrest in lymphoblastoid cell lines derived from patients with FA (Surrallés’ laboratory, Barcelona, Spain), which is important because aldehyde production in response to oxidative stress plays a significant role in the pathogenesis of BMF and development of squamous cell carcinoma in FA.^51-57 

We hypothesized that quercetin would be safe, well tolerated, feasible to take every day, and would decrease ROS levels in children with FA. We report the results of our dose-finding phase 1 open-label study of safety, tolerability, and pharmacokinetics (PKs) of quercetin in patients with FA. Additionally, we report the results of an expansion cohort in which patients were treated with quercetin at the recommended therapeutic dose identified in the phase 1 study.

This single-center, prospective, phase 1 dose-finding study of quercetin for patients with FA was followed by an expansion cohort to verify the identified optimal dose (Figure 1). The study was approved by institutional review board of Cincinnati Children’s Hospital Medical Center (CCHMC), performed under an “investigational new drug” (number 113343) from the US Food and Drug Administration (FDA) and registered at www.ClinicalTrials.gov (identifier: NCT01720147). Patients or parents/legal guardians of all minor patients gave written informed consent for study participation, and patients provided assent if age appropriate.

Patients with FA aged ≥2 years who were able to take enteral medication were eligible. Exclusion criteria included morphological evidence of myelodysplasia or leukemia, concurrent cyclosporine or digoxin therapy, those who had taken quercetin or any other antioxidant in the 30 days before enrollment, and patients who were pregnant or breastfeeding. Additionally, those with renal failure requiring dialysis or with a total bilirubin of >3 mg/dL and/or alanine aminotransferase (ALT) of >200 IU/L at the time of enrollment were excluded. Marrow failure status at baseline was defined using standard clinical criteria.^1,58,59 

Quercetin was purchased, stored, and distributed by the investigational pharmacy of CCHMC using standard operating procedures. A liquid quercetin preparation was used for the initial 4 patients enrolled. The study product was switched to a powdered formulation for the remaining patients. The FDA approved this change because of unexpected, prolonged unavailability of the liquid formulation. Doses were dispensed in individual packets for home administration. Quercetin powder was mixed with a small amount of yogurt and taken twice a day. Pediatric drug dosing was extrapolated based on body weight using allometric scaling, and was calculated as a percentage of the adult dose (supplemental Table 1).

Patients received oral quercetin twice a day for 4 months (16 weeks). An optional 20-month continuation phase was offered to those who wished to continue after completion of the first 4 months (Figure 1). The first 3 patients enrolled were aged >12 years with guidance from FDA. Initial weight-adjusted dosing of quercetin was capped at a total of 750 mg/d, because this dose was previously shown to be safe and well tolerated in adults without FA.^42,60-63 Intrapatient dose escalation was performed in the first 3 patients and patients were followed up carefully. In the absence of dose limiting toxicity (DLT), the dose was increased to a maximum dose of 1125 mg/d after 1 month and then again to the maximum adult dose of 1500 mg/d or equivalent starting month 3 in the first 3 patients. The highest recommended dose in adults without with available PK data is 1500 mg/d.⁶⁴ We assumed a similar exposure-response relationship in our pediatric and adult patients with FA because there were no prior pediatric PK studies for quercetin.

DLT was defined as: any grade ≥3 toxicity, any grade ≥2 nausea or vomiting (based on previous report in adult patients), or acute precipitous drop in blood counts (ie, if 1 of the first 3 patients developed pancytopenia within 1 week or any time during 4 months (16 weeks) of observed therapy or a decline in blood counts of all 3 lineages to <50% of baseline and below the normal range for age at any time during 4 months of therapy). Patients who wished to continue quercetin beyond 4 months were treated for an additional 20 months up to a total of 2 years, at the original assigned dose (continuation phase).

Patients were treated with quercetin for 6 months at the recommended weight-adjusted dose identified in the dose-finding phase 1 study. Those who wished to continue quercetin beyond 6 months were treated for an additional 6-month period to a total of 1 year (note: the continuation phase for expansion cohort was shorter, extended from 6 months until the 1-year time point; Figure 1).

Twenty-one patients (last 3 patients of the phase 1 cohort and 18 expansion cohort patients) treated using the recommended dosing of quercetin were included for the analyses of correlative studies in this cohort (Figure 1). Of note, last 3 patients in the phase 1 study were analyzed up to the 1-year time point only, to match with the expansion cohort’s final time point, which was a maximum of 1 year of treatment.

The primary objective of the dose-finding phase 1 study was to assess safety, feasibility, and PKs of oral quercetin and to propose the recommended dose of quercetin for children and young adults with FA. The objective of the expansion cohort was to confirm the safety of the identified recommended dose of quercetin in a larger number of patients. Finally, the objective of the analysis cohort was to perform preliminary correlative assessment of ROS reduction in patients with FA treated at the recommended dose. Optimal ROS response was defined a priori as ≥25% reduction in peripheral blood (PB) ROS level compared with baseline after quercetin treatment. We also made a preliminary assessment of the impact of quercetin on maintenance of PB counts and insulin sensitivity/glucose tolerance.

Safety was assessed using National Cancer Institute’s common terminology criteria for adverse effects version 4.0. Criteria for feasibility included absence of DLT, patient/parental withdrawal from the study, or poor adherence to study therapy. If ≥3 of the first 6 patients missed ≥2 weeks of therapy per month during 4 months of therapy, it would have been concluded that long-term oral quercetin therapy was not feasible. Compliance was assessed by weekly phone calls (phase 1 study only), review of patient’s drug diary, and counting returned empty packets of quercetin.

Total plasma quercetin levels were quantified at CCHMC using liquid chromatography–mass spectrometry (MS) assay. See details in the supplemental Methods.

Quercetin plasma PK was determined after the first dose of oral quercetin and again after 4 months (16 weeks) of quercetin given on a twice-a-day schedule. Six blood samples (predose time 0; and postdose time, ½, 1, 3, 8, and 24 hours) were collected for each PK assessment. Recommended quercetin dose was evaluated based on safety data and serial assessments of quercetin PKs for each cohort of 3 patients.

Plasma concentration data were analyzed by noncompartmental PK analysis, using the software package PKanalix (version 2023R, Lixoft, France) and a weighed least-squares algorithm. Details of generated PK parameter estimates, and PK analysis are described in the supplemental Methods.

ROS levels were measured by flow cytometry using a FACSCalibur after incubation with CM-H2DCFDA (Molecular Probes), a cell-permeable fluorescence dye that reacts with a broad spectrum of ROS.^65,66 Additionally, ROS levels in the bone marrow stem cell compartment that is CD34⁺ and CD34⁺/38⁻ were similarly measured in the flow-core at CCHMC.^65,66 Samples were run in duplicate, and an average value was used for final analyses.

See supplemental Methods for details of quantitative and qualitative assessment of hematopoietic stem cell (HSC) progenitors, along with monitoring of blood counts and growth and other endocrine parameters.

A conventional 3+3 design was used for the dose-finding study. Patient characteristics were summarized using median (range) for continuous variables and frequencies (percentages) for categorical variables. Four-, 6-, and 12-month continuous characteristic variables were compared with baseline using Wilcoxon sign-rank tests. Sample sizes are relatively small, which limited rigorous statistical testing, and some analyses are descriptive.

Demographics and disease characteristics of the dose-finding cohort are described in Table 1. Twelve patients with FA with a median age of 7 years (range, 3-21) were enrolled. No DLTs were observed at any of the quercetin doses. Median compliance during the first 4 months was 99% (range, 85%-100%).

Of 12 patients in the dose-finding study, 10 opted to participate in the voluntary continuation phase for 20 additional months. Patients were treated at the original assigned dose of quercetin for a total of 2 years. Two patients were unable to participate in the continuation phase, 1 because of the unavailability of the liquid formulation of quercetin, and a second because the need to proceed with HCT. Patients remained on the continuation phase for a median of 17 months (range, 4 months to 2 years) and had a median compliance of 99% (range, 58%-100%) during that time. Five patients completed the full 2 years of the continuation phase.

Quercetin was found to be safe at all dose levels and was well tolerated. No attributable severe adverse events (SAEs) occurred. All SAEs and any AEs possibly, probably, or definitely related to quercetin treatment are described in Table 2. Of 12 patients, 6 gained weight (3 with grade ≥2 weight gain) and 1 patient had a slight increase in his platelet function analysis that did not require any intervention. No additional grade ≥3 AEs attributed to the study drug were reported. Remaining unrelated AEs of every grade are described in supplemental Table 2. None of the patients discontinued quercetin before 4 months or during the continuation phase because of an AE. Additionally, no patients met stopping rules for lack of feasibility, including those who completed the continuation phase, confirming the feasibility of long-term quercetin administration.

Total plasma quercetin concentrations measured by tandem MS after the first dose and at 4 months showed wide interindividual variability in absorption (Figure 2A). The first 4 patients who received a liquid formulation were excluded from the final PK analyses. PK parameter estimates for the 8 patients, all aged <18 years, receiving powder formulation of quercetin on a twice-a-day schedule are summarized in Figure 2B. Data from all 12 patients are provided in the supplemental Figure 1. No significant differences were observed in the maximum concentration (Cmax), time to reach Cmax (Tmax), and half-life (t½) between day 1 after a single dose of quercetin and at 4 months after multiple doses given twice daily. The median steady-state area under the curve at the 4-month analysis was 7040 ng·h/mL (range, 2760-18 000; Figure 2B). Significantly higher apparent oral clearance (CL/F) and volume of distribution (Vd/F) were observed at 4 months than at the initial dose. Clearance increased from a median of 94 L/h (range, 37-120) at baseline to 165 L/h (range, 85-242) at 4 months (P < .01) and Vd/F increased from a median of 1380 L (range, 921-1860) at baseline to 2810 L (range, 1760-4470) at 4 months after quercetin (P < .01). Quercetin accumulation was not observed after 4 months of administration. The 3 patients who received a weight-adjusted dose with an adult maximum dose of 3000 mg/d (ie, patient numbers 7-9) reached median exposure levels of 7340 ng.h/mL (similar to reports in adults without FA, 6000 ng.h/mL [range, 2970-20880]⁶⁴; Figure 2C). The overall quercetin exposure did not increase significantly in the last 3 patients (patient numbers 10-12), who achieved a median exposure level of 6800 ng.h/mL (range, 5940-11700), despite receiving a 33% higher dose than the previous cohort (patient numbers 7-9; Figure 2C). These data indicate that further dose escalation would not result in increased quercetin exposure. The weight-adjusted daily maximum dose of 4000 mg/d was considered the recommended dose for treatment of patients with FA in the expansion cohort.

Of 12 (58%) patients in the phase 1 cohort, 7 experienced a decrease in PB ROS at 4 months and 7 of 9 patients (78%) showed decrease in PB ROS at 1 year. The final 3 patients treated at the daily maximum dose of 4000 mg/d achieved the highest and most consistent ROS reduction (Figure 3A). In this phase 1 cohort, the median total PB ROS levels decreased from 12142.5 (interquartile range [IQR], 8190.2-15681) relative fluorescent units (RFUs) at baseline to 6977 (IQR, 5293.8-8747.5) RFUs after 4 months of quercetin (reduction by 42.5%; N = 12; P = .23). In 9 patients with available results, median ROS levels decreased further to 3011 (IQR, 1231-8370) RFUs at 1 year, a 75% reduction compared with baseline (P = .04; Figure 3B).

Nineteen patients were enrolled on the expansion cohort (Figure 1). One patient never started the study drug. Demographics of this cohort are presented in Table 1. Eighteen patients completed 6 months of treatment using the weight-adjusted recommended dose of quercetin, with a median compliance of 99% (range, 70%-100%). All 18 patients voluntarily opted to participate in the continuation phase and take quercetin for an additional 6 months, to a total of 1 year (note: continuation phase for the expansion cohort was shorter, until 1-year time point). Patients remained on the continuation phase for a median of 6 months (range, 2-6). Two patients with severe marrow failure came off study during the continuation phase to move forward with HCT, and a third patient came off study to pursue additional investigational therapy. Compliance could not be confirmed reliably in 3 patients. Median compliance for the remaining 12 patients in the continuation phase was 99% (range, 42%-100%).

Quercetin was well tolerated in the expansion cohort. Of 18 patients, 3 gained weight (1 with grade ≥2 weight gain). Two patients developed diarrhea, and 1 patient developed constipation, all self-resolved without any intervention. Two patients developed gastroesophageal reflux during quercetin treatment, 1 of whom was placed on antacid treatment (Table 2). No additional attributable SAEs or AEs were identified. Remaining unrelated AEs of every grade are described in supplemental Table 2.

Correlative studies were analyzed in 21 patients who received the recommended dose of quercetin (analysis cohort; Figure 1). Changes in total PB ROS levels after quercetin are shown in Figure 3C. Median total PB ROS levels decreased from 13 806.0 (IQR, 10409.0-17211.0) RFUs at baseline to 10341.5 (IQR, 6095.5 to 18475.5) RFUs at 6 months, a 25% reduction (P = .67). At 1 year, median PB ROS decreased by 18% to a level of 11307.5 (IQR, 4723.9-15379.1) RFUs compared with baseline (P = .06; Figure 3C). PB ROS levels in individual patients of the analysis cohort are provided in supplemental Figure 2. ROS levels were also measured in the BM stem cell compartment. Univariate analyses in 18 patients with available data for BM ROS found that the median ROS levels in CD34⁺ BM cells decrease by 11% at 6 months and at 1 year after quercetin treatment compared with baseline (P = nonsignificant [NS]; supplemental Table 3). Similarly, median ROS levels in BM CD34⁺/38⁻ cells decreased by 24% at 6 months and by 22% at 1 year compared with baseline (P = NS; supplemental Table 3). Multivariate analysis was considered but was not feasible because of the small number of patients. Quantitative assessment of the bone marrow stem cell compartment and colony-forming units showed no significant change after quercetin treatment (P = NS; supplemental Table 4).

PB counts were followed up closely over the study period. Count trend for the analysis cohort are shown in Figure 4. Median platelet counts increased at 2 and 4 months, remained stable at 6 months, and increased again at 1 year (P = .06). Median absolute neutrophil count (ANC) improved at 2 and 4 months, remained stable at 6 months, and decreased at 1 year (P = .01). Median hemoglobin remained stable at 2 and 4 months and decreased at 6 months and 1 year (P < .001). Additionally, hematological responses were evaluated in patients with evidence of marrow failure at baseline according to an a priori analysis defined in the protocol. Of 21 patients in the analysis cohort, 15 had evidence of marrow failure at baseline. Of 15 patients, 8 (53%) with marrow failure met the predefined response criteria in terms of improvement in blood counts at different time points over the study period (Table 3). ANC increased in 7 patients, and 1 of these patients also showed a response in hemoglobin after quercetin treatment. An additional patient had a response in platelet counts (Table 3). Three patients (patient numbers 5, 6, and 7 in Table 3) showed evidence of a more sustained response in counts compared with the other patients in whom response was seen at limited time points. There was no correlation of hematological response with genotype or severity of marrow failure at baseline. None of the patients progressed to myelodysplastic syndrome or acute myeloid leukemia or acquired a new cytogenetic clone in serial bone marrow examinations with fluorescence in situ hybridization and karyotype testing.

Glucose tolerance tests (performed only for phase 1 cohort), serum glucose and insulin secretion, and insulin sensitivity were not affected by quercetin treatment (P = NS; data not shown). Quercetin also did not affect thyroid function or markers of growth.

This is, to our knowledge, the first study to describe PKs of a powerful antioxidant, quercetin, for children with FA. Importantly, we have proposed the recommended dose of quercetin that can be used in other studies. It is possible that baseline quercetin levels derived from normal diet were clinically important. To avoid this, we restricted intake of quercetin-containing foods around the time of PK analysis. Moreover, time 0 concentration and exposure accounted for up to 1.0% of the Cmax and mean area under the curve of 12 hours, respectively, making their impact on PK analytical results negligible. The half-life and other PK parameters for quercetin in our study were in line with the previous reports for adults without FA.^67,68 A significantly higher oral clearance (CL/F) and Vd/F were observed after multiple doses of quercetin than a single dose. Previous reports have suggested that enterohepatic recirculation may play a role in quercetin PKs.⁶⁴ Saturation of enterohepatic recirculation with high-dose quercetin could lower bioavailability and lead to an increase in CL/F and Vd/F. In addition, given the high protein binding of quercetin, saturation of protein binding capacity could lead to higher unbound fraction of quercetin concentrations and subsequent observed increase in quercetin clearance. No significant predictive covariates for clearance and Vd/F were identified in this study. Protein binding–related PK nonlinearity has been reported for other drugs ^69,70 and natural plant-based polyphenols.⁷¹ Our PK analysis indicated no further increase in quercetin exposure beyond that achieved by the dose recommended in the phase 1 dose-finding study. This is likely multifactorial, with rate-limiting absorption of quercetin by surpassing transporter kinetics, with enterohepatic circulation and first-pass metabolism being possible contributing causes.

Quercetin was well tolerated in this study as reported by others in normal volunteers.^42,60-63 The only possibly attributable AE was weight gain in 4 patients who received varying doses of quercetin. Our results align with previous reports from animal studies in which addition of quercetin helped prevent chemotherapy-induced weight loss. Mice in the chemotherapy plus quercetin group gained more weight compared with those receiving chemotherapy only.⁷² It is however unclear why this effect was seen in some and not all patients in our study. Overall, our results confirm that quercetin therapy is safe in patients with FA and biologically relevant blood levels can be achieved at the recommended dose.

An additional important question addressed by this study was whether treatment with quercetin has any impact on ROS levels in patients with FA. We found reduction in PB ROS levels and ROS levels in the bone marrow stem cell compartment in a subset of patients treated with the recommended dose of quercetin. In the phase 1 study, ROS levels declined at 4 months and 1 year after quercetin. We also report, an a priori-defined optimal response of 25% reduction in the PB ROS level compared with baseline for the analysis cohort. Animal studies have demonstrated that pharmaceutical reduction of ROS with antioxidants rescues HSC reconstitution in Fancc-knockout mice. Additionally, tumor necrosis factor-α–treated Fancc-knockout mice analyzed by HSC transplantation showed improved engraftment of transplanted FA HSCs in lethally irradiated recipient mice after antioxidant treatment^29,34.

We determined the clinical effectiveness of ROS reduction in our analysis cohort patients by measuring serial blood counts. Stable to slightly improved platelet count and a slow but consistent decline in ANC and hemoglobin were observed over the study period. A previous report that examined metformin for cytopenia (marrow failure) in FA, reported that 4 of 13 patients (31%) had a hematological response.¹⁹ 

Our data, using similar predefined criteria, show that 8 of 15 patients (53%) with cytopenia had a hematological response at some point after quercetin therapy, 3 of whom had a more sustained response in counts. However, interpretation of these data is complex. We note that small sample size related to the rarity of FA, differences in severity of baseline marrow failure, along with inherent count fluctuation in patients with FA limit assessment of response in both studies, and the results must therefore be viewed with caution.¹⁹ 

Our work has strengths and limitations. This is, to our knowledge, the first study to characterize quercetin PKs in pediatric patients. Our PK study was successful in identifying the recommended therapeutic dose of quercetin and further demonstrated safety and tolerability in a heterogenous cohort of patients with FA. Determination of efficacy was not a primary goal, but we did perform correlative studies by determining biological responses to the dosing of quercetin. The small number of patients eligible for enrollment because of the rarity of the disease is a challenge for all FA studies, as is natural variation in blood counts and in inflammatory markers such as ROS. Additionally, the impact of intercurrent infections on ROS levels and different time points for ROS assessment in the analysis cohort, might also have affected our results. It is also possible that to observe a clear clinical benefit, treatment needs to extend over several years or be initiated early in life. Randomized placebo-controlled studies would be optimal but would require a large sample size and prolonged treatment courses, making feasibility of such studies unlikely.

The authors thank the patients with Fanconi anemia and their families for participating in the study; and the authors thank the referring physicians.

This study was supported by RO1 from Office of Orphan Products Development, US Food and Drug Administration, R01 FD004383 (P.A.M) and an Aplastic Anemia and MDS International Foundation, Inc grant (P.A.M.); and research support was received from the Schubert Research Clinic at Cincinnati Children’s Hospital Medical Center (P.A.M.). Travel assistance for families was provided, in part, by the Fanconi Cancer Foundation (previously, Fanconi Anemia Research Fund).

Contribution: P.A.M. and S.M.D. conceptualized the study, designed the study, performed clinical research, analyzed results, and wrote the manuscript; S.L., S.E., E.M., and K. McIntosh assisted with study visit coordination, data collection, and contributed to the manuscript; D.L. and A.T.C. provided investigational and clinical pharmacy support for the study and contributed to the manuscript; K.D.R.S. and J.Z. designed/validated the pharmacokinetics (PK) assay, analyzed all the PK samples, and contributed to the manuscript; T.F. and K. Mizuno analyzed and interpreted the PK results and contributed to the manuscript; N.L. and K.L. performed reactive oxygen species (ROS) analysis; J.A.C. assisted with analysis and interpretation of ROS results, analyzed the hematopoietic colony assay results, and contributed to the manuscript; S.I.W. provided conceptual insights for study design, interpretation of data, and contributed to the manuscript; A.L. was responsible for statistical design, analysis, and contributed to the manuscript; T.C. performed statistical analyses and contributed to the manuscript; A.N. and K.C.M. assisted with study subject accrual, conduction of clinical study, and contributed to the manuscript; D.E. and J.H. designed and analyzed the endocrine studies and contributed to the manuscript; and all authors reviewed and edited the manuscript.

Conflict-of-interest disclosure: P.A.M. is a board member at Orthogon Therapeutics. D.E. receives research support from Dexcom and Sanofi. J.H. is a consultant for EMD Serono. K.C.M. receives research funding from Elixirgen Therapeutics and Incyte. K.D.R.S. holds equity in Asklepion Pharmaceuticals and Aliveris s.r.l.; and is a consultant for Mirum Pharmaceuticals. The remaining authors declare no competing financial interests.

Correspondence: Parinda A. Mehta, Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229; email: parinda.mehta@cchmc.org.