Phase I Clinical Trial of the Flavonoid Quercetin: Pharmacokinetics and Evidence for in Vivo Tyrosine Kinase Inhibition1

Vol. 2, 659-668, April 1996 Clinical Cancer Research 659

David R. Ferry,2 Anna Smith, Joy Malkhandi, tyrosine kinase activity, and evidence of antitumor activity David W. Fyfe, Philippa G. deTakats, was seen. David Anderson, Jim Baker, and David J. Kerr INTRODUCTION Cancer Research Campaign Institute for Cancer Studies. University of Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham Bl5 Flavonoids are found as either simple or complex glyco- 2TH, United Kingdom sides in all aerial plants (I ). Humans have been estimated to consume approximately 1 g flavonoidsiday, the most common flavonoid glycones found in the diet being quercitrin, turin, and ABSTRACT robinin (2, 3). Quercitrin and rutin are hydrolyzed to quercetin We have performed a Phase I clinical trial with the and robinin to kempferol by the 3-glucosidase activity of obli- naturally occurring flavonoid quercetin (3,3’,4’,5,7-penta- gate anaerobes in the gastrointestinal tract (4). A recent detailed hydroxyflavone). Quercetin has antiproliferative activity in estimation of free flavonoid consumption in Western diets found vitro and is known to inhibit signal transduction targets the richest sources of quercetin to be onions (347 mg/kg), apples including tyrosine kinases, protein kinase C, and phosphati- (36 mg/kg), and red wine ( 1 1 mg/kg; Ref. 5). Since the realiza- dyl inositol-3 kinase. Quercetin was administered by short tion that many folk medicines still in use contain flavones (2), i.v. infusion at escalating doses initially at 3-week intervals. interest in this class of compounds has intensified. The first dose level was 60 mg/rn2; at the 10th dose level of Quercetin (Fig. 1) has a wide range of biological activities 1700 mg/m2, dose-limiting nephrotoxicity was encountered, including inhibition of the NaKATPase (6), protein kinase C but no myelosuppression. At the preceding dose level of 1400 (7), tyrosine kinases (8), HIV reverse transcriptase (with a Km of mg/rn2, five patients were treated at 3-week intervals, and 0.08 p.M; Ref. 9), Ca2tATPase of sarcoplasmic reticulum (10), another eight patients were treated on a once-weekly sched- and pp60 kinase. Indeed, quercetin was probably the first ule; overall, 2 of 10 evaluable patients had renal toxicity, 1 at tyrosine kinase inhibitor to be described ( I 1), and is also re- grade 2 and 1 at grade 4. We therefore treated other patients ported to cause cell cycle arrest of human leukemic T cells (12) at 945 mg/m2 (eight at 3-week intervals and six at weekly and gastric cancer cells (13) in late G. More recently, quercetin intervals); 3 of 14 patients had clinically significant renal has been reported to induce apoptosis through a pathway in- toxicity, 2 patients with grade 2 and 1 patient with grade 3. volving heat shock proteins (14). Quercetin can down-regulate Patients treated on the weekly schedule did not have cumu- mutant p53 levels (15), and as mutant p53 can block apoptosis; lative renal impairment but did have a fall in the glomerular this is another mechanism through which quercetin could facil- filtration rate of 19 ± 8% in the 24 h after drug adminis- itate cell death. Quercetin is also a potent inhibitor of phosphati- tration. We recommend 1400 mg/m2 as the bolus dose, dyl-3 kinase ( 16) and l-phosphatidylinosotol-4 kinase (17), which may be given either in 3-week or weekly intervals, for important enzymes in proliferation signaling pathways ( I 8). Phase II trials. Quercetin has antiproliferative activity in vitro against Quercetin pharmacokinetics were described by a first- ovarian (19), breast (19), and stomach (20) cancer cell lines. In order two-compartment model with a median t#{189}a of 6 mm vivo synergy of quercetin with cisplatin against Walker lung and median t#{189}(1 of 43 mm. The median estimated clearance cancer xenografts in nude mice has been described (2 1 ). Fur- was 0.28 liter/minim2, and median volume of distribution at thermore, quercetin has antiproliferative activity against human steady state was 3.7 liter/rn2. In 9 of 11 patients, lymphocyte ovarian cancer primary cultures and can potentiate the action of protein tyrosine phosphorylation was inhibited following cisplatin ex vito (22). In primary cultures of human acute administration of quercetin at 1 h, which persisted to 16 h. myeloid leukemia, quercetin has been demonstrated to potenti- In one patient with ovarian cancer refractory to cisplatin, ate the cytotoxic action of 143-o-arabinofuranosylcytosine (23). following two courses of quercetin (420 mg/rn2), the CA 125 These activities of quercetin make it a promising candidate for had fallen from 295 to 55 units/mI, and in another patient Phase I evaluation in cancer patients. with hepatoma, the serum a-fetoprotein fell. In conclusion, Quercetin was administered in an ethanol vehicle to six quercetin can be safely administered by i.v. bolus at a dose normal human volunteers in 1975 at a fixed dose of 100 mg injection. The plasma levels achieved inhibited lymphocyte without side effects (24). We planned a dose escalation Phase I trial using 60 mg/m2 as the starting dose. We have developed a novel HPLC3 method of detecting quercetin in plasma (25), and

Received 8/3/95; revised 1 1/14/95: accepted I 2/1 1/95. I Supported by the Cancer Research Campaign. D. W. F. is a Cancer Research Campaign funded research fellow. P. G. d. T. is a senior 3 The abbreviations used are: HPLC, high-performance liquid chroma- Cancer Research Campaign clinical research fellow. tography; AUC, area under the curve; AFP, a-fetoprotein: lC), 50% 2 To whom requests for reprints should be addressed. inhibitory concentration: NO, nitrous oxide.

DMSO on the day of use and supplied in glass syringes. It was administered via the side arm of a polypropylene giving set through which RIMSO 50 (50% volume DMSO and 50% water) OH was being slowly injected. A total volume of 50 ml of RIMSO 50 was injected with each quercetin injection. This was essential to prevent precipitation of quercetin in the giving set. The rate of i.v. injection of quercetin at the starting dose of 60 mg/m2 was initially rapid over 30 s, but at doses above 945 mglm2, it was increased to 5 mm because of pain on injection. When the quercetin injection was completed, the drip was left in situ, and 0 200 ml saline were infused over the succeeding 30 mm. The starting dose of 60 mg/m2 was chosen on the basis of Fig. I Structure of quercetin. a previous normal volunteer study (24). Thereafter, doses were increased according to a modified Fibonacci regimen (Table 2), with three patients treated at each dose level if grade 3 or 4 toxicity was not seen. The maximum tolerated dose was con- in this article describe the results of a Phase I trial of quercetin sidered to have been reached when two of three patients or three with pharmacokinetic evaluation. In addition, because quercetin of six patients experienced dose-limiting toxicity. Dose-limiting is a tyrosine kinase inhibitor, we attempted to demonstrate that toxicity was defined as the production of grade 3 or 4 general a pharmacodynamic effect occurred in treated patients. toxicity, or grade 2 renal toxicity, cardiac toxicity, or neurotox- icity. PATIENTS AND METHODS Pharmacokinetics. Venous blood samples for pharma- Patients. Fifty-one patients were entered into this study, cokinetics were drawn at frequent intervals, placed in Li-heparmn the eligibility criteria was: microscopically confirmed diagnosis tubes, and within 15 rain separated by centrifugation. The of cancer no longer amenable to standard therapies; age 18-75 plasma was aspirated, frozen at -70#{176}C, and analyzed within 2 years; performance score of 2 on the WHO scale; life expect- weeks. In one patient, a sample of ascites was drawn before and ancy of at least 12 weeks; informed consent; no previous che- 60 mm after quercetin administration. Urine was collected for motherapy or immunotherapy in the preceding 3 weeks or 6 24 h before and after quercetin administration and stored at weeks for nitrosoureas, mitomycmn C, or extensive radiotherapy; -70#{176}Cbefore analysis. hemoglobin > 10 glliter, WBC count >4 X l09/liter, and plate- HPLC Determination of Quercetin in Plasma. Chro- let count >100 X lO9lliter; and reasonable serum biochemistry matographic analysis of flavonoids was performed using a Kon- with bilirubin <25 p.M. liver enzyme levels less than twice the tron HPLC system (Watford, United Kingdom) with a upper limit of normal, and serum creatinine 120 p.M. All p.-Bondapak C18 column equipped with a high-pressure mixing patients gave written informed consent, as approved by the solvent delivery system (HPLC 422), an automatic sample in- Ethics Committee of the Queen Elizabeth Hospital (Birming- jector (HPLC 465), and diode array spectrophotometric detector ham, United Kingdom). set to 375 mm (HPLC 440). System control, data collection, and Baseline studies before administration of quercetin includ- data evaluation were performed using an IBM PC with a 450 ed: history and physical examination, blood pressure, height, MT2 software data package (Kontron, Watford, United King- weight, performance status, blood count with differential, pro- dom). Standards and control samples were analyzed as previ- thrombin time, serum chemistry including bilirubin, alkaline ously described (25). phosphatase, aspartate aminotransferase, creatinine, urea, so- Pharmacokinetic Modeling. Quercetin plasma levels dium, and potassium, urinalysis for protein and sugar, electro- were modeled to a two-compartment open model using the cardiogram, chest X-ray, and, when appropriate, tumor mea- Statis package based on the Marqhardt algorithm. The data fitted surements. While on study, patients had a weekly review with better to a dual- than to a single-compartment model as judged clinical examination, blood count, and serum chemistry estima- by Akaike and Swartzmann’s criteria. The parameters derived tion. In the first part of this trial, patients were treated at 3-week from the model (A, a, B, and 3) allow calculation of drug intervals. Because myelosuppression was not evident with the clearance (mI/mn), volume of distribution at steady state (li- 3-week schedule, after the dose-limiting toxicity had been en- ters), and AUC. countered, weekly treatments were introduced to intensify drug Determination of Tyrosine Kinase Inhibition in Lym- delivery. phocytes. Twenty ml blood were taken at time intervals up to Quercetin Administration. Quercetin dihydrate was 16 h after quercetin administration. Blood specimens were im- supplied as a pale yellow powder of >99% purity by the Aldrich mediately placed on ice and within 30 mm layered onto 10 ml Chemical Company. It was formulated under sterile conditions ice-cold Lymphoprep (GIBCO) and centrifuged for 30 rain at in a laminar flow cabinet in analytical grade DMSO (Sigma). 500 X g at 4#{176}Cto separate the lymphocytes. The lymphocytes Quercetin was reconstituted at a concentration of 50 mg/ml for were aspirated, added to 40 ml ice-cold wash buffer [50 mti doses up to 945 mgim2 and at a concentration of 100 mg/mI for Tris-HCI (pH 7.4), 1 misi EDTA, 1 mM EGTA, 150 mr NaCl, higher doses. HPLC studies showed that quercetin remains and 100 p.M sodium o-vanadate], and centrifuged at 500 X g at stable when stored in ethanol or DMSO at 10 nmi over 21 days 4#{176}Cfor 10 mm. The supernatant was removed, and the pellet under air at 4#{176}C(data not shown). Quercetin was made up in was resuspended in another 20 ml ice-cold wash buffer and

Table 1 Patient characteristics Table 2 Number of patients and courses at each dose level Total patients S 1 Quercetin dose No. of No. of 3-Week treatment 37 Dose level (mg/rn2) patients treated courses Weekly treatment 14 1 60 3 9 Male:female 26:25 2 120 3 8 Median, yr (range) 55 (23-78) 3 200 3 5 Histological diagnosis 4 300 3 7 Large bowel 14 S 420 3 8 Ovarian 10 6 630 5 10 Pancreas 7 7 945 8 11 Melanoma 6 8 1400 5 8 Stomach S 9 2000 1 1 Hepatoma 3 10 1700 3 3 Non-small cell lung cancer 2 1 1 (weekly) 1400 8 18 Renal 2 12(weekly) 945 6 8 Other 2 Previous chemotherapy 40 Previous radiotherapy 9 No previous chemotherapy or radiotherapy 10 Performance status renal toxicity was encountered, quercetin was given without 0 6 prehydration. There was no hematological toxicity, neurological 38 toxicity, mucositis, or alopecia. 2 6 3 Toxicity during Bolus Injection. At the first dose level, the only toxicity was mild local pain on injection, which lasted for the duration of injection and for perhaps 10 s afterwards. Following injection there was a brief period of flushing that affected the whole body and was associated with sweating. This recentrifuged as described above. The supernatant was then lasted for approximately 3 mm, but was not associated with any removed, and the pellet was resuspended in 500 p.1 lysis buffer fall in blood pressure. [50 mM Tris-HC1 (pH 7.4), 1 mti EDTA, 1 ms EGTA, 100 p.M At and above quercetin doses of 945 mgim2, the concen- sodium o-vanadate, 100 p.M phenylmethylsulfonyl fluoride, 25 tration of quercetin injected was increased from 50 to 100 mM benzamine, 50 p.g/ml aprotonin, S p.giml leupeptin, and 2% mg/mi. This was done in an attempt to minimize the volume of Triton X-lOO] and incubated on ice for 15 mm prior to being DMSO injected. It was immediately apparent that at this dose centrifuged at 13,500 rpm for S mm. Lymphocyte lysates were level there was more severe pain on injection, which was local- stored at -80#{176}C. ized to the muscles in the upper arm on the side of the injection. Lymphocyte lysates from an individual patient had protein In an attempt to minimize this, the duration of quercetin slow content estimated to ensure equal loading of lanes and then were bolus injection was increased to S mm, and 10 mg morphine subjected to SDS-PAGE (8% acrylamide gel). Proteins were elixir were given before drug administration. Although no for- transferred onto polyvinylidene difluoride membranes (Milli- mal assessment of the pain score was made, this seemed more pore) over 4 h at 95 mV in blotting buffer comprised of 25 mii tolerable and decreased the subjective intensity of pain and Tris-HC1 (pH 8.3), 192 iiiM glycine, and 1% SDS in 20% (viv) flushing. methanol. After the transfer, nitrocellulose filters were washed To investigate whether bolus injection of the vehicle in PBS-Tween, and blocked for 2 h at 37#{176}Cinblocking buffer caused flushing and pain, two patients received 50 ml RIMSO containing 5% fat-free dried milk in PBS-Tween (without 50 solution by rapid injection. These patients did not experience azide). The membranes were then incubated with the horserad- any side effects, and it was therefore concluded that quercetin ish peroxidase-conjugated polyclonal antibody RC-20 (Affiniti) and not the vehicle was responsible for pain and flushing on at 4#{176}Covernight. After washing with PBS-Tween, the strips slow bolus injection. Quercetin administered to a patient at the were incubated in detection solution containing 50% luminoll 630-mgim2 dose level via a Hickman catheter resulted in no 50% Enhancer (Amersham ready-to-use ECL system) for 1 mm. pain but slight flushing on injection. The strips were then arranged in a transparency and exposed to During the dose escalation at 1400 mg/m2, patients also Hyperfilm-ECL (Amersham) for 40 mm. reported dyspnea during and for a few minutes after quercetin administration. This was difficult to quantify, but the patient RESULTS treated at 2000 mg/m2 had severe dyspnea which lasted S mm Patient Population. Table 1 lists the characteristics of following quercetin administration. We considered this unac- the patients treated in this study. The median age was 56 (range, ceptable and treated the next cohort of patients at 1700 mg/m2, 23-78) years. The tumor histologies of the treated patients were: at which dyspnea was less prominent. large bowel, 14; ovary, 10; pancreas, 7; melanoma, 6; stomach, Emesis. At the 10th dose level (1700 mg/m2), grade 4 5; renal, 2; hepatoma, 3; and non-small cell lung, 2. Two emesis was encountered. This occurred within minutes of quer- patients had other diagnoses: one with testicular teratoma and cetin administration, which has been the pattern in all patients one with gastrinoma. with quercetin-imduced vomiting. Subsequently, the 5-HT3 an- Dose Escalation and Toxicity. Table 2 shows the dose tagonist granisitron (3 mg) combined with dexamethasone (8 levels and number of patients treated at each dose level. Until mg) were given i.v. before quercetin. Comparing vomiting at

3 Quercetin-induced emesis” Table 4 Renal toxicity and dose level CTC vomiting grade CTC nephrotoxicity (creatinine) Dose level” With iv. granisitron (mg/m2) 0 1 2 3 4 Without iv. antiemetics and dexamethasone Dose level 630 4 0 0 1 0 945 3 3 1 1 - (mg/rn2) 0 1 2 3 4 0 1 2 3 4 0 1400” 3 1 0 0 0 630 8 1 1 0 0 2000 1 0 0 0 0 945 2 0 2 0 0 8 3 0 0 0 1700 0 1 1 0 1 1400 4 1 0 0 0 6 4 2 2 1 l400(weekly)c 3 1 1 0 1 1700 0 0 1 0 1 0 0 1 0 0 945 (weekly) 5 0 1 0 0 2000 1 0 0 0 0 ‘, No renal toxicity was encountered at dose levels below 630 “ The number of observations refers to courses for which data were mg/rn2.

obtained and amounts to 39 of 59, or 66%, of all potentially evaluable 1’ One patient not evaluable. courses. C Two patients not evaluable.

doses of 945 mgim2 and above, without antiemetics, 4 of 12 courses were associated with grade 2-4 vomiting versus 6 of 27 24-h creatinine clearances before and immediately after querce- with antiemetics. Thus, the antiemetics had no significant effect tin therapy. For 1 1 courses at 945 mgim2 and for 2 at 1400 (Table 3). mgim2, the mean pretreatment creatinine clearance was 69.5 Cardiovascular Toxicity. One patient with a history of mlimin, falling to 56.6 mllmin after quercetin treatment (P = hypertension had renal impairment (grade I), but her hyperten- 0.032, paired t test), i.e., a 19 ± 8% fall. Since no myelosup- sion was grade 3 with a blood pressure of 230/130 mm Hg when pression was seen with the 3-week schedule, the dose of 1400 renal impairment ensued. Her pretreatment serum creatinine was mg/rn2 was explored in a more intensive weekly schedule. All of 94 p.M and creatinine clearance was 69 ml/min. In the 24 h after the data given are for treatments given without iv. hydration. treatment, this fell to 46 ml/min, and I week later her serum The first patient treated on the weekly schedule at this dose had

creatinine was 234 p.M and creatinine clearance was 26 ml/min. an unresectable pancreatic cancer. Following quercetin therapy, Within 2 weeks, her creatinine clearance had normalized to 72 he had prolonged nausea and developed acute renal failure mI/mm, and her hypertension had been controlled with atenolol. (grade 4) requiring peritoneal dialysis. Another two of seven Two other cardiovascular events occurred, both in patients patients treated with this dose level on the weekly schedule had treated at 945 mgim2. One patient with a previous history of renal toxicity: one patient had grade 1 and one patient had angina developed central chest pain 24 h after the second course grade 2. of quercetin and died on the second day after treatment. Another Four patients on the weekly schedule had three or more patient developed grade 3 nephrotoxicity after the second course courses of quercetin. Fig. 2B shows pretreatment creatinine of quercetin, developed back pain, and died in heart failure 16 clearance versus day on study. There was no cumulative fall in days after the second course. A postmortem examination re- creatinine clearance with multiple weekly treatments, indicating vealed aortic dissection extending beyond the renal arteries. that any nephrotoxicity due to quercetin was transient and Renal Toxicity of Quercetin. Three patients were reversible within 7 days. treated at 1700 mg/rn2, all of whom experienced renal toxicity, Overall, 2 of 10 patients who were treated at 1400 mgim2 with elevation of serum creatinine (Table 4). These patients also had clinically significant nephrotoxicity (1 patient with grade 4, experienced nausea and vomiting within minutes of the querce- and 1 patient with grade 2). Since this had occurred with the first tin injection. In one patient (Q26) treated at 1 700 mgim2, nausea course of quercetin, we therefore enrolled an additional six was grade 4 (Table 3) and more prolonged. This may be because patients at the preceding dose level of 945 mg/rn2 weekly. At this patient developed grade 4 renal toxicity, with serum creat- 945 mgim2, eight patients were treated at 3-week and 6-week mine reaching a peak of 2145 p.M I I days after treatment, which intervals. Overall, combining data for both schedules, 3 of 14 returned to normal by day 34 (Fig. 2A). Two other patients were patients developed clinically significant renal impairment, 2 treated at this level: one experienced grade 2 renal toxicity and developed grade 2, and 1 developed grade 3. the other grade 1 toxicity, but in patient Q27 the serum creati- Abrogation of Renal Toxicity with i.v. Hydration. One nine remained elevated at 246 p.M on day 34 posttreatment, and patient treated at 630 mg/m2 developed grade 3 renal toxicity, in patient Q28 the serum creatinine was elevated at 149 p.M on with elevation of the serum creatinine to 420 p.M by day 10. By day 28. None of the patients who experienced renal toxicity had day 21 this patient’s serum creatinine had normalized (Fig. 2C). evidence of nephritis, infection, or obstructive uropathy on This patient was keen for additional therapy and was therefore ultrasound scanning of the abdomen. retreated, but with prehydration of 1 liter normal saline given Dose Escalation with Weekly Treatment and Nephro- over I h, and after quercetin treatment, 0.5 liter 5% dextrose was toxicity. At the highest doses of quercetin administered (2000 given; there was no fall in creatinine clearance. The next course and 1700 mgim2), myelosuppression was not seen, but there in this patient was given at 945 mg/rn2 with hydration, and no were dose-limiting toxicities. We therefore attempted to dose renal toxicity ensued. In other patients, hydration was given intensify by using a weekly schedule. During this weekly sched- before bolus injection of quercetin at 945 mg/rn2, and the ule, a concerted effort was made to monitor renal function with pretreatment clearance was 82.3 ± 6.7 mI/mm. In the 24 h after

i 10000 1 -#{149}-- Ursa -- 0- - Creatlnlne 1700 #{163}:-Q 1000 00.

. 100 0 10 I C 0 1 0 -O--Q38 I- 0 0.1 1_b 0 10 20 30 40 0 Day on study 0.01 0 40 80 120 160 200 , 10 Time (mm) : 200 Fig. 3 Pharmacokinetics of quercetin. In the key, the first number 160 refers to the patients trial number and the second to the dose in mg/rn2. :.. -.--Q35

. 120

Reduction in Serum Potassium. For 38 of S 1 patients on whom data were available, there was a statistically signifi- z; 80 cant fall in serum potassium, comparing day of treatment (4.32 0 .E 40 ± 0.07 mM) with day 8 posttreatment (3.98 ± 0. 10 mM P C 0.006). We therefore analyzed the pre- and post-quercetin urine 0 - 0 II3I040 specimens for potassium and sodium content. In 10 specimens I- a 0 10 20 available for analysis, the mean 24-h potassium excretion before Day on study treatment was 46.8 ± 4 mmol and after quercetin was 50.9 ± 6 mmol (P = 0.28). Values for sodium were: pretreatment, 76 ± 20 mmol/day; after, 80.9 ± 1 7 mmol/day (P = 0.43). Thus, no - -4-- Ssrum crsatlnlne (pM) significant changes in urine potassium or sodium excretion were --0- - Crsatlnlna clsaranci (mi/mm) detected. 630 630 945 Pharrnacokinetics. The data from I I patients were fitted 1000 0 to a two-compartment model. The distribution half-life was C $ 0.7-7.8 mm, with a median of 6 mm. The elimination half-life C was 3.8-86 mm, with a median of43 mm (Fig. 3 and Table 5). 0 0 The clearance was 0.23-0.84 liter/minIm2, with a median of

- - -- 0.28, and the median volume of distribution was 3.7 literim2. U 100 - There was an excellent correlation of 0.89 between the dose in E mg/m2 and AUC. There was no obvious relationship between 0 toxicity and kinetic parameters. Cl) C ,Y The serum levels achieved immediately after injection of 10 -10 20 50 8 0 1 1 0 quercetin were in the range of 200-400 p.M at 945 mg/rn2, with Day on study serum levels above 1 p.M being maintained up to 4 h (Fig. 3). The clearance is in the range of hepatic blood flow, and at early time points after quercetin injection, new peaks appear in the Fig. 2 Renal toxicity of quercetin. A. reversible grade 4 renal toxicity HPLC trace which may be quercetin metabolites (data not in patient Q26 treated at 1700 mg/rn2. Arrow, day of treatment. B, lack shown). In one patient, ascites fluid levels of quercetin were of cumulative nephrotoxicity in patients treated weekly. Both patients Q3S and Q38 were treated at 1400 mg/rn2. C, effect of prehydration on determined 60 mm after a dosage of 945 mg/rn2. The level in nephrotoxicity. Arrows, day of treatment at indicated doses in mg/rn2. ascitic fluid was 0.34 p.M. which is 20% of that of a parallel On day 0, quercetin was given without hydration; on days 35 and 78, blood specimen. with hydration, as described in the text. Quercetin concentration was estimated in urine from eight patients in fourteen 24-h posttreatment urine samples: seven patients were treated at 945 mg/rn2 and one at 1400 mg/m2 for quercetin treatment, the creatinine clearance was 79.5 ± 19.5 one course and then at 945 mg/rn2 for another four courses. The ml/min. Thus, a simple outpatient hydration schedule abrogated mean percentage of administered quercetin excreted unchanged falls in creatinine clearance. in the urine was 1 .97 ± 0.66% (range, 0.03-7.6%), with a range

Ta ble 5 Pharma cokineti c modeling 600 Dose AUC t112a t1123 Clearance Vd Patient (mg/rn2) (p.g/mllmin) (mm) (mm) (mI/mm) (liter)

3 60 187 3.5 NM” 0.320 1.61 0 3a 420 2760 2.0 33 0.152 4.00 : 400 7 120 100 2.0 NM 1.200 3.50 9 200 445 0.7 5.6 0.500 1.50 10 300 355 2.5 NM 0.840 3.10 11 300 1280 2.8 45 0.230 10.5 12 300 803 3.2 86 NM 18.2 200 13 420 1508 3.5 64 0.280 6.98 14 420 1185 2.5 8 0.345 1.78 15 420 528 1.5 3.8 0.800 2.20 16 630 2369 4.4 60 0.270 3.92 0 17 630 1285 2.9 49 0.490 3.70 0 50 100 150 200 250 18 630 2579 7.8 33 0.240 3.10 19 945 4139 2.9 26 0.23 2.80 Day on study 20 945 2633 3.0 43 0.26 3.90 a Patient retreated as part of a Phase I trial of quercetin combined with carboplatin.4 b NM, not modeled better by two-compartment kinetics; volume of distribution at steady state. 400 I I I 420 420 500 + AUC4 . 4 300 in concentration from 3.3 to 199 p.M, indicating considerable interpatient variability in quercetin excretion. There was no correlation between fall in creatinine clearance and quercetim 200 concentration. Antitumor Activity. No patients achieved conventional radiological responses according to WHO criteria; however, one 4 C.) 100 patient with metastatic hepatocellular carcinoma, who was treated at the first dose level (60 mg/rn2), had a sustained fall in B serum AFP and alkaline phosphatase (Fig. 4A). Another patient 0 with stage 4 metastatic ovarian cancer with extensive pelvic -50 0 50 100 150 disease and liver metastases had completed six courses of cy- Day on study clophosphamide (750 mg/rn2) and cisplatin (75 mg/rn2) 3 months before entry into the trial. Her response to this chemo- Fig. 4 Fall in tumor markers following quercetin therapy. A, patient therapy was minor, and her serum CA 125 was rising progres- Q03 with hepatocellular carcinoma and fall in AFP following treatment with quercetin at 60 mg/rn2 (arrows). B, patient QlS with ovarian cancer sively, reaching a level of 290 units/mI. Following treatment and fall in CA 125. The third and fourth treatments were administered with two courses of quercetin at 420 mg/rn2, her CA 125 fell to with carboplatin at AUC 4 as part of a parallel Phase I trial.4 55 units/ml (Fig. 4B). This patient was treated with three additional courses of quercetin at 500 mg/rn2 [combined with carboplatin AUC 4 (26) as part of a trial of quercetmn plus carbo- vehicle, DMSO, caused some hemolysis, which was most pro- platin],4 and 6 months after going on study her CA 125 was 45 units/mi, fulfilling recently suggested criteria for marker re- nounced in the samples obtained up to 30 mm after quercetin sponse in this disease (27). administration (Fig. 5, A and B) but which had largely disap- In Vivo Inhibition of Lymphocyte Tyrosine Kinase Ac- peared at the 1 h and subsequent time points. Despite rigorous washing procedures, the hemolysis contaminated the lympho- tivity. In 1 1 patients, lymphocyte tyrosine kinase activity was investigated before and at times up to 16 h after quercetin cyte lysates, resulting in a wide phosphotyrosine band of mo- administration (Table 6 and Fig. 5). In 2 of 1 1 patients, phos- lecular weight approximately Mr 20,000, which was clearly photyrosine-banding patterns were unchanged up to 60 mm after discernible from the bands attenuated by quercetin. Attenuation quercetin administration. However, for time points l h in all of phosphotyrosine bands occurred at all dose levels (Fig. 5, C-H), and the number of bands inhibited increased with time. In evaluated samples, phosphotyrosine bands of between Mr 45,000 and 60,000 were attenuated or eliminated following one patient, inhibition of phosphotyrosine banding was apparent up to 16 h after treatment (Fig. SG). quercetin treatment (Table 6). At 2 h posttreatment, inhibition of phosphotyrosine banding occurred in five of five patients. The DISCUSSION The aberrant expression of protein tyrosine kinases acti- vated by peptide growth factors can override growth regulatory

4 D. R. Ferry, D. Fyfe, A. Smith, J. Malkhandi, J. Baker, and D. J. Kerr. control and lead to cancer (28, 29). Nonreceptor tyrosine ki- Phase I trial of quercetin plus carboplatin, manuscript in preparation. nases, such as ppsrc, have SH2 domains which direct binding

Table 6 Inhibitio n of tyrosine kinase in patient lymphocytes

Phosphotyrosine bands

Quercetin

Q2l 945 + nd - nd nd Q22 1400 + + nd nd nd Q22 1400 + + nd nd nd Q22 1400 + + nd nd nd Q23 1400 + + nd nd nd Q23 1400 + + nd nd nd Q23 1400 + + nd nd nd

Q27 1700 + nd - - nd

Q28 1700 + + - - nd

Q32 1400 + nd - - nd

Q4l 945 + + - - -(4hr)

Q48 945 + nd - - - (3& l6hr) a , present; -, absent. b nd, not determined.

to phosphotyrosines of receptor tyrosine kinases, further ampli- may be a possibility. The pain was significantly reduced by fying and diversifying the growth signals. Thus, it is not sur- prolonging the infusion time and using 10 mg morphine elixir. prising that the development of tyrosine kinase inhibitors, tyr- During the injection of quercetin, all of the patients expe- phostims, is an attractive concept and a very active area (30). The rienced transient flushing for up to S mm. This was not associ- first tyrphostin discovered was the flavonoid quercetin, which ated with a fall in blood pressure, and must therefore be related

was found to inhibit ppJSC (11). Perhaps, it is therefore appro- to dilation of blood vessels smaller than resistance vessels. priate that quercetin should be the first tyrphostin to be system- Quercetin is known to have vasodilator effects, inhibiting the atically tested in a Phase I cancer trial. spontaneous myogenic contractions of rat portal vein with an

Quercetin was administered to six normal volunteers in IC50 of 9 p.M and reversing phorbol l2-myristate 13-acetate- 1975; at that time it was considered a drug with potentially induced aortic contractions with an IC50 of 1 1 p.M (33), sug- beneficial vascular effects (24). This previously published work gesting involvement of protein kimase C. Other biological ac- provided us with a safe starting dose in the absence of conven- tivities of quercetim may have contributed to the vasodilator tional preclinical animal toxicity. effect, including inhibition of phosphodiesterases (34). The limit of solubility for quercetin in 500 ml 10% ethanol No clinically significant toxicities were seen until the lOh is 100 mg, and severe alcohol intoxication would result if higher dose level (1700 mg/rn2) when renal toxicity, manifesting as doses were used. For this reason, we pursued alternative formu- elevation of serum creatinine, occurred. In one patient, this was lations which would allow dose escalation. The solubility of grade 4 reaching 2145 p.M. but without dialysis, serum creati- quercetin in DMSO at room temperature is 150 mg/ml. These nine normalized within 34 days. The mechanism of renal tox- questions then arise, how much DMSO can be administered to icity has not yet been elucidated, possibilities include alteration humans safely, and what are the potential complications? of renal blood flow or direct renal tubular damage. It is recog- DMSO is reported to be used i.v. to lower raised intracranial nized that differentiating between these mechanisms is difficult, pressure before emergency meurosurgical procedures (3 1). The requiring invasive techniques (35). One potential biochemical doses used were initially 1 g/kg infused as a 20% solution, but mechanism is impairment of NO synthase which generates the doses as high as 8 g/kg/day could be given safely. DMSO is vasodilator NO. Quercetim inhibits NO synthase with an IC50 of recognized as causing mild hemolysis, and the higher the con- 220 p.M (36). Even small reductions in the blood flow to the centration of DMSO the more hemolysis is observed (32). renal medulla, which is in a state of incipient hypoxia due to its However, administration of a 40% solution of DMSO at a dose high metabolic rate, can lead to ischemic damage, and, if this of 1 g/kg i.v. over 20 mm is reported to be well tolerated (32). persists, lead to acute tubular necrosis (37). Quercetin-induced At a quercetin dose of 1400 mg/m2, approximately 0.5 glkg, of renal impairment was investigated intensively in patients treated DMSO is given as the vehicle, which is one half of the amount on the weekly schedule. In these patients, 24-h creatinine clear- previously reported as well tolerated. ance was estimated before and after quercetmn administration. On administration of quercetin, the patients noted pain, Quercetin caused a 20% reduction in creatimine clearance, which principally in the biceps muscle on the side of injection. This recovered within 7 days. lasted throughout the injection and for a few minutes afterward. When we encountered dose-limiting grade 4 renal toxicity This pain was more severe at higher doses of quercetin and was at 1700 mg/rn2, we treated a total of five patients at 1400 mg/rn2 not due to the vehicle. The speed of onset makes ischemic pain on the 3-week schedule, no myelosuppression was seen. We perhaps unlikely, and the direct stimulation of noiceptive fibers then attempted to increase dose intensity by giving 1400 mg/rn2

Molecular B C E weight 945 mg/rn2 945 mg/rn2 Molecuhn D 945mg/rn2 1400mg/rn2 weight 60mg/rn2

97 116

45 29

pre 10mm pre 20mm

pre lh pre lh pre lh 2h

F G Molecular 945 mg/rn2 945 mg/rn2 weight

205 S....

116

45 4’ ‘S 0

Pr.

Fig. 5 1 visa tyrosine kinase inhibition with quercetin. Western blot analysis of pre- and postquercetin peripheral blood leukocytes using antiphosphotyrosine monoclonal antibody RC 20. A, DMSO caused hemolysis of patient samples, resulting in a wide band of low molecular weight, 26 p.g lysate protein/lane. B, DMSO-induced hemolysis was rapidly cleared, a reduction being apparent at 20 mm after quercetin, 35 p.g lysate protein/lane. C-E, I h after treatment, hemolysis was no longer apparent, with 45, 23, and 62 p.g lysate proteinllane, respectively. Quercetin attenuated phosphotyrosine bands of Mr 45,00060,000. The doses of quercetin and times after drug administration are indicated. F and G. 48 and 39 p.g lysate protein/lane, respectively. Time courses from two patients indicate that quercetin attenuation of phosphotyrosine bands persisted for up to 16 h after treatment.

on a weekly schedule, and encountered grade 4 toxicity in the sponse in melanoma patients treated with dirnethylphenyltria- first patient treated. In the next seven patients treated at this dose zine CB 10-227 (39), and in ovarian cancer treated with level, one patient had grade 2 renal toxicity, which was consid- cisplatin, the formation of platinum-DNA adducts in blood ered sufficiently serious to be regarded as dose limiting. We lymphocytes strongly predicts for response (40). therefore attempted to dose intensify using the previous dose We were able to demonstrate that before quercetin admim- level of 945 mg/rn2 weekly and encountered significant neph- istration the majority of patients had detectable phosphotyrosine rotoxicity in one of six patients, which we felt represented an bands, particularly in the molecular weight range Mr 40,000 acceptable level of toxicity. 60,000, and that quercetin treatment abrogated some of these Additional patients were treated at 945 mg/rn2 on a 3-week bands. This approach is open to criticism. First, the tyrosine schedule as part of a parallel trial to combine carboplatin and kinases of lymphocytes may be different than those in tumors. quercetin, and surprisingly encountered grade 3 renal toxicity. Second, we cannot be certain that tyrosine kinase inhibition is We therefore retreated the next patient at 630 mg/m2, and this the mechanism through which quercetin exerts its antiprolifera- patient had reversible grade 3 toxicity. At this stage we were tive effects. For these reasons, we have not pursued the charac- concerned that the nephrotoxicity might be unpredictable. This terization of the identified phosphotyrosine bands. patient however normalized renal function by day 14 and was This is the first reported Phase I clinical trial with a novel keen to continue therapy. She received prehydratiom with 1 liter normal saline followed by posthydration with 0.5 liters 5% class of antiturnor agents, tyrphostims, in humans. We are cur- dextrose. 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Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 1996 American Association for Cancer Research. Phase I clinical trial of the flavonoid quercetin: pharmacokinetics and evidence for in vivo tyrosine kinase inhibition.

D R Ferry, A Smith, J Malkhandi, et al.

Clin Cancer Res 1996;2:659-668.

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