phase iclinical trial oftheflavonoid quercetin: pharmacokinetics andevidence for invivo … ·...

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

Phase I Clinical Trial of the Flavonoid Quercetin: Pharmacokinetics

and Evidence for in Vivo Tyrosine Kinase Inhibition1

David R. Ferry,2 Anna Smith, Joy Malkhandi,

David W. Fyfe, Philippa G. deTakats,

David Anderson, Jim Baker, and David J. Kerr

Cancer Research Campaign Institute for Cancer Studies. University of

Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham Bl5

2TH, United Kingdom

ABSTRACT

We have performed a Phase I clinical trial with the

naturally occurring flavonoid quercetin (3,3’,4’,5,7-penta-

hydroxyflavone). Quercetin has antiproliferative activity in

vitro and is known to inhibit signal transduction targetsincluding tyrosine kinases, protein kinase C, and phosphati-

dyl inositol-3 kinase. Quercetin was administered by short

i.v. infusion at escalating doses initially at 3-week intervals.

The first dose level was 60 mg/rn2; at the 10th dose level of

1700 mg/m2, dose-limiting nephrotoxicity was encountered,

but no myelosuppression. At the preceding dose level of 1400

mg/rn2, five patients were treated at 3-week intervals, and

another eight patients were treated on a once-weekly sched-

ule; overall, 2 of 10 evaluable patients had renal toxicity, 1 at

grade 2 and 1 at grade 4. We therefore treated other patients

at 945 mg/m2 (eight at 3-week intervals and six at weekly

intervals); 3 of 14 patients had clinically significant renal

toxicity, 2 patients with grade 2 and 1 patient with grade 3.

Patients treated on the weekly schedule did not have cumu-lative renal impairment but did have a fall in the glomerular

filtration rate of 19 ± 8% in the 24 h after drug adminis-

tration. We recommend 1400 mg/m2 as the bolus dose,

which may be given either in 3-week or weekly intervals, for

Phase II trials.

Quercetin pharmacokinetics were described by a first-

order two-compartment model with a median t#{189}a of 6 mm

and median t#{189}(1 of 43 mm. The median estimated clearancewas 0.28 liter/minim2, and median volume of distribution at

steady state was 3.7 liter/rn2. In 9 of 11 patients, lymphocyteprotein tyrosine phosphorylation was inhibited following

administration of quercetin at 1 h, which persisted to 16 h.

In one patient with ovarian cancer refractory to cisplatin,following two courses of quercetin (420 mg/rn2), the CA 125

had fallen from 295 to 55 units/mI, and in another patient

with hepatoma, the serum a-fetoprotein fell. In conclusion,

quercetin can be safely administered by i.v. bolus at a dose

injection. The plasma levels achieved inhibited lymphocyte

tyrosine kinase activity, and evidence of antitumor activity

was seen.

INTRODUCTION

Flavonoids are found as either simple or complex glyco-

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

robinin (2, 3). Quercitrin and rutin are hydrolyzed to quercetin

and robinin to kempferol by the �3-glucosidase activity of obli-

gate anaerobes in the gastrointestinal tract (4). A recent detailed

estimation of free flavonoid consumption in Western diets found

the richest sources of quercetin to be onions (347 mg/kg), apples

(36 mg/kg), and red wine ( 1 1 mg/kg; Ref. 5). Since the realiza-

tion that many folk medicines still in use contain flavones (2),

interest in this class of compounds has intensified.

Quercetin (Fig. 1) has a wide range of biological activities

including inhibition of the Na�K�ATPase (6), protein kinase C

(7), tyrosine kinases (8), HIV reverse transcriptase (with a Km of

0.08 p.M; Ref. 9), Ca2tATPase of sarcoplasmic reticulum (10),

and pp60� kinase. Indeed, quercetin was probably the first

tyrosine kinase inhibitor to be described ( I 1), and is also re-

ported to cause cell cycle arrest of human leukemic T cells (12)

and gastric cancer cells (13) in late G�. More recently, quercetin

has been reported to induce apoptosis through a pathway in-

volving heat shock proteins (14). Quercetin can down-regulate

mutant p53 levels (15), and as mutant p53 can block apoptosis;

this is another mechanism through which quercetin could facil-

itate cell death. Quercetin is also a potent inhibitor of phosphati-

dyl-3 kinase ( 16) and l-phosphatidylinosotol-4 kinase (17),

important enzymes in proliferation signaling pathways ( I 8).

Quercetin has antiproliferative activity in vitro against

ovarian (19), breast (19), and stomach (20) cancer cell lines. In

vivo synergy of quercetin with cisplatin against Walker lung

cancer xenografts in nude mice has been described (2 1 ). Fur-

thermore, quercetin has antiproliferative activity against human

ovarian cancer primary cultures and can potentiate the action of

cisplatin ex vito (22). In primary cultures of human acute

myeloid leukemia, quercetin has been demonstrated to potenti-

ate the cytotoxic action of 143-o-arabinofuranosylcytosine (23).

These activities of quercetin make it a promising candidate for

Phase I evaluation in cancer patients.

Quercetin was administered in an ethanol vehicle to six

normal human volunteers in 1975 at a fixed dose of 100 mg

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

Cancer Research Campaign clinical research fellow.2 To whom requests for reprints should be addressed.

3 The abbreviations used are: HPLC, high-performance liquid chroma-

tography; AUC, area under the curve; AFP, a-fetoprotein: lC�), 50%

inhibitory concentration: NO, nitrous oxide.

Research. on January 25, 2021. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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0

Fig. I Structure of quercetin.

660 Phase I Clinical Trial of the Flavonoid Quercetin

OH

in this article describe the results of a Phase I trial of quercetin

with pharmacokinetic evaluation. In addition, because quercetin

is a tyrosine kinase inhibitor, we attempted to demonstrate that

a pharmacodynamic effect occurred in treated patients.

PATIENTS AND METHODS

Patients. Fifty-one patients were entered into this study,

the eligibility criteria was: microscopically confirmed diagnosis

of cancer no longer amenable to standard therapies; age 18-75

years; performance score of �2 on the WHO scale; life expect-

ancy of at least 12 weeks; informed consent; no previous che-

motherapy or immunotherapy in the preceding 3 weeks or 6

weeks for nitrosoureas, mitomycmn C, or extensive radiotherapy;

hemoglobin > 10 glliter, WBC count >4 X l09/liter, and plate-

let count >100 X lO9lliter; and reasonable serum biochemistry

with bilirubin <25 p.M. liver enzyme levels less than twice the

upper limit of normal, and serum creatinine �120 p.M. All

patients gave written informed consent, as approved by the

Ethics Committee of the Queen Elizabeth Hospital (Birming-

ham, United Kingdom).

Baseline studies before administration of quercetin includ-

ed: history and physical examination, blood pressure, height,

weight, performance status, blood count with differential, pro-

thrombin time, serum chemistry including bilirubin, alkaline

phosphatase, aspartate aminotransferase, creatinine, urea, so-

dium, and potassium, urinalysis for protein and sugar, electro-

cardiogram, chest X-ray, and, when appropriate, tumor mea-

surements. While on study, patients had a weekly review with

clinical examination, blood count, and serum chemistry estima-

tion. In the first part of this trial, patients were treated at 3-week

intervals. Because myelosuppression was not evident with the

3-week schedule, after the dose-limiting toxicity had been en-

countered, weekly treatments were introduced to intensify drug

delivery.

Quercetin Administration. Quercetin dihydrate was

supplied as a pale yellow powder of >99% purity by the Aldrich

Chemical Company. It was formulated under sterile conditions

in a laminar flow cabinet in analytical grade DMSO (Sigma).

Quercetin was reconstituted at a concentration of 50 mg/ml for

doses up to 945 mgim2 and at a concentration of 100 mg/mI for

higher doses. HPLC studies showed that quercetin remains

stable when stored in ethanol or DMSO at 10 nmi over 21 days

under air at 4#{176}C(data not shown). Quercetin was made up in

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)

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

200 ml saline were infused over the succeeding 30 mm.

The starting dose of 60 mg/m2 was chosen on the basis of

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-

sidered to have been reached when two of three patients or three

of six patients experienced dose-limiting toxicity. Dose-limiting

toxicity was defined as the production of grade 3 or 4 general

toxicity, or grade 2 renal toxicity, cardiac toxicity, or neurotox-

icity.

Pharmacokinetics. Venous blood samples for pharma-

cokinetics were drawn at frequent intervals, placed in Li-heparmn

tubes, and within 15 rain separated by centrifugation. The

plasma was aspirated, frozen at -70#{176}C, and analyzed within 2

weeks. In one patient, a sample of ascites was drawn before and

60 mm after quercetin administration. Urine was collected for

24 h before and after quercetin administration and stored at

-70#{176}Cbefore analysis.

HPLC Determination of Quercetin in Plasma. Chro-

matographic analysis of flavonoids was performed using a Kon-

tron HPLC system (Watford, United Kingdom) with a

p.-Bondapak C18 column equipped with a high-pressure mixing

solvent delivery system (HPLC 422), an automatic sample in-

jector (HPLC 465), and diode array spectrophotometric detector

set to 375 mm (HPLC 440). System control, data collection, and

data evaluation were performed using an IBM PC with a 450

MT2 software data package (Kontron, Watford, United King-

dom). Standards and control samples were analyzed as previ-

ously described (25).

Pharmacokinetic Modeling. Quercetin plasma levels

were modeled to a two-compartment open model using the

Statis package based on the Marqhardt algorithm. The data fitted

better to a dual- than to a single-compartment model as judged

by Akaike and Swartzmann’s criteria. The parameters derived

from the model (A, a, B, and �3) allow calculation of drug

clearance (mI/mn), volume of distribution at steady state (li-

ters), and AUC.

Determination of Tyrosine Kinase Inhibition in Lym-

phocytes. Twenty ml blood were taken at time intervals up to

16 h after quercetin administration. Blood specimens were im-

mediately placed on ice and within 30 mm layered onto 10 ml

ice-cold Lymphoprep (GIBCO) and centrifuged for 30 rain at

500 X g at 4#{176}Cto separate the lymphocytes. The lymphocytes

were aspirated, added to 40 ml ice-cold wash buffer [50 mt�i

Tris-HCI (pH 7.4), 1 misi EDTA, 1 mM EGTA, 150 mr� NaCl,

and 100 p.M sodium o-vanadate], and centrifuged at 500 X g at

4#{176}Cfor 10 mm. The supernatant was removed, and the pellet

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

S 13714

26:25

55 (23-78)

Dose levelQuercetin dose

(mg/rn2)No. of

patients treatedNo. of

courses

1

2

3

60

120

200

3

3

3

9

8

5

14

107

6

S

3

2

22

40

4

S

67

8

910

1 1 (weekly)12(weekly)

300420

630945

1400200017001400945

3

3

58

5

1

38

6

7

8

1011

8

1

318

8

910

638

62

3

Clinical Cancer Research 661

Total patients3-Week treatmentWeekly treatmentMale:female

Median, yr (range)

Histological diagnosisLarge bowelOvarianPancreasMelanoma

StomachHepatomaNon-small cell lung cancerRenalOther

Previous chemotherapy

Previous radiotherapyNo previous chemotherapy or radiotherapy

Performance status0

recentrifuged as described above. The supernatant was then

removed, and the pellet was resuspended in 500 p.1 lysis buffer

[50 mM Tris-HC1 (pH 7.4), 1 mt�i EDTA, 1 ms� EGTA, 100 p.M

sodium o-vanadate, 100 p.M phenylmethylsulfonyl fluoride, 25

mM benzamine, 50 p.g/ml aprotonin, S p.giml leupeptin, and 2%

Triton X-lOO] and incubated on ice for 15 mm prior to being

centrifuged at 13,500 rpm for S mm. Lymphocyte lysates were

stored at -80#{176}C.

Lymphocyte lysates from an individual patient had protein

content estimated to ensure equal loading of lanes and then were

subjected to SDS-PAGE (8% acrylamide gel). Proteins were

transferred onto polyvinylidene difluoride membranes (Milli-

pore) over 4 h at 95 mV in blotting buffer comprised of 25 mi�i

Tris-HC1 (pH 8.3), 192 iiiM glycine, and 1% SDS in 20% (viv)

methanol. After the transfer, nitrocellulose filters were washed

in PBS-Tween, and blocked for 2 h at 37#{176}Cin blocking buffer

containing 5% fat-free dried milk in PBS-Tween (without

azide). The membranes were then incubated with the horserad-

ish peroxidase-conjugated polyclonal antibody RC-20 (Affiniti)

at 4#{176}Covernight. After washing with PBS-Tween, the strips

were incubated in detection solution containing 50% luminoll

50% Enhancer (Amersham ready-to-use ECL system) for 1 mm.

The strips were then arranged in a transparency and exposed to

Hyperfilm-ECL (Amersham) for 40 mm.

RESULTS

Patient Population. Table 1 lists the characteristics of

the patients treated in this study. The median age was 56 (range,

23-78) years. The tumor histologies of the treated patients were:

large bowel, 14; ovary, 10; pancreas, 7; melanoma, 6; stomach,

5; renal, 2; hepatoma, 3; and non-small cell lung, 2. Two

patients had other diagnoses: one with testicular teratoma and

one with gastrinoma.

Dose Escalation and Toxicity. Table 2 shows the dose

levels and number of patients treated at each dose level. Until

renal toxicity was encountered, quercetin was given without

prehydration. There was no hematological toxicity, neurological

toxicity, mucositis, or alopecia.

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

lasted for approximately 3 mm, but was not associated with any

fall in blood pressure.

At and above quercetin doses of 945 mgim2, the concen-

tration of quercetin injected was increased from 50 to 100

mg/mi. This was done in an attempt to minimize the volume of

DMSO injected. It was immediately apparent that at this dose

level there was more severe pain on injection, which was local-

ized to the muscles in the upper arm on the side of the injection.

In an attempt to minimize this, the duration of quercetin slow

bolus injection was increased to S mm, and 10 mg morphine

elixir were given before drug administration. Although no for-

mal assessment of the pain score was made, this seemed more

tolerable and decreased the subjective intensity of pain and

flushing.

To investigate whether bolus injection of the vehicle

caused flushing and pain, two patients received 50 ml RIMSO

50 solution by rapid injection. These patients did not experience

any side effects, and it was therefore concluded that quercetin

and not the vehicle was responsible for pain and flushing on

slow bolus injection. Quercetin administered to a patient at the

630-mgim2 dose level via a Hickman catheter resulted in no

pain but slight flushing on injection.

During the dose escalation at 1400 mg/m2, patients also

reported dyspnea during and for a few minutes after quercetin

administration. This was difficult to quantify, but the patient

treated at 2000 mg/m2 had severe dyspnea which lasted S mm

following quercetin administration. We considered this unac-

ceptable and treated the next cohort of patients at 1700 mg/m2,

at which dyspnea was less prominent.

Emesis. At the 10th dose level (1700 mg/m2), grade 4

emesis was encountered. This occurred within minutes of quer-

cetin administration, which has been the pattern in all patients

with quercetin-imduced vomiting. Subsequently, the 5-HT3 an-

tagonist granisitron (3 mg) combined with dexamethasone (8

mg) were given i.v. before quercetin. Comparing vomiting at




3 Quercetin-induced emesis”

CTC vomiting grade

With iv. granisitron

Dose level

(mg/rn2)

Without iv. antiemetics and dexamethasone

0 1 2 3 4 0 1 2 3 4-630 8 1 1 0 0

945 2 0 2 0 0 8 3 0 0 0

1400 4 1 0 0 0 6 4 2 2 1

1700 0 0 1 0 1 0 0 1 0 02000 1 0 0 0 0

“ The number of observations refers to courses for which data were

obtained and amounts to 39 of 59, or 66%, of all potentially evaluablecourses.

doses of 945 mgim2 and above, without antiemetics, 4 of 12

courses were associated with grade 2-4 vomiting versus 6 of 27

with antiemetics. Thus, the antiemetics had no significant effect

(Table 3).

Cardiovascular Toxicity. One patient with a history of

hypertension had renal impairment (grade I), but her hyperten-

sion was grade 3 with a blood pressure of 230/130 mm Hg when

renal impairment ensued. Her pretreatment serum creatinine was

94 p.M and creatinine clearance was 69 ml/min. In the 24 h after

treatment, this fell to 46 ml/min, and I week later her serum

creatinine was 234 p.M and creatinine clearance was 26 ml/min.

Within 2 weeks, her creatinine clearance had normalized to 72

mI/mm, and her hypertension had been controlled with atenolol.

Two other cardiovascular events occurred, both in patients

treated at 945 mgim2. One patient with a previous history of

angina developed central chest pain 24 h after the second course

of quercetin and died on the second day after treatment. Another

patient developed grade 3 nephrotoxicity after the second course

of quercetin, developed back pain, and died in heart failure 16

days after the second course. A postmortem examination re-

vealed aortic dissection extending beyond the renal arteries.

Renal Toxicity of Quercetin. Three patients were

treated at 1700 mg/rn2, all of whom experienced renal toxicity,

with elevation of serum creatinine (Table 4). These patients also

experienced nausea and vomiting within minutes of the querce-

tin injection. In one patient (Q26) treated at 1 700 mgim2, nausea

was grade 4 (Table 3) and more prolonged. This may be because

this patient developed grade 4 renal toxicity, with serum creat-

mine reaching a peak of 2145 p.M I I days after treatment, which

returned to normal by day 34 (Fig. 2A). Two other patients were

treated at this level: one experienced grade 2 renal toxicity and

the other grade 1 toxicity, but in patient Q27 the serum creati-

nine remained elevated at 246 p.M on day 34 posttreatment, and

in patient Q28 the serum creatinine was elevated at 149 p.M on

day 28. None of the patients who experienced renal toxicity had

evidence of nephritis, infection, or obstructive uropathy on

ultrasound scanning of the abdomen.

Dose Escalation with Weekly Treatment and Nephro-

toxicity. At the highest doses of quercetin administered (2000

and 1700 mgim2), myelosuppression was not seen, but there

were dose-limiting toxicities. We therefore attempted to dose

intensify by using a weekly schedule. During this weekly sched-

ule, a concerted effort was made to monitor renal function with

Table 4 Renal toxicity and dose level

CTC nephrotoxicity (creatinine)Dose level”(mg/m2) 0 1 2 3 4

630 4 0 0 1 0

945 3 3 1 1 0

1400” 3 1 0 0 0

2000 1 0 0 0 0

1700 0 1 1 0 1

l400(weekly)c 3 1 1 0 1

945 (weekly) 5 0 1 0 0

‘, No renal toxicity was encountered at dose levels below 630mg/rn2.

1’ One patient not evaluable.C Two patients not evaluable.

24-h creatinine clearances before and immediately after querce-

tin therapy. For 1 1 courses at 945 mgim2 and for 2 at 1400

mgim2, the mean pretreatment creatinine clearance was 69.5

mlimin, falling to 56.6 mllmin after quercetin treatment (P =

0.032, paired t test), i.e., a 19 ± 8% fall. Since no myelosup-

pression was seen with the 3-week schedule, the dose of 1400

mg/rn2 was explored in a more intensive weekly schedule. All of

the data given are for treatments given without iv. hydration.

The first patient treated on the weekly schedule at this dose had

an unresectable pancreatic cancer. Following quercetin therapy,

he had prolonged nausea and developed acute renal failure

(grade 4) requiring peritoneal dialysis. Another two of seven

patients treated with this dose level on the weekly schedule had

renal toxicity: one patient had grade 1 and one patient had

grade 2.

Four patients on the weekly schedule had three or more

courses of quercetin. Fig. 2B shows pretreatment creatinine

clearance versus day on study. There was no cumulative fall in

creatinine clearance with multiple weekly treatments, indicating

that any nephrotoxicity due to quercetin was transient and

reversible within 7 days.

Overall, 2 of 10 patients who were treated at 1400 mgim2

had clinically significant nephrotoxicity (1 patient with grade 4,

and 1 patient with grade 2). Since this had occurred with the first

course of quercetin, we therefore enrolled an additional six

patients at the preceding dose level of 945 mg/rn2 weekly. At

945 mgim2, eight patients were treated at 3-week and 6-week

intervals. Overall, combining data for both schedules, 3 of 14

patients developed clinically significant renal impairment, 2

developed grade 2, and 1 developed grade 3.

Abrogation of Renal Toxicity with i.v. Hydration. One

patient treated at 630 mg/m2 developed grade 3 renal toxicity,

with elevation of the serum creatinine to 420 p.M by day 10. By

day 21 this patient’s serum creatinine had normalized (Fig. 2C).

This patient was keen for additional therapy and was therefore

retreated, but with prehydration of 1 liter normal saline given

over I h, and after quercetin treatment, 0.5 liter 5% dextrose was

given; there was no fall in creatinine clearance. The next course

in this patient was given at 945 mg/rn2 with hydration, and no

renal toxicity ensued. In other patients, hydration was given

before bolus injection of quercetin at 945 mg/rn2, and the

pretreatment clearance was 82.3 ± 6.7 mI/mm. In the 24 h after



1700

�

#{163}:��-Q0�0.

0

1� -#{149}-- Ursa

-- 0- - Creatlnlne

1000

.� 100

10

1_b 0

C

00I-

0

30 40 010 20Day on study

1

0.1

0.01

i 10000

I�, 10

:� 200

:�.. 160

�. 120

�z; 80

0

.E 40C

00I-a

10000C

C

0

0

U 100

E

0Cl)

10

-O--Q38

-.--Q35

II�3I040

0 10 20Day on study

- -4-- Ssrum crsatlnlne (pM)

--0- - Crsatlnlna clsaranci (mi/mm)

630 630 945

,Y

-10 20 50Day on study

Fig. 2 Renal toxicity of quercetin. A. reversible grade 4 renal toxicityin patient Q26 treated at 1700 mg/rn2. Arrow, day of treatment. B, lackof cumulative nephrotoxicity in patients treated weekly. Both patientsQ3S and Q38 were treated at 1400 mg/rn2. C, effect of prehydration onnephrotoxicity. Arrows, day of treatment at indicated doses in mg/rn2.On day 0, quercetin was given without hydration; on days 35 and 78,with hydration, as described in the text.

quercetin treatment, the creatinine clearance was 79.5 ± 19.5

ml/min. Thus, a simple outpatient hydration schedule abrogated

falls in creatinine clearance.


0 40 80 120 160 200Time (mm)

Fig. 3 Pharmacokinetics of quercetin. In the key, the first numberrefers to the patients� trial number and the second to the dose in mg/rn2.

Reduction in Serum Potassium. For 38 of S 1 patientson whom data were available, there was a statistically signifi-

cant fall in serum potassium, comparing day of treatment (4.32

± 0.07 mM) with day 8 posttreatment (3.98 ± 0. 10 mM� P

0.006). We therefore analyzed the pre- and post-quercetin urine

- specimens for potassium and sodium content. In 10 specimens

available for analysis, the mean 24-h potassium excretion before

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

significant changes in urine potassium or sodium excretion were

detected.

Pharrnacokinetics. The data from I I patients were fitted

to a two-compartment model. The distribution half-life was$ 0.7-7.8 mm, with a median of 6 mm. The elimination half-life

was 3.8-86 mm, with a median of43 mm (Fig. 3 and Table 5).

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.

� - There was an excellent correlation of 0.89 between the dose in

mg/m2 and AUC. There was no obvious relationship between

C toxicity and kinetic parameters.

The serum levels achieved immediately after injection of

8 0 1 1 0 quercetin were in the range of 200-400 p.M at 945 mg/rn2, withserum 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

HPLC trace which may be quercetin metabolites (data not

shown). In one patient, ascites fluid levels of quercetin were

determined 60 mm after a dosage of 945 mg/rn2. The level in

ascitic fluid was 0.34 p.M. which is 20% of that of a parallel

blood specimen.

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

one course and then at 945 mg/rn2 for another four courses. The

mean percentage of administered quercetin excreted unchanged

in the urine was 1 .97 ± 0.66% (range, 0.03-7.6%), with a range



600

0

400

0 50 100 150 200 250Day on study

I I I

420 420 500 + AUC4. � 4 �

-500

B

0 50Day on study

100 150

Fig. 4 Fall in tumor markers following quercetin therapy. A, patientQ03 with hepatocellular carcinoma and fall in AFP following treatmentwith quercetin at 60 mg/rn2 (arrows). B, patient QlS with ovarian cancerand fall in CA 125. The third and fourth treatments were administeredwith carboplatin at AUC 4 as part of a parallel Phase I trial.4


4 D. R. Ferry, D. Fyfe, A. Smith, J. Malkhandi, J. Baker, and D. J. Kerr.Phase I trial of quercetin plus carboplatin, manuscript in preparation.

Ta ble 5 Pharma cokineti c modeling

Dose AUC t112a t112�3 Clearance Vd��Patient (mg/rn2) (p.g/mllmin) (mm) (mm) (mI/mm) (liter)

3 60 187 3.5 NM” 0.320 1.613a 420 2760 2.0 33 0.152 4.007 120 100 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

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 2.2016 630 2369 4.4 60 0.270 3.9217 630 1285 2.9 49 0.490 3.70

18 630 2579 7.8 33 0.240 3.10

19 945 4139 2.9 26 0.23 2.80

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.4b NM, not modeled better by two-compartment kinetics; �

volume of distribution at steady state.

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

concentration.

Antitumor Activity. No patients achieved conventional

radiological responses according to WHO criteria; however, one

patient with metastatic hepatocellular carcinoma, who was

treated at the first dose level (60 mg/rn2), had a sustained fall in

serum AFP and alkaline phosphatase (Fig. 4A). Another patient

with stage 4 metastatic ovarian cancer with extensive pelvic

disease and liver metastases had completed six courses of cy-

clophosphamide (750 mg/rn2) and cisplatin (75 mg/rn2) 3

months before entry into the trial. Her response to this chemo-

therapy was minor, and her serum CA 125 was rising progres-

sively, reaching a level of 290 units/mI. Following treatment

with two courses of quercetin at 420 mg/rn2, her CA 125 fell to

55 units/ml (Fig. 4B). This patient was treated with three addi-

tional courses of quercetin at 500 mg/rn2 [combined with car-

boplatin AUC 4 (26) as part of a trial of quercetmn plus carbo-

platin],4 and 6 months after going on study her CA 125 was 45

units/mi, fulfilling recently suggested criteria for marker re-

sponse in this disease (27).

In Vivo Inhibition of Lymphocyte Tyrosine Kinase Ac-tivity. In 1 1 patients, lymphocyte tyrosine kinase activity was

investigated before and at times up to 16 h after quercetin

administration (Table 6 and Fig. 5). In 2 of 1 1 patients, phos-

photyrosine-banding patterns were unchanged up to 60 mm after

quercetin administration. However, for time points �l h in all

evaluated samples, phosphotyrosine bands of between Mr

45,000 and 60,000 were attenuated or eliminated following

quercetin treatment (Table 6). At 2 h posttreatment, inhibition of

phosphotyrosine banding occurred in five of five patients. The

0

:� 400

� 200

� 300

� 200

4C.) 100

vehicle, DMSO, caused some hemolysis, which was most pro-

nounced in the samples obtained up to 30 mm after quercetin

administration (Fig. 5, A and B) but which had largely disap-

peared at the 1 h and subsequent time points. Despite rigorous

washing procedures, the hemolysis contaminated the lympho-

cyte lysates, resulting in a wide phosphotyrosine band of mo-

lecular weight approximately Mr 20,000, which was clearly

discernible from the bands attenuated by quercetin. Attenuation

of phosphotyrosine bands occurred at all dose levels (Fig. 5,

C-H), and the number of bands inhibited increased with time. In

one patient, inhibition of phosphotyrosine banding was apparent

up to 16 h after treatment (Fig. SG).

DISCUSSION

The aberrant expression of protein tyrosine kinases acti-

vated by peptide growth factors can override growth regulatory

control and lead to cancer (28, 29). Nonreceptor tyrosine ki-

nases, such as pp�src, have SH2 domains which direct binding




Table 6 Inhibitio n of tyrosine kinase in patient lymphocytes

Quercetin

Phosphotyrosine bands

<I h 1 h 2 h

Patient (mg/m2� Pretreatment postquercetin postquercetin postquercetin Other

Q03 60 mcI” nd ndQ2l 945 + nd - nd ndQ22 1400 + + nd nd ndQ22 1400 + + nd nd ndQ22 1400 + + nd nd ndQ23 1400 + + nd nd ndQ23 1400 + + nd nd ndQ23 1400 + + nd nd ndQ27 1700 + nd - - ndQ28 1700 + + - - ndQ32 1400 + nd - - ndQ4l 945 + + - - -(4hr)Q48 945 + nd - - -

(3& l6hr)

a� , present; - , absent.b nd, not determined.

to phosphotyrosines of receptor tyrosine kinases, further ampli-

fying and diversifying the growth signals. Thus, it is not sur-

prising that the development of tyrosine kinase inhibitors, tyr-

phostims, is an attractive concept and a very active area (30). The

first tyrphostin discovered was the flavonoid quercetin, which

was found to inhibit pp�JS�C (11). Perhaps, it is therefore appro-

priate that quercetin should be the first tyrphostin to be system-

atically tested in a Phase I cancer trial.

Quercetin was administered to six normal volunteers in

1975; at that time it was considered a drug with potentially

beneficial vascular effects (24). This previously published work

provided us with a safe starting dose in the absence of conven-

tional preclinical animal toxicity.

The limit of solubility for quercetin in 500 ml 10% ethanol

is 100 mg, and severe alcohol intoxication would result if higher

doses were used. For this reason, we pursued alternative formu-

lations which would allow dose escalation. The solubility of

quercetin in DMSO at room temperature is 150 mg/ml. These

questions then arise, how much DMSO can be administered to

humans safely, and what are the potential complications?

DMSO is reported to be used i.v. to lower raised intracranial

pressure before emergency meurosurgical procedures (3 1). The

doses used were initially 1 g/kg infused as a 20% solution, but

doses as high as 8 g/kg/day could be given safely. DMSO is

recognized as causing mild hemolysis, and the higher the con-

centration of DMSO the more hemolysis is observed (32).

However, administration of a 40% solution of DMSO at a dose

of 1 g/kg i.v. over 20 mm is reported to be well tolerated (32).

At a quercetin dose of 1400 mg/m2, approximately 0.5 glkg, of

DMSO is given as the vehicle, which is one half of the amount

previously reported as well tolerated.

On administration of quercetin, the patients noted pain,

principally in the biceps muscle on the side of injection. This

lasted throughout the injection and for a few minutes afterward.

This pain was more severe at higher doses of quercetin and was

not due to the vehicle. The speed of onset makes ischemic pain

perhaps unlikely, and the direct stimulation of noiceptive fibers

may be a possibility. The pain was significantly reduced by

prolonging the infusion time and using 10 mg morphine elixir.

During the injection of quercetin, all of the patients expe-

rienced transient flushing for up to S mm. This was not associ-

ated with a fall in blood pressure, and must therefore be related

to dilation of blood vessels smaller than resistance vessels.

Quercetin is known to have vasodilator effects, inhibiting the

spontaneous myogenic contractions of rat portal vein with an

IC50 of 9 p.M and reversing phorbol l2-myristate 13-acetate-

induced aortic contractions with an IC50 of 1 1 p.M (33), sug-

gesting involvement of protein kimase C. Other biological ac-

tivities of quercetim may have contributed to the vasodilator

effect, including inhibition of phosphodiesterases (34).

No clinically significant toxicities were seen until the lOh

dose level (1700 mg/rn2) when renal toxicity, manifesting as

elevation of serum creatinine, occurred. In one patient, this was

grade 4 reaching 2145 p.M. but without dialysis, serum creati-

nine normalized within 34 days. The mechanism of renal tox-

icity has not yet been elucidated, possibilities include alteration

of renal blood flow or direct renal tubular damage. It is recog-

nized that differentiating between these mechanisms is difficult,

requiring invasive techniques (35). One potential biochemical

mechanism is impairment of NO synthase which generates the

vasodilator NO. Quercetim inhibits NO synthase with an IC50 of

220 p.M (36). Even small reductions in the blood flow to the

renal medulla, which is in a state of incipient hypoxia due to its

high metabolic rate, can lead to ischemic damage, and, if this

persists, lead to acute tubular necrosis (37). Quercetin-induced

renal impairment was investigated intensively in patients treated

on the weekly schedule. In these patients, 24-h creatinine clear-

ance was estimated before and after quercetmn administration.

Quercetin caused a 20% reduction in creatimine clearance, which

recovered within 7 days.

When we encountered dose-limiting grade 4 renal toxicity

at 1700 mg/rn2, we treated a total of five patients at 1400 mg/rn2

on the 3-week schedule, no myelosuppression was seen. We

then attempted to increase dose intensity by giving 1400 mg/rn2



C D E60mg/rn2 945mg/rn2 1400mg/rn2

97

66

45

29

Molecuhnweight

116

97

66

45

29

F945 mg/rn2

pre lh pre lh

G945 mg/rn2

pre lh 2h

205

116

97

66

S....

4’45

29

‘S0


Molecular � Bweight 945 mg/rn2 945 mg/rn2

pre 10mm pre 20mm

Molecularweight

Pr. <lb 2h 4b pre lb lb 4b 16h

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 lysateprotein/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

first patient treated. In the next seven patients treated at this dose

level, one patient had grade 2 renal toxicity, which was consid-

ered sufficiently serious to be regarded as dose limiting. We

therefore attempted to dose intensify using the previous dose

level of 945 mg/rn2 weekly and encountered significant neph-

rotoxicity in one of six patients, which we felt represented an

acceptable level of toxicity.

Additional patients were treated at 945 mg/rn2 on a 3-week

schedule as part of a parallel trial to combine carboplatin and

quercetin, and surprisingly encountered grade 3 renal toxicity.

We therefore retreated the next patient at 630 mg/m2, and this

patient had reversible grade 3 toxicity. At this stage we were

concerned that the nephrotoxicity might be unpredictable. This

patient however normalized renal function by day 14 and was

keen to continue therapy. She received prehydratiom with 1 liter

normal saline followed by posthydration with 0.5 liters 5%

dextrose. This prevented renal toxicity in this patient, and in an

additional six courses of quercetin given with hydration at 945

mg/rn2 to five patients, no falls in creatinine clearance were

seen.

Conventionally Phase I trials should lead to a recommen-

dation of the dose to be explored in Phase II trials. Nephrotox-

icity seem at 1700 mg/rn2 was dose limiting, but we also saw

grade 3 nephrotoxicity in one patient treated at 630 mg/rn2.

Susequently, as other patients were studied, it became clear that

simple i.v. prehydration could at least partially abrogate the

nephrotoxicity of quercetim. With this in mind, the ernesis and

the pain on injection, we believe that 1400 mg/rn2 given weekly

or every 3 weeks can be recommended for Phase II trials.

In the literature, quercetin has been demonstrated to have

antiproliferative effects against rnultidrug resistant MCF-7 hu-

man breast cancer cells with a 50% effective dose of 10 p.M (38),

against ovarian cancer cells with an IC50 of S p.M (22), to

synergize with cisplatin in a mouse xenograft model at a dose of

10 mg/kg (21), and to potentiate the actions of l-�3-D-arabino-

furanosylcytosine to kill human AML cells at 1 p.M (23). At

doses above 630 mg/rn2, quercetin plasma levels were in excess

of 1 p.M for at least 3 h.

In this Phase I trial, one patient with cisplatin refractory

ovarian cancer had a large and sustained fall in serum CA 125

levels. Criteria have been proposed for the use of CA 125 as a

surrogate marker of response (27), which our patient fulfills.

Another patient with hepatocellular carcinoma had a fall of

a-fetoprotein from 460 to 40. Again, this fall was sustained, but

not associated with radiological regression of measurable dis-

ease to be counted as a standard partial response.

If one takes a mechanistic approach to cancer drug devel-

oprnent, then it is essential to attempt to demonstrate pharma-

codymamic effects and correlate these with the drug’s pharma-

cokinetics. Ideally, this would involve biopsy of cancers before

and after drug administration. However, human tumors can be

hazardous to biopsy, and the ethics and discomfort make this

approach untenable. The other option open is to use patient

lymphocytes as surrogate indicators. This has been applied

successfully in other areas of signal transduction research, e.g.,

down-regulation of 0 protein-coupled adremoceptors. In blood

leukocytes, measurement of 06-alkylguanine-DNA-alkyltrans-

ferase activity inversely correlated with the probability of re-

sponse in melanoma patients treated with dirnethylphenyltria-

zine CB 10-227 (39), and in ovarian cancer treated with

cisplatin, the formation of platinum-DNA adducts in blood

lymphocytes strongly predicts for response (40).

We were able to demonstrate that before quercetin admim-

istration the majority of patients had detectable phosphotyrosine

bands, particularly in the molecular weight range Mr 40,000

60,000, and that quercetin treatment abrogated some of these

bands. This approach is open to criticism. First, the tyrosine

kinases of lymphocytes may be different than those in tumors.

Second, we cannot be certain that tyrosine kinase inhibition is

the mechanism through which quercetin exerts its antiprolifera-

tive effects. For these reasons, we have not pursued the charac-

terization of the identified phosphotyrosine bands.

This is the first reported Phase I clinical trial with a novel

class of antiturnor agents, tyrphostims, in humans. We are cur-

rently refining the methodology to allow specific targets of

tyrosime kinase inhibition to be identified and investigated in

solid tumors and leukernic cells in ongoing clinical trials.

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1996;2:659-668. Clin Cancer Res D R Ferry, A Smith, J Malkhandi, et al. inhibition.pharmacokinetics and evidence for in vivo tyrosine kinase Phase I clinical trial of the flavonoid quercetin:

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