BIOPHARMACEUTICS & DRUG DISPOSITION, VOL.

17,493499 (1 996)

A PHARMACOKINETIC STUDY WITH THE

HIGH-DOSE ANTICANCER AGENT
MENADIONE IN RABBITS
OLIVER YOA-PU H U * ~ CHIH-YUAN
, W U ~ WIN-KAI
, CHANT,
FELICIA Y.-H. WUt AND JACQUELINE WHANG-PENGt
*School of Pharmacy. National Defense Medical Centre, Taipei 100, Taiwan, Republic of China
Institute of Biomedical Sciences, Academia Sinica. Taipei 11529, Taiwan, Republic of China

ABSTRACT
The aim of this investigation was to assess the pharmacokinetic properties of high-dose
menadione (VK,), as an anticancer agent, in plasma and red blood cells (RBCs) in
rabbits. An extremely high dose of 75 mg menadiol sodium diphosphate (Synkayvite)
was intravenously injected. HPLC analysis was applied to measure the major
metabolite, menadione, VK,. The kinetic properties of VK3 in both plasma and red
blood cells showed a short elimination half-life, high clearance, and large volume of
distribution in plasma and RBCs. The mean elimination tl,* values of menadione in
plasma and in RBCs were 27.17+ 10.49min and 35.22+ 1142min, respectively. The
plasma clearance (CLIF) of VK, was 0.822 & 0.254 L min-I. The systemic clearance in
RBCs was 0.407 & 0.152 L min- l . The apparent volume of distribution (Vd/F)in plasma
was 30.833+12.835L and that in RBCs 20.488+9.401L. The plasma AUC was
+
32-453 9.785 pg min mL- and that of RBCs 67.2 19 24.449 pg min mL-'. Menadiol
was rapidly biotransformed to menadione in blood. The formation rate constant (k') of
menadione in plasma was 0.589 & 0.246 min-I, and that of RBCs 1.520_+ 1.345min-I.
Through this study the estimated menadione dosage needed to maintain a plasma level
of 1 pgmL-' for anticancer purposes was 19.7mgkg-' every hour.
KEY WORDS: pharmacokinetics;menadiol sodium diphosphate (synkayvite);menadione

INTRODUCTION

Vitamin K3 (VK3,2-methyl-l,4-naphthoquinone, or menadione) is a synthetic

vitamin K congener and has been used to treat the bleeding tendency
associated with its deficiency.' In addition to its antihaemorrhagic function,
VK3 has been shown since 1958 to have anticancer activity. Chlebowski2and
0thers~9~have done a great deal of research on the effect of VK3 on human and
animal cell lines. In these studies, VK3 was cytotoxic in 86% of human tumours
tested at 1 pgmL-1.2 Also, VK3 (4pgmL-') was found to have a greater
cytotoxicity (>70% inhibition of control colony formation) than VK,

*Addresseefor correspondence:Dr. Oliver Yoa-Pu Hu, Professor, School of Pharmacy, National

Defense Medical Centre, P.O. Box 90048-508, Taipei, Taiwan, Republic of China.

CCC 0142-2782/96/060493-07 Received 15 June 1995

01996 by John Wiley & Sons, Ltd. Accepted 1 November 1995
494 0. Y.-P. HU ET AL.

(75pgm-l) in L1210 leukaemia cells2 and also in neuroblastoma cell lines.3

Potential mechanisms of VK3 antineoplastic activity include generation of
semiquinone, free radicals, and s ~ p e r o x i d e , ~
causing
.~ glutathion depletion,
lipid per~xidation,~ and single- (double-) strand DNA breaks.*
The cytotoxicity mechanism of menadione is relatively complicated. In liver,
VK3 combines with glutathione to form thiodione (2-methyl-3-glutathionyl- 1,
4-naphthoquinone) catalysed by GSH S-transferase or n~nenzymatically.~
Thiodione then enters the redox cycling and is excreted in plasma causing
oxidative stress, and then excreted to the bile.*OJ1Also, VK3 can be reduced by
DT-diaphorase12or one-electron transformation, producing a semiquinone free
radical; the possible enzymes involved include NADPH-cytochrome P450
reductase, NADH-cytochrome b5 reductase, and NADH-ubiquinone oxido-
reductase, resulting in formation of their corresponding semiquinone and
hydroquinone. Menadione semiquinone free radicals dismutate rapidly and may
generate other radicals such as superoxide. Indirect evidence suggests that VK3
cytotoxicity may be a result, in part, of the generation of these toxic species.13
In humans, menadiol is metabolized by phase I metabolism into
menadione1&l6and conjugated by phase I1 metabolism into glucuronide, as
well as sulphate ~0njugates.I~ Differing from humans, VK3 is also metabolised
to VK2 (menaquinone-4) in animals.'*
The distribution and metabolism of labelled menadione and menadiol were
examined in rats,15J8 and, generally, menadione was uniformly distributed
throughout all organs. No distinctive distribution of remaining radioactivity
was noted for either compound at 17-18 h post-injection. Menadiol sodium
diphosphate is excreted by both urinary and biliary-faecal routes in rats.
There is little kinetic data reported regarding a high dose of menadione or its
water-soluble forms. In 1969, Thierry and Suttie15 studied radioactive
Synkayvite in rats with a dose of 1-6-2.0pg/rat (160-21OpCi). Recently,
Akman and c o - w ~ r k e r s ,treating
~~ two cancer patients with Synkayvite,
reported that concentrations of VK3 in plasma were measured (lasting approx-
imately 4h) after parenteral administration of 20mgm-2 i.m. by using a
differential pulse polarographic determination.
The purpose of this pharmacokinetic investigation in rabbits was to
characterize the pharmacokinetics of menadione following administration of
a high single-dose menadiol sodium diphosphate (Synkayvite, 75 mg) i.v.
injection. The pharmacokinetic properties will then serve as a guide for further
study in phase I clinical trials.

MATERIALS AND METHODS

Pharmacokinetic studies
Seven New Zealand White rabbits between the ages of 1 and 2 years, with a
weight of between 2.3 and 3.8 kg, were included in this study. Before drug
 HIGH-DOSE ANTICANCER DRUG MENADIONE IN RABBITS 495

administration, a lOmL blood sample was collected for the individual

calibration curve. Samples were collected in a 1.5mL heparin rinsed
Eppendorp vial. After administration of a high dose of 75mg menadiol
sodium diphosphate by i.v. bolus, serial blood samples were drawn at 1,2, 3,4,
5,7, 10, 12, 15,20, 25, 30,45,60,90, and 120min. One millilitre blood samples
were obtained at each time point except at 60,90, and 120 min (3 mL). In order
to avoid exposure to light, all tubes were wrapped with aluminium paper.

Drug analysis
Both the plasma and RBC concentrations of menadione were determined by
C18 reverse-phase high-performance liquid chromatography as previously
described.20Briefly, carbazole, 1.75pg (internal standard), was added to 0.5 mL
plasma. Samples were extracted by 6 mL n-hexane, then evaporated under
nitrogen gas. Separation was performed on a precolumn (10 pm Bondapack@,
CI8) and a Beckman Ultrasphere ODS HPLC column (5 pm, 250 x 4.6mm)
with a mobile phase of methanol/purified water (70: 30) at a flow rate of
0.8mLmin-I. An ABI Model 78314 UV detector (Karots, Ramsey, NJ,
U.S.A.) was set at 265 nm at a sensitivity of 0.0005 a.u.f.s. To the RBC samples,
the same volume of purified water was added to dilute sample viscosity. The
retention times of menadione and carbazole were 8.8 and 12.5min,
respectively. To prevent menadiol conversion to menadione, n-hexane was
immediately added to the blood sample after it was withdrawn; the test tube
and n-hexane were maintained at 0 "C to stop any possible conversion during
the sampling period. All blood samples were stored at - 84 "C until analysis.
The limit of quantitation was 10 ng mL- in plasma. The absolute recovery was
82.5%.

Pharmacokinetic analysis
The menadione plasma concentration obtained from HPLC was fitted to a
three-exponential equation:
= A e-"
~,(t> + B e-Pt + c e-kft
where Cp(t) is the plasma concentration at time t after drug administration and
A , B, and C are intercepts on the y axis for each exponential segment of the
curve in units of concentration, with the use of PCNONLIN,21 a nonlinear
regression computer program. A weight function of l/Ci(t) was used. The
Akaike information criteria,22weighted residual sum of squares, correlations,
and the residual plots were used to obtain the best estimates of the parameters.
The estimates on the initial parameters which were required for nonlinear
regression were obtained with the use of a linear regression computer program
(CSTRIP).23The weighted residual sum of squares, correlation, and residual
496 0.Y.-P. HU ET AL.

plots were used to obtain the best estimates of A , B, C, a,and p, area under the
plasma concentration-time curve to time infinity (AUC,), and transformation
and elimination half-lives (tlI2(kf)and t1,*(P)) were calculated for each
individual subject according to the standard formula.24 The volume of
distribution and total plasma and RBC clearance were presented as Vd/F
and CLIF, respectively. F is the fraction of menadiol that converts to
menadione in v i v a

RESULTS AND DISCUSSION

Plasma (n=7) and RBC (n=4) levels and pharmacokinetic parameters of

VK3 are shown in Figure 1 and Table 1 respectively. The peak plasma men-
adione concentration (Cmax)of 2.102k0.494 pgmL-' (range, 1.3-2.7 pgmL-')
occurred at 4.29 k 1.58min (range, 4 7 min) after administration of menadiol
sodium diphosphate i.v. Almost at the same time, 4.75k3.86min (range, 1-
lOmin), the C,, in RBCs was reached, but the concentration was slightly
higher, 3.228 &- 1.500 pgmL-' (range, 1.7-5.2 pgmL-'). The VK3 biotrans-
formation half-life (tl/2(kf)) in plasma was 1.44k0.72 min, indicating that
rnenadiol rapidly converted to menadione in plasma. The same phenomenon
was observed in RBCs. When the water-soluble form VK, was administered,
the diphosphate ester was rapidly hydrolysed to lipophilic VK,, the major
metabolite, menadione. VK, or the glutathion conjugated form may be further

10 Obs. plasma conc. (n=7)

-Cal. plasma conc. (n=7)
Obs. RBC conc. (n=4)
---- Cal. RBC conc. ( n 4 )
z 1
9
s
.s
B
g .1

.01
0 20 40 60 80 100 120 140
Time (min)
Figure 1 . Concentration-time curves of menadione in plasma (mean S.D.) and RBCs after i.v.
administration of high-dose menadiol sodium diphosphate 75 mg (Synkayvite") in seven rabbits.
The symbols represent the observed data, and the line indicates the calculated data according to the
estimated pharmacokineticparameters
 HIGH-DOSE ANTICANCER DRUG MENADIONE IN RABBITS 497

Table 1. Pharmacokinetic parameters of menadione after i.v. administration of 75.0 mg

Synkayvite (menadiol sodium diphosphate) in rabbits. C@)=A ecar+Be-P'+ Cevkf',
where A , B, and C are intercepts

Pharmacokinetic parameters Plasma (n = 7) RBC (n=4)

k , , (min-I) 0.1 17& 0.050 0.077 f0.028

k l Z(min-I) 0.038 50.031 0.169 f0.166
k,, (min-I) 0.042 & 0.022 0.075 f0.05 1
aa (min-I) 0.168+0*096 0.301 & 0.236
p" (min-I) 0-030&0.012 0.02 1 & 0.007
k p (min-I) 0.589 k0.246 1.520f 1.345
?I/, (a)b (mi4 5.37 f2.72 4.19 f3.91
fl/2 (P)" (mi4 27.175 10.49 35.225 11.82
tl/z(kf)db i n ) 1.44k0.72 l.00f 1.05
V d / F (L) 304333f12.835 20.488 f9.401
L a x (min) 4.29 f 1.58 4.75 f3.86
Cmax (pg mL- '1 2.102 50.494 3.228 f 1.500
AUC,' (pg minmL-') 32.453 5 9.785 67.219 & 24.449
CL,,/P (Lmin-I) 0.822 f0.254 0.407 & 0.152

aRate constants for distribution phase, elimination phase, and transformation phase, respectively.
bThe half-life of drug in the distribution phase.
T h e half-life of drug in the elimination phase.
dThe half-life of drug in the transformation phase.
"Apparent volume of the distribution.
'Area under plasma concentration-time curve to time infinity.
Total plasma clearance, where F is the fraction of menadiol converted to menadione.

enzymatically reduced by both one- and two-electron reductions. VK3 or

glucuronide form is then excreted in the urine or faeces.17 The apparent
volumes of distribution ( V d / q were 30.833& 12.835 and 20.488 k9.401 L, and
the clearances (CL/F) 0.822 f0.254 and 0-407k 0.1 52 L min- in plasma and
RBCs, respectively. The high value of clearance (19.7 L h-1 kg-') as well as the
large volume of distribution (12.33 L kg-I) indicated that VK3 did have
extensive tissue distribution and possible binding in rabbits.
The concentration of VK3 then declined rapidly in a biphase fashion, with a
terminal elimination half-life of 27.17 f 10.49 and 35.22+ 11.82min in plasma
and RBCs, respectively. The longer half-life of VK3 in RBCs might reflect a
slower removal of VK3 from RBCs, although not significantly. This was also
shown in the case of RBC/plasma concentration ratios which were increased with
time. It took an average of around 3-01h to eliminate99% VK3from plasma after
administration and the level was not detectable around 2 h postdose. The
respective areas under the concentration curves (AUC,) in plasma and RBCs
were 32.453k9.785 and 69.219 f24.449 pg min rnL-'. The greater volume of
distribution and the larger area under the curve of VK3in RBCs than in plasma
showed that RBCs have a higher affinity to VK3 than plasma.
Pilot phase I clinical trials of VK3 have been carried out in the U.S.A. The
published pharmacokinetic data is relatively limited, in the short-report or
498 0. Y.-P. HU ET AL.

abstract f ~ r m . 'Advanced
~ ? ~ ~ cancer patients (40) were treated with Synkayvite
given via a small i.v. infusion (1-5h) starting at 40mgm-2 and escalating to
1360mgm-2. No haematological toxicity was observed. VK3 plasma
concentrations were in the range of 0.2-7.4 pM (0.034-1.273 pgmL-') during
the 1360mgm-2 infusi0n.~5Because no dose limited toxicity was reported and
to achieve plasma menadione concentration x time indices which are associated
with in vitro tumoricidal activity (5G100 pM), the author suggested prolonged
infusion (48-96 h) with an increasing dosage starting at 4gm-2/24h.25 No
further kinetic analysis data was reported in this study. The same suggestions
have been confirmed in the Akem et al. study in high-dose infusion of
Synkay~ite.'~
The present report is to evaluate the pharmacokinetic properties of VK3
in rabbits after a single-high-dose 75 rng i.v. Synkayvite injection. Our
pharmacokinetic data demonstrated that menadiol is rapidly converted to
menadione after i.v. injection, then menadione is further metabolized and
completely excreted within 3 h. According to the high clearance, large volume
of distribution, and rapid elimination of VK3, a high dose of VK3 dose will be
needed to maintain the postulated effective concentration of 1 pgmL-l. The
maintenance dose of 1182mgh-l VK3 is suggested for a 60kg subject in a
future clinical study.

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