[CANCER RESEARCH 50, 4539-4545, August I. 1990]
In Vitro Biotransformations
of Tetrachloro(d,l-fra/«)-l,2-
diaminocyclohexaneplatinum(IV)
Rat Plasma1
(Tetraplatin) in
Stephen G. Chancy,2 Steven Wyrick, and Gail Kaun Till
Department of Biochemistry and Biophysics, School of Medicine [S. G. C., G. K. T.], and Division of Medicinal Chemistry and Natural Products, School of Pharmacy
[S. W.I, University of North Carolina, Chapel Hill, North Carolina 27599
that c/i-dichlorobis(isopropylamine)platinum(II)
is a major
plasma metabolite of iproplatin,1 although their data suggest
ABSTRACT
The in vitro biotransformations of tetrachloro(d,l-frans)-l,2,-diaminocyclohexaneplatinum(IV) (tetraplatin) in the plasma of Fischer 344
rats were studied by the two-column high-performance liquid chromatography technique described previously (Mauldin et al., Cancer Res., 48:
5136-5144,1988). The reduction of tetraplatin to dichloro(d,l-rranî)-l,2diaminocyclohexaneplatinum(II) [PtCl2(dach)| was extremely rapid.
From experiments with diluted plasma, it was possible to estimate a t,,
for tetraplatin of approximately 3 s at 37°Cin undiluted plasma. By
titrating with /V-ethylmaleimide, it was possible to show that sulfhydryl
groups were responsible for 70-80% of the total reducing potential of
plasma. The rapid reduction of tetraplatin to PtCl2(dach) was followed
by slower substitution reactions involving the chloro ligands of
PtCl2(dach). The fw for PtCl2(dach) in plasma at 37°Cwas 1.5 h. The
monoaquamonochloro complex was an important biotransformation prod
uct at early times, reaching 10 to 12% of the total platinum present from
15 min to 2 h, when it was gradually replaced with more stable biotrans
formation products. Three major stable biotransformation products ac
cumulated in the plasma. One of these biotransformation products was
identified as the Pt(methionine)(dach) complex. The other two were
tentatively identified as the Pt(cysteine)(dach) or Pt(ornithine)(dach)
complex and the Pt(urea)(dach) or Pt(citrato)(dach) complex on the basis
of coelution in two different high-performance liquid chromatography
separation systems. These biotransformation products could play a role
in tetraplatin effectiveness and/or toxicity.
INTRODUCTION
Although the biotransformation products of platinum anticancer agents may well be responsible for some of their thera
peutic and toxic effects, relatively little is currently known about
the biotransformations
of this clinically important class of
compounds. On the basis of model reactions, platinum(IV)
complexes are thought to be reduced to their platinum(II)
analogues by biological reducing agents (1, 2). Platinum(II)
complexes with chloro leaving ligands appear to undergo a two
step reaction sequence, with an intermediate aquation (hydrol
ysis) step preceding the actual substitution reactions (3-6). The
reactivity of incoming ligands in the substitution reaction ap
pears to be in the order CN~ > NH3 ~ OH~ > I~ > SCN~ >
Br > Cl' » F' ~ H2O ~ methanol (4, 7). Thioether (R2S)
compounds are also particularly reactive (4). Thus, it is clearly
possible to predict likely biotransformation products of plati
num complexes in blood plasma and other biological fluids.
However, while platinum(IV) and platinum(II) reactions are
well understood on a theoretical basis, the actual biotransfor
mation products of these platinum complexes in blood remain
poorly characterized. Pendyala and coworkers (8,9) have shown
Received 12/27/89; revised 4/9/90.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This research was supported in part by Grant CH-393 from the American
Cancer Society.
2To whom requests for reprints should be addressed, at Department of
Biochemistry and Biophysics, FLOB, Campus Box 7260, School of Medicine,
University of North Carolina. Chapel Hill, NC 27599-7260.
that the platinum(IV) to platinum(II) reduction actually occurs
intracellularly. At least one polar metabolite of iproplatin was
also observed but not identified (8,9). Daley-Yates and McBrien
(10) and Mistry et al. (11) have resolved at least 7 plasma
biotransformation products of cisplatin and have identified one
of them as a hydrolysis product, presumably the aquachloro
complex. Similarly, Elferink et al. (12) have reported detection
of hydrolysis products of spiroplatin in plasma. Daley-Yates
and McBrien (10) tentatively identified two of the other cispla
tin biotransformation products in plasma as the mono- and bismethionine complexes. Long and Repta (13) have also reported
a methionine biotransformation product of cisplatin, but Mistry
et al. (11) were not able to confirm the presence of methionine
biotransformation products by another HPLC technique. Even
though relatively little is known about these plasma biotrans
formations, the available evidence suggests that they may be
important. Reduction of platinum(IV) complexes to their platinum(II) analogues appears to be essential for their biological
activity (2, 14). In addition, Daley-Yates and McBrien (10) have
suggested that the Pt(II)-methionine biotransformation prod
ucts of cisplatin may be biologically inert, while the aquated
derivatives are considerably more nephrotoxic than cisplatin
itself. Similarly Elferink et al. (15) have shown that hydrolysis
products of spiroplatin were more toxic than the parent com
pound.
There has been considerable interest in platinum complexes
with the dach carrier ligand because (a) they are often effective
against cell lines with either natural or acquired resistance to
cisplatin (16-18); and (b) they appear to have reduced nephrotoxicity compared with cisplatin (19, 20). Recently one of these
compounds, tetraplatin, has been characterized (21-23) and
approved for Phase I/II clinical trials.
We have recently developed a two-column HPLC separation
for resolving and identifying the biotransformation products of
platinum complexes with the dach carrier ligand (24) and have
used this technique to characterize the biotransformations of
tetraplatin in tissue culture medium (25, 26). In RPMI-1640
medium, tetraplatin was reduced to its platinum(II) analogue,
PtCl2(dach), with a tv, of 5 to 15 min at 37°C,depending on the
protein sulfhydryl level in the medium (15). The major reducing
3 The abbreviations and trivial names used are: iproplatin. c/s-dichloro-rransdihydroxybis(isopropylamine)platinum(I V); cisplatin, a's-diamminedichloroplatinum(II); spiroplatin, aqua[ 1.1-bis(aminomethyl)cyclohexane]sulfatoplatinum(ll);
Pt.platinum; dach.(d,l-rranj)-1.2-diaminocyclohexane;
tetraplatin, tetrachloro( d,lrrani)-l,2-diaminocyclohexaneplatinum(IV);
PtCI2(dach), dichloro(d,l-franj)-l,2-diaminocyclohexaneplatinum(II); |Pt(H2O)(CI)(dach)]*, aquachloro(d,l-/rans)l,2-diaminocyclohexaneplatinum(H);
HPLC, high-performance liquid chroma
tography; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); NEM, /V-ethylmaleimide;
Pt(methionine)(dach),
(d,l-rrani)-l,2-diaminocyclohexanemethionineplatinum(II); Pt(citrato)(dach), citrato(d,l-rrans)-l,2-diaminocyclohexaneplatinum(II);
Pt
(cysteine)(dach),cysteine(d,l-rrans)-l,2-diaminocyclohexaneplatinum(II);Pt(ornithine)(dach), (d,\-trans)-1,2-diaminocyclohexaneornithineplatinum(H);
Pt(mal)(dach), (d,\-trans)-1,2-diaminocyclohexanemalonatoplatinum(II);
Pt(urea)(dach),
(d,l-rrani-l,2-diaminocyclohexaneureaplatinum(II).
4539
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BIOTRANSFORMATIONS
OF TETRAPLATIN
agent in tissue culture medium was protein sulfhydryl, with
glutathione and glucose making only minor contributions (25).
The rapid reduction of tetraplatin to PtCl2(dach) was followed
by much slower substitution reactions [/,/, for PtCl2(dach) in
complete medium was 7.5 h] (25). These substitution reactions
appeared to occur via intermediate formation of aquated and
bicarbonato intermediates (26). In fact, transient formation of
the aquachloro complex was shown both in tissue culture me
dium (26) and intracellularly (27). The major biotransformation
product which accumulated in tissue culture medium was the
monomethionine complex, although other amino acid com
plexes were found in lesser amounts (25, 26). The methionine
complex appeared to be inert in tissue culture, since there were
a very low rate of uptake and little or no binding of
Pt(methionine)(dach) to the cell membrane of L1210 cells (26).
We report here on the in vitro biotransformations of tetraplatin
in plasma from Fischer 344 rats. We have resolved at least five
major biotransformation products. We have provided strong
identification for two of them and tentative identification for
the other three.
MATERIALS
AND METHODS
IN RAT PLASMA
bound platinum was calculated from the difference in radioactivity
before and after filtration. Tetraplatin was resolved from PtCI2(dach)
on a Zorbax 7 ODS column (Phenomenex, Rancho Palos Verdes, CA)
(25), and PtCl2(dach) was resolved from other biotransformation prod
ucts on the two-column HPLC system described previously (24, 25).
Following HPLC separation, aliquots from each fraction were counted.
Data from the scintillation counter were collected and converted to
ASCI files with the Ultraterm program (Pharmacia-LKB, Piscataway,
NJ). Peaks were identified and quantitated with the Chromatochart
program (Interactive Microware, State College, PA). Reactivity of the
biotransformation products isolated by HPLC was determined by the
DNA-binding assay described previously (27). Total sulfhydryl concen
trations in the plasma preparations were determined by the DTNB
assay described by Ellman (30). The actual sulfhydryl levels are reported
in the figure and table legends for each experiment.
RESULTS
The initial goal of these experiments was to measure the
conversion rate of tetraplatin to PtCl2(dach) in rat plasma.
Previous in vivo studies had shown that maximum platinum
concentrations of 5 fig/ml (25 ÕIM)
were attained in rat plasma
at therapeutic doses of tetraplatin (23). Thus, our in vitro
incubations were carried out with 10 to 50 ^M tetraplatin.
Initially, 11 /¿M
tetraplatin was incubated at 37°Cwith freshly
Materials. Tetraplatin, PtCl2(dach), and Pt(mal)(dach) were prepared
with high specific activity, nonexchangeable tritium in position 4,5 of prepared rat plasma, and tetraplatin was resolved from
the diaminocyclohexane ring. The synthesis and purity of these com
PtCl2(dach) on a Zorbax HPLC column as described previously
pounds as well as the starting material, [4,5-3H2(n)](d,l-Ã-ranÃ-)l,2-dia-
(25). Peak b, is tetraplatin, and Peak b2 is PtCl2(dach) (25).
Peak a is unidentified. Surprisingly, complete conversion of
tetraplatin to PtCl2(dach) was repeatedly observed even in the
control samples without incubation (Fig. \B). This suggested
that reduction of tetraplatin to PtCl2(dach) in plasma was much
more rapid than in tissue culture medium (25). Consequently,
total sulfhydryl levels were measured by the DTNB assay (30).
use. The stock solutions were stable for at least 6 mo under these
Sulfhydryl levels in freshly prepared rat plasma ranged from
conditions as determined by reverse-phase HPLC analysis on both
212 to 256 /UM,which is 7 to 8 times greater than the usual
Whatman Partasi! ODS-3 and Zorbax 7 ODS columns using the elution
levels in tissue culture medium. Based on previous reports (31,
conditions previously described (24, 25). Other platinum standards for
32), most of these sulfhydryl groups were probably associated
HPLC were obtained as described previously (16, 24). Briefly, 20 /¿g/ with the plasma protein fraction. Thus, various dilutions of
ml [3H]Pt(mal)(dach) was incubated with 1 to 10 mM concentrations of
plasma into 0.15 M NaCl were made to determine the dilution
different potential ligands for 24 h at 37°Cin H2O. With Pt(mal)(dach)
as the starting material, the substitution reactions generally proceed via of plasma needed to accurately measure the rate of the tetrapla
tin reduction reaction. From the data in Fig. 1, it was clear that
direct displacement of the malonate ligand rather than through the
at least a 1:20 dilution was required to obtain a good 0 time
intermediate formation of an aquated species (26). Thus, side reactions
value for the incubation.
are minimized, and one obtains only Pt(mal)(dach) and the platinum
complex to be characterized. These can usually be readily resolved by
Accordingly, the time course of the tetraplatin to PtCl2(dach)
HPLC. While this procedure does not provide direct information on
conversion was measured at 1:25 and 1:50 dilutions (Fig. 2).
the stoichiometry of the complexes formed, it does allow assignment
As expected, the extent of tetraplatin reduction to PtCl2(dach)
of HPLC retention times for platinum(II) complexes formed with
was dependent on the amount of sulfhydryl present in the
compounds of biological significance. DTNB and NEM were obtained
incubation. The tv, for tetraplatin in the reduction reaction was
from Sigma Chemical Co. (St. Louis, MO). Adult Fischer 344 rats
approximately 1.2 min at the 1:25 dilution and 2.5 min at the
were obtained from Charles River Breeding Laboratories (Raleigh, NC).
1:50 dilution. Previous studies in tissue culture medium showed
The rats weighed 170 to 250 g and were housed on a 12-h light/dark
that
protein sulfhydryl was the major reducing agent for tetra
cycle in stainless steel cages with room temperature maintained at
22°C.The animals were allowed Purina rodent chow ad libitum.
platin (25). However, rat plasma might be expected to contain
significant concentrations of ascorbate and other non-sulfhyMethods. Rat blood was obtained from the tail vein and collected in
dryl-reducing agents. Thus, the 1:25 dilution of plasma was
heparinized tubes under light vacuum. The animals were anesthetized
with Ketamine/Rompun (25 and 5 mg/kg, respectively) prior to bleed
also pretreated with NEM before incubation with tetraplatin.
ing, and at least 3 wk were allowed between bleedings. Control incu
The NEM pretreatment completely eliminated detectable
bations showed that heparin did not react appreciably with either
sulfhydryl and decreased the rate and extent of the tetraplatin
tetraplatin or PtCl2(dach) under our assay conditions. Following col
to PtCl2(dach) conversion 3- to 5-fold (Fig. 2), but did not
lection, the blood was immediately cooled to 4°Cand centrifuged at
eliminate it completely. Control experiments showed that NEM
4°Cfor 10 min in an IEC Model PR-J clinical centrifuge at 2800 rpm
had no effect on the stability of tetraplatin or PtCl2(dach). Since
(1960 x g) to obtain plasma. Following incubation of freshly isolated
these data indicated that more than one reducing agent was
plasma with [3H]tetraplatin, samples were diluted 1:20 into cold 0.15
M NaCl to stop the reaction. The plasma ultrafiltrate was obtained by present, it was not possible to determine second-order rate
constants for the reduction reaction for comparison with pre
filtration through an Amicon YMT membrane with a M, 30,000 cutoff
(Amicon Corp., Danvers, MA) as described previously (27). Proteinviously determined values (25).
minocyclohexane, are described elsewhere (28). However, subsequent
experience has shown that the l,2-bis-azidocarbonylcyclohex-4-ene in
termediate in the described synthesis is extremely explosive, and we no
longer recommend that protocol (29). Stock solutions (100 iig/ml) of
tetraplatin and PtCl2(dach) were prepared in 0.15 M NaCl, while stock
solutions of Pt(mal)(dach) were prepared in water. All stock solutions
were stored in small aliquots at —80°C
and thawed immediately before
4540
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BIOTRANSFORMATIONS
20
B
b2
OF TETRAPLATIN
IN RAT PLASMA
8
15
6
10
4
l5
2 *-^
Ö
o -£
£o
5 8
6 o.
l
ae
O
y 4
LU
CD
2
Q
Ul
2
I5
30 0
RETENTION
I5
30
TIME (min.)
Fig. 1. Rapid reduction of tetraplatin to PtCI2(dach) in vitro. [3H]Tetraplatin
(11 /.\i i was added to various dilutions of rat plasma at 4°C.immediately diluted
1:10 into cold 0.15 M NaCl, and filtered through an Amicon YMT membrane
filter at 4°C.Tetraplatin was resolved from PtCl2(dach) with a Zorbax C-18
column as described by Anderson et al. (21). Peak b,, tetraplatin; Peak b2,
PtCl2(dach); and Peak a, unknown. A, tetraplatin alone; others, tetraplatin added
to undiluted rat plasma (B) or plasma diluted 1:10 (C), 1:20 (D), 1:50 (£),or
1:100 (F). All dilutions were in 0.15 M NaCl. Total sulfhydryl in the undiluted
rat plasma was 212 ^M as measured by the DTNB assay (30).
20
40
0
20
40
RETENTION TIME (min.)
Fig. 3. HPLC analysis of tetraplatin biotransformations in rat plasma in vitro.
[3H]Tetraplatin (44 ^M) was incubated in undiluted rat plasma at 37"C. At the
indicated times aliquots were removed, diluted 1:20 in cold 0.15 M NaCl, filtered
through an Amicon YMT membrane, and frozen. Platinum biotransformation
products were analyzed by reverse-phase HPLC as described previously (24): A,
0-min incubation (•,profile obtained with tetraplatin standard): B, 15 min; C,
30 min; D, l h; E, 2 h; F, 3 h; G, 6 h; H, 12 h.
similar to that seen when Pt(mal)(dach) or PtCl2(dach) was
incubated with tissue culture medium (26), except that Peak e
was more prominent in plasma and there did not appear to be
a distinct Peak h. The time course for these biotransformations
is shown in Fig. 4. PtCl2(dach) disappeared with a ty, of ap
proximately 1.5 h at 37°C.Most of the platinum rapidly became
0
5
10
15
20
25
TIME OF INCUBATION (min.)
Fig. 2. Effect of sulfhydryl on the reduction of tetraplatin to PtCl2(dach). [3H]Tetraplatin (7 UM) was incubated at 37°Cwith diluted rat plasma containing
different levels of sulfhydryl measured by the DTNB assay. Tetraplatin remaining
at different times was determined as described in Fig. 1: O, 1:25 dilution of
plasma, sulfhydryl = 4.0 UM;A, 1:50 dilution of plasma, sulfhydryl = 1.6 >IM;•,
1:25 dilution of plasma pretreated for 15 min at room temperature with 10 MM
NEM, sulfhydryl = 0 <iM.
Since the substitution reactions with PtCl2(dach) were ex
pected to be much slower than the reduction of tetraplatin,
these were studied in undiluted plasma. PtCl2(dach) was initially
resolved from its plasma biotransformation
products on a
Whatman ODS-3 HPLC column as described previously (24).
The results obtained with 44 ¿IM
tetraplatin are shown in Fig.
3. At least 4 major peaks of biotransformation products could
be resolved by reverse-phase HPLC.4 The pattern obtained was
4 The nomenclature used to describe the peaks in this separation was based on
the patterns originally observed in tissue culture studies with Pt(mal)(dach) and
PtCl2(dach) (26. 27). Peak a represents material eluting in the void volume and
has not been characterized. The elution position of PtCMdach) was originally
protein bound. Peak f represented the major ultrafilterable
biotransformation product at early times but was transient,
with Peaks e and g accumulating at later times. At 11 MM
tetraplatin, the same biotransformation products were observed
(data not shown). Under these conditions Peak f was more
persistent, plateauing between 2 and 3 h before decreasing
significantly at 6 h. When 44 /¿M
tetraplatin was incubated with
a 1:1 dilution of rat plasma in 0.15 NaCl, the formation of all
the biotransformation products was significantly slower, and
Peak f was the major ultrafilterable biotransformation product
through 6 h (data not shown).
Previous experience has shown that each peak isolated by
reverse-phase HPLC may represent two or more biotransfor
mation products. Thus, each of these peak fractions was further
described as Peak b. Since tetraplatin élûtes
just prior to PtClj(dach) in these
studies, we have described the peaks corresponding to tetraplatin and PtCl2(dach)
as b, and b, respectively. Peak r corresponds to Pt(mal)(dach), and Peak d
corresponds to one or more intracellular platinum biotransformation products.
Neither Peak c nor d was seen in these studies. Peak h appears to represent
platinum complexed with basic amino acids and was not seen in these studies.
The likely identities of Peaks e,f, and g are discussed in the text.
4541
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BIOTRANSFORMATIONS
OF TETRAPLATIN
100
013345
0123456
TIME OF INCUBATION(hr)
Fig. 4. Time course of tetraplatin biotransformations in rat plasma in vitro.
The amount of 3H-labeled platinum biotransformation products was determined
from the experiment described in Fig. 3. A, percentage of protein-bound platinum.
B, fiM concentration of platinum biotransformation products. O, total free plati
num; •protein-bound platinum; •,PtCl2(dach); A, Peak e; A. Peak f: D, Peak
Table 1 Reactivity toward DNA of rat plasma in vitro biotransformation products
The peak fractions from reverse-phase HPLC analysis of experiments such as
the one described in Fig. 3 were incubated with salmon sperm DNA for 60 min
at 30°Cas described previously (27) to determine their reactivity. The assay
measures the percentage of the input radioactivity which reacts with the DNA.
The monoaquamonochloro complex gives 43.6 ±5.9% (n = 10) DNA binding
under these reaction conditions.
IN RAT PLASMA
incubation. The cation exchange pattern for Peak g was clearly
time dependent, however (Fig. 5, C to F). At early times, two
peaks were observed, one with a retention time of 32-35 min
(Peak g,) and a second with a retention time of 48 to 51 min
(Peak g2). While Peak g2 was quite prominent at 3 to 6 h, it
had usually disappeared by 12 h.
Tentative identification of these biotransformation products
was obtained by comparing their retention times on both HPLC
columns with the retention times of a series of standards run
on the same HPLC columns. The original set of standards for
these columns, primarily Pt(dach)-amino acid complexes, was
chosen on the basis of the composition of tissue culture medium
(26). For these experiments several other compounds present
in blood were tested for their ability to form platinum(II)
complexes, and the retention times of the complexes formed
were determined on both HPLC columns (Table 2). The char
acteristics of some of the Pt(dach)-amino acid standards which
had similar retention times to one of the plasma biotransfor
mation products are also shown in Table 2. The characterization
of 8 other Pt(dach)-amino acid standards has been reported
previously (24). We have also characterized an additional 13
platinum standards [Pt(dach) complexes with acetate, pyruvate,
láclate, ascorbate, NADH, glucose 6-phosphate, phosphoenol
pyruvate, AMP, ADP, ATP, GTP, UTP, and dTTP] for use
with intracellular studies, but none of these showed similar
retention times to the plasma biotransformation products (data
not shown). The cation exchange profiles of these standards
bindingPeakf39
of DNA
Time of
incubation30
eND°
min
Ih
2h
3h6h
ND
ND
ND2.0(1)
45
40
35
12hPeak
2.6 ±1.2(3)%
" ND, not determined because insufficient
g11(1)
±1.6*(3)c
14.5 ±0.7 (3)
±5(3)
±5 (4)
14 ±2(3)
6.1 ±1.5(3)
±3 (3)
ND
5.6(1)
NDPeak
1.2(1)
quantities ofthat peak were available
0.8
at that time.
* Mean ±SD.
c Numbers in parentheses, number of determinations.
characterized. The transient nature of Peak f suggested that it
might be a fairly reactive intermediate. We have previously
shown that Peak f may contain [Pt(H2O)(CI)(dach)]+ as well as
Pt(dach) complexes with the neutral and acidic amino acids
serine, threonine, glutamine, asparagine, glutamate, and aspartate (24-27). The aquachloro complex can best be distinguished
from the coeluting biotransformation products on the basis of
its reactivity toward DNA (27). Hence, the reactivity of each of
the peak fractions obtained by reverse-phase HPLC was deter
mined (Table 1). Based on these reactivity measurements, it
would appear that Peak f was largely the aquachloro complex
through 2 h. By 3 h the percentage of aquachloro in Peak f was
approximately 80%. No distinct Peak f could be resolved at 6
and 12 h. However, those fractions were included in Peaks e
and g, and they showed little or no reactivity toward DNA at
those times. Peak e showed minimal reactivity toward DNA at
any time. Peak g showed some reactivity at early time points,
but that most likely represented some overlap from Peak f.
Each of the reverse-phase HPLC fractions was further char
acterized by cation exchange HPLC at pH 4 (24). As described
previously, the exact patterns and retention times are somewhat
more variable with cation exchange than with reverse-phase
HPLC (24). However, the results shown in Fig. 5 are typical of
the patterns observed. The patterns observed for Peaks e (Fig.
5/1) and f (Fig. 5B) did not change appreciably with time of
50
RETENTION TIME (min.)
Fig. 5. Cation exchange HPLC analysis of in vitro biotransformation products.
Peak fractions from the reverse-phase HPLC separation shown in Fig. 3 were
analyzed by cation exchange HPLC at pH 4 as described previously (24): A, Peak
e at 6 h; B, Peak f at 3 h; C Peak g at 30 min; D. Peak g at 3 h; E, Peak g at 6
h; F, Peak g at 12 h. Free (d.l-/rans)-l,2-diaminocyclohexane
was used to
standardize the columns. Its elution position is shown by the arrow.
4542
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BIOTRANSFORMATIONS
OF TETRAPLATIN
.i11hv11«1
standardsPlatinum
Table 2 Characterization of platinum
previously(24).standards were prepared and characterized as described
platinumcomplexes
The reverse phase and cation exchange elution positions of these
usedto
were determined with the same columns and elution conditions
peakswere
characterize the plasma biotransformation products. Where two or more
detected by HPLC, the major one
underlined.%
is
of
displacementof
fromApproximate
mal
Cationconcentration
Pt (mal) (dach)
exchangePotential
bylOmM
retentionligand in plasma"
ligand in
(min)Cysteine
(mM)
24 h*
Reversephase
elution
position
9,11Ornithine
+ cystine
8Aspartate
0.14
0.12
87
21
Peak e
Peak e
24Citrulline
16,23Glutamate
18,27Serine
12,16Threonine
14,20(H2O)(C1)
6,10,15Creatinine
0.05
0.06
0.05
0.19
0.40
43
71
37
10
10
Peak f
Peak f
Peak f
Peak f
Peak f
Peakf
17,
44Glutathione
15,23,33Methionine
35Taurine
37Urea
38,48Citrate
0.05
0.03
0.06
0.14
3.2
60
97
98
23
19
Peak
Peak
Peak
Peak
Peak
17,
51Histidine
40Arginine
0.14
0.06
55
8
Peak g/h
Peak g/h
51Lysine
0.18
4
38°
0.40
4
(33).*
From Dittmer
thereaction
Incubations were carried out in H2O at 37°Cfor
reverse-phase
was quantitated from the disappearance of Pt
HPLC (24).1
g
g
g
g
g
IN RAT PLASMA
T1tn1IT[11I
2l.f—0.90.60.5
0.3Ç 1fJ
11
times
«aC1l1Ilo
*ta-
nI
00
0.3^Q
Ö0
°??Jul
ftaiDV1iiPbto.
i \^—
F2.0
V1I1.5
^o10
0.2LU_JU
SLUlâ€
V1 fe
O.I<_J1
010
i
^0¡¡—
05
fi1
(lLIII
<Lj
_ ,R __. ,
¡^Pt
n^i^^^^yypi
IÕÕMI•W•
^»^^rtftEr
15105n-
'0.6
in040.2n
0v
iilltinL
Peak h
Peak h
24 h. The extent of
(mal) (dach) by
bJv\U/^pPwr
«hu*
•Bu
which most closely resembled the plasma biotransformation
products are shown in Fig. 6. Of the 37 standards tested
to date, the Pt(cysteine)(dach) (24) and Pt(ornithine)(dach)
(Fig. 6A) complexes most closely resembled Peak e. The
[Pt(H2O)(Cl)(dach)]+ standard was prepared by incubating
PtCl2(dach) in H2O (24) and was analyzed directly on the cation
exchange column without prior purification on reverse phase
(Fig. 6C). The first peak is PtCl2(dach), and the last peak is
[Pt(H2O)2(dach)]2+ (24). The second and third peaks are char
acteristic of [Pt(H2O)(Cl)(dach)]+ and probably represent a mix
ture of [Pt(H2O)(Cl)(dach)r and Pt(OH)(Cl)(dach) (24). Those
peaks closely resemble the profile seen with Peak f. Peak gì
has
retention times identical to the Pt(methionine)(dach) standard
(Fig. 6B), while Peak g2 most closely resembles the Pt(citrato)(dach) (Fig. 6D) and Pt(urea)(dach) (Fig. 6F) standards.
DISCUSSION
Most platinum anticancer agents studied to date appear to
be converted to between 3 and 7 distinct biotransformation
products in plasma (8-12). We feel that it is of both theoretical
and practical significance to determine the identity, toxicity,
and therapeutic effectiveness of these biotransformation prod
ucts. Such knowledge might well lead to the design of alterna
tive treatment protocols or to the use of more effective modi
fying agents. Since tetraplatin is a platinum(IV) complex that
is about to enter Phase I/II clinical trials, this research was
designed to characterize the biotransformations of tetraplatin
in rat plasma. We have utilized a two-column HPLC technique
which provides excellent resolution of platinum biotransfor
mation products and a large number of Pt(dach) standards to
allow tentative identification of these compounds.
Platinum(IV) complexes are generally considered prodrugs
that require reduction to the platinum(II) level for activity (2,
14, 21). Our data show that the reduction of tetraplatin to
25
500
RETENTION
25
W1.00.80.60.40.2
50
TIME (min.)
Fig. 6. Cation exchange HPLC analysis of selected standards. With the
exception of (Pt(H2O)(Cl)(dach)]*, platinum standards were prepared and purified
by reverse-phase HPLC as described previously (24). The peak fractions from
reverse phase were analyzed by cation exchange HPLC as described in Fig. 5.
[Pt(H2O)(Cl)(dach)r was prepared by incubating PtCI2(dach) in H2O for 24 h at
37°C(24) and was analyzed by cation exchange HPLC without prior purification
on reverse phase. A, Pt(ornithine)(dach); B, Pt(methionine)(dach); C, [Pt(H2O)(Cl)(dach)n D, Pt(citrato)(dach); E, Pt(serine)(dach); F, Pt(urea)(dach).
PtCl2(dach) in rat plasma is very rapid. Based on the /./,for this
reaction at the 1:25 and 1:50 dilutions, the estimated tv, for
tetraplatin in undiluted rat plasma would be approximately 3 s
at 37°C.Given the speed of the reaction at 37°C,it is in
retrospect not surprising that we observed complete conversion
of tetraplatin to PtCl2(dach) in the control samples at 4°C
during the time required (1 h) to prepare the plasma ultrafil
trate. The reduction of tetraplatin to PtCl2(dach) is more rapid
in rat plasma than in tissue culture medium (25). This probably
reflects the much greater concentrations of sulfhydryl and the
other reducing agents in plasma.
The data with NEM pretreatment suggest that sulfhydryl
groups are the major reducing agent in rat plasma (Fig. 2).
Since protein sulfhydryl is present at higher concentrations
than low-molecular-weight sulfhydryls in plasma (31, 32), and
our previous data show that protein sulfhydryls are almost as
effective as glutathione or cysteine at reducing tetraplatin (25),
it is likely that protein sulfhydryl is the major reducing agent
for tetraplatin in plasma. However, our data clearly show that
reducing agents which do not react with NEM contribute 20 to
30% of the reducing potential of rat plasma. We have not
attempted to identify the nonsulfhydryl reducing agents in
plasma. However, ascorbate concentrations are about 20-fold
lower than protein sulfhydryl levels in rat plasma (32, 33), and
ascorbate is significantly less effective as a reducing agent for
4543
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BIOTRANSFORMATIONS OF TETRAPLATIN IN RAT PLASMA
tetraplatin (25), so one would not expect ascorbate to make a
significant contribution to the reduction observed.
Once formed, PtCl2(dach) undergoes a series of slower sub
stitution reactions which appear to be similar to the ones
previously reported for cisplatin (10, 11). However, since we
were working with a radiolabeled platinum complex, we were
able to characterize these biotransformations in more detail.
The tv, for PtCl2(dach) in rat plasma in vitro is 1.5 h, and the
major biotransformation product is protein-bound platinum
(Fig. 4). These results are qualitatively similar to the data of
LeRoy and Thompson (34) with human plasma, although they
reported a tv,of 0.875 h for tetraplatin binding to human plasma
proteins at 37°C.This could represent a difference between
human and rat plasma, but most likely reflects a difference in
technique. The technique they used measured binding to all
proteins with a molecular weight of 10,000 or greater, while
the filter binding technique we used measures binding to pro
teins with a molecular weight of 30,000 or greater.
Our data provide clear evidence for the existence of the
aquachloro biotransformation product in rat plasma. Not only
is the retention time of Peak f similar to [Pt(H2O)(Cl)(dach)]+
on both reverse-phase and cation exchange HPLC, but Peak f
shows the same reactivity toward DNA as [Pt(H2O)(Cl)(dach)]+. Of the 37 standards tested to date, only 9 have reten
tion times similar to Peak fon reverse-phase HPLC, and none
of these has reactivities similar to [Pt(H2O)(Cl)(dach)]+ in our
DNA binding assay (Refs. 26 and 27; Table 1). While it is still
possible that some unknown biotransformation product may
have similar retention times and reactivity as the aquachloro
complex, the chances for that occurring would appear to be
remote because of the large number of platinum standards
characterized. Of course, the presence of the aquachloro com
plex in rat plasma was expected. Aquated biotransformation
products have been reported for cisplatin in rat plasma (10, 11)
and spiroplatin in human plasma (12). The presence of these
aquated biotransformation products is likely to be significant,
as they appear to be considerably more toxic than the parent
compounds (10, 15).
The kinetic data suggest that the aquachloro complex forms
more rapidly than the other biotransformation products (Fig.
4). However, it is present only transiently, eventually giving
way to more stable biotransformation products. This is fully
consistent with current models suggesting that substitution
reactions for platinum! 11)complexes with chloro leaving groups
are a two-step process, consisting of a rate-limiting aquation
reaction followed by a very rapid bimolecular (SN2) reaction
between the aquated complex and the incoming ligand (3, 4).
In fact, the time course for [Pt(H2O)(Cl)(dach)]+ appearance
and disappearance closely follows the kinetics predicted for
such a reaction sequence (34). Our other data are also consistent
with such a model. A decreased initial concentration of
PtCl2(dach) would be expected to decrease the rate of both
reactions. Thus, the time course for the formation and disap
pearance of Pt(H2O)(CI)(dach) would be prolonged, but the
steady-state fraction would be unchanged. A decreased concen
tration of plasma would be expected to decrease the rate of the
second reaction only, leading to an increase in the steady-state
levels and a decreased rate of disappearance of the aquachloro
complex. Both predictions match our observations.
There appear to be 3 other major plasma biotransformation
products. The biotransformation product represented by Peak
g2 was clearly more stable than the aquachloro complex, but it
too was transient. Peaks e and gì,
on the other hand, appeared
to be stable. Peak g, is most likely the Pt(methionine)(dach)
complex. Our tentative identification is based on the similarity
of the retention times of Peak g, with the Pt(methionine)(dach)
standard on both reverse-phase and cation exchange HPLC,
and on the known reactivity of methionine toward platinum(II)
complexes (4). Daley-Yates and McBrien (10) have previously
suggested the formation of a cisplatin-methionine complex in
rat plasma, but Mistry et al. (11) were unable to confirm its
presence by a second HPLC system. Our data suggest that their
original observation was probably correct. The effect of the
Pt(methionine)(dach) complex on tetraplatin is difficult to pre
dict. Mauldin et al. (26) have shown that Pt(methionine)(dach)
is not taken up by LI210 cells, and Daley-Yates and McBrien
(10) have shown that the 1:1 cisplatin:methionine complex has
neither toxicity nor therapeutic benefit in rats. However, DaleyYates and McBrien (35) have also reported that the 1:1 cisplatin:methionine complex inhibits renal ATPase activity in vitro,
and Alden and Repta (36) have shown that methionine exac
erbates cisplatin toxicity.
Peak e most closely resembles the Pt(cysteine)(dach) or
Pt(ornithine)(dach) complexes by HPLC, while Peak g2 could
be either the Pt(urea)(dach) or Pt(citrato)(dach) complex. Of
course, these identifications are tentative at present. We have
characterized 37 potential biotransformation products to date
(Ref. 24; Table 2), but we cannot exclude the possible formation
of other platinum complexes. Very little is known about the
cysteine, ornithine, or urea complexes, but the formation of the
citrato complex, if it is confirmed by subsequent studies, would
be interesting. The Pt(citrato)(dach) complex is known to have
significant therapeutic potential (37, 38), so the conversion of
tetraplatin to Pt(citrato)(dach) would represent an activation
pathway. The citrato complex is also known to be somewhat
unstable, which would be consistent with our kinetic data for
Peak g2. We think that PtCl2(dach), [Pt(H2O)(Cl)(dach)]+,
Pt(methionine)(dach),
Peak e, and Peak g2 represent all the
major biotransformation products of tetraplatin in rat plasma
in vitro. Free (d,l-fra/is)-l,2-diaminocyclohexane
(dach) is read
ily resolved from the biotransformation products detected in
these experiments and was not evident in any of the HPLC
runs. Thus, there would appear to be very little fraws-labilization
of our ['Hjdach carrier ligand and, consequently, no undetected
platinum complexes in our HPLC separations.
In summary, we have shown that tetraplatin is very rapidly
reduced to PtCl2(dach) in rat plasma in vitro, and that
PtCl2(dach) is converted to 4 other major biotransformation
products with a tv, of 1.5 h. We have provided a strong identi
fication of two of these biotransformation products, one of
which is known to be highly toxic and the other inert. We have
provided tentative identification of the other two, with the
possibility that one of them may be a platinum complex with
known therapeutic activity. Obviously, once we have a more
complete knowledge of the biotransformation products formed
and their characteristics, it may be possible to modify the
treatment conditions to minimize the toxic and maximize the
therapeutic biotransformation products. Research is currently
under way in our laboratory to confirm the identity of these
biotransformation products and to determine what effect agents
known to modify cisplatin toxicity have on the biotransformation of tetraplatin in plasma.
ACKNOWLEDGMENTS
The authors would like to thank Dr. David Holbrook for scholarly
discussions and critical review of the manuscript.
4544
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BIOTRANSFORMATIONS
OF TETRAPLATIN
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4545
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In Vitro Biotransformations of Tetrachloro(d,l-trans
)-1,2-diaminocyclohexaneplatinum(IV) (Tetraplatin) in Rat
Plasma
Stephen G. Chaney, Steven Wyrick and Gail Kaun Till
Cancer Res 1990;50:4539-4545.
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