cis-Amminedichloro(2-methylpyridine) Platinum(II) (AMD473 ... · histochemical staining for...
Transcript of cis-Amminedichloro(2-methylpyridine) Platinum(II) (AMD473 ... · histochemical staining for...
Vol. 3, 2063-2074, Noi’e,nber /997 Clinical Cancer Research 2063
cis-Amminedichloro(2-methylpyridine) Platinum(II) (AMD473), a
Novel Sterically Hindered Platinum Complex: In Vivo Activity,
Toxicology, and Pharmacokinetics in Mice’
Florence I. Raynaud,2 Frances E. Boxall,
Phyllis M. Goddard, Melanie Valenti,
Mervyn Jones, Barry A. Murrer,
Michael Abrams, and Lloyd R. Kelland
Cancer Research Campaign Centre for Cancer Therapeutics. TheInstitute of Cancer Research, Sutton, Surrey SM2 5NG, United
Kingdom [F. I. R., F. E. B., P. M. G., M. V.. M. J., L. R. K.]; Johnson
Matthey Technology Centre, Sonning Common. Reading, United
Kingdom [B. A. Ml; and AnorMED Inc.. Langley, British Columbia,
Canada [M. A.]
ABSTRACT
A novel sterically hindered platinum complex, AMD473
[cis-amminedichboro(2-methylpyridine) platinum(II)J, de-
signed primarily to be less susceptible to inactivation by
thiobs, has shown in vitro activity against several ovarian
carcinoma cell lines. Notably, AMD473 has shown activity invitro in human carcinoma cells that have acquired cisplatin
resistance due to reduced drug transport (41M/4lMcisR) or
enhanced DNA repair/increased tolerance of platinum-DNA
adducts (CH1/CHlcisR). In this study, we show that
AMD473, at its maximum tolerated dose of 35-40 mg/kg i.p.
administration, produced marked in vivo antitumor activity
against a variety of murine (ADJIPC6 plasmacytoma, L1210
leukemia) and human ovarian carcinoma xenograft models,
including several possessing acquired resistance to cisplatin
[ADJ/PC6cisR, Ll2lOcisR, CHlcisR, and HX11O (carbopla-
tin-resistant)]. In the ADJIPC6 model, an increased thera-
peutic index was noted following oral as opposed to i.p.
administration. In a head-to-head comparison using
CHlcisR xenografts and equitoxic doses (q7d x4 schedule),
comparative growth delays were as follows: AMD473, 34
days; cisplatin, 10.4 days; carboplatin, 6.4 days; and JM216
(p.o. administration), 3.5 days (in a previous experiment, the
trans-platinum complex JM335 induced a growth delay of
5.4 days against this model). In this model, oral activity was
also noted with a growth delay of 34 days at 400 mg/kg every
Received 4/10/97; revised 7/I 1/97: accepted 7/1 1/97.
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.
I This study was supported by grants to the Institute of Cancer Research
from the Cancer Research Campaign and the Medical Research Council.
2 To whom requests for reprints should be addressed, at Cancer Re-search Campaign Centre for Cancer Therapeutics, The Institute of
Cancer Research, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, UnitedKingdom. Phone: 44-181-643-8901; Fax: 44-181-770-7885: E-mail:
7 days (total of four doses). In addition, AMD473 showed
promising activity against CH1 xenografts that had regrown
following initial treatment with cisplatin (additional growth
delay of 30 days over that observed for retreatment with
cisplatin). Across the whole panel of cisplatin-sensitive to
cisplatin-resistant human ovarian carcinoma xenografts,
AMD473 showed improved or at least comparable activity
to that observed for an equitoxic dose (4 mg/kg) and sched-
ule of cisplatin.
Platinum pharmacokinetics showed that following i.v.
administration of 20 mg/kg AMD473 in saline to Balb/c
mice bearing murine plasmacytoma (ADJIPC6), a biexpo-
nential decay was observed in the plasma with a rapid
distribution tl/2a of 24 mm followed by a slow elimination
ti,213 of 44 h. Platinum accumulated in various organs with
platinum tissue to plasma area under the curve ratios of 8.6
for liver and kidney, 5.7 for spleen, 3.7 for heart, 5.2 for
lung, and 5 for tumor. The plasma and tissue concentration
time curve following i.p. administration was similar to that
observed following i.v. administration, with a bioavailability
of 89%. When AMD473 was given p.o., the platinum ab-
sorption was rapid (K01 of 30 mm) and the bioavailability
was 40%. A less than proportional increase in area under
the curve and Cmax was noted in tissue, plasma, and plasma
ultrafiltrate following increasing oral doses of AMD473. In
vitro, with AMD473, the rate of binding to different plasma
proteins was approximately half of that of cisplatin. Follow-
ing administration of 45 mg/kg i.p. in oil, 33% of the ad-
ministered platinum was eliminated in the urine after 24 h,
and 40% was eliminated after 72 h. Fecal recovery repre-
sented 13% of the administered dose after 3 days. Similar
results were observed following oral and i.v. administration
of 20 mg/kg, but significantly more was excreted in the feces
(over 50% of the administered dose) following oral admin-
istration of 400 mgfkg, showing that absorption might be a
limiting factor by this route of administration. The dose-
limiting toxicity for AMD473 in mice was myebosuppression,
and no renal toxicity was observed. The promising antitu-
mor activity of AMD473, together with its lack of nephro-
toxicity and favorable pharmacokinetic profile, suggests
that AMD473 is a good candidate for clinical development.
AMD473 is entering Phase I clinical trials under the aus-
pices of the United Kingdom Cancer Research Campaign in
1997.
INTRODUCTION
The platinum coordination complex cisplatin, cis-diam-
minedichloroplatinum(II) has played a major robe in the chemo-
therapeutic treatment of a variety of neoplasms over the past 25
years. However, the drug possesses significant limitations in
being markedly toxic to many normal tissues (especially neph-
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2064 AMD473, A Novel Platinum Complex
4 Holford et al. , submitted for publication.
rotoxicities, neurotoxicities, and gastrointestinal tract toxicities)
and in that many tumors are either intrinsically resistant or
acquire resistance to the antitumor effects of the drug. These
limitations have driven intensive synthetic efforts to discover
new platinum-based drugs with reduced toxicity profiles and
especially those possessing activity against resistant disease.
Consequently, since 1971, over 20 cisplatin analogues have
entered clinical trial (reviewed in Ref. 1)
Our platinum-based drug discovery program in collab-
oration with the Johnson Matthey Technology Center (and in
part with Bristol-Myers Squibb) has thus far resulted in the
successful world-wide introduction of the less toxic ana-
logue, carboplatin (Paraplatin; Ref. 2). In addition we re-
ported the discovery of the ammine/amine platinum(IV) di-
carboxylates (3), which led to the introduction of the first p.o.
bioavailable platinum complex, JM216 [bis-acetato-ammine-
dichboro-cyclohexylamine platinum(IV)], which is now in
Phase II clinical trial (4, 5). More recently, we have described
the identification of a trans-platinum complex possessing at
least some in vivo antitumor activity against a range of
murine and human tumor models, JM335 [trans-ammine
(cyclohexylaminedichlorodihydroxo) platinum(IV)] (6- 8).
However, although carboplatin (and JM216) have made (or
may make) a substantial impact in improving the quality of
life for patients undergoing platinum-based chemotherapy,
there remains an overwhelming need to broaden the activity
of platinum-based drugs so as to induce responses against
currently resistant tumors.
One of the early, and thus far few, leads to platinum
complexes possessing activity against cisplatin-resistant tu-
mors is those based upon the DACH3 carrier ligand (9). Of
particular note, DACH-platinum complexes were shown to
retain activity against acquired cisplatin-resistant murine
Ll2lO leukemia tumors. However, other studies (including
our own) in many additional preclinical models of acquired
cisplatin resistance, including human ovarian carcinoma xc-
nografts, have shown that circumvention of acquired cisplatin
resistance by DACH-platinum complexes is not universal
(10-1 1). Recent clinical trials with the DACH-platinum
complexes tetraplatin (Ormaplatin, [tetrachloro-1,2-diamino-
cyclohexane platinum(IV)}; Ref. 12) and oxaliplatin (Eloxa-
tin, [oxabato- 1 ,2-diaminocyclohexane platinum(II)]; Refs. 13
and 14) and related complexes, such as Lobaplatin ([1,2-
diaminomethyl cyclobutane-platinum(II) lactate; Refs. 15
and 16), have shown a range of dose-limiting toxicities
(especially neurotoxicity with tetraplatin and oxaliplatin), but
thus far there has been no compelling evidence of activity in
cisplatin-resistant disease.
In recent years, considerably more has been elucidated
concerning the mechanisms underlying tumor resistance to
cisplatin (reviewed in Ref. 17). One or more of four mech-
anisms of resistance predominate: reduced platinum trans-
port, increased cytoplasmic detoxification via elevated glu-
tathione and/or metallothionein levels, increased DNA repair
of platinum-DNA adducts, and increased cellular tolerance to
platinum-DNA adducts. With these mechanisms of resistance
in mind, especially drug inactivation via interaction with
thiols, and building upon the promising structural features of
the previously described asymmetric ammine/amine dicar-
boxylates (3), a new class of sterically hindered platinum
complex centered upon AMD473 [cis-amminedichloro(2-
methylpyridine) platinum(II)] has been designed and synthe-
sized. Introduction of steric bulk at the platinum center (using
2-methylpyridine) predicts for favoring a dissociative mech-
anism of substitution rather than the associative mechanism
that predominates with cisplatin. Recent clinical studies have
described a significant positive correlation between immuno-
histochemical staining for glutathione S-transferase in head
and neck cancer and failure to respond to platinum-based
chemotherapy (18). Our previous studies with AMD473 have
shown it to be less susceptible than cisplatin to detoxification
by glutathione, and AMD473 has shown activity in in vitro
human ovarian carcinoma cell lines that have acquired cis-
platin resistance due to reduced drug transport (4lM/
4lMcisR) or enhanced DNA repair/increased tolerance of/to
platinum-DNA adducts (CH1/CHlcisR; Ref. 19).�
In this study, the in vivo antitumor effects of AMD473 (in
comparison to cisplatin) are reported in both murine tumors
(ADJ/PC6 plasmacytoma s.c. model and Ll210 leukemia; Ref.
10) and a range of cisplatin-responsive and -unresponsive hu-
man ovarian carcinoma xenografts (1 1), including tumors pos-
sessing acquired resistance to cisplatin (20). The antitumor
activity of AMD473 following oral administration is also pre-
sented. The toxicity profile and the disposition and excretion of
AMD473 following different routes of administration (i.p., iv.,
and p.o.) to mice are also evaluated.
MATERIALS AND METHODS
Platinum Drugs
Cisplatin, carboplatin, JM216, JM335, and AMD473 were
synthesized by and obtained from the Johnson Matthey Tech-
nology Center; structures of these agents are shown in Fig. 1.
In Vivo Activity Studies
Tumor Lines
Two murine tumor models plus their respective cisplatin-
resistant variants have been used; the s.c. solid ADJ/PC6 plas-
macytoma and ADJ/PC6cisR and ascitic i.p. L1210 leukemia
and Ll2lOcisR. The derivation of these models and their cali-
bration with “standard” platinum drugs has been described
previously (10). ADJIPC6 tumors were grown in syngeneic
female Balb/c mice and Ll2lO in DBA, mice.
A series of human ovarian tumor xenografts has also been
used as described previously ( 1 1). These lines, grown s.c. in
female nude (nu/nu) mice, were selected to encompass a broad
I The abbreviations used are: DACH. 1 .2-diaminocyclohexane; MTD,
maximum tolerated dose; ALT, alkaline transaminase: ALP, alkaline
phosphatase; ED,�), dose in mg/kg required to reduce tumor mass by
90%; q7dX4, once every 7 days for a total of 4 weeks.
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A�MD473
Fig. 1 Structures of the platinum complexes cisplatin, carboplatin,
JM216, JM335, and AMD473.
Clinical Cancer Research 2065
H3N\ /Cl
H3N/ Pt\ci
CISPLATIN
OCOCHNil J Cl3
3\ /
� �2I �Cl
OCOCH�
JM216
H3N\ /oc�\ //\
H3N / �CARBOPLATIN
OHCl � Nil
\/ 3
QN112 �Cl
JM335
groups (n = 6) or control groups (n = 10); the day of random-
ization was termed day 0. Drugs were administered at MTDs
based on previous determinations and/or ADJIPC6 tumor data
(approximately MTD/lO% lethal LD,0 dose). Unless otherwise
stated, drugs were administered on days 0, 7, 14, and 21.
Tumors were measured weekly using calipers, and volumes
were determined (4, 6, 1 1) until the tumors had at least doubled
their starting volume. Responses were compared in terms of
growth delays: the difference in time taken for control and
treated tumors to double in volume.
Pharmacokinetics StudiesNil
3\ /Cl
Pt
�/ �Cl
spectrum in responsiveness to cisplatin and carboplatin and
ranged from relatively sensitive to cisplatin [PXN/65 and CH1
(formerly also termed PXN/lO9T/C)] to intermediate sensitivity
(HX/l 10) to refractory (SKOV-3 and HX/62). Two xenograft
models of acquired cisplatin/carboplatin resistance were also
included as described previously (20); CHlcisR (derived from
the corresponding cell line and also formerly termed PXN/l09/
T/CC) and HX/l lOP (derived through repeated treatment with
carboplatin of animals bearing HX/l 10 tumors).
Assessment of Antitumor Activity
Platinum drugs were administered i.p. or p.o. according to
the stated schedules either in saline (cisplatin and carboplatin) or
as sonicated suspensions in arachis oil.
ADJ/PC6 and ADJIPC6cisR. Assessment of antitumor
activity was as described previously (4, 6, 10). Briefly, 20 days
following s.c. implantation of 1-mm3 tumor fragments, drugs
were administered (at halving doses) as single i.p. or p.o. doses.
Three animals were used at each dose level, and 10 control
animals were used. Ten days later, tumors were removed, and
the weights of control and treated groups were compared. As
previously, antitumor efficacy has been defined in terms of a
“therapeutic index”: the ratio of the MTD in mg/kg to the ED�).
L1210/Ll2lOcisR. Antitumor activity was assessed as
described previously (4, 10) using an increase in life span.
Briefly, 5 mice/treated group and 10 untreated controls were
implanted i.p. with 1 X l0� cells. Platinum drugs were then
administered on days 1, 5, and 9. Animals were not permitted to
die in these survival end point assays, but were sacrificed at the
onset of moribundity.
Human Ovarian Carcinoma Xenografts
Nude mice bearing comparably sized s.c. xenografts (typ-
ically 6-8 mm diameter) were randomized into either treatment
Animals
Female Babb/c mice (6 weeks of age) were acclimatized
to the laboratory conditions 2 weeks prior to the experiment.
They were allowed food (SDS expanded rodent diet) and water
ad libitum. The animals weighed 20 ± 1 .2 g at the time of
treatment.
Experiment 1
The animals were implanted s.c. in the right flank with I
mm3 ADJIPC6 fragments. The tumor was left to grow for 20
days. Animals of equal tumor sizes were then randomized into
the different groups. Animals with large or small tumors were
excluded from the experiment.
Group 1: the animals were injected iv. in the tail vein with
20 mg/kg (0.1 mI/lO g) AMD473 following transient hyperther-
mia to induce vasodilation. Group 2: the animals were given 20
mg/kg (0.1 mI/lO g) AMD473 i.p in saline. Group 3: the animals
were given 20 mg/kg AMD473 p.o. by gavage.
The animals were anesthetized with halothane, and blood
was collected in heparinized syringes following severing of the
axillic vessels. Blood was centrifuged for 10 mm at 1000 X g.
and the plasma was decanted and frozen at -70#{176}Cuntil analy-
sis. An abiquot of the plasma was ultrafibtered upon collection
through Amicon 10,000 MW exclusion membranes by centrif-
ugation at 1500 x g for 45 mm. Tissues (liver, kidney, spleen,
heart, lung, brain, skin, and tumors) were collected as quickly as
possible following cervical dislocation of the animals and snap
frozen in liquid nitrogen.
Blood and tissues were collected 5 mm, 15 mm, 30 mm,
1 h, 2 h, 4 h, 6 h, 24 h, 48 h, and 72 h postadministration (n =
4 animals per time point).
Experiment 2
Balb/c mice were given 25, 50, 100, 200, and 400 mg/kg
AMD473 p.o. in saline. Blood, liver, kidney, spleen, heart, and
lung were collected 1, 2, 4, 6, 24, 46, and 72 h (n = 3 animals
per time point) postadministration and treated as described
above.
Experiment 3
Animals were placed in metabolic cages for 3 days and
treated with AMD473 on day 1 . The urine and feces were
collected daily and frozen at -70#{176}Cuntil analysis.
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AMD473
2066 AMD473, A Novel Platinum Complex
Table I In vivo antitumor efficacy of AMD473 versus cisplatin against murine tumors, ADJIPC6 and the corresponding cisR s.c.
plasmacytomas
Cisplatin
MTD (mg/kg) ED�) TIMID (mg/kg) ED�) TI”
ADJ/PC6 i.p. 43 3 14.3 11.3 1.6 7.1ADJ/PC6 p.o.
ADJ/PC6cisR i.p.560
356.2
-25”90.3
1.4140
1124 5.8
ADJIPC6cisR p.o. 560 200 2.8 ND ND ND
“ TI. therapeutic index: MTD/ED�1. ND. not determined.
I, 87% inhibition.
Table 2 Maximum increase in life span (%) in L1210 and cisR i.p.leukemias
AMD473 (dose) Cisplatin (dose)
L12l0 54(32mg/kg) 79(4mg/kg)
Ll2lOcisR 29 0
Protein BindingfPlasma Incubation
Fresh human plasma was incubated with 5 �iM AMD473 or
cisplatin at 37#{176}Cfor 2 mm, S mm, 15 mm, 30 mm, 1 h, 2 h, 6 h,
and 24 h and subsequently ultrafiltered as described previously.
and the free and total platinum was measured in the samples by
atomic absorption spectrophotometry.
Analytical Conditions
A Perkin-Elmer (model 1 100) atomic absorption spectro-
photometer with a graphite furnace (model 700) was used for
furnace atomic absorption spectrophotometry. A 5-7-stage tern-
perature program was used according to the type of tissue.
Fifty-p.l samples were introduced into the furnace at 60#{176}C.The
absorption of platinum was recorded at 265.9 nm. Platinum was
quantified in the samples using an external standard calibration
method with platinum standards between 0 and 100 ng/ml for
plasma ultrafiltrates and between 0 and 400 mg/kg for tissue and
plasma platinum. Quality controls were included in duplicate at
the beginning and end of each run at the level of 20 and 50
ng/ml and 100 ng/ml for tissue samples. To 200 mg tissue or
feces was added 0.5 ml of hyamine (Sigma Chemical Co.,
United Kingdom), and the mixture was incubated at 50#{176}Cover-
night. HC1 (0. 1 M; S ml) was then added, and the diluted samples
were analyzed by furnace atomic absorption spectrophotometry.
Plasma and plasma ultrafiltrates were diluted as required prior to
analysis.
Toxicology Studies
All toxicities studies were performed under strict control.
Animals were checked several times per day and sacrificed at
the onset of moribundity.
Hematological Toxicity
Female Balb/c’ received a single dose of either AMD473
(45 mg/kg i.p. in oil) or control arachis oil (n = S animals per
group). Mice were bled as described previously over 28 days
(days 1, 2, 4, 7, 14, 22, and 28). The blood was placed in
heparinized tubes; WBC, platelets, and hemoglobin were eval-
uated.
Disaccharidase Activity
Using the same group of mice as for the hematology study,
a 5-cm section of jejunum was removed, and gut mucosa was
gently scraped and frozen until analyzed. The disaccharide
(sucrose, maltose, and trehalose) content was measured with
Bio-Rad assays as described previously (21).
ALT, ALP, Creatinine, and Urea
Female Balb/c mice received a single dose of drug i.p. in
oil (n = 3 animals per control group and 6 animals in the treated
group). At 2 h, 2 days, 6 days, and 10 days, they were bled by
axillary incision under halothane and plasma decanted following
centrifugation. Urea, creatinine, ALT, and ALP were analyzed
at the Royal Marsden Hospital by conventional methods.
Histopathology Examination
Using the same group of mice and the same time point as
for the hematology study, liver, kidney, spleen, gut, heart, and
lung were removed and fixed in metharcan until they were
sliced and stained for microscopic examination.
Inulin Clearance
Glomerular filtration rate was determined in mice (10
control animals and 10 animals treated with 45 mg/kg AMD473
i.p in oil) after treatment with ‘4C-inulin as described previously
(22).
Neurotoxicity Assessment in the Rat
The effect of chronic treatment over 6 weeks with
AMD473 (8 or 12 mg/kg i.p. in oil twice weekly) or cisplatin (2
mg/kg i.p. in saline) on nerve conduction velocity was evaluated
using the previously described method (23).
Statistics
Results were expressed as means ± SD. The differences
between groups were assessed with Student’s t test for unpaired
samples, ANOVA, and the Mann-Whitney test, as appropriate.
Pharmacokinetic parameters were calculated with the PC-
NONLIN software (Lexington, KY) with compartmental anal-
ysis. Functions consisting of 1 , 2, or 3 exponential components
were fitted to the data by the least squares method. Each set of
data was analyzed with 1 , 2, or 3 compartments and the best fit
Research. on January 23, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
500
A
�
l0
B
600
� 500aa
I- 400
I- 300
.�
.� 200
101
Clinical Cancer Research 2067
Table 3 In vivo antitumor ef ficacy of AMD473 ye rsus cisplatin against human ovarian carcinoma xenografts”
Growth delay (days)
AMD473
(30-40 mg/kg i.p.)
Cisplatin
(4 mg/kg i.p.)
Carboplatin”
(80 mg/kg i.p.)
JM216”
(90 mg/kg: p.o.)JM335’
(4 mg/kg i.p.)
PXN/65HXJ11O
HX/llOPCHICHlcisRSKOV-3HX/62
>21564
24>139
346.80.1
2094V
T3410.4
0-2.9
ND”47
1443
6.42
ND
ND36
ND43
3.56
ND
ND25.5
ND17.5
5.46.8
ND
“ All drugs were given on a q7d X 4 schedule. AMD473, cisplatin. and JM335, i.p. administration: JM216, p.o. administration.I, Data taken from Refs. 4. 6. and 20 except for CHlcisR data.“ Data taken from Ref. 7.“ ND, not determined.(. AMD473 and cisplatin were not compared in head-to-head experiments.
was adopted. For example, it was shown that the best fit for the
plasma iv. data was a two-compartment model (model 7),
whereas the best fit for oral plasma level at 20 mg/kg was a
model 4 (one compartment with first-order absorption). Tissue
to plasma ratios were calculated using the relative AUC calcu-
lated to the last point with the trapezoidal method. Bioavailabil-
ity following i.p. and oral administration was calculated as the
ratio of total platinum AUC versus the iv. administration total
platinum AUC.
RESULTS
In Vivo Antitumor Activity. Tables 1-3 summarize the
antitumor efficacy data for AMD473 (compared wherever pos-
sible to cisplatin) for a variety of murine and human tumor
models. Against the murine ADJ/PC6 plasmacytorna, AMD473
by single i.p. administration showed antitumor efficacy similar
to that observed for cisplatin. Moreover, AMD473 exhibited
some retention of activity against a variant of the ADJ/PC6
model made resistant to cisplatin by in vivo selection. Notably,
antiturnor activity was retained with oral administration but with
a substantial reduction in toxicity, resulting in an increased
therapeutic index of 90. As shown previously (4) no improve-
ment in therapeutic index and a loss in antitumor activity was
observed following oral administration of cisplatin. With
AMD473, antitumor activity by the oral route was also observed
against the acquired cisplatin-resistant subline. Against the i.p.
ascitic L1210 murine leukemia, AMD473 showed efficacy sirn-
ilar to cisplatin and retained some activity against the cisplatin-
resistant variant.
AMD473 exhibited improved antitumor activity compared
to cisplatin when used against either the cisplatin-sensitive
PXN/65 human ovarian carcinoma xenograft (Fig. 14) or the
xenograft derived from the CH1 cell line (Fig. 2B). Whereas
PXN/65 tumors eventually regrew following 4 mg/kg cisplatin
q7dX4, AMD473 was curative against this xenograft. Against
the CH1 xenograft, cisplatin (4 mg/kg q7dX4) typically induces
growth delays of 25-35 days (Ref. 4 and this study). In contrast,
AMD473 at 40 mg/kg q7dX4 was curative; AMD473 used at 30
mg/kg q7dX4 also induced a substantial growth delay (approx-
imately 80 days).
A head-to-head experiment comparing AMD473 with cis-
j 400
I� 300
� 200
ZD )U I) 11,11) IZ� I)U I 11 LOU 12)
DAY
0 25 50 75 100 125 150 75
DAY
Fig. 2 In vito antitumor activity of AMD473 versus cisplatin (q7dX4
i.p. schedule) against PXN/65 human ovarian carcinoma xenograft (A)
or CH1 human ovarian carcinoma xenograft (B). fl. controls; S. 4
mg/kg cisplatin: V. 30 mg/kg AMD473: A. 40 mg/kg AMD473. Data
points, means: bars. SD.
platin, carboplatin, and JM2I6 was performed using the xc-
nograft derived from the acquired cisplatin-resistant CH lcisR
cell line (Fig. 3). Results show that AMD473 (especially at 40
mg/kg) shows an encouraging level of in vito activity against
this acquired cisplatin-resistant tumor, inducing a growth delay
of 29-34 days, about 3-fold greater than that observed with
cisplatin (10.4 days) and much greater than that observed for
carboplatin (6.4 days), JM216 (3.5 days), or JM335 (5.4 days).
Research. on January 23, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
(5E
0>
0E
I-.(5>15
(5
Fig. 4 In 100 antitumor activity of AMD473
lersus cisplatin (each given on days 42. 49. and
56 following initial treatments) against CH1
human ovarian carcinoma xenografts at re-
growth after treatment with cisplatin (3-4
mg/kg on days 0. 7. 14. and 21). fl. controls:
., 4 mg/kg cisplatin: A. 35 mg/kg AMD473.
Data points. means: l)arS. SD. Arrows, days of
drug administration.
DAY
IDAY
2068 AMD473. A Novel Platinum Complex
Fig. 3 In rho antitumor activ-
ity of AMD473 versus cisplatin
and carboplatin (q7dX4 i.p.
schedule) and JM216 (q7dX4
P.O. schedule) against CHlcisR
human ovarian carcinoma xc-nograft. E, controls; #{149},4
mg/kg cisplatin; X, 80 mg/kg
carboplatin; 0. 90 mg/kgJM2I6: V, 35 mg/kg AMD473:
A. 40 mg/kg AMD473. Data
points, means; bars, SD.
Across the whole range of human ovarian carcinoma xc-
nografts used in this study (Table 3), AMD473 showed activity
that was improved over (e.g., CH1 and CHlcisR) or at least
comparable to (e.g., HX/62 and SKOV-3) that observed for
cisplatin. Evidence of activity for AMD473 against another
model of acquired platinum drug resistance [HX/l lOP with in
vito-derived resistance to carboplatin (20)] was also apparent.
The antitumor activity of AMD473 was compared to that
of cisplatin against CHI human ovarian carcinoma xenografts
that had regrown following initial treatment with cisplatin (Fig.
4). Importantly, at the time of retreatment (42 days after first
cisplatin dose), tumors were comparably large, being approxi-
mately twice their volume at the start of treatment (day 0).
Retreatment with cisplatin induced a plateau in tumor growth
and a gain of about 20 days growth delay before rapid regrowth
of the tumors. In contrast, AMD473 administered at 35 mg/kg
on days 42, 49, and 56 induced a marked reduction in relative
tumor volume and a gain of approximately 50 days growth delay
before reemergence of the tumors.
Following the observation of antitumor activity by the oral
route in the ADJIPC6 model, oral antitumor studies with
AMD473 were also performed using the CHI/cisR xenograft
tumor. Comparable activity was obtained to that observed by the
i.p. route of administration (e.g. , maximum of 34.5 days with
400 mg/kg p.o. q7dX4). In addition, and in contrast to results
with JM216 (24), no schedule dependency was noted; similar
growth delays were observed with a daily schedule (5 days per
week; 60 mg/kg/day for 4 weeks; growth delay of 22.4 days)
versus a weekly schedule (300 mg/kg q7dX4; growth delay of
25.9 days; Fig. 5).
Pharmacokinetics. The time course of platinum in
plasma and plasma ultrafiltrates following iv., i.p., and oral
Research. on January 23, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
DAY
10000
1000
100
10
10000
1000
100
10
E
C
0.
Ea)C
a-
I i,�,ll-
02468 10 20 30 40 50
Time (h)60 70 80
1 11111 I i j , I
02468 10 20 30 40 50 60 70 80
Time (h)
Fig. 6 Time course of plasma platinum (top) and plasma ultrafiltrableplatinum (bottom) following 20 mg/kg AMD473. Data points. means:bars, SE.
Clinical Cancer Research 2069
Fig. 5 Schedule dependency of antitu-
mor activity of AMD473 following oral
administration to mice bearing the
CHlcisR human ovarian carcinoma xc-nograft. E�. controls: A, 300 mg/kg
AMD473 (q7dX4 p.o. schedule): #{149}.60
mg/kg/day AMD473 (p.o. for 5 days per
week for 4 weeks). Data points, means:
bars, SD.
administration of AMD473 to ADJ/PC6 tumor-bearing mice is
shown in Fig. 6. Following iv. administration, the total platinum
levels fitted a two-compartment model with half-lives of 24 mm
and 44 h (Table 4). Following i.p. administration of AMD473,
maximum platinum levels in the plasma were reached by 0.5 h,
and the plasma decay was very similar following i.v. and i.p.
administration with a bioavailability of 89%. The concentration
versus time course of ultrafiltrable platinum also followed a
two-compartment model with a terminal half-life of 6 h. When
given p.o., AMD473 was rapidly absorbed; Cmax occurred
within 1 h postadministration and showed an elimination half-
life similar to that observed iv. (3 1 h). The overall bioavail-
ability was 40% (Table 4).
The different platinum levels in various tissues and
organs are shown in Fig. 7, and the pharmacokinetics param-
eters are shown in Table 5. Platinum was widely and rapidly
distributed with accumulation occurring in the liver and
kidney. Tissue exposure was prolonged, with elimination
half-lives often greater than 100 h, and sometimes the half-
life could not be determined because no significant decrease
in platinum in the tissues could be observed over the time
course studied. As a result, the liver to plasma and kidney to
plasma ratios were high with the three schedules (8.6 follow-
ing iv. administration). Platinum could not be detected in the
brain, and the lowest levels were found in skin and muscle.
With all three routes of administration, tumor levels were in
the cytotoxic range. Following i.v. administration, the tumor
to plasma ratio was 5; following oral administration, it was
only 1.5.
The pharmacokinetics parameters following increasing oral
doses of AMD473 are shown Table 6. A less than proportional
increase in AUC, Crnax, was seen in plasma, ultrafiltrable
plasma, and all tissues examined (liver, kidney, and spleen; Fig.
8). A significant increase in the platinum half-life of elimination
was registered in plasma and plasma ultrafiltrate with increasing
doses (P < 0.01). However, Cn�ax was always reached at 1 h
(Table 6).
.
-5-- iv plasma
...-. �0 plasma
-,-- ip plasma
-0- iv UF
-o�poUF
� -c-ipUF
� �-
Following administration of 20 mg/kg AMD473 i.v or 45
mg/kg i.p, approximately 33% of the total platinum was elimi-
nated in the 24-h urine and 8% in the 24-h feces (Table 7).
Following oral administration, the 24-h fecal recovery increased
Research. on January 23, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Fig. 7 Time course of platinum in tissues follow-ing 20 mg/kg AMD473 iv. Data points, means:bars, SE.
Time (h)
2070 AMD473. A Novel Platinum Complex
Table 4 Plasma and ultrafiltrable plasma pharmacokinetic parameters evaluated with PCNONLIN compartmental analysis followingadministration of 20 mg/kg AMD473 to Babb-C mice bearing ADJIPC6 plasmacytoma tumors
SEs on estimated pharmacokinctic parameters were <30%.
AUC (ngPt . ml� . h) Cmax (ng . ml’’) T,nax (h) K01 (h) t#{189}Q (mm) 1#{189}01(h)Clearance(ml . h’)
Volume ofdistribution (ml)
Plasma iv. 37532 6936 0.25 24 44 0.24 28Plasma i.p. 33315 7044 0.5 19 50
Plasma p.o. 15025 1308 0.5 0.5 31UF iv. 4922 4278 0.25 8.4 9.66 67 617
UF i.p. 4550 1325 0.5 2.4 9.14UF p.o. 2677 � 201 0.99 0.57 66 26
0I
a-
to 25% after 20 mg/kg and 58% after 400 mg/kg, reflecting the
unabsorbed platinum. The 16% urinary recovery after 20 mg/kg
AMD473 further confirms the 40% bioavailablility, because
33% was eliminated following 20 mg/kg iv.
The protein binding of AMD473 and cisplatin is shown
Table 8. AMD473 and cisplatin both bound to albumin more
readily than globulin. However, AMD473 showed signifi-
cantly less affinity for proteins than cisplatin; AMD473 had
initial half-lives of binding to albumin, globulin IV, and
plasma of 6, 12, and 6.2 h, respectively, compared to 2.5, 8,
and 3 h for cisplatin. The globulin II binding was signifi-
cantly slower, with less than 50% of platinum bound 24 h
postincubation.
Murine Toxicology. Toxicology studies conducted at
the predetermined mouse MTD of 45 mg/kg (i.p.) revealed that
myelotoxicity was the dose-limiting toxicity in mice, with both
leukopenia and thrombocytopenia occurring by 10 days after
treatment and reversing after 2 weeks. A decrease in platelets
was also registered at the same time but it was not quite
significant (Fig. 9).
Histological examination showed no clear signs of organ
toxicity. A transient gut toxicity was observed on day 4 in one
of three animals with villi atrophy in the gut crypt (data not
shown). However, there was no significant difference in the
disaccharide levels over the 28 days (data not shown), implying
that gut toxicity is not significant. Spleen inflammation (red
pulp) was observed after 2 weeks but reversed after 28 days. No
sign of liver or cardiac toxicity was noted; this was further
confirmed by measurements of ALTs and ALPs, which were not
significantly different in the treated groups compared to the
control group (data not shown).
The urea and creatinine levels were not significantly
different in the treated group compared to the control group
over the time course studied. The inulin clearance was
(18.6 ± 2.4 mlmin’kg’) versus (17.1 ± 1.4
ml-min � Lkg 1) in the control group, showing that AMD473
has no renal toxicity in the mouse. Additional studies in the
rat have also shown no significant neurotoxicity following
chronic dosing over 6-8 weeks of 8 and 12 mg/kg AMD473
(i.p.) and assessment by the previously described sensory
nerve conduction velocity model (23). The nerve conduction
velocities following AMD473 treatment were 44 ± 20 m-s
after 6 mg/kg and 50 ± 20 ms ‘ following 8 mg/kg as
opposed to 22 ± 9 ms ‘ for the cisplatin-treated group (2
mg/kg) and 38 ± 9 ms ‘ for the control group.
Research. on January 23, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Table 5 Tissue pharmacokinetic parameters evaluated with
PCNONLIN compartmental analysis following administration of 20
mg/kg AMD473 to Balb C mice bearing the ADJIPC6
plasmacytoma tumors
SEs on t/ were <40%.
Tissue AUC (jig . g�) � h Cmax (�g � g1) T,,,ax (h) tv� (h)
Table 6 Plasma and ultrafiltrable plasma platinum pharmacokinetic
parameters following oral administration of AMD473 to mice
SEs on estimate parameters were <40%.
i.P.
(‘ P < 0.05 compared to the 50 mg/kg group.
I’ p < 0.01 compared to the 50 mg/kg group.
C� NE. not evaluated.
Clinical Cancer Research 2071
i.v.
Liver 322.4 10.6 1 85
Kidney 322.2 11.6 1 85
Spleen 213.1 4.5 2 NE”Heart 141.1 6.1 2 NE
Lung 195.4 2.1 3 NE
Tumor 200.5 7.5 0.5 20
Muscle 80.6 3.7 2.6 13
Skin 94.7 5.9 2 NE
Liver 334.7 14.3 4 97
Kidney 217.9 5.1 1 NE
Spleen 202.9 7.3 2 NE
Heart 130.2 7.3 1.9 8.8
Lung 147 8.2 2 NE
Tumor 126.5 5 2.6 16.8
Muscle 25 5.1 4 13.4
Skin 26 1 .5 0.6 NE
P.O.
Liver 86.6 5.3 4 NE
Kidney 66.4 2.9 1 87
Spleen 39.3 1.3 1 104Heart 43 3.4 4.8 31
Lung 46.3 0.5 36 NE
Tumor 37 1.6 1.5 74
Muscle 14 1.6 4 NE
Skin 47 4 0.2 119
“ NE, not evaluated.
DISCUSSION
A collaborative effort between the Cancer Research
Campaign Center for Cancer Therapeutics at the Institute of
Cancer Research and the Johnson Matthey Technology Co.
was established many years ago with the aim of discovering
and developing platinum drugs with a toxicological and/or
antitumor profile improved over that of cisplatin. From well
over 500 compounds studied, the following lead complexes
have emerged: carboplatin, the only cisplatin analogue to be
registered worldwide (2); JM216, now in Phase II clinical
trials as the first p.o. bioavailable platinum drug (4); JM335,
the first trans-platinum analogue to demonstrate attractive in
vis’o antitumor activity against a range of s.c. preclinical
tumor models (6); and the sterically hindered complex
AMD473 (herein). In contrast to the development of carbo-
platin and JM2I6, which was predominantly on the basis of
improving patient quality of life during platinum-based
chemotherapy, AMD473 was designed in recognition of an
increasing awareness of the mechanisms by which tumors
might become resistant to cisplatin/carboplatin in the clinic.
In vitro, AMD473 seems to be able to overcome all types of
known platinum resistance in cell lines known to have de-
creased cisplatin accumulation/uptake, increased DNA re-
pair, and reduced detoxification by thiols (19).
In this study, we have shown that AMD473 also exhibits a
remarkable level of activity against acquired cisplatin-resistant
tumors in vivo. At least some evidence of in vivo antitumor
Dose AUC(mg/kg) (ngPt - mI� . h)
C,�
(ng . ii�I’) (h)
t1/2(�
(h)‘1/2)3
(h)
Plasma
25 26752 1380 1 0.5 50
50 59287 2684 1 0.44 24
100 79799 5538 1 0.82 42
200 102375 7370 1 1.48 96”
400 166167 11114 1 1.7 87”
Plasma ultrafiltrate
25 3409 532 1 1.7 0.04
50 6068 920 1 1.3 6
100 4701 985 1 1.47 22”
200 6388 1047 1 1 33”
400 16058 1069 1 2 NE’
activity was observed in four models of acquired cisplatin
resistance: ADJ/PC6cisR, L 12 1OcisR, HX/l I 0 (carboplatin-re-
sistant), and, in particular, CHlcisR (for which a growth delay
of around 30 days was obtained). In t’is’o activity at least
comparable to that observed for cisplatin was found for
AMD473 for all xenografts studied. Tetraplatin was inactive in
vivo against the ADJ/PC6 cisR tumor ( 10) and induced signif-
icant growth delays in only 2 of 16 human ovarian tumor
xenografts (those were also sensitive to cisplatin: Ref. 1 1 ). In
xenograft models corresponding to two cell lines with acquired
resistance to cisplatin, derived from the A2780 cell line, growth
delays of 1 1 and 13 days (parent line) and 9 and I 2 days
(acquired cisplatin-resistant line) for cisplatin and lobaplatin.
respectively, were observed (20. 25). Although the previously
described trans-platinum(IV) complex JM335 also showed
promising circumvention of acquired cisplatin resistance in vitro
(6), it was less active than cisplatin in vito against the panel of
human ovarian carcinoma xenografts (Refs. 6 and 7 and data
herein for CHlcisR).
The activity of AMD473 was also retained p.o. with a
high therapeutic index of 90 in the ADJ/PC6 model and with
some retention of efficacy against the acquired cisplatin-
resistant ADJ/PC6cisR. Against the CHlcisR acquired cis-
platin-resistant human ovarian carcinoma xenograft, maximal
growth delay by the oral route (34 days) was comparable to
that observed by the i.p. route and considerably greater than
that previously obtained for the oral platinum drug JM21#{212}in
this model. Moreover, in contrast to JM216 (24) there was no
apparent schedule dependency for AMD473: similar growth
delays were observed for the same total weekly dose given
once a week or split over 5 days.
The pharmacokinetic and tissue binding experiments
showed that the behavior of AMD473 is in many ways inter-
mediate between that of cisplatin and carboplatin. Qualitatively.
protein binding was similar to that of cisplatin, with greater
affinity to albumin than globulin. However, quantitatively, it
was only half of that observed with cisplatin at equimolar
concentrations. The slower reactivity of AMD473 with protein
Research. on January 23, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Fig. 8 AUC versus dose inplasma (0), plasma ultrafiltrate
(0). liver (#{149}),kidney (A), andlung (#{149})following administra-tion of 25, 50, 100, 200, and400 mg/kg AMD473. Valuesare expressed in p.g ofplatinum-m1 -h in plasma and�sg of platinumg ‘�h in tissues.
Dose AMD473 (mg/kgJ
2072 AMD473, A Novel Platinum Complex
5 Personal communication.
.C
EI-0at
at
a.
L)
Table 7 P ercentage of p latinum administered recovered in the urine and feces follow ing administration of AMD473
Urinary recovery, % dose (Pt) Fecal recovery, % dose (Pt)
24h 48h 72h24h 48h 72h
2Omg/kgi.v.
2Omg/kgp.o.
45mg/kgi.p.
400mg/kgp.o.
33±7
16±2
34±7
13±1
38±4
17±2
38±8
14±1
NE�
NE
40±8
NE
8±2
25±10
7.5±15
58±2
15±1
26±10
10±7
59±6
NE
NE
12±7
NE
“ NE, not evaluable.
Table 8 Protein binding of AMD473 versus cisplatin to human plasma and plasma proteins
Half-lives are expressed in h and in percentage of free platinum remaining 24 h postincubation. The SE of % offreeplatinum was <20%.
AMD473CDDP
tI,2
(h) % free platinum at 24 h
11/2
(h) % free platinum at 24 h
Human albuminHuman globulin IIHuman globulin IVHuman plasma
2.515
83
5
522420
61216
6.2
20644425
compared with that of cisplatin is the result of a slower aquation
rate.5 The cisplatin protein binding compares well with values
published in the literature (26, 27).
The plasma platinum concentration versus time curve showed
a classical bi-exponential decay with long elimination half-
lives (30-45 h) after all routes of administration. This long
terminal half-life corresponds to the protein elimination half-
life as covalent binding occurs. The platinum tissue distribu-
tion of AMD473 was wide, which is classical for a platinum
complex with high liver, kidney, and tumor platinum expo-
sure (tissue to plasma ratio of 8.6, 8.6, and 5, respectively, in
the iv. schedule). Tissue absorption was rapid, with tmax
occurring mainly 1 h postadministration. The absolute tissue
platinum levels observed following AMD473 administration
were between those reported after cisplatin and JM216 at
equitoxic doses (28, 29). No platinum could be detected in
the brain. The bioavailability following i.p. administration
was 89%, which showed that AMD473 is absorbed well i.p.
Research. on January 23, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
200
150
Fig. 9 Hematological toxicity following 45mg/kg AMD473 i.p. Results are expressed aspercentage of controls. #{149},leukocytes; A, he-
moglobin; 0, platelets. *, P < 0.002.
50
0 5 1#{149}0 15 20 25 30
DAY
Clinical Cancer Research 2073
zCU
This finding justifies the choice of the i.p. route for toxicol-
ogy/in vivo antitumor evaluation, because the relatively poor
aqueous solubility of the drug limits the dose that can be
administered iv. to 20 mg/kg. At this dose, the oral bioavail-
ability of AMD473 was 40%. The percentage of platinum
administered that was recovered in the urine was signifi-
cantly lower than what was observed following equitoxic
doses of cisplatin (Ref. 28; 33% after AMD473 versus 50%
following cisplatin). In view of the fact that AMD473 binds
proteins less than cisplatin, the opposite would be expected.
This suggests that more platinum may have entered the tissue
following AMD473 when compared with cisplatin.
Given p.o., both the AUC and Cmax showed a less than
proportional increase at doses above 100 mg/kg. Although
there was no change in the tmax, there was an increased
elimination half-life with 400 mg/kg, suggesting that late
absorption might still occur at high doses but that the absorp-
tion peak was not delayed. The terminal half-life for ultra-
filtrable platinum was significantly higher at higher doses
compared to 25 and 50 mg/kg, suggesting that platinum
species arc still being absorbed at late time points. However,
the ratio of intratumor platinum exposure following oral
administration to exposure following i.p. administration was
1.5. In view of the improved therapeutic index after oral
administration, it is possible that oral administration results
in some sort of metabolic activation of AMD473, which is
presently under investigation.
The fact that the percentage of platinum excreted in the feces
increases from iv. to oral administration and also increases with
higher doses confirms that absorption is a limiting factor by this
route of administration. When compared with the other oral plati-
num analogue JM216, AMD473 showed more urinary and fecal
excretion than JM216 at equitoxic doses [40% total platinum re-
covered following the MTD of JM216 (29) versus 72% for
AMD473�. This is comparable to what was observed with JM216,
for which absorption proved to be a limiting factor and a fraction-
ated daily schedule (for S days) proved to be more successful both
preclinically and in the Phase I trial (24).
The toxicity study of AMD473 was very similar to that
previously reported for JM216 (29): the dose-limiting toxicity
was myclosuppression, with leukopenia occurring 10 days post-
treatment and reversing after 2 weeks. Histological examination
revealed no toxicity apart from mild gut toxicity, which is
characteristic of antiproliferative agents. There was no sign of
renal toxicity as observed histologically, biochemically with
urea and creatinine levels, or by measurement of the gbomerular
filtration rates by inulin clearance. This is comparable to previ-
ous observations with carboplatin and JM216 (30, 31). Prelim-
mary data also suggest that AMD473 does not induce peripheral
neurotoxicity following chronic i.p. treatment to rats.
In summary, AMD473 appears to possess a significant level
of in vivo antitumor activity against some acquired cis-platin-
resistant tumors. In common with JM216, activity by the oral route
was also observed. Combined with a favorable pharmacokinetic/
toxicological profile (myclosuppression was the dose-limiting tox-
icity observed in mice, and there was no evidence of nephrotoxic-
ity), AMD473 is a promising candidate for clinical evaluation in
the cisplatin resistance setting and has recently been selected for
clinical study under the auspices of the United Kingdom Cancer
Research Campaign Phase I/H Committee. It is planned that studies
will have begun in mid-1997.
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