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:

[email protected].

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.



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.



AMD473


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



500

A

�

l0

B

600

� 500aa

I- 400

I- 300

.�

.� 200

101


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).



(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



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.


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



Fig. 7 Time course of platinum in tissues follow-ing 20 mg/kg AMD473 iv. Data points, means:bars, SE.

Time (h)


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.



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.


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



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


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.



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


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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