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[CANCER RESEARCH 48, 4509-4512, August 15, 1988)
Increased Cytotoxicity and Reversal of Resistance to c/f-Diamminedichloroplatinum(II) with Entrapment of c/s-Bis-neodecanoato-f raws-/?,/?-! ,2diaminocyclohexaneplatinum(II) in Multilamellar Lipid Vesicles1
Roman Perez-Soler,2
Li Y. Yang, Benjamin Drewinko, Julio Lauterzstain,
and Abdul R. Khokhar
Immunobiology and Drug Carriers Section, Department of Clinical Immunology and Biological Therapy [R. P. S., J. L.J, and the Departments of Laboratory Medicine
IL. Y. Y., B. D.], and Medical Oncology [A. R. K.¡,The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030
ABSTRACT
The role of liposome entrapment in modulating the cytotoxicity of a
lipophilic cisplatin derivative was assessed. ctVBis-neodecanoato-fransÄ,Ä-l,2-diaminocyclohexaneplatinum(II)
(NDDP) wa§tested in suspen
sion (free NDDP) or entrapped in multilamellar vesicles composed of
dimyristoylphosphatidyl
choline and dimyristoylphosphatidyl
glycerol
(L-NDDP). Against LoVo colon carcinoma cells sensitive to cisplatin, I .NDDP was two times more cytotoxic in vitro than free NDDP and
cisplatin (Do 7 ut* for L-NDDP, 15 uM for free NDDP, and 16 MMfor
cisplatin). Against LoVo cells resistant to a concentration of 3 Mg/ml of
cisplatin, L-NDDP was three times more cytotoxic than free NDDP and
cisplatin (Do 14 u\t for L-NDDP, 45 u\l for free NDDP, and 48 >iMfor
cisplatin). In in vivo studies, free NDDP was less potent and less active
than L-NDDP against i.p. I -12111 leukemia (free NDDP, optimum %T/
C 148 at a dose of 75 mg/kg; L-NDDP, optimum %T/C 185 at a dose of
25 mg/kg) and i.p. L1210/PDD leukemia (free NDDP, optimum %T/C
128 at a dose of 50 mg/kg on Days 1, 5, and 9; L-NDDP, optimum %T/
C 200 at a dose of 12.5 mg/kg on Days 1, 5, and 9). Free NDDP
administered i.v. was inactive against liver métastasesof M5076 reticulosarcoma (%T/C 102) while L-NDDP showed significant activity (%T/
C 140). The single dose i.v. I ,!><„
in mice of free NDDP and L-NDDP
were similar (79.4 mg/kg for free NDDP and 64.5 mg/kg for L-NDDP).
These studies show that NDDP is a liposome-dependent
drug since it
can only be satisfactorily formulated in the liposomal form and since the
liposomal carrier plays a crucial role in determining its antitumor activity.
INTRODUCTION
Intrinsic or acquired resistance to the available anticancer
agents is one of the main limitations of cancer chemotherapy
(1). Cisplatin is one of the most effective antitumor agents
against certain solid tumors in humans (2). However, drug
resistance may develop after several courses of therapy in pa
tients with sensitive tumors such as testicular and ovarian
carcinoma. Cisplatin has significant in vitro cytotoxic activity
against human cell lines of colon carcinoma (3), but it has
limited efficacy against metastatic colon carcinoma in patients
(1). Tumor heterogeneity and inadequate delivery of the drug
to the tumor are likely explanations for this discrepancy.
The mechanisms of acquired resistance to cisplatin are mul
tiple. Mechanisms involving a wide variety of cellular functions
have been implicated, such as drug uptake, interaction with
cellular detoxifying compounds, decreased DNA platination,
and increased DNA repair activity among others (4-6).
During the last two decades, extensive work has been done
on the synthesis and development of new platinum complexes
Received 2/3/88; revised 5/10/88; accepted 5/19/88.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by NIH grants 1-RO1 CA41581 to ARK
and 1-RO1 CA23272 to BD, and by a grant from The Liposome Co., Inc.,
Princeton, NJ.
2To whom requests for reprints should be addressed, at Department of Clinical
Immunology and Biological Therapy, Immunobiology and Drug Carriers Section,
The University of Texas System Cancer Center, M. D. Anderson Hospital and
Tumor Institute, 1515 Holcombe Boulevard, Houston, TX 77030.
with a broader spectrum of antitumor activity (7). The attach
ment of a cyclohexane group to the two amino groups has been
shown to result in less nephrotoxic platinum complexes that
are active against LI 210 leukemia cells resistant to cisplatin
both in vitro and in vivo (8, 9). These characteristics have
directed considerable attention to the so-called diaminocyclohexane cisplatin analogues. Some of these analogues reached
the clinical evaluation stage but they were not adequately tested
because of synthetic or stability problems (10).
We have previously reported on the biological activity of
different new lipophilic cisplatin analogues designed to be en
trapped in liposomes (11, 12). Platinum complexes that can be
efficiently entrapped in liposomes are easy to design and syn
thesize. The liposomal carrier may protect the complex from
chemical decomposition by the aqueous milieu until the vesicles
are degraded in vivo or the drug is directly delivered to the
target cell. In addition, liposomes may enhance the intracellular
delivery of the agent in organs with fenestrated capillaries by
directly interacting with cell membranes. These new platinum
complexes can be adequately formulated only in the liposomal
form because they are highly lipophilic and insoluble in water
solutions. One of the most promising liposomal-platinum for
mulations, L-NDDP3, was shown to be significantly more ef
fective than cisplatin in vivo against LI 210 leukemia and liver
métastasesof M5076 reticulosarcoma (12).
We recently began to study the role of liposome entrapment
in the biological activity of L-NDDP. We assessed the in vitro
cytotoxic activity of L-NDDP, free NDDP, and cisplatin
against LoVo carcinoma cells and LoVo carcinoma cells devel
oped in our laboratories that are resistant to a cisplatin concen
tration of 3.0 M§/ml.We also studied the organ distribution,
toxicity, and in vivo antitumor activity of L-NDDP and free
NDDP in different murine tumor models. In all these studies,
free NDDP was prepared as a suspension in saline and Tween
80, or saline, ethanol, and Tween 20. Our results show that
NDDP is a liposome-dependent drug since entrapment in li
posomes permits satisfactory delivery of the drug and also plays
a major role in determining its antitumor activity.
MATERIALS AND METHODS
Materials
was purchased from Aesar (Johnson Matthey, Inc., Seabrook, NH). rra/w-/J,/i-l,2-Diaminocyclohexane
was purchased from
Morton Thiokol, Inc. (Danvers, MA). Neodecanoic acid was obtained
from Exxon Chemical Co. (Houston, TX). Chromatographically pure
(thin layer chromatography) DMPC and DMPG were obtained from
Avanti Polar Lipids (Birmingham, AL).
3 The abbreviations used are: L-NDDP, liposome entrapped NDDP; NDDP,
a's-bis-neodecanoato-fra/is-Ä.Ä-1,2-diaminocyclohexaneplatinum(II);
Da, mean
lethal dose equal to the concentration required to reduce survival by 63% on
exponential part of the survival curve; T/C, median survival of treated mice
divided by median survival of control mice; DMPC, dimyristoyl phosphatidyl
choline; DMPG, dimyristoyl phosphatidyl glycerol; I .Dm. 1.1>•.„.
and LD«o,the
dose lethal to 10, 50, or 90%, respectively, of the animals tested.
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INCREASED CYTOTOXICITY BY ENTRAPMENT OF NDDP IN LIPOSOMES
Synthesis of NDDP
NDDP was synthesized as previously described (12). The complex
was characterized by elemental analysis, infrared spectroscopy, and
nuclear magnetic resonance spectrometry. NDDP was found to be more
than 95% pure by high-performance liquid chromatography analysis
using two protein pak 160 columns and 100% methanol. The chemical
structure of NDDP is shown in Fig. 1.
Preparation of L-NDDP and Free NDDP
NDDP was entrapped in multilamellar vesicles composed of DMPC
and DMPG at a 7:3 molar ratio by reconstituting a lyophilized powder
containing NDDP, DMPC, and DMPG with 0.9% NaCl solution in
water and handshaking for 1 min. The final drug to lipid weight ratio
was 1:15, and the entrapment efficiency was more than 98%.
Free NDDP was prepared as a suspension in 0.9% NaCl solution in
water and 1% Tween 80 for the in vitro studies. For the in vivo studies,
NDDP was suspended in 0.9% NaCl solution in water with 1% ethanol
and 2% Tween 20 at a final concentration of 1 mg/ml. The suspension
was sized in a Coulter counter (Coulter Electronics, Hialeah, FL). Free
NDDP was shown to be stable at room temperature for at least 6 h as
assessed by thin layer chromatography.
Cell Lines
LoVo Cells and LoVo/DDP 3.0 Cells. LoVo cells are derived from a
carcinocmbryonic antigen-producing human colon carcinoma cell line.
The biological properties of this cell line have been extensively described
previously (13).
LoVo/DDP 3.0 cells were developed from LoVo cells by stepwise
increment of cisplatin concentrations up to 3.0 //g/ml. After the devel
opment of resistance, the resistant cells were maintained in drug-free
(Ham's F-10 supplement with 5% fetal calf serum) or drug-containing
medium (the drug-free medium containing 3.0 »Kml of cisplatin) in
alternate passages.
I 121(1/0Leukemia, L1210/PDD Leukemia, and M5076 Reticulosarcoma. LI210/0 and L1210/PDD cells were obtained from the DCT
tumor repository'. National Cancer Institute, Frederick Cancer Re
search Facility, Frederick, MD. The cell lines were grown in vivo in the
peritoneal cavity of BDFi mice and transplanted weekly. Animals
bearing L1210/PDD leukemia were treated on Day 5 with 5 mg/kg of
cisplatin.
M 5076 is a mouse reticulosarcoma that metastasizes predominantly
to the liver when cells are inoculated i.v. M5076 cells were obtained
from the Department of Cell Biology at The University of Texas, M.
D. Anderson Hospital and Tumor Institute at Houston, and were kept
in vivo as an ascitic tumor in C57BL/6 mice and transplanted every 3
weeks.
Cytotoxic Activity against LoVo and LoVo/DDP 3.0 Cells
Thecytotoxic activity of L-NDDP, free NDDP, and cisplatin against
LoVo and LoVo/DDP 3.0 cells was assessed by the colony inhibition
technique as described previously (14). Briefly, 5 x 10* LoVo cells in
Ham's F-10 medium were seeded in 60-mm Petri dishes. The super
natant was decanted 48 h later and the cells were exposed to different
drug concentrations for l h at 37°C.At the completion of the incubation
period, cells were harvested as a single cell suspension and counted in
R'
H2
N
Pt — O-C
C
R
N ^
l
H2
Fig. 1. Chemical structure of NDDP. R. R', and R" can be an aliphatic chain
of two to six carbons. Combined with the carboxyl group, the neodecanoic moiety
has an empirical formula <>lC,,,I!,.,<>.
an electronic particle counter (Coulter Counter Model ZM; Coulter
Electronics, Inc., Hialeah, FL). Known cell aliquots were then plated
in 60-mm Petri dishes so that the number of colonies developed in each
dish ranged from 50 to 100. Cells were then incubated for 14 to 21
days at 37°Cin a 5% COj humidified atmosphere. At the completion
of the experiment, colonies were stained with 2% crystal violet in 95%
ethanol and counted with a dissecting microscope. Viability was defined
as the ability of a single cell to give rise to a colony of 50 cells. Each
experiment contained six control dishes in which cells were treated in
the same manner but without drug exposure. The survival fractions
were normalized with respect to the controls for each drug at the
concentration required to reduce survival by 63% on the exponential
part of the survival curve (Do).
Organ Distribution of L-NDDP and Free NDDP
CD-I swiss mice were treated with a dose of 25 mg/kg of either LNDDP or free-NDDP. Animals were sacrificed 2 h after the adminis
tration of the drugs. Blood, liver, spleen, and kidney were resected and
assayed for elemental platinum content by X-ray fluorescence as pre
viously described (12). Results were expressed as MgPt/g dry tissue or
milliliter of blood.
Single Dose i.v. Toxicity of L-NDDP and Free NDDP in Mice
Groups of six CD-I swiss mice were administered different i.v. doses
of L-NDDP or free NDDP. Animals were observed on a daily basis to
calculate the LDio, 1 I KM.and LD<x>of each preparation.
In Vivo Antitumor Activity against 1,1210/0 Leukemia, L1210/PDD
Leukemia, and Liver Métastases
of M5076 Reticulosarcoma
BDFi mice, weighing 20-25 g, were inoculated with IO6LI210/0 or
L1210/PDD cells i.p. on Day 0. Treatment with L-NDDP or free
NDDP was given i.p. starting on Day I. The treatment schedules used
were as follows: single dose i.p. on Day 1 for the LI210/0 model, and
triple dose i.p. on Days 1, 5, and 9 for the L1210/PDD model. Unless
indicated otherwise, the doses used were the maximum tolerated doses
for each preparation.
C57BL/6 mice, weighing 20-25 g, were inoculated i.v. on Day 0
with 2 x IO4 M5076 cells obtained from the peritoneal cavity of tumor
bearing animals. Treatment with L-NDDP or free NDDP was admin
istered i.v. using a triple dose schedule on Days 4, 11, and 18.
Results were expressed as %T/C (median survival of treated animals:median survival of control animals x 100). Results presented are
the mean of at least two experiments.
RESULTS
Size Distribution of L-NDDP and Free NDDP. The size
distribution of L-NDDP and free NDDP was assessed in a
Coulter counter and channelizer. The vesicle size of L-NDDP
ranged from 1 to 5 ^m, with 90% of vesicles measuring between
2 and 5 ^m in diameter. The particle size of free NDDP in
suspension ranged from 1 to 5 \im with 95% of particles smaller
than 3 urn.
Cytotoxic Activity of L-NDDP and Free NDDP against LoVo
and LoVo/DDP Cells. Fig. 2 shows the results of three different
experiments in which the cytotoxic effect of equimolar concen
trations of L-NDDP, free NDDP, and cisplatin against LoVo
cells was assessed. L-NDDP was twice as cytotoxic as free
NDDP and cisplatin (Do 7 MMfor L-NDDP versus 15 fiM for
free NDDP and 16 J/M for cisplatin).
Fig. 3 shows the cytotoxic effect of the three drugs against
LoVo/DDP 3.0 cells in three different experiments. L-NDDP
was three times as cytotoxic as free NDDP and cisplatin (Do
14 MMfor L-NDDP versus 45 MMfor free NDDP and 48 nM
for cisplatin). The cytotoxic effect of L-NDDP against LoVo
cells relatively resistant to cisplatin was, therefore, reduced by
half when compared with the cytotoxic effect against the parent
4510
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INCREASED CYTOTOXICITY
BY ENTRAPMENT
100
¿ 10
10
20
30
40
50
Drug Concentration (uM)
Fig. 2. Survival of LoVo cells treated with cisplatin (•),L-NDDP (D), and
free NDDP (A).
100
8\
f
10
OF NDDP IN LIPOSOMES
with L-NDDP were uniformly several times higher than those
achieved with free NDDP in all organs in which they were
measured (blood, 5.98 versus 2.08 ßg/m\,liver 118.22 versus
43.34 ng/g dry tissue, spleen 52.55 versus 21.74 /ug/g dry tissue,
and kidney 61.34 versus 14.31 Mg/g dry tissue).
Single Dose i.v. Toxicity of L-NDDP and Free NDDP. Table
2 shows the LD,0, LD50, and LD90 of L-NDDP and free NDDP
after i.v. administration in swiss mice. L-NDDP was slightly
more toxic than free NDDP (LD,0, 36.3 mg/kg for L-NDDP
versus 54.9 mg/kg for free NDDP; LD50, 64.5 mg/kg for LNDDP versus 79.4 mg/kg for free NDDP; LD90, 93.0 mg/kg
for L-NDDP versus 97.7 mg/kg for free NDDP).
In Vivo Antitumor Activity of L-NDDP and Free NDDP
against L1210/0 and L1210/PDD Leukemia. Results of the in
vivo antitumor activity of L-NDDP and free NDDP against
L1210/0 and L1210/PDD leukemias are shown in Table 3.
Against LI210 leukemia, the optimal dose of L-NDDP (25
mg/kg i.p. on Day 1) resulted in a %T/C of 185, whereas free
NDDP at the maximum dose tested (75 mg/kg i.p. on Day 1)
was less effective (%T/C 146).
Against L1210/PDD leukemia, L-NDDP (12.5 mg/kg i.p.
on Days 1, 5, and 9) resulted in a %T/C of 200, whereas free
NDDP at a dose of 50 mg/kg i.p. on Days 1, 5, and 9 had no
significant antitumor activity (%T/C 128).
In Vivo Antitumor Activity of L-NDDP and Free NDDP
against Liver Métastasesof M5076 Reticulosarcoma. Table 3
shows also the results of the in vivo antitumor activity of LNDDP and free NDDP against liver métastasesof M5076
reticulosarcoma. L-NDDP (12.5 mg/kg i.v. on Days 4, 11, and
18) significantly prolonged the survival of animals bearing liver
métastasesof M5076 reticulosarcoma in two different experi
ments (%T/C 140), while free NDDP at the maximum tolerated
dose (25 mg/kg i.v. on Days 4, 11, and 18) was completely
ineffective (%T/C 102).
DISCUSSION
This study shows that liposome entrapment plays an impor
tant role in increasing the cytotoxicity of a lipophilic cisplatin
Table 2 Single dose i.v. toxicity of L-NDDP and free NDDP in mice
10
20
30
Drug Concentration
Fig. 3. Survival of LoVo/DDP
(U), and free NDDP (A).
40
50
PreparationL-NDDP (mg/kg)36.3
(uM)
Free NDDPLD,o
3.0 cells treated with cisplatin (•),L-NDDP
Table 3 Antitumor activity of L-NDDP and free NDDP against LI 210/0
leukemia, LI2IO/PDD leukemia, and liver métastasesof A/5076 reticulosarcoma
Empty liposomes are devoid of antitumor activity against all three tumor
systems.
Table 1 Organ distribution of L-NDDP and free NDDP 2 h after i.v.
administration in mice
Dose of L-NDDP and free NDDP = 25 mg/kg.
Dose
Elemental platinum Gig/ml or ¿ig/gdry
tissue)
OrganBlood
±1.23
Liver
118.22 ±16.68
Spleen
52.55 ±8.68
KidneyL-NDDP5.98 61.34 ±10.05Free
79.4LD»(mg/kg)93.0
97.7
54.9LD50(mg/kg)64.5
(mg/kg)L1210/0
Preparation
leukemia"L-NDDP
12.525Free
NDDP2.08
±0.22
43.34 ±2.66
21.74 ±8.50
14.31 ±2.94
Route
(day)i.p.i.p.i.p.i.p.i.p.i.p.
255075L1210/PDD
NDDP
line (Do 14 pM against LoVo/DDP 3.0 versus 1 ^M against
LoVo) but identical to that of cisplatin against LoVo cells
sensitive to cisplatin (Z)0of L-NDDP against LoVo/DDP 3.0
15 MMversus D0 of cisplatin against LoVo 14 ^M).
Organ Distribution of L-NDDP and Free NDDP. Table 1
shows the elemental platinum levels in blood and different
tissues 2 h after the i.v. administration of 25 mg/kg of L-NDDP
and free NDDP to swiss mice. Mean platinum levels achieved
leukemia*L-NDDP
12.5Free
2550M5076
NDDP
1,5,9i.p.
1,5,9i.p.
1,5,9i.v.
reticulosarcomarL-NDDP
12.5Free
4,11,18i.v.
NDDP
2550Schedule 4,11,18¡.v.
4,11,18%T/C171185100128146200107128140
°Inoculum: 1 x 10*cells i.p. on Day 0.
* Inoculum: 1 x 10' cells i.p. on Day 0.
c Inoculum: 2 x IO4cells i.v. on Day 0.
451
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INCREASED CYTOTOXICITY
BY ENTRAPMENT
analogue, NDDP, against two cell lines of human colon carci
noma in vitro, and three murine tumor models in vivo. In spite
of the increased antitumor activity, liposome entrapped NDDP
was only slightly more toxic than nonentrapped NDDP in mice.
Against LoVo cells sensitive to cisplatin, the in vitro cytotoxic
effect of free NDDP was similar to that of cisplatin at equimolar
concentrations whereas L-NDDP was two times more cyto
toxic. LoVo/DDP 3.0 cells were three times as resistant to
cisplatin and free NDDP as the parental line but only twice as
resistant to L-NDDP. In the in vivo studies, free NDDP had
minimal antitumor activity against i.p. 1.1210/0 and 1.1210'
PDD leukemias when administered i.p. and had no activity
against liver métastasesof M5076 reticulosarcoma when ad
ministered i.v. On the other hand, L-NDDP was significantly
active against all three tumor models.
The mechanisms that make L-NDDP more cytotoxic than
free NDDP against tumor cells either sensitive or resistant to
cisplatin are unknown. This increased cytotoxic effect may be
secondary to an increased delivery of active drug to the molec
ular targets. Liposome entrapment may result in such increased
delivery of active drug by altering one or more of the following:
intraccllular delivery of the drug, intracellular distribution of
the drug, or drug metabolism (protective effect).
Liposomes have been previously shown to alter significantly
the cellular uptake of drugs //; vitro and the organ distribution
of drugs in vivo (15, 16). Drugs entrapped in liposomes may be
delivered intracellularly by different mechanisms. One mecha
nism, release of the drug from the lipid vesicles and subsequent
uptake of the free drug by the cells, appears very unlikely at
least in our in vitro studies, since L-NDDP vesicles are stable
for up to 24 h at 37°Cand the time of drug exposure was 1 h.
Phagocytosis of vesicles measuring several ^m in diameter
occurs in cells of the monocyte-macrophage lineage, like the
M5076 cells, but probably not in LoVo or LI210 cells. Endocytosis involves smaller vesicles than the multilamellar vesicles
used as a carrier system for NDDP. Direct liposome to cell
membrane interaction with rapid exchange of membrane con
stituents including NDDP appears to be the most likely mech
anism involved in the cellular uptake of NDDP.
Changes in subcellular distribution and in extracellular and
intracellular drug metabolism secondary to liposome entrap
ment have not been extensively studied but may play a role in
increasing the delivery of active drug to the cellular molecular
targets. In our case, liposome entrapment of NDDP may pre
vent drug degradation by preventing its contact with the extra
cellular milieu and secondarily may increase the intracellular
delivery of bioactive compound.
Several hydrosoluble cisplatin analogues with a diaminocyclohexane group have been shown to be not cross-resistant with
cisplatin against murine leukemias (8). Some of these com
pounds were introduced in the clinic but were not completely
evaluated. The mechanisms involved in this lack of crossresistance with cisplatin are unknown. Our results do not sug
gest a role for the diaminocyclohexane group in the reversal of
resistance observed with liposome-entrapped NDDP against
OF NDDP IN LIPOSOMES
LI 210/PDD leukemia in vivo. Otherwise, we should have found
that free NDDP had significant activity against L1210/PDD
in vivo.
In summary, we have shown that liposome entrapment
changes the distribution and increases the cytotoxicity of a
lipophilic cisplatin analogue without resulting in major changes
in the LD5o of the drug. Our results do not suggest that the
partial reversal of resistance is secondary to the diaminocyclo
hexane group as has been previously reported (8). NDDP is a
liposome-dependent drug since it can only be satisfactorily
formulated in the liposomal form and since the liposomal
carrier plays a crucial role in determining its biological activity.
ACKNOWLEDGMENTS
We wish to thank Trellis Brown and Karen Francis for excellent
technical assistance.
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Increased Cytotoxicity and Reversal of Resistance to cis
-Diamminedichloroplatinum(II) with Entrapment of cis
-Bis-neodecanoato-trans-R,R
-1,2-diaminocyclohexaneplatinum(II) in Multilamellar Lipid
Vesicles
Roman Perez-Soler, Li Y. Yang, Benjamin Drewinko, et al.
Cancer Res 1988;48:4509-4512.
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