Note: Descriptions are shown in the official language in which they were submitted.
:
~3~938 ;~
.,;
: :; ` . .
~ ~-
HYDROPHOBIC CIS-]?LATINUM COMPLEXES
EFFICIENTI.Y INCORPORATED INTO ~IPOSOMES
~ 10 ,.,,~
This application is a divisional application
~: of parent application Serial No. 520,893 filed : -
20 October, 1986.
; This invention relates to newly synthesized platinum -
complexes with hydrophobic properties. The use of
liposomes incorporating these new and previously `~
synthesized complexes in anti-tumor chemotherapy is also `~ -
described.
; Cis-platinum (CDDP) is a highly effective drug in the
treatment of several neoplastic diseases in humans
(Loehrer et al (1984) Ann. Int. Med. V 100, pp 704-713).
However, its use is l~imited by severe systemic toxicity,
particularly nephrotoxlcity and neurotoxicity ~Zwelling~et
al Platinum Complexes. In: Pharmacologic principles of
~; cancer treatment (1982) Ed by B.A. Chabner, Saunders,
Philadelphia, PA). In an~attempt to modify the~
30 therapeutic index of CDDP,~ new derivatives have been ~ ;
synthesized during the last decade. However, the
~ ~ development~of some promising analogues has been
!` ~ prevented by their low hydrosolubility, which decreases ~-~
their potential for clinical use (Burchenal et al ~1979)
Cancer Treat~. Rep. V 63, pp 1493-1497).
::
133~38
-2-
Liposomes are lipid vesicles which may form
spontaneously upon addition of an aqueous solution to a --
dry lipid film (Mayhew et al, In: Liposomes (1983) Ed by
Marc J. Ostro, Marcel Dekker, Inc., New York, N.Y.).
Liposomes may be used as drug carriers of hydrophobic or
hydrophilic drugs entrapped in their hydrophobic or
hydrophilic compartments respectively. Multilamellar ~ ~
liposomes are multilayer lipid vesicles (MLV) that are ;~--
particularly suited for carrying hydrophobic drugs since
their hydrophobic compartment is larger than their
hydrophilic compartment. When injected intravenously (iv)
in animals, (Kasi et al (1984) Int. J. Nucl. Med. Biol. V ~ -
11 pp 35-37, Lopez-Berestein et al. (1)(1984) Cancer Drug
Deliv. V 1, pp 199-205) and humans (Lopez-Berestein et al
(2)(1984), Cancer Res. V 44, pp 375-378), MLV concentrate
in the liver, spleen and other organs rich in
reticuloendothelial (RES) cells. ;
Liposomes have been previously used in vitro to
deliver chemotherapeutic agents, (Mayhew et al, Liposomes
(1983), Ed by Ostro, Marcel Dekker, Inc., New York, N.Y.)
and immunomodulators and anti-fungal agents in vitro -~
(Mehta et al (1984), Immunology V 51 pp 517-527, and in
vivo in animals (Lopez-Berestein et al ~4)(1984) Clin Exp
Metastasis V 2 pp 127-137 and Lopez-Berestein et al
(1983), J Inf Dis V 147, pp 937-945) and in humans
(Lopez-Berestein et al (1985) J. Inf. Dis. V 151 pp 704-
710).
Recent studies show that liposomes can reduce certain -
types of drug-related toxicities such as doxorubicin -~
cardiotoxicity (Forssen et al (1981) Proc. Natl. Acad.
Sci. V 78 pp 1873-1877, Olson et al (1982), Eur. J. Cancer
Clin. Oncol. V 18 pp 167-176, Gabizon et al (1982) Cancer
Res. V 42 pp 4734-4739, Herman et al (1983) Cancer Res. V
43 pp 5427-5432) and CDDP nephrotoxicity, (Freise et al
; '
~3~3~
.. . .
--3--
:,
~1982), Arch. Int. Pharmacodynamie Therapie V 258 pp 180- ~
192) and may increase antitumor activity as a result of a ~ ~-
slow release mechanism (Mayhew et al ~1978) Ann. N.Y.
Acad~ Sci. V 308, pp 371-386, Patel et al (1984~ Int. J.
Cancer V 34 pp 717-723) a higher drug uptake by tumor
cells or due to a more selective or~an distribution
~Gabizon et al (1983) Cancer Res. V 43, pp 4730-4735 and
Mayhew et al (1983), Cancer Drug Deliv. V 1 pp 43-58). In
U.S. Patent No. 4,330,534 N4-acylcytosine arabinoside
incorporated into liposomes, for example, was found to be
therapeutically effective when administered to tumor~
bearing animals. In spite of these promisîng results, the
clinical application of antitumor agents encapsulated in
liposomes has been del`ayed, mainly~due to formulation,
drug stability and large scale production problems.
CDDP has been previously encapsulated in MLV but with
a very low encapsulation efficiency (7.4%) and poor
stability (75% at 48 hours in 0.9% NaCl solution) (~reise
~; 20 et al (1982) Arch. Int. Pharmacodynamie Therapie V 258 pp
180-192).
.,
In V.S. Patent No. 4,256,652 are described certain
platinum compounds comprising resolved stereoisomers of
1,2 diaminocyclohexane (DACH). The isomers utilized were
cis-DACH, trans-RR-DACH and trans-SS-DACH. The platinum ~ i
compounds described therein contained, in addition to a
resolved DACH isomer, two hydrophilic platinum ligands
such as bromide, iodide, nitrate, bromoacetate, sulfate or
glucuronate. The platinum compounds comprising the
trans-RR-DACH were described as often more therapeutically
effective than those bearing cis-DACH.
In European Patent Application No. 83305726.7 published
35 on 18 July, 15~84 under Publication No. 0113508 certain platinum
compounds are described which may comprise diaminocy¢lohexane
(non-stereochemically resolved) and do ~
: ~::
3 3 ~ 9 3 ~
-4~
comprise phosphatidyl groups having fatty acid substituents.
These compounds are described as largely insoluble in plasma and ;~
preferably employed with lipid vesicle carriers. The platinum ~ -
compound-phospholipid vesicles were pre~erably prepared by a
sonic oscillation procedure which characteristically yields
, . .
unilamellar vesicles.
In accordance with the embodiment of the invention
which is the subject of this application there is provided a
limpsome comprising a four co-ordinate platinum complex having
~, . . :.
the formula~
R3 R
: .
Pt (II)
~;~ 15 R4 R2
' '..::
wherein Rl and R2 are each carboxylato bearing a
hydrophobic radical function or when linked together are a
dicarboxylato bearing a hydrophobic radical function, and wherein
R3 and R4 are each: ~
~ , .,: .
H
N Rs
H ;~
~
and R5 is hydrogen, alkyl having between about 1 and 20 -
carbon atoms, or cycloalkyl having between about 3 and 12 carbon
atoms; or wherein R3 and R4 when linked together are cycloalkyl-
1,2-diamino having between about 3 and 7 carbon atoms, or
, ' ~ ~,,
1 3 ~ 9 ~ 3 ~
~` .
5-
alkenyl-1,2-diamino having between about 2 and 12 carbon atoms.
The complex is defined further as being substantially soluble in -~
methanol or chloroform and substantially insoluble in water, and
a phospholipid.
Also in accordance with the invention claimed herein
a method of producing a liposome is provided. A platinum (II)
four co-ordinate complex having the foregoing formula is
provided.
~'''' ' ~
R3 R1
Pt/ (II) ~;
R4 R2
~15 The said complex is mixed with phospholipid at a ratio of about
15 in a hydrophobic solvent. The hydrophobic solvent is
evaporated to produce a film of phospholipid and complex or
lyophilizing the mixture to form a powder~ The film or powder
is dispersed in an aqueous solution to produce liposomes. -
Important aspects of the present invention involve
liposomes comprising fatty substances such as phospholipids,
optionally cholesterol, and the four co-ordinate platinum
complexes described above,~ as well as the preparation and uses
of these liposomes. Liposomes of the present invention contain
the platinum complex and the phospholipid in a preferred ratio
between about 1 to 10 and about 1 to 30, a more preferred ratio
being 1 to 15.
Preferred phospholipids of these liposomes include
phosphatidylglycerol, phosphatidylcholine, sphingomyelin,
'
- ~ 3 3 ~ ~ 3 ~
-6-
phosphatidic acid or phosphatidylserine, the more preferred
phospholipids being phosphatidylglycerol, phosphatidylcholine
or a combination thereof. The most preferred phosphatidyl~
glycerol is one consisting essentially of dimyristoylphospha~
tidylglycerol and the most preferred phosphatidylcholine is
one consisting essentially of dimyristoylphosphatidylcholine. ;
When the liposomes of the present invention comprise
dimyristoylphosphatidylglycerol and dimyristoylphosphatidyl- -
choline they are preferably in a ratio between about 1-10 and
10-1, more preferably in a ratio of about 3 to 7.
The liposomes of the present invention may be
multilamellar, unilamellar or have an undefined lamellar
construction. A pharmaceutical composition comprising the
liposomes of and a pharmaceutically acceptable carrier or diluent
may be used for the therapy of disease conditions such as cancer.
A focal point of the present invention involves a
method of treating a host animal afflicted with tumor cells
sensitive to the presence of a platinum (II) four co-ordinate ;~
complex. This method comprises administering to the host an
amount of the platinum (II) four co-ordinate complex described
above or a liposome of the present invention comprising a
phospholipid and a tumor cell-inhibiting effective amount of said
platinum complex. The administering step is preferably parenteral
and by intravenous, intraarterial, intramuscular, intralymphatic,
intraperitoneal, subcutaneous, intrapleural or intrathecal
injection or by topical application or oral dosage. Such
administration is preferably repeated on a timed schedule, for
example twice daily for a period of two weeks. The treatment may
:
~ 133~3~
be maintained until tumor regression or disappearance has been
achieved and may be used in conjunction with other forms of tumor
therapy such as surgery or chemotherapy with different agents.
These antitumor methods may also be used to inhibit the
metastic spread of tumors such as reticulosarcoma. A
preventative pretreatment with the p:Latinum (II) complexes or
liposomes comprising those complexes may be used to preclude
metastatic spread in a vaccination-like manner.
Platinum (II) four co-ordinate complexes were prepared
utilizing racemic (unresolved) DACH, trans-RR-DACH or trans-SS-
DACH. It was found that platinum complexes comprising trans-RR-
DACH or trans-SS-DACH and two hydrophobic ligands such as
cyclopentene-carboxylato were more efficiently incorporated into
~15 liposomes than were the analogous complexes comprising racemic
DACH.
Liposomes incorporating these platinum complexes were
found to be stable in an aqueous milieu, non-nephrotoxic and
active against murine leukemia L-1210.
In a general sense, the square-planar platinum (II)
four co-ordinate complexes of the present invention have the
f OrlllUlA:
' ','~ '
- ` 133~3~
.~" .- :.
. ,
Rl
3 t( ) ~:
R2
where Rl and R2 are preferably carboxylato monoanions~ ~;
bearing a hydrophobic radical function. R1 and R2 may
also be a single carboxylato dianion where the carboxylato
10 groups are bound to an interlinking atom which in turn is ~-
bound to a radical function. Additionally, R3 is a
; vicinal diaminoalkane or vicinal diaminocycloalkane. It
is contemplated that R3 may be composed of two independent
alkylamines, cycloalkylamines or ammonia. These
15 components confer upon the complex a substantial -
solubility (normally greater than about 5.0 mg/ml) in
methanol or chloroform at ambient temperatures and a
substantial insolubility (less than about 0.5 mg/ml) in
aqueous solutions at ambient temperatures. -;~
~` 20 ~ ,
The hydrophobic radicaI function, that function
covalently pendant from the~carboxyl group or from an
intermediate or linking group, may be an alkyl,
~ substituted aryl, aryl, alkenyl, cycloalkyl or~ ~ 25 cycloalkenyl group or even combinations of these functions
such~as alkylaryl or arylalkenyl, ~to name but two of the
many possible hydrophob~ic~combinations. The hydrophobic
radical function characteristically has between 5 and 20
carbon atoms. When this hydrophobic radical function
comprises an alkyl or alkenyl group, this group may be
straight or branched.~ Polar functions such as hydroxyl
groups, for example, substituted on the hydrophobic ~ -
radica}s would tend to lessen their~hydrophobicity and may `~
render them less useful for the purposes of the present
35 invention. `
'~ '. ',' ,~.';,`,, '
. :., . .:, .:
~ 3 ~
. ,. g : .
In certain cases the Rl and R2 functions may be
interlinked, for example when two acetate or propionate
functions are bound together by a nitrogen atom. In these
cases the Rl and R2 functions are a single carboxylato
dianion. An alkyl hydrophobic unction having between six
and twenty carbon atoms, such as n-decane, for example,
may be bound to the interlinking nitrogen and the
resu~tant compound utilized in the production of the
platinum (II) four coordinate complex of the present
invention.
With certain hydrophobic radical functions
(cyclopentene, for example) present on the carboxylato
monoanion it was found that efficient incorporation of PT
complexes into phospholipid liposomes was dependent on
characteristics of the R3 function. For example, when R3
was 1, 2 diaminocyclohexane (DACH) the amino groups may be
in several relative stereochemical configurations, cis, ;-
trans-RR and trans-SS (a mixture of these being termed -~
"racemic"). With platinum (II) complexes containing
cyclopentene radical functions (Rl and R2) and various
stereochemical types of D~CH it was found that the trans-
RR-DACH and trans-SS-DACH-complexes were more efficiently
incorporated into phospholipid liposomes (vesicles being a
term equivalent to liposomes) than were the analogous
racemic DACH complexes.
When a hydrophobic radical function was a branched
alkyl containing nine carbon atoms such as that in
neodecanoato, (CloH20O2, empirical formula) the
incorporation efficiency of a platinum (II) complex
comprising racemic DACH or trans-RR-DACH was maximal
(100%). Thus, linear or branched alkyl radical functions
having from about 6 to about 12 carbon atoms confer
35 properties of efficient phospholipid liposome ~ ~-
incorporation upon any DACH-containing Pt (II) complex.
~, ' `'''"'' .
' . '.
- ~33~3~-
. . . - . . ,
-10~
Liposomes containing the platinum (II) complexes
described herein may be prepared from various amphipathic
substances including natural or synthetic phospholipids. -~
The phospholipids usable to produce liposomes are numerous
and not exhaustively listed herein since they are
generally well known in the art. These phospholipids
include but are not limited to: lecithin, phosphati-
dylethanolamine, lysolecithin, lysophatidylethanolamine,
phosphatidylserine, phosphatidylinositol, sphingomyelin,
cardiolipin, phosphatidic acid and the cerebrosides. Most
preferable phospholipids for the practice of aspects of
the present invention include dimyristoylphosphatidyl-
glycerol (DMPG) and dimyristoylphosphatidylcholine (DMPC). --~
Cholesterol in minor proportions ranging from less than 1~ ~;
15 to about 50~ may be included with phospholipids and -
platinum (II) complexes to produce liposomes of the
present invention. A preferable but not limiting
combination of `DMPG and DMPC has been found to be a ratio ~ -
of 3 to 7 although ratios between 1:10 and 10:1 are
contemplated as satisfactory. Ratios of platinum
complex to phospholipid between about l to lO and about l ;~
to 30 are contemplated as generally satisfactory although
a l to 15 ratio was primarily used in studies thus far.
Either unilamellar or multilamellar or other platinum
(II) complex - containing liposomes~may be used in the
~; practice of the present invention. Multilamellar ~-
liposomes are presently preferred since the platinum (II)
complexes of the present invention are substantially water
- insoluble and they appear to be incorporated into the
phospholipid bilayers of the liposome lamellae. -
.,... ~, :
Generally, the procedure for synthesis of the
platinum II compounds of the present invention may be
3S described on one scale as follows- ~l) about lO mmole -~
vlcinal diamino cycloalkane or alkane are added to 50 ml
. ,~;
' ~', ;' `~"'
~3~3~
.. ~ 11-
aqueous solution of K2Pt C14 (3.5 9) and stirred for six
to eight hours at room temperature. The yellow solid
formed comprising cis-bis-dichloro-1,2-diamine-Pt(II) may
be removed by filtration and washed with fluids such as
water, methanol or acetone. The solid may then be
suspended in about 20 ml H2O and an aqueous solution
containing about 0.75 g Ag2SO4 added thereto. After
stirring for about 24 hours in the dark, precipitated Ag
Cl may be removed by filtration. The sulfato-vicinal
diamine-Pt may then be dissolved in about 100 ml H~O and
added to about 2 m mol of alkali earth metal salt of a
carboxylato anion prepared in situ, and stirred therewith
for about 30 minutes. After removal of BaSO4 by
filtration, the platinum II complex of the present
invention may be obtained, for example by crystallization
or removal of the solvent by evaporation.
The methods of preparation of particular platinum
(II) complexes and chemotherapeutic treatment with
particular platinum (II) complexes described in the
Examples contained later herein are readily adapted to the
production and use of analogously described and claimed
complexes by simple substitutions of appropriate vicinal
diamines or hydrophobic radical-containing carboxylato
monoanions.
Liposomes comprising phospholipids and platinum
complexes (Pt-liposomes) of the present invention are
useful in inhibiting both the growth and metastatic spread
of tumors.
Such Pt-liposomes may be administered parenterally, c
topically or orally. Oral or parenteral dosages of these
Pt-liposomes between about 2.5 mg/kg body weight and 25
35 mg/kg body weight are contemplated as adequate in most
conditions. The paxticular dosages, if a tumor-bearing
~3~93~
, . . .
12
.- ",
human is being treated may vary in each case according to -
the condition of the patient, the type and extent of
tumor, and particular Pt-liposome toxicity.
The amount of liposomal-platinum included in the
pharmaceutical composition and the dosage utilized in the
method of treatment of the invention will vary depending
in each case upon the conditions of the patients, the
nature of the tumor undergoing treatment, antitumor
10 activity of liposomal-platinum, the toxicity and - ~-
solubility characteristics thereof, etc. Liposomal-
platinum may also be administered in combination with
other antitumor asents in a combined therapeutic regimen.
Parenteral administration may be intraperitoneal,
subcutaneous, intrapleural, intrathecal, intraurethral,
intravenous, intraarterial, intramuscular or intralym~
phatic. Such parenteral administration preferably
involves Pt-liposome suspensions in pharmaceutically
acceptable solutions such as sterile isotonic aqueous
solutions. These suspensions may be obtained fully
prepared or may be prepared from preformed components. As ;~ `
known to those skiIled in the art, Pt-liposomes may be
, : : `, ! :~
prepared as pellets or powders. These pellets or powders ;
may be mixed with pharmaceutically acceptable solutions to
form suspensions for parenteral administration.
Topical administration of Pt-liposomes may involve ~-
pharmaceutical compositions such as suspensions, creams or
ointments which may be obtained fully prepared or prepared
from Pt-liposome powders or pellets. Such topical
administration may be near to sites of cancerous lesions
such as the epithel`ium or mucosa for example.
Oral administrations of Pt-liposomes preferably
involve encapsulation of Pt-liposome powder or pellets
1~3~3~
.
-13- -
,
whereby the Pt-liposomes are protected from much gastric
and intestinal digestive activity before release from the
encapsulation.
When desired, Pt-liposomes may be prepared to
contain, for example, other therapeutic agents for
treatment of tumors or anti-oxidants to aid in liposome
stabilization.
Use of the complexes of the present invention,
particularly as a component of liposomes, focuses upon the ;
inhibition of tumor growth and prevention of the
metastatic spread of tumors. For example, first a host is
identified as bearing a tumor type known to generally
contain cells whose growth is often inhibited by platinum
~; ~ (II) complexes. Tumor growth in the host may be inhibited
~- by administering to the host the PT-containing liposomes
of the present invention.
Similarly, the metastatic spread of tumors in a host
may be inhibited. A host bearing metastatic or poten-
tially metastatic tumors of a type noted often to be
sensitive to platinum tII) complexes, would first be
identified. The administration of the PT-containing
liposomes of the present invention to that host would
serve to inhibit metastatic spread.
The following examples are presented to further
illustrate preferred embodiments of the present invention;
30 they are not intended to limit the invention unle~s ~;
otherwise so stated in the accompanying claims. ~ ~
.,~ ~'
~33~n?3~
-14-
:
~,
EXAMPLE 1 -~
~ , . .~;
Materials and AnaIYses ~
:: ., .
K2PtC14 was purchased from AESAR (Johnson Matthey,
Inc. Seabrook, NH). Cyclopentenecarboxylic acid was ~-
purchased from Pfaltz and Bauer, Inc., Stamford, CT; 1,2~
, .
diaminocyclohexane (DACH) from Aldrich Chemical Co.,
Milwaukee, WI., trans-RR-DACH and trans-SS-DACH from
Mortol Thiokol, Inc., Danvers, MO., and neodecanoic acid
from Exxon Chemical Co., Houston, Texas. Elemental ~-~
analyses on the platinum complexes were performed by
Integral Microanalytical Laboratories, Inc., Raleigh, NC
and by Robertson Laboratory, Inc., Florham Park, N.J.
Infrared spectra of the complexes (as KBr pellets) were
measured in the range of 600-4000 cm using a Nicolet
6000 Eourier transform infrared spectrophotometer.
~ Chromatographically pure tthin-layer chromatography) -
; ~ 20 dimyristoylphosphatidylcholine (DMPC) and dimyristoyl-
phosphatidylglycerol (DMPG) used in this study were :
obtained from Avanti Polar Lipids (Birmingham, AL).
Cholesterol was purchased from Sigma Chemical Co. (St. `
Louis, MO).
EXAMPLE 2 ~ -~
Synthesis of cis-bis-cyclopentenecarboxylato-
_ 1,2-DACH-platinum (II~
,, ~ :
Cis-bis-cyclopentenecarboxylato-1,2-DACH-platinum
~II) was one cis-platinum (CDDP) hydrophobic analogue used
as a prototype to develop liposomal-platinum (L-PT)
preparations. The general structure of this particular
complex was as follows~
. ,.
:- - :~:
- ~33~32
--15--
This prototype was synthesized using a multi-step
procedure as described for racemic DACH (Khokhar et al.
~;~ Inorg. Chem. Acta. Bioinorganic Section, V 108, p 63
(1985) follows: 0.96 9 of DACH were added to a filtered
aqueous solution of K2PtCl4 (3.5 9 in 50 ml H2O) and the
mixture was stirred for six to eight hours at room
temperature. The yellow solid containing cis-bis~
dichloro-DACH-Pt (II) was removed by filtration and washed
with H2O, methanol, and finally acetone. After the final
product was dried under vacuum, the yield was calculated
to be 56~. Subsequently, l.0 g of cis-bis-dichloro-DACH-
Pt (II) was suspended in 20 ml H2O and an aqueous solutionof Ag2S04 (0.75 g in 150 ml H20) was added to obtain water
soluble sulfato-DACH-Pt H20. The reaction mixture was
stirred in the dark for 24 hours and the precipitated AgCl
was removed by~filtration. The~yellow solution was
evaporated to dryness at 45-50`C under reduced pressure
and the yellow roduct was further dried over P205 in
vacuum. The yield of sulfato-DACH-~t (II) H2O was 90%.
~inally, 0.423 9 (l mmol) of sulfato-DACH-Pt (II~ wàs ;;~
dissolved in 100 ml of H20, and the barium cyc~opentene- ; -
carboxylato was prepared in situ by the addition of 0.3 g
` ~ of Ba(OHj2 to 0.226 9 (2 mmol) of cyclopentenecarboxylic
acid in H20. These components were mixed and the reaction
mixture was st~irred for 30 minutes at room temperature.
The ~aSO4 precipitate was filtered off, and the yellow
filtrate was evaporated to dryness at 45 C under reduced
pressure using a rotary evaporator. A yellow solid was
-
~ ~'''':''
.: ~ -:;~. . .
3 ~
~16- -
obtained, which was purified from methanol. The product
was finally dried under vacuum. Yield was 70%. Analysis~
C 40.26%, H 5.65~ and N 5.05~ C18H30N204Pt (PT)
(theoretically C 40.67~, H 5.65% and N 5.27%. The
5 infrared spectrum for the complex (as KBr pellet) /C=0 ~-
1632 cm } and /C-0 1398 cm 1.
,i . ~::
Cis-bis-cyclopentenecarboxylato-1,2-DACH-Pt (II) is
highly soluble in methanol and chloroform and only ~ r~
slightly soluble in water (less than 0.5 mg/ml). The
above-described synthetic procedure was carried out with
both trans-RR-DACH and trans SS-DACH to produce the
trans-RR and trans-SS isomers of cis-bis-cyclopentene-
carboxylato-1,2-DACH-Pt (II). The analysis of cis-bis-
cyclopentenecarboxylato-1,2-trans-RR-DACH-Pt (II)-3H2O
was: C-37.36~, H-5.50%, N-5.12% ~theoretically C-37.93%,
H-5.82~, N-4.80%).
EXAMPLE 3
Synthesis of Cis-Bis-Neodecanoato-
DACH-Platinum ~
.. . .
..
Sulfato-racemic-DACH-platinum (II) (0.423 9),
prepared as described in Example 2, was dissolved in 10 ml
water. A potassium salt of neodecanoic acid (0.420 q) was
added to this solution and the reaction mixture stirred
for 30 minutes at room temperature. A gummy mass was
obtained which was extracted in chloroform and the
chloroform extract was dried over anhydrous MgSO4. The
MgSO4 was separated by filtration and the filtrate
evaporated to dryness. An off-white solid product was
obtained which was dried in vacuo and over P2O5. This
final product was stored at OC.
'~"' ",
13~3~
-17~
The elemental analysis of the final product was: C-
48.30%; H-8.10% and N-3.92%. The calculated elemental -~
values for a compound of the empirical formula;
C26H52N2O4Pt- is: C-47.93%; H 8.00% and
The structural formula of the cis-bis-neodecanoato-
DACH-platinum II was as follows:
R
10 f\1-- NH2 l ' '.,~
P t o c---- C R
~NH2 --
R ~ ~
:~ 1 5 ~ 2
where R,R',R" can be: CH3, C2H5 or C3H7 to give a ~
; carboxylato radical function CloH19O2 (MW = 172) as ~-
empirical formula.
'~
Cis,bis-neodecanoato-DACH-platinum (II) was highly
soluble in methanol and chloroform and insoluble in water. -
The above described procedure was carried out with
trans-RR-DACH to produce the trans-RR isomer of cis-bis-
neodecanoato-DACH-Platinum (II). The analysis of cis-
bis-neodecanoato-RR-DACH-Platinum (II) was: C-47.75%, H~
8.16%, N-3.98% (theoretically C-47.93%, H-8.00%, N-4.30%).
EXAMPLE 4
Synthesis of Cis-Bis-n-Decyliminodiacetato-
DACH-Platinum (II~
Sulfato-DACH-platinum (II) H2O (0.423 9), prepared as
described in Example 2, was dissolved in 10 ml of H2O.
~ , . ,~
'. ~,'., .
i3~Q~
,,
--18-
: ~'.'''~,.. '
The sodium salt of n-decyliminodiacetic acid was prepared
in situ by the addition of NaOH (0.08 g) to N-decylimino~
diacetic acid (0.273 9) in 50 ml of water. This aqueous
solution of sodium N-decyliminodiacetato was added to the
sulfato-DACH-platinum solution and stirred for 30 minutes
at room temperature. The resultlng solution was evapo-
rated to dryness on a rotary evaporator at 40~C under
reduced pressure. The yellow so:Lid thus obtained was
dissolved in methyl alcohol and filtered through Celite. - ~ ;
The yellow filtrate was evaporated to dryness in a rotary
evaporator under reduced pressure. A yellow crystalline
product was obtained, which was further purified from 1-
propanol, yield 80%. -~
, ., ::
An elemental analysis of the product showed: C-39.28; ;
H-7.12% and N-6.68%. The theoretical composition for a
compound of the empirical formula, C2oH39N3O4Pt.2H2O, was:
C-38.95%; H-7.02% and N-6.81%. The infrared spectrum for
the complex (as ~Br pellet) / C=0 1580cm 1 and / C-0
1410cm
The structural formula of the cis-bis-n-decylimino- .
,
diaceto-DACH-platinum (II) is shown as follows:
~ NH2 OOC CH2 ~;
~ Pt N R
/ ~ / / , .:,
~ NH2 OOC CH2 ;~
.`
* Trade Mark
~L~3~3~
:
--lg-- :
where R = n-decyl or a longer straight or branched chain
alkyl radical.
Cis-bis-n-decyliminodiacetato-DACH-Platinum (II) is
S highly soluble in methanol and chloroform and insoluble in
water. The above described synthetic procedure was
carried out with trans-RR-D~CH to produce the trans-RR
isomer of cis-bis-n-decyliminodiacetato-DACH-Pt ~II). The
analysis of cis-bis-n-decyliminocliacetato-trans-R~-DACH-Pt
(II) was: C-38.60%, H-6.87%, N-6.82% ttheoretically C-
38.95%, H-7.02%; N-6.81%).
.
The above described compound represents a structure
where Rl and R2 of the earlier described general structure c~
are one and the same and the carboxylato monoanions are
bound together by a linking atom bearing an alkyl
hydrophobic radical to form, in fact, a carboxylato
dianion.
.
EXAMPLE 5
:
Platinum-Containina Li~osomes ~L-PT)
:
Multilamellar Iipid vesicles (MLV) or liposomes
containing incorporated platinum complexes (PT) of the
above descriptions were prepared~as previously described
for other compounds (Lopez-Berestein et al (4)(1984) Clin. ~1
Exp. Metastasis V 2 pp 127-137 and Lopez-Berestein et al
(1983) J Inf Dis V 147 pp 937-945). In brief, chloroform
solutions of lipids (at the desired molar ratio) and PT
were mixed at a lipid-PT ratio of 15:1 and the chloroform
was evaporated in a rotary evaporator (Buchi, Brinkmann
Instruments, Westbury, NY). The dried lipid film
obtained, containing PT, was then dispersed with an
35 aqueous solution (0.9% NaCl in water) by vigorous ~
handshaking. The suspension was subsequently centrifuged ;
' ' ``' "''.`
", ~ 3 g . ~
- 2 0
, . , . ~.
at 30,0Q0 x 9 for 45 minutes, the supernatant was dis~
carded, and the pellet containing PT was resuspended in
o.9% NaCl solution. ~ ~
~ .:. :.~:,
MLV or liposomes containing platinum complexes may
also be prepared from a lyophilized powder containing
lipid and platinum compound. The lipid and platinum
compound are dissolved in the hydrophobic solvent tertiary
butanol (M.P. 26C) at the ratios described above. The
solution is freeze-dried and a white powder obtained. MLV
containing the platinum compound are formed upon the -~
addition of 0.9~ NaCL solution in water to the yophilized ~
powder with mild shaking by hand. ~ ;
, '
EXAMPLE 6 :
Calculation of Encapsulation
Efficiency (EE) and Stabilit
Elemental platinum (Pt) was determined in the
liposome suspension and the pellet by x-ray fluorescence
as previously reported (Seifert et al (1979) Proc. Amer.
Ass'n. Cancer Res. V 20 p. 168) in the Department of
Analytical Chemistry, The University of Texas Medical
School at Houston, TX. The amount of platinum complex
(PT) was determined in the supernatant by ultraviolet (UV)
spectrophotometry using a wavelength of 224 nm. The EE
was initially calculated with the two following formulas~
.. - . ~ -:
1. EE = Pt in pellet/total Pt in the initial
liposome suspension
2. EE = Total Pt initially added - Pt in
supernatant/total Pt initially added ;~
Since the results obtained by these two methods were
highly comparable and the second method only requires PT
:, .
133~3~ :
--21--
determination by UV spectrophotometry, most EE determina-
tions were calculated with the second method.
The stability of the different liposome-PT (L-PT)
preparations in 0.9% NaCl solution at 4C, and 50% human
(AB) serum in 0.9% NaCl solution at 37C was determlned
using the followin~ formula: -
Stability at x hours = ~ EE at x hours x 100
% EE at 0 hours
The EE values used in the stability determinations ;~
were obtained by measuring PT in the supernatants by UV --
spectrophotometry (0.9% NaCl solution) or x-ray
fluorescence (50% human AB serum). The stability in 0.9%
NaCl solution was determined up to 14 days after the
initial preparation. In addition, the L-PT preparations
were observed microscopically on day 14 to check the `~
morphology of the vesicles. The stability in 50% human AB
serum was determined up to 18 hours after incubation.
Different cis-platinum analogues were encapsulated in
multilamellar vesicles composed of DMPC:DMPG 7:3. The
encapsulation efficiency for Pt-cyclopentenecarboxylato
racemic-DACH was 66%. The encapsulation efficiency was
significantly increased when the pure trans RR DACH or
trans-SS-DACH isomers were used (88% and 90%, respective-
ly~. The encapsulation efficiency for Pt-neodecanoato- ~ d~
racemic-DACH, Pt-neodecanoato trans-RR-DACH, Pt-n-
decyliminodiacetato racemic DACH complexes are presented
in Table I, shown below.
..: .
' '' ~ " '~ ~
: .
,``` 13~9~3~
~. ~ ` . ..
-22~ ' ~ ~
':
TABLE 1 ;~
ENCAPSULATION EFFICIENCY OF
L-PT USING DIFFERENT ANALOGUES :
:~ -
Platinum Analogue% Encapsulation Efficiency
Pt-cyclopentenecarboxylato ~;
racemic-DACH mixture 66
Pt-cyclopentenecarboxylato
trans-RR-DACH 88
Pt-cyclopentenecarboxylato
trans-SS-DACH 90
Pt-neodecanoato-racemic . `-
DACH-mixture 99
~ 20 '"'"'~'~''`
: : Pt-neodecanoato-trans-RR-DACH 99 :-~
: Pt-N-decyliminodiaceto-racemic~
DACH mixture 100
:Pt-N-decyliminodiacetato-trans-RR-DACH 98
. :, ~ . ... .
: ~ 1 Mean of at leaat three experiments. Liposome .
composition : DMPC:DMPG 7-3
... .. ::
Analogous experiments (not shown) with DMPC alone,
: DMPG alone, cholesterol alone, DMPC and DPMG combined in
other concentrations, with or without cholesterol showed
no better encapsulation efficiency and, most frequentlyj a .
si~nificantly decreased EE.
.
~3~3~, -
. ~ ~
-Z3-
EXAMPLE 7
StabilitY of L-PT in Aqueous Milieu
Preparations were suspended in 0.9% saline and
incubated at 4C for 14 days. The liposome compositions ;
were observed by light microscopy and the amount of free
PT in the saline determined. Liposomes containing PT and
composed of DMPG as the only phospholipid showed '
10 significant microscopically determined loss of structure. ~ ~,
Free PT was determined and stability was calculated as the ~ `
percentage PT remaining in the liposomes. The results of -
these measurements are presented in Table 2 below. ~ ;- '
. . -., " . ~
TABLE 2
~. . . - .- .
STABILITY OF L-PT IN SALINE L-PT AT 14 DAYS~
20 Platinum Analog Stability %
,''.'~... ~.,~
Pt-cyclopentenecarboxylato-racemic-DACH 89 -
Pt-cyclopentenecarboxylato-trans-RR-DACH 94
25 Pt-cyclopentenecarboxylato-trans-SS-DACH
Pt-neodecanoato-racemic-DACH 100 ,~
Pt-neodecanoato-trans-RR-DACH ~ 99
Pt-iminodiacetato-racemic-DACH 100
Pt-iminodiacetato-trans-RR-DACH 100
1 Liposome composition : DMPC:DMPG 7:3
As may be seen in the above data, both the Pt
cyclopentenecarboxylato trans-RR-DACH and the Pt~
neodecanoato-racemic-DACH exhibit greater stability than
the Pt cyclopentenecarboxylato-racemic-DACH.
~ 3 ~ ~ ~ 3 ~
,~. . . .
-24-
EXAMPLE 8
Toxicity Studies of L-PT
(Cvclo~entenecarboxvlato-racemic-DACH) ~
-
Toxicology studies were carried out in 6-8 weeks old
CD-l Swiss mice weighing 22-25 gm and purchased from The ~ -
University of Texas Science Park (Bastrop, TX). Free PT
in suspension in hydroxypropylcellulose, DMPG, DMPG-PT,
DMPC:DMPG 7:3 and DMPC:DMPG 7:3-PT were administered
intraperitoneally ~ip) in volumes ranging between 0.1 and
0.3 ml. DMPC:DMPG 7:3-PT was also administered
intravenously ~iv) in one single injection or in 3 daily
injections. The clinical behavior and the survival times
were monitored on a daily basis. The LDlol LD50 and LD
were calculated considering the deaths occurring up to 14
days after injection.
PT in suspension in hydroxypropylcellulose, DMPG-PT
20 and DMPC:DMPG 7:3-PT had a similar LD50 dose leveI when - ; -
given in a single intraperitoneal (ip) injection (91
mg/kg, 86 mg/kg and 75 mg/kg respectively) (Table 3). The
amount of PT that would be encapsulated at the LD50 dose ~` -
level of empty;liposomes composed of DMPG and DMPC:DMPG
25 7:3 was higher (183 mg/kg and~>304 mg/kg respectively). -~.
The LD50 dose for DMPC:DMPG 7:3-PT injected iv~was similar
for the two schedules used: single~inj~ection and daily x
3 injections (82 mg/kg vs 96 mg/kg~ Most deaths in both
the ip and iv toxicity studies occurred between day 5 and
10 after injection. ~he results of these studies are
;presented in Table 3.
,.........
1 3 3 ~ r~ 3 8
-25-
:;: ,. : ~ , :
TABLE 3
TOXICITY OF DIFFERENT L-PT PREPARATIONS
(CYCLOPENTENECARBOXYLATO-RACEMIC-DACH)
5ADMINISTERED IP AND IV ~
.:
L-PT Route of ~Dlo LD50 LDg
PreparationAdministrationmg/kg mg/kg mg/kg
Free PT ip x l 61 91 125
DMPG-PT ip x 1 56 86 133
DMPC:DPMG 7:3-PT ip x 1 . 54 75 94 :
iv x 1 -- 82 100
: iv qd x 3 63 86 107 ,~
DMPG* ip x 1 158 183 228 i~
DMPC:DMPG 7:3*ip x 1 >304 >304 >304
* Results expressed in mg/kg of PT that would be
encapsulated at the LDlo/ LD50 and LDgo dose levels of
empty liposomes.
EXAMPLE 9 `
: 25
NEPHROTOXICITY .
Blood urea nitrogen~(BUN) was determined in samples
obtained from the retroorbital plexus of CDl Swiss mice ~ `
weighing 22-25 gm 96 hours after the ip injection (single
dose) of CDDP, PT cyclopentenecar~oxylato racemic DACH in
~hydroxypropylcellulose, DMPG-PT or DMPC:DMPG 7:3-PT at ;~
doses correspond;ng to the previously determined LD50.
All L-PT`preparations teste~d for toxicity w~ere prepared ~ t~
under sterile conditions on the same day of the
experiments. There were no significant 9UN elevations
13~3~
,
-26- ;
~,, ,",
after the ip administration of the LD50 dose of PT in
suspension in hydroxypropylcellulose, DMPG-PT and
DMPC;DMPG 7:3-PT ~BUN at 96 hours 34.4 + 9.6 mg%, 30.0 +
4.6 mg~ and 32.0 + 2.3 mg% respecti.vely). The results are
shown below in Table 4.
TAsLE 4
ACUTE NEP~ROTOXICITY OF CDDP, ~ :
10FREE PT (CYCLOPENTENECAR~OXYLATO-RACEMIC-DACH) ~ :
AND L-PT AT THE LD50 DOSE IN CDl MICE
LD50 DoseBUN at 96 hours
15 Preparation ip Single Injection (mg~
tmg/kg) ,,~
CDDP 17 78.3 + 8.0
Free PT 91 34.4 + 9.6
20 DMPG-PT 86 30.0 + 4.6
DMPC:DMPG 7:3 75 3 a .0 ~ 2.3 `~
l Normal=27.2 ~ mg%
EXAMPLE 10 , - -
In Vitro Antitumor Activity of
Cyclopentenecarboxylato-Racemic~
DACH-Pt Aaainst Ll210 Cells
L1210 leukemic cells were grown in a suspension
culture in McCoy's 5A medium (Gibco Laboratories, Grand
Island, NY) supplemented with 10% horse serum, glutamine, ;~
streptomycin and penicillin at 37C, 95~ relative humidity
in a 5% CO2 atmosphere. Four ml of cell suspension were
added to culture tubes and the appropriate concentration
of free-PT or L-PT was added (from 0.01 micro g/ml to 10
~ 3 ~ 2
; -27-
micro g/ml final concentration). After 96 hours, the cell
concentration of control and experimental cultures were
calculated with a Coulter Counter ~Coulter Electronics,
Hialeah, FLA) and the percent inhibition calculated. The
following preparations were tested: free PT, DMPG,
DMPC:DMPG 7:3, DMPG-PT and DMPC:DMPG 7:3-PT. Results were -~
expressed as the dose achieving a 50% growth inhibition
(ID50). Results for empty liposomes were expressed as the
amount of PT that would have been encapsulated at the ID50
concentration.
The ID50 for free cyclopentenecarboxylato-racemic
DACH was 1.3 micro g/ml ~Table 5). O the two L-PT
preparations tested, DMPG-PT was slightly more active than
DM~C:DMPG 7:3-PT (mean ID50 of three experiments; 0.7 and
1.6 micro g/ml respectively). Empty liposomes composed of
DMPG had an ID50 of 3.7 micro g/ml while the ID50 for
empty liposomes composed of DMPC:DMPG 7:3 was >10 micro
g/ml (Table 5).
: . .. :.,:, .,
: :: . : ~
' ' ~
~33~s~
-28- ;;
TABLE 5
:, .
IN VITRO ANTITUMOR ACTIVITY AGAINST L1210 CELLS OF
FREE PT, L-PT AND EMPTY LIPOSOMES
~where PT is cy~lo~entenecarboxvlato-racemic-DACH-PT)
ID50 (micro g/ml)~
Preparation - ~::
~:
Exp. l Exp. 2 Exp. 3 Mean
Free PT 1.3
DMPG-PT 0.7 1.0 1.4 0.7
DMPG2 3.0 3.0 5.0 3.7
DMPC:DMPG 7:3-PT 1.9 2.0 0.9 1.6 -~
DMPC:DMPG 7:32>10.0 --
. . . .. .. .
1 CDDP ID50=0.1 micro g/ml
ID50 expressed as micro g/ml of PT that would be
encapsulated at the ID50 concentration of empty liposomes.
~XAMPLE ll
In Vivo Antitumor Activity of L-PT -~
Against L1210 Mouse~Leukemia where
PT is cYclopentenecarboxylato-racemic-DACH .
:.:, . ::
The in vivo antitumor activity of CDDP, PT in
hydroxypropylcellulose, DMPG, DMPG-PT, DMPC:DMPG 7:3, and
DMPC:DMPG 7:3-PT was tested in an L1210-BDFl mouse model
~25). BDFl mice were purchased from Charles River ::~e~
(Wilmington, MA). Groups of 6-8 mice weighing 18-22 gm :~ 9;~
were inoculated ip with 1 x 106 L1210 leukemia cells on
day 0. L1210 cells were kept in DBA2 mice were weekly
passages between the different experiments. All L-PT
preparations were injected ip in volumes of 0.1 to 0.3 ml
24 hours after tumor inoculation. Two different schedules
~ ' , "
~33~3~
-29-
of administration were used: a single injection on day 1
or an injection on days 1, 5 and 9 (multiple). The doses
of CDDP used were the ones that had resulted in a maximum
antitumor activity in previous experiments. The doses of
PT, DMPG-PT and DMPC:DMPG 7:3-PT used ranged from 3.125 ~;
mg/kg to 50 mg/kg ~approximate LDlo). Clinical behavior
and survival times were monitored until all animals had
died. Results were expressed as % T/C (median survival
time of treated mice/median survival time o control mice
x 100~ and number of long-term survivors. Mice living
more than 30 days and more than 60 days were considered to
be long-term survivors for the single and multiple
injection schedule respectively. All L-PT preparations
tested for antitumor activity were prepared under sterile
15 conditions on the same day of the experiment. In the -~
first set of experiments, the effect of a single ip dose
of CDDP, free PT, DMPG-PT and DMPC:DMPG 7:3-PT in the -~
treatment of L1210 leukemia was tested. Mice treated with
free PT at doses of 12.5 mg/kg and 25 mg/kg had a % T/C
comparable to those treated with 10 mg/kg of CDDP (162 vs
178, means of three experiments) (Table 6). DMPG-PT at
doses of 12.5 mg/kg, 6.25 mg/kg and 3.125 mg/kg had an
antitumor activity comparable to free PT and CDDP (mean %
T/C=175 for 12.5 mg/kg; 158 for 6.25 mg/kg; and 163 for ~;
3.125 mg/kg) (Table 6). Doses of DMPG-PT of 25 mg~kg or
more were toxic for L1210 leukemia-bearing BDFl mice.
Mice treated with DMPC:DMPG 7:3-PT at doses of 25 mg/kg,
12.5 mg/kg and 6.25 mg/kg had a similar or slightly higher
% T/C than those obtained with CDDP, free PT and DMPG-PT
(mean: % T/C=215 for 25 mg/kg; 178 for 12.5 mg/kg; and 200
for 6.25 mg/kg)~(Table 6). DMPC:DMPG 7:3-PT at a dose of
50 mgjkg was toxic for L1210 leukemia-bearing BDPl mice.
Empty liposomes composed of DMPG and DMPC:DMPG 7:3, at
doses equivalent to the optimal ones of loaded vesicles
(L-PT) did not shown antitumor activity (% T/C=105 for
DMPG and 93 for DMPC:DMPG 7~3). Long-term survivors (one
1 3 ~
:' -30-
or two mice of six) were seen in the groups treated with
CDDP, DMPG-PT and DMPC:DMPG 7:3-PT (Table 6).
In the experiment using a multiple dose schedule, the
highest ~ T/C obtained was 253 for CDDP 7.5 mg/kg x 3, 284
for free PT 12.S mg/kg x 3, 179 for DMPG-PT 6.25 mg/kg x 3
and 403 for DMPC:DMPG 7:3-PT 12.5 mg/kg x 3. Long-term
survivors (l of 6 mice) were only observed in the group
treated with DMPC:DMPG 7:3 PT (Table 7). ~ .
''',`, ~;~ '"
' ~'"'' ;;~;' ' '
133~3~
3l ~ ~
TABLE 6
.- - . .
IN VIVO ANTITUMOR ACTIVITY AGAINST MOUSE L1210 LEUKEMIA ::~
OF CDDP, FREE PT AND L-PT AD~INISTERED IP (DAY 1)
where PT is cvcloPentenecarboxvlato-racemic-DACH
' .- ; ~
% T/C
Group(Number of Survivors on Day 30)
Number Preparation4 Exp.l Exp.2 Exp. 3
1 CDDP 10 mg/kg 167 -- 189 (1/6)
2 Free PT 25 mg/kg -- -- 168 .-~
3 12.5 mg/kg 173 136 179
4 DMPG-PT 25 mg~kg 100 -- -~
12.5 mg/kg 160 209 (2/6) 158 ~ .
66.25 mg/kg ~ 158 :~
73.12 mg/kg -- -- 163 (2/6)
8DMPC:DMPG 7:3-PT 25 mg/kg 213 200 ~1/6) 232 (1/6)
912.5 mg/kg 167 (1/6) -- 189 (1/6)
106.25 mg/kg -- -- 200
.,.
1 median survival of control = 7.5 days
2 median survival of control = 11 days
3 median survival of control = 9 days
4 All preparations were injected ip in 0.1-0.3 ml 24 hours after the
ip inoculation of 1 x 106 L1210 cells. :
'''....
1 3 ~
, ~, ,
~3
TABLE 7
lN VIVO ANTITUMOR ACTIVITY AGAINST MOUSE
5 L1210 LEUKEMIA OF CDDP, FREE PT AND L-PT
ADMINISTERED IP (DAYS 1, 5 and 9) WHERE ~4:
PT IS CYCLOPENTENECARBOXYLATO-RACEMIC-DACH
~ ' : ,' :'.~ :" '
% T/C
(Number of ::~
GroupSurvivors
Number Preparationl Dose on Day 60)
1 CDDP 7.5 mg/kg x 3 253
: : 2 Free PT 25 mg/kg x 3 158
3 12.5 mg/kg x 3 284
4 : ~ 6.25 mg/kg x 3 168
DMPG-PT 12.5 mg/kg x 3 105 ;~
6 6.25 mg/kg x 3 179
7 3.12 mg/kg x 3 168
: 8 1.56 mg/kg x 3 1}5 ;.
: 9 DMPC:DMPG 7:3-PT 12.5 mg/kg x 3 403 (1/6)
6.25 mg/kg x 3 253
25 11 ` 3.125 mg/kg x 3 210 (1/6)
-- - . ~ .:
All preparations were injected ip in volumes of 0.1 to
0.3 ml 24 hours, 5 days and 9 days after tumor .
inoculation
i ~ 30
EXAMPLE 12 :~
In Vivo:Antitumor Activity of Different
L-PT Analoqs Aqainst L-1210 Leukemia .
The antitumor activity against L-1210 murine `. .:
leukemia of the different L-PT analogs was assessed in an `:~
., ;, '"'~',:
:..,, -, ~ :.:
- ~33~3~
~ 33.
'
~-1210-BDF, mouse model ttumor inoculation day 0,
treatment day 1, ip) as described earlier herein.
Compared with CDDP ~% T/C=175), the L-PT analogues showed -
equivalent or slightly higher anti-tumor activity, with
the exceptions of L-PT (cyclopentenecarboxylato trans-SS-
DACH = 155 and N-decyliminodiaceto racemic-DACH = 150 and
N-decyliminodiacetato trans-RR-DACH = 162). These results ~ :
are shown in Table 8. ..
TA~LE 8
ANTITUMO~ EFFECT AGAINST L1210
LEUKEMIA OF CIS~PLATINUM AND l -
DIFFERENT LIPOSOMAL-PLATINUM ANALOGUES
-~ :
Platinum Analog Dose4
mg/k9 % T/C
.
PT-cyclopentenecarboxylato- :
racemic-DACH 25 213 ~ .
- ` :
25 PT-cyclopentenecarboxylato- -
trans-RR-DACH 25 187 .;~
PT-cyclopentenecarboxylato~
trans-SS-DACH 25 155 ..
.
PT-neodecanoato-racemic-DACH 50 187
:, . . :. ~ -:
,`"',''.''";~'',''-,''''','~',`
"';"''','~' '` '',~
~ 1 3 3 Q 9 3 ~
:~ 3 4
PT-neodecanoato-trans-RR-~ACH 25 175
PT-N-decyliminodiacetato-racemic-DACH 25 150
5 PT-N-decyliminodiacetato trans-RR-DACH 25 162
Cis-platinum (CDDP)5 10 175
~
1 Liposome composition : DMPC:DMPG 7:3 : ~ :
10 2 Median survival of treated mice :::
Median survival of control mice x 100 :
3 1 x 106 L1210 cells inoculated ip on day 0.
15 4 ~reatment : ip on day 1 :~
: 5 Dissolved in saline, 1 mg/ml
~ '~, '; ': `~
EXAMPLE 13 .
In Vivo Antitumor Activity of L-PT-NEODECANOATO-
RACEMIC-D.~CH Against Liver Metastases of Mouse
M5d76 Reticulosarcoma
. .,
The potential antitumor effect of L-PT against liver
metastases was tested using L-PT-NEODECANOATO-RACEMIC-DACH
~, ,
prepared as described in Example 6 and the~mouse M5076 - .. ~.
reticulosarcoma, which is a tumor that metastasizes
exclusively to the liveF. .~
L-PT-NEODECANOATO-RACEMIC-DACH was more active than
cis-platinum in the treatment of established liver
metastases of M5076 using two different tumor inocula and
schedules of administration ~Table 9 and 10). L-PT-
NEODECANOATO-RACEMIC-DACH (25 mg/kg on days, 8, 12, 16)
resulted in a more than two fold reduction in the number ~ : :
~33~3~ :
-3s-
of liver metastases compared with cis-platinum (7.5 mg/kg - ~-on days, 8, 12, 16) on day 30 after the intravenous
inoculation of 105 M5076 cells ~mean number of liver
metastases + SD = 39 + 21 for L-PT VS 114 + 38 for cis-
5 platinum (Table 9). L-PT-NEODECANOATO-RACEMIC-DACH (25 -.
mg/kg on days 4, 8, 12) resulted in the complete
inhibition of liver metastases of M5076 on day 45 after
the inoculation of 104 M5076 cells, while 4/6 animals
treated with cis-platinum (7.5 mg/kg on days 4, 8, 12) had ~:
175 or more liver metastases (Table 10).
L-PT-NEODECANOATO-RACEMIC-DACH was effective in the ~ -
prophylaxis of liver metastases of M5076 reticulosarcoma. ~::
L-PT-N~ODECANOATO-RACEMIC-DACH (37.5 mg/kg on day -1)
resulted in a five-fold decrease in the number of liver
metastases on d~y 21 after the intravenous inoculation of
104 M5076 cells (-inoculation day 0) compared with cis- -~
platinum (10 mg/kg on day -1) and untreated animals (mean -
number of liver metastases + SD = 52 + 20 for L-PT, 256 +
20 54 for cis-platinum, and 226 + 21 for control) (Table 11). ;; ~
'-''. " ~.. ' ,' ., ~,
~ .,.., ~,,,,,,,""., ,,~,.
'~' ',. '',`".',.,' '
', '.
~ ~33~3~
--3 6--
TABLE 9 ~ ~-
'.
Treatment of Liver Metastases of M5076 Reticulosarcoma ~ ~
with L-PT-NEODECANOATO-RACEMIC-DAC~ ~ -
Dose Schedule No. Liver Metastases on
Day 30
Treatment mg/kg day mean + SD
1 0
Cis-platinum7.5 8,12,16 114 ~ 38
15 L-P~-NEODECANOATO- 25 8,12,16 39 + 21
RACEMIC-DACH
*Groups of 10 C57BL/6 mice were inoculated on day 0 with -
105 M5076 cells intravenously. Animals were treated on
days 8, 12, and 16 with cis-platinum or L-PT-
NEODECANOATO-RACEMIC-DACH. The median survival of
untreated animals was 19 days. Treated animals were
sacrificed on day 30, the livers dissected, placed in
Bouin's fixative, and the number of liver-tumor colonies
25 counted. -
` ' ~ ' "', .'"~
~33a~3~ :
-37-
TA~LE 10
Treatment of Liver Metastases of M5076 Reticulosarcoma
with L-PT-NEODECANOATO-RACEMIC-DACH
S
Dose Schedule No. Liver Metastases on ~ -
Day 45
Treatment mg/kg day mean f SD ~
''"
:,
Cis-platinum7.5 24,8,12 0,0,175,>200,>200,>200
~'",".'~,' , "'"~ .
15 L-PT-NEODECANOATO- 25 4,8,12 0,0,0,0,0,1
RACEMIC-DACH -,:; ; "
*Groups of 6 C57BL/6 mice were inoculated on day 0 with
104 M5076 cells intravenously. Animals were treated on ; ~
20 days 4, 8, and 12 with cis-platinum or L-PT-NEODECANOATO- i~` -
RACEMIC-DACH. The median survival~of untreated animals ~ ` ;
was 30 days. Treated animals were sacrificed on day 45,
the livers dissected, placed in 8Ouin's fixa~tive, and the
number of liver-tumor colonies counted. Two of the
25 animals treated with cls-platinum died before day 45 ; ~
versus none in the group~treated with L-PT. ` `
; ,, . . . ~,- ,.~, .
''i `.'..'' ',, ~','
~'.,, ~, ' i ~ ' .
- ~33~<~3~
... ~ :
- 3 8-
TABLE 11
Prophylaxis of Liver Metastases of M5076 Reticulosarcoma ~ -
with L-PT-NEODECANOATO-RACEMIC-DACH
, ,
Dose Schedule No. Liver Metastases on
Day 21 -
Treatment mg/kg day mean + SD
1 0
'~: " "" ''
None -- -- 226 + 21 -~
Cis-platinum 10 - 1 256 + 54
L-PT-NEOD~CANOATO- 37.5 - 1 52 + 20
RACEMIC-DAC~
*Groups of 6 C57BL/6 mice were treated on day
- 1 with cis-platinum or L-PT Neodecanoato- - - -
Racemic-DACH. Animals were inoculated on day
0 with 104 M5076 cells intravenously. The
median survival of untreated animals was 21
days. Treated animals were sacrificed on day
21, the livers dissected, placed in Bouin's
fixative, and the number of tumor colonies
counted.
3 ~ :
-39- :
EXAMPLE l4
Preparation of cis-bis-neodecanoato-
cis-diamine-~latinum (II).
Cis-diamine-diiodo-platinum ~II) (NH3)2 Pt-I2 was
first prepared by the following method: ~ -
'-,,''' .',' ' . ~ '' ,:'
K2 PtCl4 (59) was dissolved in water (20ml). An aqueous
solution of KI (3g) was added and a dark brown solution
was obtained. Aqueous concentrated ammonia (2ml) was - -
added to the mixture which was stirred for 2-3 hr. at room
temperature. The mixture was filtered and the solid was -
washed with an excess of water, ethanol and ether. The ` -~
product was dried over P205 under vacuum. Yield 4.5g
(77~
The compound according to the invention was then
prepared as follows~
(NH3)2-Pt-I2 (l.Og) was suspended in water (50ml),
and aqueous solutions of AgN03 (0.68g 20ml~H20) was added ~ ``
thereto. The reaction mixture was stirred~overnight at
room temperature in the dark. The AgI precipitate was
25 filtered and filtrate was concentrated by rotary `~
evaporation. To the concentrated;solution~a solution of
sodium neodecanoato prepared in situ, (neodecanoic acid
0.688g in 20ml methanol and lN NaOH, 4ml) was added. The
, j reaction mixture was stirred overnight at room tempera~
ture. The yellow reaction mixture was evaporated to
. .
dryness at 40C under reduced pressure using a rotary ;~
evaporator. A gummy product was obtained which was ;~
extracted in chloroform and the chloroform extract was
dried over anhydrous MgS04. MgS04 was separated by
filtration and filtratè was evaporated to dryness under
reduced pressure using a rotary èvaporator. A cream-color
~L 3 3 9 .~
- ; . ,
--40--
. ; -'
solid was obtained which was dried over P205 in vacuo.
The final product was stored at 0C. ~ ~-
.,,- .:
The elemental analysis of the final product was ~ ~ ~
C4}.58, H8.03; N 4.45%. The calculated values for -
C20H44N24Pt. is C42.00; H7.7, N4.90%.
,:,. . .
The structural formula of the cis-bis-neodecanoato-
bis-diamine-platinum (II), is as follows~
1 0 , ' '' ',', . .'. ' ''.,
[H3N]2 - Pt - [OOC - CR'RR ]2
where R, R', R" can be CH3, C2 H5 or C3H7 to give a
carboxylato radical function with CloH19O2 (MW = 171) as
empirical formula.
Cis-bis-neodecanoato-bis-diamine-platinum tII) is highly
soluble in chloroform, methanol and other common organic `~
solvents, but insoluble in water.
EXAMPLE 15
, . ,~,: ~
Preparation of cis-bis-neodecanoato-bis
cvclohexvlamine-platinum (II) dihydrate
The method of Example 14 was -followed, using the
cyclohexylamine ligand in place of ammonia. The complex
is highly soluble in chloroform, methanol and other
organic solvents, but insoluble in water.
~ ~ ~
Elemental Analysis; Calculated ~or C32H64N2O4.2H2O, ~ ; ;
C49.71; H8.80, N3.62%, found C49.31: H8.39; N3.16%
The structural formula of the cis-bis-neodecanoato~
bis-cyclohexylamine-platinum (II) is as follows~
;~
. ~ - , .
- 1 ~ 3 ~ 9 3 8
-41-
, '' "' -
E 6H11 NH2]2 Pt [OOc-c-R'RRll~2 ~
where R, R', R" can be CH3, C2H5 or C3H7 to give a ;
carboxylato radical functions C1oH19O2 (MW = 171) as -~
empirical formula.
EXMPLE 16
Preparation of cis-bis-neodecanoato- i
ethvlenediamine-~latinum (II) H2O. ~ ;~
, ~,, ~.. ,, ~...
Cis-diiodo-ethylenediamine-platinum (II), was first
prepared by the method of Example I, using ethylenediamine ~ `~
ligand in place of ammonia and with a 96% yield of
product.
Sulfato-ethylenediamine-platinum (II). H2O was
prepared by the following method. ~ :
Cis-bis-diiodo-ethylenediamine-platinum (II), (3.9)i
was suspended in 10ml H2O and an aqueous solutions of ~ -
Ag2SO4 ~2.29 in 200 ml H2O) was added thereto. The
reaction mixture was stirred in the dark overnight at room
temperature. AgI was removed by~filtration and the yellow 5
filtrate was evaporated to dryness at 40-45C under
reduced pressure using rotary evaporation. The final
product was dried over P2O5 under vacuum. The product ~ 9 ~-
yield was 2.359 181~
.. ..
~ The compound according to the invention was then
prepared as follows~
Sulfato-ethylenediamine-platinum (II), H2O (0.369 9)
was dissolved in H2O t20ml) and a solution of sodium
neodecanoato prepared in situ (neodecanoic acid, 0.3449 in
20 ml methanol and IN NaOH, 2ml) was added thereto. The
~;
reaction mixture was stirred for 2-3 hr. at room
temperature. The reaction mixture was evaporated to
dryness at 40-45C under reduced pressure using rotary
evaporation. A gummy product was obtained which was
extracted in chloroform and the chloroform extract was
dried over anhydrous MgSO4. The MgSO4 was separated by
filtration and the filtrate was evaporated to dryness
under reduced pressure using a rotary evaporator. The
final product was dried over P2O5 in vacuo. The product
10 was stored at OC. ;~
Elemental analysis; calculated for C22H46N~O4Pt. H2O,
C42.86; H7.79; N 4.54~, found C43.07; H 7.32; N 4.71%.
Cis-bis-neodecanoato-ethylenediamine-platinum tII) is
highly soluble in chloroform, methanol and other organic ~ -
- solvents, but insoluble in water. -~
The structural formula of the cis-bis-neodecanoato- ~ ~-
ethylenediamine-platinum (II) is as follows~
CH2 NH2 ; '
Pt [OOC-C-R'RR"]
CH2 NH~
where R, R', R" can be CH3, C2H5 or C3H7 to give a
carboxylato radical function CloH19O2 (MW = 171) as
empirical formula.
:., "; ,
~ 3 ~ Q ~ 3 ~
~43~ ~ -
EXAMPLE 17
Preparation of cis-bis-neodecanoato- i
Sbis-iso~ro~vlamine-~l~tinum (II) - -
Cis-bis-neodecanoato-bis-isopropylamine-platinum (II)
and sulfato-bis-isopropylamineplatinum (II) were first
prepared by the method of Example 16, using isopropylamine
ligand in place of ethylenediamine~
The compound according to the invention was then
prepared as follows~
15Sulfato-bis-isopropylamine-platinum (II). H2O
~0.4279) was dissolved in H2O (50ml) and a solution of ;-
sodium-neodecanoato prepared :n situ (neodecanoic acid -~
0.3449 in 20 ml of methanol and lN NaOH, 2 ml) was added
thereto. The reaction mixture was stirrèd for 2-3 hr. at
room tempeature. The reaction mixture was evaporated to
dryness at 40-45C under reduced pressure usin~ rotary
evaporator. A gummy mass~was obtained which was extracted
in chloroform and the chloroform extract was dried over
anhydrous MgSO4. The MgSO4 was separated by filtration
and filtrate was evaporated to dryness under reduced
~-~pressure using a rotary evaporator. The~product was dried ~ ~
over P2O5 in V2CUO. The ;inal~product was stored at OC. ~ '
Elemental analysis, calculated for C26H56N2O4Pt,
C47.63; H8.53; N4.26%; found C 47.74; H8.47i N 3.93
The structural formula of the cis-bis-neodecanoato~
bis-isopropylamine-platinum~(II) is~as follows~
9 3 ~
-44-
([H3C]2CH-NH2)2 Pt [OOC-C-R'RR"]2
where R, R', R" can be CH3, C2H5 or C3H7 to give a
carboxylato radical function with C10~192 (MW = 171) as
an empirical formula.
Cis-bis-neodecanoato-bis-isopropylamine-platinum
(II), is highly soluble in chloroform, methanol and other
organic solvents, but insoluble in water.
1 0
EXAMPLE 18
Preparation of cis-bis-decanoato-trans- `
R,R-1,2-diaminocYclohexane-platinum II.
Sulfato-trans-R,R-1,2-diaminocyclohexane-platinum II
H2O (0.423g) was dissolved in H2O (20ml) and a solution of
sodium-decanoato prepared in situ (0.344g decanoic acid in
20ml methanol and 1~ NaOH 2ml) was added thereto. The
reaction mixture was stirred for 2-3 hr. at room
temperature. The reaction mixture was evaporated to
dryness at 40-45~C under reduced pressure using a rotary
evaporator. A solid was obtained which was extracted in
chloroform and chloroform extract was dried over anhydrous
MgSO4. The MgSO4 was removed by filtration and filtrate
was evaporated to dryness under reduced pressure using
rotary evaporator. The product was dried over P2O5 ln
vacuo. The final product was stored at OC.
Elemental analysis - calculated for C26H52N2O4Pt.H2O
C46.59; H8.06 N 4.18%, found C 45.94; H7.87 N 4.31%.
Cis-bis-decanoato-trans-R,R-1,2-diaminocyclohexane~
platinum II is highly soluble in chloroform and other
organic solvents, but insoluble in water.
413~38
: . :.,~, . . ...
The structural formula of the cis-bis-decanoato~
trans-R,R-1,2-diaminocyclohexane-platinum II is as
follows~
: ., . ~ . , ,; : . ~
~ PT - [ooc (c~2~8 C~3]2
~\ / - ~ .
~ 2
' ,' ' . ::.
EXAMPLE 19
Cis-bis-neopentanoato-trans-R,R-l,
2-diaminoc~clohexane-~latinum II.
Sulfato-trans-R,R-1,2-diaminocyclohexane-platinum II. ~ -~
H2O ~0.423g) was dissolved in water (20ml) and a solution
of barium-neopentanoato prepared in situ (neopentanoic
acid, 0.204g, in 5 ml of~methanol and Ba (OH)2.8H2O 0.39
in 50 ml H2O combined together) was added thereto. The
reaction mixture was stirred for 2-3 hr. at room
temperature. The reaction mixture was evaporated to ~ ~ ~
dryness at 40-45C under reduced pressure using a rotary ~ ;
evaporator. The residue was extracted with methanol,
filtered and the filtratè was evaporated to dryness. A
solid was obtained which was further extracted with
chloroform. The chloroform extract was evaporated to `~
dryness and a cream-color product was obtained. The
30 product was dried over P2O5 under vacuum. ;~
Elemental analysis calculated for C16H32N2O4Pt.2H2O;
C35.00; H6.57; N5.11%, found C35.16; H6.17; N5.27%.
.'``'~.,''"'''~`"`'",''~''''".
. ,, ~ ' ' .' .'''~, ~
1 3 3 13 r9 3 ~
-~6~
Cis-bis-Neopentanoato-trans-R,R-1,2-dlaminocyclo-
hexane-platinum II, is highly soluble in chloroform,
methanol and other common organic solvents.
The structural formula of the cis-bis-neopentanoato-
trans-R,R-1,2-diaminocyclohexane-platinum II is as
follows: .
~ NH2 :::
~ 1
Pt [OOC~C~(c~3)3]2 ~ :
\ / ~
~ _~' NH2
EXAMPLE 20
Encapsulation efficiency and antitumor
activitv of li~o~hilic cis~latin analoqs
The compounds prepared in Examples 14~19 were tested
for their efficiency of encapulation in liposomes by the : ~;
methods described in Example 6. These liposome-
encapsulated compounds were tested to determine their
optimal dose and effectiveness as inhibitors of in vivo
tumor growth as described in Example 12. The data in
Table 12 reveal the resultant measurements.
:~
. ~,: .:
~331~8 ::
- 4 7-
n r) ~ o n
. ~. ,,. ,.. ,.. ,..................................... .: .- ' ~
a cr D~ ~ :: :
,.. ,,. ,,. ,.. ,.. ,~. . .. -
l l l l l l "~
O n O & - ~ ..
tD ~ D (D
n ~ n
c~ l 2 Dl
O ~ 0~ 3o
, C ': , . ::
u~ ~ :1 : :
r -:
, rloJ 3 ~ D
D~ _- ~u
1 3 3 tD D/ P)
~ 3~ _. ~ ~ :
Dl O ~ 3 \- : . :
3 ,< ,~ 3
g ". 3 ~ ~ ~o 0'
~- x c -- :~ ~, ~ :~;; " :' '' ''
X I H _
: _ o n 3
3. ~
,o,3
~ b
_-~
r
DW ~ o O .
w C~ , . ~ O r~
D oD ~ OD ~ ~-C
~c O
~ ''~'~ '~,:'' ~"'`''''~"'
~ ~ o~ ."~
V~ O O Vl oO O ~ ID 3 .~
;.'"".'~
co ul a ~ o ~~ o ~ : .
o _~ o a v~ o Cb ~
O ~. .,.. ,.,~
,;""., ~ ''` ."'
133~3~
,.~. .
-48-
EXAMPLE 21
In Vitro cytotoxicity aaainst
human malianant cell lines
Cis-bis-neodecanoato-1,2-diaminocyclohexane platinum `:
(II) was tested in the liposomal-form against three human
malignant cell lines of colon carcinoma (LoVo, SW620, and
SW403) using a colony formation inhibition assay. The
drug concentration that resulted in a 50% inhibition of
colony formation (ICS0) ranged from 4 to 8 uM. The IC50
of cisplatinum for these same cell lines ranged from 3 to
7 uM.
'~
~: x x x x x x x x x x -'
: Changes may be made in the various compositions, -~
: elements, steps and procedures described herein without
20 :departure from the concept and scope of the invention as :~
defined in the following claims.
',..'',.` .' '';', ,`.''''~''' ".
