Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02206333 1997-OS-28
NOVEL ANTHRACYCLINE COMPOUND DERIVATIVE AND PHARMACEUTICAL
PREPARATION CONTAINING THE SAME
TECHNICAL FIELD
This invention relates to a novel anthracycline compound
derivative and a high molecular block copolymer-drug complex
pharmaceutical preparation which contains the derivative.
BACKGROUND ART
Daunomycin (British Patent 1003383, U.S. Patent 3616242),
adriamycin (U. S. Patent 3590028, U.S. Patent 3803124) and the like
obtained from culture liquids of actinomycetes are known as
anthracycline anticancer agents. They have. broad anticancer
spectrums against experimental tumors and also are clinically used
widely as cancer chemotherapeutics. On the contrary, however, it is
known that they frequently cause serious side effects, such as
leukopenia, alopecia, myocardial disorder and the like.
In order to resolve this problem, various derivatives have _,
been proposed. For example, pirarubicin (common name) aims at
reducing its toxicity by introducing a tetrahydropyranyl group into
the 4' position of the sugar moiety of adriamycin.
Also, epirubicin (common name) is a compound in which a
hydroxyl group on the 4' position of the sugar moiety of adriamycin
is bound to the a position, thereby attempting to reduce its toxicity.
However, though these drugs have lower toxicities in
comparison with adriamycin, their problems such as limited total -
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doses and the like are not completely settled yet.
On the other hand, it is a well known technique to make use
of a high molecular micelle formed from a block copolymer to improve
solubility of drugs which are slightly soluble in water. And it has
been confirmed that high molecular block copolymer-drug complex
pharmaceutical preparations obtained in Japanese Patent Application
(Kokai) No. 2-300133, Japanese Patent Application (Kokai) No. 6-
107565, Japanese Patent Application (Kokai) No. 5-955, Japanese
Patent Application (Kokai) No. 5-124969, Japanese Patent Application
(Kokai) No. 5-117385, Japanese Patent Application (Kokai) No. 6-
206830, Japanese Patent Application (Kokai) No. 7-69900 and Japanese
Patent Application (Kokai) No. 6-206815 are possessed of anticancer
effects which are superior to those of adriamycin.
DISCLOSURE OF THE INVENTION
The inventors of the present invention have conducted
intensive studies on a high molecular block copolymer-drug complex
pharmaceutical preparation which is possessed of both higher effect
and lower toxicity in comparison with the prior art high molecular
block copolymer-drug complex pharmaceutical preparations, and have
accomplished the present invention as a result of the efforts.
Accordingly, the present invention relates to:
-( 1 ) a dimer, trimer or tetramer of anthracycline compound which can
be obtained by directly, chemically bonding anthracycline compound
or compounds having anticancer activities to each other by an alkali
treatment;
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CA 02206333 1997-OS-28
-(2) the dimer, trimer or tetramer of anthracycline compound
according to the aforementioned item (1) wherein the anthracycline
compound or compounds comprise at least one kind of compound selected
from adriamycin, daunomycin, pirarubicin, epirubicin and acid salts
thereof;
-(3) a dimer of anthracycline compound which can be obtained by
directly, ch~ni.cally bonding adriamycin molecules or acid salts
thereof to each other, or by directly, ch~nically bonding adriamycin
or an acid salt thereof to daunomycin or an acid salt thereof, by an
alkali treatment;
-(4) the dimer, trimer or tetramer of anthracycline compound
according to the aforementioned item (1), (2) or (3) wherein the
mutual binding mode of anthracycline compounds is Schiff base
bonding;
-(5) the dimer of adriamycin having the structure of the following
formula (AA): O
O HO
I I ~ OH CH20H
H3C0 O HO p
H3C ~O~
OH N
o HO ~~ II
C-C-H
I I ~ off (AA)
H3C0 O HO
H3C ~O
NH2 _'
OH
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CA 02206333 1997-OS-28
-(6) a trimer of adriamycin which can be obtained by directly,
chemically bonding adriamycin molecules or acid salts thereof to each
other by an alkali treatment and has the mass spectrum shown in Fig.
7:
-(7) a high molecular block copolymer-drug complex pharmaceutical
preparation in which the high molecular block copolymer having a
hydrophilic polymer segment and a hydrophobic polymer segment forms
a micelle having the hydrophilic segment as its outer shell and
contains in its hydrophobic inner core a dimer, trimer or tetramer
of anthracycline compound, if necessary together with other drugs;
-(8) the high molecular block copolymer-drug complex pharmaceutical
preparation according to the aforementioned item (7) wherein the
dimer, trimer or tetramer of anthracycline compound is the dimer,
trimer or tetramer of anthracycline compound of the aforementioned
item (1), (2), (3), (4), (5) or (6);
-(9) the high molecular block copolymer-drug complex pharmaceutical
preparation according to the aforementioned it~n (7) or (8) wherein
the high molecular block copolymer has a structure of the following
formula (1) or (2):
Ri ( OCHZCHZ ) n O-R2-CO- ( NHCHCO ),o-X ( NHR3 ~ CO ) X ~a ( 1 )
3p O
R1- ( OCHZCHZ ) n O-R2-NH- ( COCHNH ),~-X ( COR3 ~ ) X RS ( 2 )
1R3
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wherein R1 represents a hydrogen atom or a lower alkyl group, R2
represents a binding group, R, represents a methylene or ethylene
group, ~ independently represents a hydroxyl group or a residue of
an anthracycline compound having anticancer activity, RS represents
a hydrogen atom or a protecting group, n is an integer of 5 to 1, 000,
m is an integer of 2 to 300 and x is an integer of 0 to 300, with the
proviso that x is not larger than m;
-(10) the high molecular block copolymer-drug complex pharmaceutical
preparation according to the aforementioned item (9) wherein the
residue of the anthracycline compound having anticancer activity is
a group represented by the following formula (3):
O Ot~ O
Y
I ~ off
H3C0 O Ot-i O C 3 )
O
HsC ~ NH -.
O~
wherein Y represents -CHZOH or -CH3 and Z represents H or
-(11) the high molecular block copolymer-drug complex pharmaceutical
preparation according to the aforementioned item (7), (8), (9) or
(10) wherein it contains in the inner core of micelle formed by the
high molecular block copolymer a dimer, trimer or tetramer of
CA 02206333 1997-OS-28
anthracycline compound in an amount of from 2 to 60~ by weight based
on the high molecular block copolymer;
-(12) the high molecular block copolymer-drug complex pharmaceutical
preparation according to the aforementioned item ( 7 ) , ( 8 ) , ( 9 ) , ( 10
)
or (11) wherein the dimer, trimer or tetramer of anthracycline
compound is the dimer of adriamycin;
-(13) the high molecular block copolymer-drug complex pharmaceutical
preparation according to the aforementioned item ( 7 ) , ( 8 ) , ( 9 ) , ( 10
) ,
( 11 ) or ( 12 ) wherein it contains in the inner core of micelle formed
by the high molecular block copolymer the anthracycline anticancer
agent and the dimer, trimer or tetramer of anthracycline compound at
the ratio of 1:0.5-20 by weight;
-(14) the high molecular block copolymer-drug complex pharmaceutical
preparation according to the aforementioned item (13) wherein the
anthracycline anticancer agent and the dimer, trimer or tetramer of
anthracycline compound are contained at the ratio of 1:0.7-10 by
weight;
-( 15 ) the high molecular block copolymer-drug complex pharmaceutical
preparation according to the aforementioned item (13) wherein the
anthracycline anticancer agent and the dimer, trimer or tetramer of
anthracycline compound are contained at the ratio of 1:1-5 by weight;
-(16) the high molecular block copolymer-drug complex pharmaceutical
preparation according to the aforementioned item (13), (14) or (15)
wherein the anthracycline anticancer agent is at least one agent
selected from adriamycin, daunomycin; pirarubicin, epirubicin and
acid salts thereof;
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-(17) a high molecular block copolymer-drug complex pharmaceutical
preparation in which the high molecular block copolymer having a
hydrophilic polymer segment and a hydrophobic polymer segment forms
a micelle having the hydrophilic segment as its outer shell and
contains in its hydrophobic inner core an anthracycline anticancer
agent, wherein, when the pharmaceutical preparation is intravenously
administered to a CDF1 mouse of 7 to 9 week's age, the amount of the
anthracycline anticancer agent in 1 ml of the mouse blood plasma after
1 hour of its administration becomes 10 ($ of dose/ml) or more,
provided that the amount of anthracycline anticancer agent in the
administered preparation is defined as 100;
-(18) the pharmaceutical preparation according to the aforementioned
item (17) wherein the amount of anthracycline anticancer agent in 1
ml of the mouse blood plasma after 1 hour of its administration
becomes 20 to 60 (~ of dose/ml);
-(19) the pharmaceutical preparation according to the aforementioned
item (17) or {18) wherein the anthracycline anticancer agent is at
least one agent selected from adriamycin, daunomycin, pirarubicin,
epirubicin and acid salts thereof; and
-(20) the pharmaceutical preparation according to any one of the
afor~nentioned iteqns ( 7 ) to ( 19 ) wherein it is used for the treatment
of a solid cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a graph of infrared absorption spectrum of the
dimer of adriamycin.
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Fig. 2 shows a graph of ultraviolet spectrum of the dimer of
adriamycin.
Fig. 3 shows a graph of mass spectrum of the dimer of
adriamycin.
Fig. 4 shows a graph of infrared absorption spectrum of the
compound presumably having the structure of formula (4), which is
generated together with adriamycin when the dimer of adriamycin is
treated with an acid.
Fig. 5 shows a graph of ultraviolet spectrum of the compound
presumably having the structure of formula (4), which is generated
together with adriamycin when the dimer of adriamycin is treated with
an acid.
Fig. 6 shows a graph of mass chromatogram (m/z = 560) of the
compound presumably having the structure of formula (4), which is
generated together with adriamycin when the dimer of adriamycin is
treated with an acid.
Fig. 7 shows a graph of mass spectrum of the trimer of
adriamycin.
Fig. 8, 9, 10 or 11 shows a graph of 1H one-dimensional spectrum,
1'C one-dimensional spectrum, COSY spectrum or CH COSY spectrum
respectively, obtained by the NMR analysis of the separated component
from Example 1 (2).
Fig. 12 shows a graph of mass spectrum of the dimer of
daunomycin with adriamycin.
Fig. 13 shows a graph of HPLC chromatogram of the
pharmaceutical pr-eparation obtained from Example 3.
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Fig. 14 shows a graph of HPLC chromatogram of the
pharmaceutical preparation obtained from Example 4.
Fig. 15 shows a graph of HPLC chromatogram of the
pharmaceutical preparation obtained from Example 5.
Fig. 16 shows a graph of tumor growth curve of mouse Colon 26
adenocarcinoma when the mouse was administered with adriamycin
hydrochloride in Application Example 1.
Fig. 17 shows a graph of tumor growth curve of mouse Colon 26
adenocarcinoma when the mouse was administered with the
pharmaceutical preparation of Example 3 in Application Example 1.
Fig. 18 shows a graph of HPLC chromatogram of the
pharmaceutical preparation obtained from Example 6.
Fig. 19 shows a graph of HPLC chromatogram of the
pharmaceutical preparation obtained from Example 7.
Fig. 20 shows a graph of HPLC chromatogram of the
pharmaceutical preparation obtained from Example 8.
Fig. 21 shows a graph of HPLC chromatogram of the
phazmaceutical preparation obtained from Example 9.
Fig. 22 shows a graph of HPLC chromatogram of the
pharmaceutical preparation obtained from Example 10.
Fig. 23 shows a graph of HPLC chromatogram of the
pharmaceutical preparation obtained from Example 11.
Fig. 24 shows a graph of HPLC chromatogram of the
pharmaceutical preparation obtained from Example 12.
Fig. 25, 26 or 27 shows a graph of tumor growth curve of mouse
Colon 26 adenocarcinoma when the mouse was administered with
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adriamycin hydrochloride, the pharmaceutical preparation from
Example 8 or the pharmaceutical preparation from Example 12
respectively in Application Example 3.
BEST MODE OF CARRYING OUT THE INVENTION
The following describes the present invention in detail.
According to the present invention, it is able to obtain
pharmaceutical preparations which have higher effect and lower
toxicity in comparison with conventional anthracycline anticancer
agents or high molecular block copolymer-drug complex pharmaceutical
preparations of the prior art.
Examples of the anthracycline compound having an anticancer
activity to be used in the present invention include adriamycin,
daunomycin, pirarubicin, epirubicin and acid salts thereof.
The method for preparing the dimer, trimer or tetramer of
anthracycline compound of the present invention is not particularly
limited, and the said dimer, trimer or tetramer can be obtained, for
example, by subjecting an anthracycline compound having an anticancer
activity to an alkali treatment. By the alkali treatment, the dimer,
trimer or tetramer of the anthracycline compound is obtained through
mutual direct chemical bonding (that is, not by a chemical bonding
via a cross-linking agent but by a bonding formed from a reaction
between functional groups of the anthracycline compounds).
The dimer, trimer or tetramer of anthracycline compound may
be obtained by bonding molecules of the same compound or different
compounds.
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As the alkali treatment, a method in which an anthracycline
compound is dissolved in a solvent and a base is added thereto can
be exemplified. The solvent to be used is not particularly limited,
provided that it can dissolve said compound, and its examples include
water, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),
dioxane, tetrahydrofuran (THF), methanol, acetonitrile and a mixture
solvent thereof.
As the base to be added, any one of inorganic bases, organic
bases and salts thereof can be used with no particular limitation,
with the proviso that it is soluble in said solvent and that the
solution after addition of the base shows a pH value of exceeding 7
and up to 14. The base concentration is also not particularly limited.
Examples of the desirable bases include sodium hydroxide, potassium
hydroxide, sodium carbonate, sodium hydrogencarbonate, potassium
carbonate, potassium hydrogencarbonate, sodium phosphate, disodium
hydrogenphosphate, sodium dihydrogenphosphate, potassium phosphate,
dipotassium hydrogenphosphate, potassium dihydrogenphosphate,
secondary and tertiary amines having 2 to 20 carbon atoms, and an acid
salt adduct thereof. The pH value in the alkali treatment is
exceeding 7 and up to 14, and preferably 8 to 10.
The alkali treatment temperature is not particularly limited,
provided that the solution does not freeze or boil, and it is
preferably 0 to 50°C, more preferably 0 to 40°C. The treatment
time
is 1 minute to 120 hours, preferably 10 minutes to 24 hours.
The thus obtained dimer, trimer or tetramer of anthracycline
compound can be purified by employing a known purification technique..
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For example, a solid substance may be obtained by freeze-drying,
precipitation or the like, or by exchanging the solvent by dialysis
or ultrafiltration followed by freeze-drying, precipitation or the
like. When further purification of the thus obtained solid substance
is required, thin layer chromatography, liquid chromatography or the
like can be used.
When a compound having both a substituent of carbonyl
structure and an amino group is used as the anthracycline compound,
or a compound having a substituent of carbonyl structure is used in
combination with another compound having an amino group, a dimer,
trimer or tetramer in which the anthracycline compounds are
chemically bound to each other via Schiff base bonding is obtained
by the aforementioned alkali treatment. In consequence, it is
desirable to use as the anthracycline compound a compound having both
a substituent of carbonyl structure and an amino group or a compound
having a substituent of carbonyl structure in combination with a
compound having an amino group. In this connection, examples of the
substituent having carbonyl structure include an aryl group having
2 to 5 carbon atoms which may be substituted with a hydroxyl group
or a halogen atom, such as fluorine, chlorine, bromine and iodine atom
or the like; or an acylalkyl group having 3 to 10 carbon atoms which
may be substituted with a hydroxyl group, a halogen atom or the like.
When the dimer, trimer or tetramer in which the anthracycline
compound are mutually bound via Schiff base bonding is treated with
an acid, at least the anthracycline compound which has been used as
a raw material is 3enerated. As the acid treatment, a method in which
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the dimer, trimer or tetramer of anthracycline compound is dissolved
in a solvent and an acid is added thereto can be exemplified. The
solvent to be used is not particularly limited, provided that it can
dissolve said compound, and its examples include water, N,N-
dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dioxane,
tetrahydrofuran (THF), methanol, acetonitrile and a mixture solvent
thereof. As the acid to be added, any one of inorganic acids, such
as hydrochloric, nitric, sulfuric and phosphoric acid, and organic
acids, such as formic., acetic and trifluoroacetic acid, can be used.
The pH value in the acid treatment is preferably from 2 to 4,
and the treating temperature is not particularly limited, provided
that the solution does not freeze or boil, and is preferably from 0
to 50°C, more preferably from 20 to 40°C. The treatment time is
within
the range of from 1 minute to 120 hours, preferably from 24 to 72
hours.
As an example of the dimer, trimer or tetramer of anthracycline
compound of the present invention which can be obtained through
directly, chemically bonding anthracycline compounds by the
aforementioned alkali treatment, the dimer of adriamycin having the
infrared absorption spectrum shown in Fig. 1 and the ultraviolet
spectrum shown in Fig. 2 can be ex~nplified. This adriamycin dimer
also has the mass spectrum shown in Fig. 3. This adriamycin dimer
has the structure represented by the aforeqnentioned formula (AA).
Infrared absorption spectrum: 1676, 1417, 1217, 1158 clril;
Ultraviolet spectrum: ~, mas = 486 nm;
Mass spectrum (ESI), m/z (%): 1067 (100), 964 (10), 938 (15), 921 (13),
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653 (20), 524 (20), 506 (50), 488 (98).
In this connection, the instruments and measuring conditions
used for the measurement of these spectra are as follows. The
infrared absorption spectrum was measured by the KBr tablet method
using System 2000 manufactured by Perkin-Elmer Co. The ultraviolet
spectrum was measured in methanol solution using U 3200
Spectrophotometer manufactured by Hitachi Co. The mass spectrum was
measured by the electrospray method using QUATTRO 2 Mass Spectrometer
manufactured by VG Co.
When treated with the acid, this adriamycin dimer generates
adriamycin, together with a compound presumably having a structure
represented by the following formula (4):
O OH
CH(OH)COOH
~ / OH
H3C0 O OH O
(4)
NH2
OH
The infrared absorption spectrum, ultraviolet spectrum or mass
chromatogram at m/z = 560 by LC/MS of the compound presumably having
the structure of formula (4) is shown in Fig. 4, Fig. 5 or Fig. 6,
respectively.
In this connection, instruments and measuring conditions used
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for the measurement of these spectra and chromatogram are as follows .
The infrared absorption spectrum was measured using the same
instrument under the same conditions as those used in the measurement
of the spectrum of Fig. 1. The ultraviolet spectrum was measured in
benzyl alcohol solution using U 3200 manufactured by Hitachi Co.
Instruments and measuring conditions for the measurement of mass
chromatogram by LC/MS are as follows.
LC:
Column: C4~300 angstrom/5 Fun, manufactured by Waters Co.;
Fluent: acetonitrile/0.1~ trifluoroacetic acid + 0.05 MS 7
(MS 7; manufactured by Gasukuro Kogyo Co.);
Gradient elution:
Time (minute) 0 20 25 30 35 36 40;
Acetonitrile concentration (~) 22 40 50 90 90 22 22;
Flow rate: 1 ml/min;
MS: QUATTRO 2 (electrospray method) manufactured by VG Co.
As another example of the dimer, trimer or tetramer of
anthracycline compound of the present invention which can be obtained
through directly, chemically bonding anthracycline compounds by the
aforementioned alkali treatment, the trimer of adriamycin, which has
the mass spectrum shown in Fig. 7 and generates adriamycin when
treated with the acid, can be exemplified.
The structure of the hydrophilic polymer segment of the high
molecular block copolymer to be used in the high molecular block
copolymer-drug complex pharmaceutical preparation of the present
invention can include the structure of polyethylene glycol,
CA 02206333 1997-OS-28
polysaccharide, polyacrylamide, polymethacrylamide, polyvinyl
alcohol, polyvinyl pyrrolidone, chitosan or the like, though it is
not particularly limited thereto with the proviso that it has a
hydrophilic polymer structure. The particularly preferred structure
is a polyethylene glycol structure.
The structure of the hydrophobic high polymer segment can
include the structure of polystyrene, polyamino acids (polyaspartic
acid, polyglutamic acid, polylysine and the like), polyacrylic acid,
polymethacrylic acid, polymaleic acid, or a derivative thereof or a
salt thereof or the like, though it is not particularly limited
thereto with the proviso that it has a hydrophobic polymer structure.
Of these, a polyamino acid, a derivative thereof or a salt thereof
is preferred, and polyaspartic acid, polyglutamic acid, a derivative
thereof or a salt thereof is particularly preferred. Though not
particularly limited, examples of the salt include a sodium salt,
potassium salt and the like.
Examples of the derivatives of polyamino acid structure
include those in which hydrophobic compounds such as an aromatic
amine, an aliphatic amine, an aromatic alcohol, an aliphatic alcohol,
an aromatic thiol, an aliphatic thiol and the like are linked to their
side chains, and the hydrophobic groups to be linked to the side
chains are not particularly limited with the proviso that they are
able to link to the side chains and can make the polyamino acid segment
into hydrophobicity. Preferred of these is a polyaspartic or
polyglutamic acid derivative in which an amine having an aromatic
ring is linked.to its side chain.
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Preferred examples of the high molecular block copolymer
include those which have the aforementioned chemical structure
represented by the formula (1) or (2).
In the aforementioned formulae (1) and (2), R1 represents a
hydrogen atom or a lower alkyl group, in which lower alkyl groups
having 1 to 3 carbon atoms, preferably methyl group, can be
exemplified as the lower alkyl group.
The binding group represented by R2 has a structure which
corresponds to the method and compound used in forming the polyamino
acid segment at the terminal of the polyethylene glycol segment, in
order to convert the terminal of the compound that forms the
polyethylene glycol segment into a structure suited for said
formation. Its examples include alkylene groups having 1 to 8 carbon
atoms, such as a methylene, ethylene, propylene, trimethylene and
isobutylene group, of which a trimethylene group is preferred.
R, represents a methylene or ethylene group, preferably a
methylene group.
1~, independently represents a hydroxyl group or a residue of
an anthracycline compound having an anticancer activity. As the
residue of the anthracycline compound having an anticancer activity,
various residues can be used with no particular limitation, and
preferably the group represented by the formula (3) can be used.
Illustrative examples of the group represented by the formula (3)
include the residues of adriamycin, daunomycin, pirarubicin and
epirubicin.
Though R, independently represents a hydroxyl group or the
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CA 02206333 1997-OS-28
anthracycline compound having an anticancer activity, it is desirable
that at least a portion, preferably 5 to 80$, of the total ~ groups
present in the high molecular block copolymer are residues of the
anthracycline compound having an anticancer activity, more
preferably 20 to 60$ thereof are said residues of anthracycline
compound.
Though RQ independently represents a hydroxyl group or the
residue of the anthracycline compound having an anticancer activity,
a group having anthracene skeleton or anthraquinone skeleton, such
as the substituent having anthracene skeleton or anthraquinor~e
skeleton disclosed in Japanese Patent Application (Kokai) No. 6-
206830, may be used in stead of said residue of anthracycline
compound.
RS represents a hydrogen atom or a protecting group, in which
an aliphatic or aromatic acyl group can be exemplified as the
protecting group. Though not particularly limited, the protecting
group can be introduced by a known method such as the method effected
by an acid anhydride or the method effected by an acid halide. A
hydrogen atom or an acetyl group is desirable as Rs.
In addition, n is 5 to 1,000, preferably 15 to 400, m is 2 to
300, preferably 10 to 100, and x is 0 to 300, preferably 0 to 100.
The high molecular block copolymer has a molecular weight of
preferably from 1,000 to 100,000, more preferably from 5,000 to
50,000, although the molecular weight is not particularly limited
when the block copolymer is soluble in water. The ratio of the
hydrophilic polymer segment to the hydrophobic polymer segment in the
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CA 02206333 1997-OS-28
high molecular block copolymer is not particularly limited, provided
that the water solubility of the pharmaceutical preparation of the
present invention is maintained, but is preferably 1:0.1-10 (by
weight), more preferably 1:0.1--5 (by weight).
Though the drug other than the dimer, trimer or tetramer of
anthracycline compound, which may be contained in the inner core of
micelle of the high molecular block copolymer, is not necessarily an
essential component, its examples include anticancer agents such as
adriamycin, daunomycin, pirarubicin, epirubicin, methotrexate,
mitomycin C, etoposide, cisplatin and a derivative thereof, of which
anthracycline anticancer agents are preferred and adriamycin,
daunomycin, pirarubicin, epirubicin or an acid salt thereof is
particularly preferred.
The amount of the dimer, trimer or tetramer of anthracycline
compound to be contained in the high molecular block copolymer-drug
complex pharmaceutical preparation is preferably from 1 to 100% by
weight, more preferably from 2 to 60% by weight, based on the high
molecular block copolymer. However, the dimer, trimer or tetramer
can be used in an amount as large as possible with no problems,
provided that it does not spoil the micelle forming ability of the
high molecular block copolymer-drug complex pharmaceutical
preparation.
The amount of other drug than the dimer, trimer or tetramer
of anthracycline compound to be contained in the high molecular block
copolymer-drug complex pharmaceutical preparation is preferably from
0 to 100% by weight, more preferably from 2 to 60% by weight, based .
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CA 02206333 1997-OS-28
on the high molecular block copolymer. However, said other drug can
be used in an amount as large as possible with no problems, provided
that it does not spoil the micelle forming ability of the high
molecular block copolymer-drug complex pharmaceutical preparation.
When the high molecular block copolymer-drug complex
pharmaceutical preparation contains "other drug than the dimer,
trimer or tetramer of anthracycline compound", the ratio of "other
drug than the dimer, trimer or tetramer of anthracycline compound"
to "the dimer, trimer or tetramer of anthracycline compound" is
generally 1:0.05-100 by weight, preferably 1:0.5-20 by weight, more
preferably 1:0.7-10 by weight, most preferably 1:1-5 by weight.
Though the dimer, trimer or tetramer of anthracycline compound
to be contained in the inner core of micelle of the high molecular
block copolymer is not particularly limited, the dimers, trimers or
tetramers of the anthracycline compound described in the
aforementioned items (1) to (6) are desirable. Only one of these
dimers, trimers or tetramers may be contained in the inner core of
micelle, or two or more of then may be contained in the inner core
of micelle.
The method for the preparation of the high molecular block
copolymer is well known, and it can be prepared for example in the
following manner. That is, it can be prepared by allowing a compound
which will constitute the hydrophilic polymer segment (for example,
polyethylene glycol, polysaccharide, polyacrylamide,
polymethacrylamide, polyvinyl alcohol, polyvinyl pyrrolidone,
chitosan or-a derivative thereof) or its terminal-modified product
CA 02206333 1997-OS-28
to react with a polymer compound that will constitute the hydrophobic
polymer segment, or by allowing a compound which will constitute the
hydrophilic polymer segment or its terminal-modified product to react
with a polymerizable monomer and then, if necessary, carrying out a
chemical reaction such as a derivative formation.
As an example of the derivative formation, when the
hydrophobic polymer segment has a polymeric carboxylic acid structure,
it may be mentioned to react a hydrophobic compound therewith in order
to increase the hydrophobic property. The hydrophobic compound forms
an ester bond, amide bond or the like and thereby binds to the high
molecular block copolymer. These reactions can be effected by
commonly known methods such as esterification, amidation and the like.
For example, when a hydrophobic compound is linked by an amide bonding
to a high molecular block copolymer having a hydrophilic polymer
segment and a polymeric carboxylic acid segment (raw material
copolymer), the reaction can be carried out in accordance with a
conventional method known as peptide bond formation method. For
example, an acid halide method, an acid anhydride method, a coupling .,
method and the like can be used, of which a coupling method in which
a condensing agent is used is desirable. with regard to the
condensing agent, 1-ethyl-(3-dimethylaminopropyl)carbodiimide
(EDC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC~HC1), dicyclohexylcarbodiimide (DCC), carbonylimidazole (CDI),
1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (EEDQ),
diphenylphosphorylazide (DPPA) and the like can be used. The
condensing agent may be used in an amount of preferably from 0.5 to
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20 moles, more preferably from 1 to 10 moles, per mole of the
hydrophobic compound. In this case, N-hydroxysuccinimide (HONSu),
1-hydroxybenzotriazole (HOBt), N-hydroxy-5-norbornene-2,3-
dicarboxylic acid imide (HONB) or the like may be allowed to coexist.
When a reaction is carried out to link the hydrophobic compound
to the raw material copolymer, the amount of the hydrophobic compound
to be used is not particularly limited, and it is used generally in
an amount of from 0.1 to 2 moles based on one equivalent of carboxyl
group of the raw material copolymer.
It is desirable to carry out the condensation reaction,in a
solvent. For example, N,N-dimethylfozmamide (DMF), dimethyl
sulfoxide (DMSO), dioxane, tetrahydrofuran (THF), water or a mixture
solvent thereof may be used as the solvent with no particular
limitation. Though not particularly limited, the solvent may be
generally used in an amount of from 1 to 500 times by weight more than
the raw material copolymer.
The condensation reaction may be carried out at a t~nperature
of preferably from -10 to 50°C, more preferably from -5 to 40°C.
The
reaction may be sufficient when carried out for 2 to 48 hours.
The high molecular block copolymer-drug complex
pharmaceutical preparation of the present invention can be prepared
for example by the following methods. In a first method, the thus
obtained high molecular block copolymer is dissolved in a solvent.
Examples of the solvent to be used can include N,N-dimethylformamide
(DMF), dimethyl sulfoxide (DMSO), dioxane, tetrahydrofuran (THF),
water, a-mixture solvent thereof and the like, of which DNA' or a
22
CA 02206333 1997-OS-28
mixture solvent of DMF and water is preferred. The dimer, trimer or
tetramer of anthracycline compound is added to this solution in an
amount of from 1 to 200 (by weight) based on the high molecular block
copolymer, and the mixture is stirred. By replacing the solvent of
the mixture solution with water by means of dialysis, ultrafiltration
or the like, the high molecular block copolymer-drug complex
pharmaceutical preparation of interest is obtained. When other drugs
are also to be contained, they may be added together with the dimer,
trimer or tetramer of anthracycline compound in an amount of from 1
to 200$ by weight based on the high molecular block copolymer.
In a second method, the high molecular block copolymer-drug
complex pharmaceutical preparation can also be prepared by carrying
out synthesis of the dimer, trimer or tetramer of anthracycline
compound simultaneously with introducing it into the high molecular
block copolymer. For example, the high molecular block copolymer is
dissolved in a solvent. Examples of the solvent to be used can include
N,N-dimethylformamide (DID'), dimethyl sulfoxide (DMSO), dioxane,
tetrahydrofuran ( THF ) , water, a mixture solvent thereof and the like,
of which DNA' or a mixture solvent of DMF and water is preferred. The
anthracycline compound or a salt thereof (for example, the
aforementioned anthracycline anticancer agent) is dissolved in the
solution, added with a base and then stirred. By replacing the
solvent of the mixture solution with water by means of dialysis,
ultrafiltration or the like, the high molecular block copolymer-drug
complex pharmaceutical preparation of interest is obtained.
In the second method, the compositional ratio of the dimer,
23
CA 02206333 1997-OS-28
trimer or tetramer of anthracycline compound to the other drugs in
the high molecular block copolymer-drug complex pharmaceutical
preparation can be controlled in the following manner. For example,
the compositional ratio of the dimer, trimer or tetramer of
anthracycline compound to the anthracycline compound (the
anthracycline anticancer drug) in the high molecular block
copolymer-drug complex pharmaceutical preparation to be obtained can
be controlled by varying the charge of the anthracycline compound or
a salt thereof (an anthracycline anticancer drug) based on the high
molecular block copolymer, or by varying the pH value.
The present invention also relates to a high molecular block
copolymer-drug complex pharmaceutical preparation in which a high
molecular block copolymer having a hydrophilic polymer segment and
a hydrophobic polymer segment forms a micelle having the hydrophilic
segment as its outer shell, and contains in its hydrophobic inner core
an anthracycline anticancer agent, wherein, when the pharmaceutical
preparation is intravenously administered to a CDF1 mouse of 7 to 9
week's age the amount of the anthracycline anticancer agent in 1 ml
of the mouse blood plasma after 1 hour of its administration becomes
(~ of dose/ml) or more, preferably 20 to 60, provided that the
amount of anthracycline anticancer agent in the administered
preparation is defined as 100.
The aforementioned high molecular block copolymer-drug
complex pharmaceutical preparation which contains in the inner core
of its micelle the dimer, trimer or tetramer of anthracycline
compound together with the anthracycline anticancer agent can be
24
CA 02206333 1997-OS-28
exemplified as such the pharmaceutical preparation.
When a commercially available prior art anticancer agent such
as adriamycin is intravenously administered to the human body, its
blood level is reduced within an extremely short period of time. On
the contrary, when the pharmaceutical preparation of the present
invention is intravenously administered, its blood concentration is
maintained at a high level for a prolonged period of time, so that
anthracycline anticancer agents can be incorporated into tumor
tissues in a large quantity, and treatment of cancers therefore can
be made effectively.
Since the high molecular block copolymer-drug complex
pharmaceutical preparation of the present invention are possessed of
high pharmacological effects, when it is used as an anticancer agent
for example, its anticancer activity is outstandingly higher than
adriamycin whereas their doses are hardly different from each other.
In particular, it can show markedly significant action of effecting
disappearance of solid cancers. In consequence, it is particularly
effective for treating patients of solid cancers such as lung cancer, _,
digestive system cancer, breast cancer, bladder cancer, osteogenic
sarcoma and the like. In addition, the high molecular block
copolymer-drug complex pharmaceutical preparation of the present
invention exerts an excellent effect of being low in toxicity.
The pharmaceutical preparation of the present invention can
be used in the conventionally used various dosage forms such as solid
formulations, ointments, liquid formulations and the like, which are
obtained by mixing it with a pharmaceutically acceptable additive,
CA 02206333 1997-OS-28
such as a carrier, filler, diluent, solubilizing agent and the like,
and, when used as an anticancer agent, it is generally used in the
form of injection which preferably contains 99.99 to 1$ of additives
based on the total formulation. Its dose is about 10 to 200 mg/m2/week
as the total amount of the dimer, trimer or tetramer of anthracycline
compound and the other drugs, and it is applied by 1 to 3 divided
administration a week.
EXAMPLES
The present invention is described illustratively with,
reference to the following examples.
Example 1
( 1 ) A 10 mg portion of adriamycin hydrochloride was dissolved
in a mixture solvent consisting of 3 ml of DMF, 1 ml of water and IO
~1 of triethylamine and allowed to react for 12 hours at 28°C in the
dark. Using a dialysis membrane having a nominal molecular weight
cutoff of_1,000, the reaction solution was dialyzed against water to
exchange the solvent with water. By separating and purifying this
by HPLC, an aqueous solution of a dimer of adriamycin was obtained.
This was then freeze-dried to obtain as a solid form the dimer of
adriamycin having the structure of formula (AA).
This adriamycin dimer showed the aforementioned infrared
absorption spectrum, ultraviolet spectrum and LC/MS mass spectrum.
The thus obtained dimer of adriamycin was acid-treated with 1~ acetic
acid. The mass spectrogram of the resulting product is shown in Fig.
26
CA 02206333 1997-OS-28
6.
In this connection, the instruments and conditions used for
the separation and purification by HPLC and the measurement of the
spectra and chromatogram are as described in the foregoing.
( 2 ) A 500 mg portion of adriamycin hydrochloride was dissolved
in a mixture solvent consisting of 40 ml of DMF and 40 ml of methanol,
and the solution was added with 1.2 ml of triethylamine and allowed
to react for 12 hours at 25°C. The reaction solution was purified
using a column which has been prepared by packing 350 ml of LH-20
(manufactured by Pharmacia) in a glass tube of 26 mm in inner diameter
and 65 cm in length. Methanol was used as the mobile phase in the
column purification and was passed through at a flow rate of 5 ml/min.
Fractions were collected in 5 ml aliquots, and the fractions of 11th
to 25th were pooled, evaporated to dryness and analyzed by a mass
spectrometer to confirm the formation of a dimer of adriamycin. As
a result of analysis by a high resolution mass spectrometry, its
molecular formula was found to be C~,H~N202~.
A 10 mg portion of the thus obtained adriamycin dimer was ._
dissolved in 2 ml of methanol, and the solution was added with 1 mg
of NaBH,CN, allowed to react for 12 hours at room temperature, mixed
with 3 ml of 1 N hydrochloric acid and then reacted for additional
12 hours. The reaction solution was analyzed by LC/MS, and a peak
of m/z 524 was separated and analyzed by NMR. The results of NMR
analysis resulted in the 1H one-dimensional spectrum shown in Fig.
8, the 13C one-dimensional spectrum shown in Fig. 9, the COSY spectrum
shown in Fig. 10 and the CH COSY spectrum shown in Fig. 11. -
27
CA 02206333 1997-OS-28
On the basis of these results, it was confirmed to be the dimer
of adriamycin having the structure of formula (AA).
( 3 ) A 500 mg portion of adriamycin hydrochloride was dissolved
in a mixture solvent consisting of 40 ml of DMF and 40 ml of methanol,
and the solution was added with 1.2 ml of triethylamine and allowed
to react for 12 hours at 25°C. The reaction solution was purified
using a column which has been prepared by packing 350 ml of LH-20
(manufactured by Pharrnacia) in a glass tube of 26 mm in inner diameter
and 65 cm in length. Methanol was used as the mobile phase in the
column purification and was passed through at a flow rate of 5 ml/min.
Fractions were collected in 5 ml aliquots, and the fractions of 5th
to 9th were pooled, evaporated to dryness and analyzed by a mass
spectrometer to obtain the mass spectrum shown in Fig. 7. The result
confirmed the formation of a trimer of adriamycin.
Example 2
A 5 mg portion of adriamycin hydrochloride and 5 mg of
daunomyc.~n hydrochloride were dissolved in a mixture solvent
consisting of 3 ml of DMF, 1 ml of water and 10 E.~,1 of triethylamine
and allowed to react for 12 hours at 28°C in the dark. Without
purification, the reaction solution was subjected to a mass spectrum
analysis by LC/MS to confirm the formation of a dimer of adriamycin
and the formation of a dimer of daunomycin with adriamycin.
Of these products, the adriamycin dimer showed the same mass
spectrum described in the foregoing. The mass spectrum of the dimer
of daunomycin with adriamycin is shown in Fig. 12. Under these
28
CA 02206333 1997-OS-28
reaction conditions, a daunomycin dimer was not formed. In this
connection, the instruments and conditions for obtaining these
spectra are as described in the foregoing.
Mass spectrum of the dimer of daunomycin with adriamycin ( ESI ) ,
m/z ($): 1051 (90), 948 (15), 922 (20), 904 (10), 653 {20), 524 (30),
506 {50), 488 (100).
Example 3
A 20.0 g portion of polyethylene glycol having a methoxy group
on one terminal and an 3-aminopropyl group on the other terminal
(PEGNHz) (molecular weight 13,900) was dissolved in 100 ml of
N,N-dimethylformamide (DMF). To this solution was added 15.0 g of
~-benzyl-L-aspartate-N-carboxylic anhydride (BLA-NCA). With
stirring in a water bath of 35°C, the polymerization reaction was
carried out for 24 hours. Next, while stirring in an ice bath, the
polymerization solution was added to an aqueous 0.5 N sodium
hydroxide solution and stirred for 20 minutes. Next, this was
adjusted to a pH value of approximately 4 by adding 2 N hydrochloric
acid, diluted with distilled water to a total volume of 20 liters and
then adjusted to pH 4 . Next, a procedure of concentrating and washing
it was repeated using a hollow fiber type ultrafiltration apparatus
(Amicon CH2, molecular weight cutoff after ultrafiltration = 10, 000 ) .
Next, the thus concentrated solution was purified using a sulfonic
acid type ion exchange resin (Amberlite IR-120B) column. The
resulting eluate was concentrated under a reduced pressure and then
freeze-dried to obtain 19.58 g of a polyethylene glycol-polyaspartic
29
CA 02206333 1997-OS-28
acid block copolymer (PEG-P(Asp.)). A 5.008 g portion of the
PEG-P(Asp.) was dissolved in 83 ml of DMF to which was subsequently
added 83 ml of acetonitrile. This was added with 8.979 g of
dicyclohexylcarbodiimide (DCC), stirred for 5 minutes and then added
with a solution which has been prepared by dissolving 2.528 g of
adriamycin hydrochloride in 167 ml of DMF and adding 786 ~,1 of
triethylamine thereto. Next, this was allowed to react for 4 hours
while stirring at room temperature. After the reaction, this was
added with 16.7 ml of an aqueous 1% phosphoric acid solution and
stirred for 5 minutes. After its dialysis using a dialysis membrane
(molecular weight cutoff = 12,000 - 14,000), the precipitate
originated from DCC was removed by filtration. The resulting
filtrate was purified using a hollow fiber type ultrafiltration
apparatus (Amicon CH2, molecular weight cutoff of ultrafiltration
membrane = 10, 000 ) . This was further concentrated by ultrafiltration
using an ultrafiltration membrane of ADVANTEC UK-50 (molecular weight
cutoff = 50,000) to obtain 177 ml of an aqueous solution having a
concentration of 12 mg/ml as adriamycin (calculated from its
absorbance at 485 nm measured by an ultraviolet ray
spectrophotometer). The thus obtained PEG-P(Asp.)ADR has the
structure of the aforementioned formula (2) in which R1 is a methyl
group, R2 is a trimethylene group, R3 is a methylene group, a portion
of 1~, is a hydroxyl group and the rest thereof is the aforementioned
residue of formula ( 3 ) [Y is CHZOH, Z is H] , RS is hydrogen, n = 315,
m = 30 and x = 8. The adriamycin content was 32.3% by weight, and
it showed appropriate water solubility. A 20 ml portion of the
CA 02206333 1997-OS-28
aqueous solution containing 12 mg/ml (as adriamycin) of the thus
obtained PEG-P(Asp.)ADR was mixed with a solution which has been
prepared by dissolving 258.8 mg of adriamycin hydrochloride in 60 ml
of DMF and adding 100 ~1 of triethylamine thereto, and the mixture
was stirred at room temperature for 2 hours in the dark. After
dialysis using a dialysis membrane (molecular weight cutoff = 12,000
- 14,000), the resulting solution was freeze-dried. This was
purified and concentrated by redissolving it in water and carrying
out ultrafiltration using an ultrafiltration membrane of ADVANTEC
UK-50 (molecular weight cutoff = 50, 000 ) . This was further filtered
using a 0.45 Fun filter to obtain 25.3 ml of an aqueous solution of
a block copolymer-drug complex pharmaceutical preparation. The thus
obtained aqueous solution was found to have the HPLC chromatogram
shown in Fig. 13. In the drawing, the peak ~ is adriamycin, the peak
~ is the dimer of adriamycin and the broad peak ~3 is adriamycin
linked to the polymer. The concentration of adriamycin (peak ~ )
was 3.06 mg/ml, and the concentration of the adriamycin dimer (peak
~ ) was 3.18 mg/ml. The weight ratio of adriamycin to the adriamycin
dimer was 1:1.04.
The measuring conditions of HPLC are as follows.
Column: C4~300 angstrom/5 E.~m, manufactured by Waters
Fluent: acetonitrile/1~ acetic acid + 40 mM sodium dodecyl
sulfate
Gradient elution
Time (minute) 0 4 12 25 30 31
Acetonitrile concentration (~) 15 35 35 85 85 15~
31
CA 02206333 1997-OS-28
Detection: 485 nm
Flow rate: 1 ml/min
Example 4
A 20 ml portion of the aqueous solution containing 12 mg/ml
(as adriamycin) of PEG-P(Asp.)ADR prepared in Example 3 was mixed
with a solution which has been prepared by dissolving 102.4 mg of
adriamycin hydrochloride in 60 ml of DMF and adding 32 ~.1 of
triethylamine thereto, and the mixture was stirred at room
temperature for 2 hours in the dark. After dialysis using a dialysis
membrane (molecular weight cutoff = 12,000 - 14,000), the resulting
solution was freeze-dried. This was purified and concentrated by
redissolving it in water and carrying out ultrafiltration using an
ultrafiltration membrane of ADVANTEC UK-50 (molecular weight cutoff
= 50, 000 ) . This was further filtered using a 0. 45 E.~m filter to obtain
20.4 ml of an aqueous solution of a block copolymer-drug complex
pharmaceutical preparation. The thus obtained aqueous solution was
found to have the HPLC chromatogram shown in Fig. 14. In the drawing,
the peak ~l is adriamycin, the peak ~ is the dimer of adriamycin and
the broad peak ~3 is adriamycin linked to the polymer. The
concentration of adriamycin (peak ~1 ) was 3.01 mg/ml, and the
concentration of the adriamycin dimer (peak ~ ) was 0.39 mg/ml. The
weight ratio of adriamycin to the adriamycin dimer was 1:0.13. The
HPLC measuring conditions are as described in Example 3.
Exanple 5
32
CA 02206333 1997-OS-28
A 20 ml portion of the aqueous solution containing 12 mg/ml
(as adriamycin) of PEG-P(Asp.)ADR prepared in Example 3 was diluted
with 20 ml of water and then freeze-dried. This was redissolved in
20 ml of water, and pH value of the solution was adjusted with acetic
acid and an aqueous sodium acetate solution in such amounts that the
solution finally became 40 ml of 30 mM acetate buffer (pH 5.0)
solution. The thus prepared solution was mixed with 128.0 mg of
adriamycin hydrochloride and stirred at room temperature for 2 days
in the dark. After dialysis using a dialysis membrane (molecular
weight cutoff = 12, 000 - 14, 000 ) , this was purified and concentrated
by carrying out ultrafiltration using an ultrafiltration membrane of
ADVANTEC UK-50 (molecular weight cutoff = 50,000) to obtain 16.7 ml
of an aqueous solution of a block copolymer-drug complex
pharmaceutical preparation. The thus obtained aqueous solution was
found to have the HPLC chromatogram shown in Fig. 15. In the drawing,
the peak ~l is adriamycin, the peak ~ is the dimer of adriamycin and
the broad peak ~3 is adriamycin linked to the polymer. The
concentration of adriamycin (peak ~1 ) was 2.99 mg/ml, and the ,
concentration of the adriamycin dimer (peak ~2 ) was 0.27 mg/ml. The
weight ratio of adriamycin to the adriamycin dimer was 1:0.09. The
HPLC measuring conditions are as described in Example 3.
Application Example 1
Colon 26 adenocarcinoma cells were transplanted
subcutaneously in the subaxillary region of each CDF1 female mouse.
When the volume of the tumor reached around 100 mm', the block
33
CA 02206333 1997-OS-28
copolymer-drug complex pharmaceutical preparation synthesized in
Example 3, 4 or 5 or adriamycin hydrochloride was intravenously
administered once a day every 4th day in total of three times
(indicated by arrows in the drawings) to examine their antitumor
effects. Each drug was used by diluting it in physiological saline
prior to its use. Each dose was determined as the amount of adriamycin
of the peak ~1 of the HPLC chromatogram. The antitumor effects of
each drug were estimated based on the tumor growth curve, the number
of mice in which the tumor disappeared and the chemotherapeutic index.
The results are shown in Tables 1 and 2 and Figs. 16 and 17. ,In
comparison with the case of the administration of adriamycin
hydrochloride, larger number of tumor-disappeared mice was observed
with broader range of dose when the block copolymer-drug complex
pharmaceutical preparations of Examples 3 to 5 were administered.
Particularly, the pharmaceutical preparation of Example 3 having a
higher adriamycin dimer content showed the most excellent results in
the complete cure ratio and the chemotherapeutic index.
34
CA 02206333 1997-OS-28
Table 1 Antitumor activity on mouse Colon 26 adenocarcinoma
Sample Dose (mg/kg) Tumor-disappeared mice
Adriamycin 5 0/5
hydrochloride 10 2/5
Pharmaceutical 2.5 1/4
preparation of 5 5/5
Example 3 10 3/5
Pharmaceutical 2.5 0/5
preparation of 5 1/5
Example 4 10 4/5
Pharmaceutical 2.5 0/5
preparation of 5 2/5
Example 5 10 3/5
Table 2 Comparison of chemotherapeutic index
Sample LDSo Min T/C4z 1~ C. index z'
Adriamycin hydrochloride 15 5.95 2.52
Pharmaceutical preparation of 15 2.59 5.78
Example 3
Pharmaceutical preparation of 15 4.88 3.08
Example 4
Pharmaceutical preparation of 15 6.02 2.49
Example 5
1 ) Minimum dose when T/C ( % ) becomes 42$ or less on the 14th day after
the initial administration
2 ) Chernotherapeutic index: LDSO/ (Min T/C4z )
CA 02206333 1997-OS-28
Example 6
A 20.0 g portion of polyethylene glycol having a methoxy group
on one terminal and an 3-aminopropyl group on the other terminal
(PEGNHz) (molecular weight 14,200) was dissolved in 100 ml of
N,N-dimethylformamide (DMF). To this solution was added 15.0 g of
(3-benzyl-L-aspartate-N-carboxylic anhydride (BLA-NCA), followed by
the polymerization reaction being carried out for 24 hours with
stirring in a water bath of 35°C. Next, while stirring in an ice bath,
the polymerization solution was added to an aqueous 0.5 N sodium
hydroxide solution and stirred for 20 minutes. This was adjusted to
a pH value of approximately 4 by adding 2 N hydrochloric acid, diluted
with distilled water to a total volume of 20 liters and then adjusted
to pH 4. A procedure of concentrating and washing it was repeated
using a hollow fiber type ultrafiltration apparatus (Amicon CH2,
molecular weight cutoff of ultrafiltration membrane = 10,000) . Next,
the thus concentrated solution was purified using a sulfonic acid
type ion exchange resin (Amberlite IR-120B) column. The resulting
eluate_.was concentrated under a reduced pressure and then freeze-
dried to obtain 21.26 g of a polyethylene glycol-polyaspartatic acid
block copolymer (PEGP(Asp: ) ) . A 7.501 g portion of the PEGP(Asp. )
was dissolved in 125 ml of DMF to which was subsequently added 125
ml of acetonitrile. Next, this was added with 12.992 g of
dicyclohexylcarbodiimide (DCC), stirred for 5 minutes and then added
with a solution which has been prepared by dissolving 3.654 g of
adriamycin hydrochloride in 250 ml of DMF and adding 1.14 ml of
triethylamine thereto. Next, this was allowed to react for 4 hours
36
CA 02206333 1997-OS-28
while stirring at room temperature. After the reaction, this was
added with 25 ml of an aqueous 1~ phosphoric acid solution and stirred
for 5 minutes. After its dialysis using a dialysis membrane
(molecular weight cutoff = 12,000 - 14,000), the precipitate
originated from DCC was removed by filtration. The resulting
filtrate was purified using a hollow fiber type ultrafiltration
apparatus (Amicon CH2, molecular weight cutoff of ultrafiltration
membrane = 10, 000 ) and then concentrated by ultrafiltration using an
ultrafiltration membrane of ADVANTEC UK-50 (molecular weight cutoff
- 50,000) to obtain 270 ml of an aqueous solution having a
concentration of 12 mg/ml as adriamycin (calculated from its
absorbance at 485 nm measured by an ultraviolet ray
spectrophotometer). The thus obtained PEG-P(Asp.)ADR has the
structure of the aforementioned formula (2) in which R1 is a methyl
group, Rz is a trimethylene group, R3 is a methylene group, a portion
of I~ is a hydroxyl group and the rest thereof is the aforementioned
residue of formula ( 3 ) (Y is CHZOH, Z is H] , R5 is hydrogen, n = 325,
m = 30 and x = 8. The adriamycin content was 32.4, and it showed,
appropriate water solubility. A 1 ml portion of the aqueous solution
containing 12 mg/ml (as adriamycin) of the thus obtained PEG-
P(Asp.)ADR was mixed with a solution which has been prepared by
dissolving 11.93 mg of "C-labeled adriamycin hydrochloride in 3 ml
of DNA' and adding 4.9 ~,1 of triethylamine thereto, and the mixture
was stirred at room temperature for 2 hours in the dark. After
dialysis using a dialysis membrane (molecular weight cutoff = 12,000
- 14,000), the resulting solution was purified and concentrated by
37
CA 02206333 1997-OS-28
carrying out ultrafiltration using an ultrafiltration membrane of
ADVANTEC UK-50 (molecular weight cutoff = 50,000) to obtain 3.0 ml
of an aqueous solution of a block copolymer-drug complex
pharmaceutical preparation. The thus obtained aqueous solution was
found to have the HPLC chromatogram shown in Fig. 18. In the drawing,
the peak ~ is adriamycin, the peak ~ is the dimer of adriamycin and
the broad peak 3~ is adriamycin linked to the polymer. The
concentration of adriamycin (peak ~ ) was 1.29 mg/ml, and the
concentration of the adriamycin dimer (peak ~ ) was 1.36 mg/ml. The
weight ratio of adriamycin to the adriamycin dimer was 1:1.05., The
measuring conditions of HPLC are as described in Example 3.
Example 7
A 2.08 ml portion of the aqueous solution containing 12 mg/ml
(as adriamycin) of the PEG-P(Asp. )ADR prepared in Example 6 was mixed
with a solution which has been prepared by dissolving 9.86 mg of
1°C-labeled adriamycin hydrochloride in 6.25 ml of DMF and adding 3.3
~1 of triethylamine thereto, and the mixture was stirred at room
temperature for 2 hours in the dark. After dialysis using a dialysis
membrane (molecular weight cutoff = 12,000 _ 14,000), the resulting
solution was purified and concentrated by carrying out
ultrafiltration using an ultrafiltration membrane of ADVANTEC UK-
50 (molecular weight cutoff = 50, 000 ) to obtain 2.3 ml of an aqueous
solution of a block copolymer-drug complex pharmaceutical
preparation. The thus obtained aqueous solution was found to have
the HPLC chromatogram shown in Fig. 19. In the drawing, the peak
38
CA 02206333 1997-OS-28
is adriamycin, the peak ~ is the dimer of adriamycin and the broad
peak ~3 is adriamycin linked to the polymer. The concentration of
adriamycin (peak ~1 ) was 3.41 mg/ml, and the concentration of the
adriamycin dimer (peak ~ ) was 0.95 mg/ml. The weight ratio of
adriamycin to the adriamycin dimer was 1:0.28. The measuring
conditions of HPLC are as described in Example 3.
Example 8
A 20.0 g portion of polyethylene glycol having a methoxy group
on one terminal and an 3-aminopropyl group on the other terminal
(PEG-NHz) (molecular weight 14,500) was dissolved in 100 ml of
N,N-dimethylformamide (DMF). To this solution was added 15.0 g of
~-benzyl-L-aspartate-N-carboxylic anhydride (BLA-NCA), followed by
the polymerization reaction being carried out for 24 hours with
stirring in a water bath of 35°C. Next, while stirring in an ice bath,
the polymerization solution was added to an aqueous 0.5 N sodium
hydroxide solution and stirred for 20 minutes . This was adjusted to
a pH value of approximately 4 by adding 2 N hydrochloric acid, diluted
with distilled water to a total volume of 20 liters and then adjusted
to pH 4. A procedure of concentrating and washing it with water was
repeated using a hollow fiber type ultrafiltration apparatus (Amicon
CH2, molecular weight cutoff of ultrafiltration membrane = 10,000).
Next, the thus concentrated solution was purified using a sulfonic
acid type ion exchange resin (Amberlite IR-120B) column. The
resulting eluate was concentrated under a reduced pressure and then
freeze-dried to obtain 19.01 g of a polyethylene glycol-
39
CA 02206333 1997-OS-28
polyaspartatic acid block copolymer (PEGP( Asp. ) ) . A 5.010 g portion
of the PEGP(Asp.) was dissolved in 83 ml of DMF to which was
subsequently added 83 ml of acetonitrile. Next, this was mixed with
8 . 693 g of dicyclohexylcarbodiimide ( DCC ) , stirred for 5 minutes and
then added with a solution which has been prepared by dissolving 2.445
g of adriamycin hydrochloride in 167 ml of DMF and adding 759 ~1 of
triethylamine thereto. Next, this was allowed to react for 4 hours
while stirring at room t~nperature. After the reaction, this was
added with 16.7 ml of an aqueous 0.5~ phosphoric acid solution and
stirred for 5 minutes. After its dialysis using a dialysis membrane
(molecular weight cutoff = 12,000 - 14,000), the precipitate
originated from DCC was removed by filtration. The resulting
filtrate was concentrated by carrying out ultrafiltration using an
ultrafiltration membrane of ADVANTEC UK-50 (molecular weight cutoff
- 50,000) to obtain 185 ml of an aqueous solution having a
concentration of 12 mg/ml as adriamycin (calculated from its
absorbance at 485 nm measured by an ultraviolet ray
spectrophotometer). The thus obtained PEGP(Asp.)ADR has the
structure of the afor~nentioned formula ( 2 ) in which R1 is a methyl
group, R2 is a trimethylene group, R3 is a methylene group, a portion
of R4 is a hydroxyl group and the rest thereof is the aforementioned
residue of formula ( 3 ) [ Y is CHZOH, Z is H ] , RS is hydrogen, n = 350,
m = 32 and x = 8. The adriamycin content was 30.2, and it showed
appropriate water solubility. A 20 ml portion of the aqueous solution
containing 12 mg/ml (as adriamycin) of the thus obtained PEG
P(Asp.)ADR was mixed with a solution which has been prepared by
CA 02206333 1997-OS-28
dissolving 564.0 mg of adriamycin hydrochloride in 60 ml of DMF and
adding 176 ~,1 of triethylamine thereto, and the mixture was stirred
at room temperature for 2 hours in the dark. After dialysis using
a dialysis membrane (molecular weight cutoff = 12, 000 - 14, 000 ) , the
resulting solution was freeze-dried. This was purified and
concentrated by redissolving it in water and carrying out
ultrafiltration using an ultrafiltration membrane of ADVANhEC UK-
50 (molecular weight cutoff = 50,000). This was further filtered
using a 0.45 ~u.filter to obtain 59.4 ml of an aqueous solution of a
block copolymer-drug complex pharmaceutical preparation. The thus
obtained aqueous solution was found to have the HPLC chromatogram
shown in Fig. 20. In the drawing, the peak ~ is adriamycin, the peak
~ is the dimer of adriamycin and the broad peak ~ is adriamycin
linked to the polymer. The concentration of adriamycin (peak ~ )
was 1.10 mg/ml, and the concentration of the adriamycin dimer (peak
~ ) was 3.07 mg/ml. The weight ratio of adriamycin to the adriamycin
dimer was 1:2.79. The measuring conditions of HPLC are as described
in Example 3.
Example 9
A 10 ml portion of the aqueous solution containing 12 mg/ml
(as adriamycin) of the PEG-P(Asp. )ADR prepared in Example 8 was mixed
with a solution which has been prepared by dissolving 32.0 mg of
adriamycin hydrochloride in 30 m1 of DMF and adding 10.0 ~1 of
triethylamine thereto, and the mixture was stirred at room
temperature for 2 hours in the dark. After dialysis using a dialysis
41
CA 02206333 1997-OS-28
membrane (molecular weight cutoff = 12,000 - 14,000), the resulting
solution was freeze-dried. This was redissolved in water and
purified and concentrated by carrying out ultrafiltration using an
ultrafiltration membrane of ADVAN~I~EC UK-50 (molecular weight cutoff
= 50, 000 ) . This was further filtered using a 0.45 ~, filter to obtain
9.1 ml of an aqueous solution of a block copolymer-drug complex
pharmaceutical preparation. The thus obtained aqueous solution was
found to have the HPLC chromatogram shown in Fig. 21. In the drawing,
the peak 01 is adriamycin, the peak ~ is the dimer of adriamycin and
the broad peak 3~ is adriamycin linked to the polymer. The
concentration of adriamycin (peak ~1 ) was 1.98 mg/ml, and the
concentration of the adriamycin dimer (peak ~2 ) was 0.12 mg/ml. The
weight ratio of adriamycin to the adriamycin dimer was 1:0.06. The
measuring conditions of HPLC are as described in Example 3.
Example 10
A 5 ml portion of the aqueous solution containing 12 mg/ml ( as
adriamycin) of the PEGP(Asp.)ADR prepared in Example 8 was mixed
with a solution which has been prepared by dissolving 51.4 mg of
adriamycin hydrochloride in 15 ml of DMF and adding 16.0 ~1 of
triethylamine thereto, and the mixture was stirred at room
t~nperature for 2 hours in the dark. After dialysis using a dialysis
membrane (molecular weight cutoff = 12,000 - 14,000), the resulting
solution was freeze-dried. This was redissolved in water and
purified and concentrated by carrying out ultrafiltration using an
ultrafiltration membrane of ADVANTEC UK-50 (molecular weight cutoff
42
CA 02206333 1997-OS-28
= 50, 000 ) . This was further filtered using a 0 . 45 ~ filter to obtain
15.2 ml of an aqueous solution of a block copolymer-drug complex
pharmaceutical preparation. The thus obtained aqueous solution was
found to have the HPLC chromatogram shown in Fig. 22. In the drawing,
the peak ~ is adriamycin, the peak ~ is the dimer of adriamycin and
the broad peak 3~ is adriamycin linked to the polymer. The
concentration of adriamycin (peak ~1 ) was 1.04 mg/ml, and the
concentration of the adriamycin dimer (peak ~ ) was 0.88 mg/ml. The
weight ratio of adriamycin to the adriamycin dimer was 1:0.85. The
measuring conditions of HPLC are as described in Example 3.
Example 11
A solution which has been prepared by dissolving 103.5 mg of
the adriamycin dimer prepared in Example 1 (2) and 49.1 mg of
adriamycin hydrochloride in 65 ml of DMF was mixed with 23 ml of the
aqueous solution containing 12 mg/ml (as adriamycin) of the PEG
P(Asp. )ADR prepared in Example 8, and the mixture was stirred at room
temperature for 2 hours in the dark. After dialysis using a dialysis
membrane (molecular weight cutoff = 12,000 - 14,000), the resulting
solution was purified and concentrated by carrying out
ultrafiltration using an ultrafiltration membrane of ADVAN~I~EC UK-
50 (molecular weight cutoff = 50,000). This was further filtered
using a 0.45 w filter to obtain 14.9 ml of an aqueous solution of a
block copolymer-drug complex pharmaceutical preparation. The thus
obtained aqueous solution was found to have the HPLC chromatogram
shown in Fig. 23. In the drawing, the peak ~l is adriamycin, the peak
43
CA 02206333 1997-OS-28
is the dimer of adriamycin and the broad peak 0 is adriamycin
linked to the polymer. The concentration of adriamycin (peak ~ )
was 1.13 mg/ml, and the concentration of the adriamycin dimer (peak
was 2.76 mg/ml. The weight ratio of adriamycin to the adriamycin
dimer was 1:2.44. The measuring conditions of HPLC are as described
in Example 3.
Example 12
A 1.0034 g portion of the PEG-P(Asp.) prepared in Example 8
was dissolved in 16 . 7 ml of DMF to which was subsequently added ,16 . 7
ml of acetonitrile. This was added with 1.7504 g of dicyclohexyl-
carbodiimide (DCC), stirred for 5 minutes and then mixed with a
solution which has been prepared by dissolving 474.4 mg of daunomycin
hydrochloride in 33.3 ml of DMF and adding 152 ~,1 of triethylamine
thereto, and the mixture was allowed to reacted for 4 hours at room
temperature. After the reaction, this was added with 3.3 ml of an
aqueous 0.5$ phosphoric acid solution and stirred for 5 minutes.
After its dialysis using a dialysis membrane (molecular weight cutoff
= 12, 000 -- 14, 000 ) , the precipitate originated from DCC was removed
by filtration. The resulting filtrate was concentrated by carrying
out ultrafiltration using an ultrafiltration membrane of ADVANTEC
UK-50 (molecular weight cutoff = 50, 000 ) to obtain 36 ml of an aqueous
solution having a concentration of 12 mg/ml as daunomycin (calculated
from its absorbance at 485 nm measured by an ultraviolet ray
spectrophotometer). The thus obtained PEG-P(Asp.)DAM has the
structure of the aforementioned formula (2) in which R1 is a methyl
44
CA 02206333 1997-OS-28
group, R2 is a trimethylene group, R, is a methyl group, a portion of
Rn is a hydroxyl group and the rest thereof is the aforementioned
residue of formula (3) [Y is CH" Z is H], RS is hydrogen, n = 350,
m = 32 and x = 8. The daunomycin content was 30.3, and it showed
appropriate water solubility. A 7 ml portion of the aqueous solution
containing 12 mg/ml (as daunomycin) of the thus obtained PEG-
P(Asp.)DAM was mixed with a solution which has been prepared by
dissolving 179.8 mg of adriamycin hydrochloride in 21 ml of DMF and
adding 55.9 ~1 of triethylamine thereto, and the mixture was stirred
at room temperature for 2 hours in the dark. After dialysis using
a dialysis membrane (molecular weight cutoff = 12, 000 - 14, 000 ) , the
resulting solution was freeze-dried. This was redissolved in water,
purified and concentrated by carrying out ultrafiltration using an
ultrafiltration membrane of ADVANTEC UK-50 (molecular weight cutoff
= 50,000) and then further filtered using a 0.45 ,filter to obtain
16.5 ml of an aqueous solution of a block copolymer-drug complex
pharmaceutical preparation. The thus obtained aqueous solution was
found to have the HPLC chromatogram shown in Fig. 24. In the drawing,
the peak ~l is adriamycin, the peak ~ is the dimer of adriamycin and
the broad peak 3~ is daunomycin linked to the polymer. The
concentration of adriamycin (peak ~1 ) was 1.07 mg/ml, and the
concentration of the adriamycin dimer (peak ~2 ) was 3.26 mg/ml. The
weight ratio of adriamycin to the adriamycin dimer was 1:3.05. The
measuring conditions of HPLC are as described in Example 3.
lication Example 2
CA 02206333 1997-OS-28
Colon 26 adenocarcinoma cells were transplanted
subcutaneously in the subaxillary region of each CDF1 female mouse,
to which the block copolymer-drug complex pharmaceutical preparation
prepared in Example 6 or 7 or 1°C-labeled adriamycin hydrochloride was
intravenously administered 12 days after transplanting. Each drug
was used by diluting it in physiological saline prior to its use.
After 15 minutes and 1, 4, 24 and 48 hours of the administration, blood
samples were collected and various organs were excised. The drug
concentrations in the blood plasma and each internal organ were
determined by measuring radioactivity thereof using a liquid ,
scintillation counter. In this test, both adriamycin and adriamycin
dimer are labeled with 1°C. Periodical variations in the total amount
( $ of dose/ml ) of adriamycin and adriamycin dimer in 1 ml of the blood
plasma when the total amount of adriamycin and adriamycin dimer in
the administered drug preparation is defined as 100 are shown in Table
3. While the drug quickly disappeared from blood plasma after its
administration when adriamycin hydrochloride alone was administered,
the drug remained in blood plasma at a higher level for a prolonged
period of time in the case of the pharmaceutical preparation of the
present invention. Improvement of the drug-retentivity was
particularly significant in the case of the pharmaceutical
preparation of Example 6 comprising the adriamycin dimer at a higher
level. Periodical variations in the total amount (% of dose/g) of
adriamycin and adriamycin dimer in 1 g of the tumor tissue when the
total amount of adriamycin and adriamycin dimer in the administered
drug preparation is defined as 100 are shown in Table 4. Also,
46
CA 02206333 1997-OS-28
periodical variations in said total amount ( ~ of dose/g ) in the heart
are shown in Table 5. In comparison with the case of the single
administration of adriamycin hydrochloride, the drug administered as
the pharmaceutical preparation of the present invention was slightly
lower in its initial concentration and decreased in the course of time
in the heart, but was accumulated in a several times higher
concentration and increased in the course of time in the tumor portion.
The drug-accumulation in tumor was particularly significant in the
case of the pharmaceutical preparation of Example 6 comprising the
adriamycin dimer at a higher level.
Application Example 3
Colon 26 adenocarcinoma cells were transplanted
subcutaneously in the subaxillary region of each CDF1 female mouse.
When volume of the tumor reached around 100 mm3, the block
copolymer-drug complex pharmaceutical preparation prepared in
Example 8 or 12 or adriamycin hydrochloride was intravenously
administered once a day every 4th day in total of three M mes
(indicated by arrows in the drawings) to examine their antitumor
effects. In the case of the pharmaceutical preparation of Example
8, its effect obtained from the only first administration was also
examined. Each drug was used by diluting it in physiological saline
prior to its use. Each dose was determined as the amount of adriamycin
of the peak ~1 of the HPLC chromatogram. The antitumor effects of
each drug were estimated based on the tumor growth curve and the
number of mice in which the tumor disappeared. The results are shown
47
CA 02206333 1997-OS-28
in Table 6 and Figs. 25, 26 and 27. In comparison with the case of
the administration of adriamycin hydrochloride, larger number of
tumor-disappeared mice was observed with broader range of dose when
the pharmaceutical preparation of Example 8 or 12 was administered.
Application Example 4
Colon 26 adenocarcinoma cells were transplanted
subcutaneously in the subaxillary region of each CDF1 female mouse,
to which the block copolymer-drug complex pharmaceutical preparation
prepared in Example 8 or 9 was intravenously administered 8 days after
transplanting. Each drug was used by diluting it in physiological
saline prior to its use. After 15 minutes and 1, 4, 24 and 48 hours
of the administration, blood samples were collected and various
organs were excised. The concentrations of adriamycin and adriamycin
dimer in the blood plasma and the tumor were determined by extracting
the drugs with an organic solvent and measuring them by HPLC.
Periodical variations in the amount (g of dose/ml) of adriamycin in
1 ml of the blood plasma when the amount of adriamycin in the
administered drug preparation is defined as 100, and periodical
variations in the amount ($ of dose/ml) of adriamycin dimer in 1 ml
of the blood plasma when the amount of adriamycin dimer in the
administered drug preparation is defined as 100 are shown in Table
7. Improvement of the drug-retentivity in blood was particularly
significant in the case of the pharmaceutical preparation of Example
8 comprising the adriamycin dimer at a higher level. Periodical
variations in the amount (~ of dose/g) of adriamycin in 1 g of the
48
CA 02206333 1997-OS-28
tumor tissue when the amount of adriamycin in the administered drug
preparation is defined as 100, and periodical variations in the
amount (~ of dose/g) of adriamycin dimer in 1 g of the tumor tissue
when the amount of adriamycin dimer in the administered drug
preparation is defined as 100 are shown in Table 8. The drug-
accumulation in tumor was markedly improved in the case of the
pharmaceutical preparation of Example 8 comprising the adriamycin
dimer at a higher level.
At~plication Example 5
Colon 26 adenocarcinoma cells were transplanted
subcutaneously in the subaxillary region of each CDF1 female mouse,
to which the block copolymer-drug complex pharmaceutical preparation
prepared in Example 8, 10 or 12 was intravenously administered 11 days
after transplanting. Each drug was used by diluting it in
physiological saline prior to its use. After 1 or 24 hours of the
administration, blood samples were collected and various organs were
excised. The concentrations of adriamycin and adriamycin dimer in
the blood plasma and the tumor were determined by extracting the drugs
with an organic solvent and measuring them by HPLC. Periodical
variations in the amount (~ of dose/ml) of adriamycin in 1 ml of the
blood plasma when the amount of adriamycin in the administered drug
preparation is defined as 100, and periodical variations in the
amount ( $ of dose/ml ) of adriamycin dimer in 1 ml of the blood plasma
when the amount of adriamycin dimer in the administered drug
preparation is defined as 100 are shown in Table 9. Results of the
49
CA 02206333 1997-OS-28
pharmaceutical preparation of Example 8 were almost the same as those
obtained from Application Example 4. The drug-retentivity in blood
was slightly lower in the case of the pharmaceutical preparation of
Example 10 in which the ratio of the adriamycin dimer was lower than
that of the pharmaceutical preparation of Example 8. Periodical
variations in the amount (~ of dose/g) of adriamycin in 1 g of the
tumor tissue when the amount of adriamycin in the administered drug
preparation is defined as 100, and periodical variations in the
amount ($ of dose/g) of adriamycin dimer in 1 g of the tumor tissue
when the amount of adriamycin dimer in the administered drug ,
preparation is defined as 100 are shown in Table 10. With regard to
the pharmaceutical preparation of Example 8, almost the same results
as those obtained from Application Example 4 were obtained. The
drug-accumulation in tumor was also slightly lower in the case of the
pharmaceutical preparation of Example 10 in which the ratio of the
adriamycin dimer was lower than that of the pharmaceutical
preparation of Example 8.
Table 3 Periodical variations in $ of dose/ml (blood plasma)
Sample Time after administration
15 minutes1 hour 4 hours 48 hours
24 hours
(A) 0.47 0.29 0.36 0.10 0.07
(B) 61.6 53.7 39.2 15.9 4.7
(C) 41.7 29.2 21.3 8.2 2.2
(A) Adriamycinhydrochloride
(B) Pharmaceutical Example 6
preparation
of
(C) Pharmaceutical Example 7
preparation
of
CA 02206333 1997-OS-28
Table 4 Periodical variations in ~ of dose/g (tumor)
Sample Time after
administration
15 minutes hour 4 hours 48 hours
1 24 hours
(A) 2.3 2.4 1.7 1.3 1.0
(B) 2.3 3.8 4.9 9.6 9.1
(C) 2.3 2.7 4.9 7.0 4.0
(A) Adriamycin rochloride
hyd
(B) Pharmaceuticalpreparation Example 6
of
(C) Pharmaceuticalpreparation Example 7
of
Table 5 Periodical variations in ~ of dose/g (heart)
Sample Time after administration
15 minutes1 hour 4 hours
24 hours 48 hours
(A) 10.5 7.0 4.6 1.1 0.5
(B) 4.9 4.2 3.3 1.6 1.0
(C) 6.5 5.4 3.8 1.4 0.7
(A) Adriamycinhydrochloride
(B) Pharmaceutical Example 6
preparation
of
(C) Pharmaceutical Example 7
preparation
of
Table 6 Antitumor activity on mouse Colon 26 adenocarcinoma
Sample Dose (mg/kg) Tumor-disappeared mice
Adriamycin hydrochloride 5 0/5
10 2/5
Pharmaceutical preparation5 5/5
of Example 8 10* 5/5
Pharmaceutical preparation1.25 0/5
of Example 12 2.5 0/5
5 4/5
10 5/5
(* first administration only)
51
CA 02206333 1997-OS-28
Table 7 Periodical variations in ~ of dose/ml (blood plasma)
Sample Time after administration
15 minutes 1 hour 4 hours 48 hours
24
hours
(A) ~ 44.9 32.9 20.73.8 2.0
67.2 69.7 61.914.2 3.9
(B) O1 27.7 12.3 5.3 1.9 0.5
23.0 22.5 19.411.1 7.0
(A) Pharmaceutical Example 8
preparation
of
(B) Pharmaceutical Example 9
preparation
of
~1 Adriamycin
02 Adriamycin dimer
Table 8 Periodical variations in ~ of dose/g (tumor)
Sample Time after administration
15 minutes 1 hour 4 hours 24 hours 48 hours
(A) ~1 4.0 3.1 6.8 19.0 20.4
02 1.1 1.0 6.4 14.1 12.2
(B) ~1 1.3 2.1 1.9 2.2 1.2
~ 0.0 8.1 2.8 3.9 4.9
(A) Pharmaceutical preparation of Example 8
(B) Pharmaceutical preparation of Example 9
~1 Adriamycin
20 Adriamycin dimer
52
CA 02206333 1997-OS-28
Table 9 Periodical variations in ~ of dose/ml (blood plasma)
Sample Time after administration
1 hour 24 hours
(A) 0 36.7 6.8
83.5 17.9
(B) ~ 11.0 4.1
65.3 16.2
(C) ~ 40.6 10.2
~ 100.2 24.5
(A) Pharmaceutical preparation of Example 8
(B) Pharmaceutical preparation of Example 10
(C) Pharmaceutical preparation of Example 12
Adriamycin
~ Adriamycin dimer
Table 10 Periodical variations in ~ of dose/g (tumor)
Sample Time after administration
1 hour 24 hours
(A) ~1 3.4 21.0
~ 1.8 16.1
(B) ~1 2.6 4.5
~ 0.1 3.0
(C) O1 5.0 9.1
~2 2.1 6.3
(A) Pharmaceutical preparation of Example 8
(B) Pharmaceutical preparation of Example 10
(C) Pharmaceutical preparation of Example 12
Adriamycin
~ Adriamycin dimer
53
CA 02206333 1997-OS-28
EFFECTS OF THE INVENTION
It was successful to endow the dimer-, trimer- or
tetramer-containing high molecular block copolymer-drug complex
pharmaceutical preparation of the present invention with higher drug
effects and lower toxicity by incorporating anticancer agents and the
like into the inner core of micelle of the block copolymer. In
consequence, the present invention can provide markedly useful
pharmaceutical preparations.
54
