U.S. patent application number 15/793501 was filed with the patent office on 2018-09-27 for inclusion compounds of fumagillol derivative or its salt, and pharmaceutical compositions comprising the same.
The applicant listed for this patent is Chong Kun Dang Pharmaceutical Corporation. Invention is credited to Soon Kil Ahn, Won Kyu Choi, Chung Il Hong, Jae Hyun Kim, Su Kyung Lee, Jong Lae Lim, Hee Jong Shin.
Application Number | 20180271999 15/793501 |
Document ID | / |
Family ID | 28673032 |
Filed Date | 2018-09-27 |
United States Patent
Application |
20180271999 |
Kind Code |
A1 |
Kim; Jae Hyun ; et
al. |
September 27, 2018 |
Inclusion Compounds of Fumagillol Derivative or its Salt, and
Pharmaceutical Compositions Comprising the Same
Abstract
The present invention relates to an inclusion compound of
fumagillol derivative or its salt with
hydroxypropyl-.beta.-cyclodextrin or
sulfobutylether-7-.beta.-cyclodextrin, and pharmaceutical
compositions comprising the same. The inclusion compound according
to the present invention has superior water solubility and
stability while exhibiting low toxicity, rendering it valuable as
an anticancer agent or inhibitor of tumor metastasis.
Inventors: |
Kim; Jae Hyun; (Seoul,
KR) ; Lee; Su Kyung; (Chungcheongnam-Do, KP) ;
Choi; Won Kyu; (Seoul, KR) ; Lim; Jong Lae;
(Chungcheongnam-do, KR) ; Ahn; Soon Kil; (Seoul,
KR) ; Shin; Hee Jong; (Kyunggi-Do, KR) ; Hong;
Chung Il; (Chicago, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chong Kun Dang Pharmaceutical Corporation |
Seoul |
|
KR |
|
|
Family ID: |
28673032 |
Appl. No.: |
15/793501 |
Filed: |
October 25, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15252625 |
Aug 31, 2016 |
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15793501 |
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14502101 |
Sep 30, 2014 |
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15252625 |
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13924768 |
Jun 24, 2013 |
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14502101 |
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13414215 |
Mar 7, 2012 |
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13924768 |
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12756372 |
Apr 8, 2010 |
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13414215 |
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11828025 |
Jul 25, 2007 |
7718695 |
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12756372 |
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10951164 |
Sep 27, 2004 |
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11828025 |
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PCT/KR2002/000583 |
Apr 3, 2002 |
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10951164 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C08L 5/16 20130101; C08B
37/0015 20130101; A61K 31/336 20130101; A61P 35/04 20180101; C07D
303/20 20130101; A61K 47/6951 20170801; B82Y 5/00 20130101; C07D
407/14 20130101; C07D 303/28 20130101; A61K 47/02 20130101; C07D
303/16 20130101; A61P 35/00 20180101; A61K 9/0019 20130101 |
International
Class: |
A61K 47/69 20170101
A61K047/69; C08L 5/16 20060101 C08L005/16; C08B 37/16 20060101
C08B037/16; C07D 407/14 20060101 C07D407/14; C07D 303/28 20060101
C07D303/28; A61K 9/00 20060101 A61K009/00; C07D 303/16 20060101
C07D303/16; B82Y 5/00 20110101 B82Y005/00; A61K 47/02 20060101
A61K047/02; A61K 31/336 20060101 A61K031/336; C07D 303/20 20060101
C07D303/20 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 28, 2002 |
KR |
2002/0016946 |
Claims
1. An inclusion compound of a fumagillol derivative or a salt
thereof with hydroxypropyl-.beta.-cyclodextrin, wherein the
fumagillol derivative is
O-(4-dimethylaminoethoxycinnamoyl)fumagillol.
2.-4. (canceled)
5. The inclusion compound of claim 1 characterized in that said
fumagillol derivative salt is selected from a group consisting of
salts of fumagillol derivative with hydrochloric acid, bromic acid,
sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic
acid, trifluoroacetic acid, oxalic acid, fumaric acid, tartaric
acid, maleic acid, methanesulfonic acid, benzenesulfonic acid or
para-toluenesulfonic acid.
6. The inclusion compound in claim 1 characterized in preparing by
dissolving hydroxypropyl-.beta.-cyclodextrin in distilled water,
and adding fumagillol derivative or its salt under stirring.
7. The inclusion compound in claim 1 characterized in that the
inclusion compound is prepared by dissolving
hydroxypropyl-.beta.-cyclodextrin in distilled water, and adding
fumagillol derivative or its salt under stirring after adjusting
the solution pH in a range of 6-8 with dilute hydrochloric acid or
sodium hydroxide solution.
8. The inclusion compound in claim 1 characterized in that the
inclusion compound is prepared by dissolving
hydroxypropyl-.beta.-cyclodextrin in buffer solution which pH was
pre-adjusted in a range of 6-8 with phosphate, and adding
fumagillol derivative or its salt under stirring.
9. The inclusion compound of claim 6 characterized in that the
inclusion compound is obtained by further lyophilization step for
the final solution obtained after stirring.
10. The inclusion compound of claim 6 characterized in that molar
ratio of fumagillol derivative or its salt to
hydroxypropyl-.beta.-cyclodextrin is 1:1 to 1:10.
11. A pharmaceutical composition comprising the inclusion compound
in claim 1 and pharmaceutically acceptable additives.
12. The pharmaceutical composition in claim 11 characterized in
that the pharmaceutically acceptable additive is at least one
selected from a group consisting of pharmaceutically acceptable
diluents, buffers, flavors, binders, thickening agent, lubricants
and preservatives.
13. The pharmaceutical composition in claim 12 characterized in
that said buffer is phosphate buffer.
14. The pharmaceutical composition in claim 11 characterized in
that it is formulated in oral or parenteral preparation.
15. The pharmaceutical composition in claim 14 characterized in
that said parenteral preparation is injection.
16. The pharmaceutical composition in claim 11 characterized in
that it is formulated in sustained-release dosage form.
17.-18. (canceled)
19. A lyophilized composition comprising the inclusion compound of
claim 1.
Description
TECHNICAL FIELD
[0001] The present invention relates to an inclusion compound of
fumagillol derivative or its salt with
hydroxypropyl-.beta.-cyclodextrin or
sulfobutylether-7-.beta.-cyclodextrin, and pharmaceutical
compositions comprising the same.
BACKGROUND ART
[0002] Cyclodextrins are cyclic compounds consisting of
glucopyranose units through .alpha.-1,4-glycosidic linkages. The
exterior surface of the cyclodextrin ring is hydrophilic, whereas
the inside cavity thereof exhibits a hydrophobic character.
Therefore, it is possible that other molecules referred to as
"guest molecule" or part thereof, which are less polar than water
(hydrophobic molecules) and have suitable dimensions to be required
to fit into the cyclodextrin cavity, are included in the
hydrophobic cavity of the cyclodextrin molecule and form inclusion
compounds. The pharmaceutical applications with cyclodextrins are
disclosed in many articles (Journal of Parenteral Science &
Technology 43(5), pp 231-240 (1989) and Pharmaceutical Research
14(5), pp 556-567 (1997)).
[0003] Cyclodextrins consisting of 6, 7 or 8 glucopyranose units
are generally referred to as .alpha.-, .beta.- and
.gamma.-cyclodextrin, respectively. Although .beta.-cyclodextrin is
the most useful one of the above natural cyclodextrins for
pharmaceutical preparations in terms of inclusion capacity and
economical efficiency, it is not always ideal for drug formulations
due to its relatively low aqueous solubility (1.8 g per 100 ml of
water), serious renal toxicity and biological membrane
incompatibility after parenteral administration. Therefore, its
application is limited to merely food products or oral
pharmaceutical preparations.
[0004] Recently, a number of chemically modified cyclodextrins such
as alkylated-, hydroxyalkylated-, carboxyethylated- and
sulfoalkylether-cyclodextrins have been prepared to improve the
inclusion capacity and physicochemical properties of natural
cyclodextrins.
[0005] Among them, as hydroxyalkylated group, C.sub.1-6 alkyl group
is preferable, and hydroxymethyl, hydroxyethyl, hydroxypropyl and
hydroxybutyl group can be enumerated. In particular, hydroxypropyl
group is preferable. In addition, as sulfoalkylated group,
C.sub.1-6 alkyl group is preferred, and sulfomethyl, sulfoethyl,
sulfopropyl and sulfobutyl group can be enumerated. In particular,
sulfobutyl group is preferable. The specific product in the
hydroxyalkylated cyclodextrins includes
2-hydroxypropyl-.beta.-cyclodextrin, and the specific product in
the sulfoalkylether cyclodextrins includes
sufobutylether-7-.beta.-cyclodextrin.
Hydroxypropyl-.beta.-cyclodextrin and
sulfobutylether-7-.beta.-cyclodextrin are especially suitable for
the parenteral application because of their high water-solubility
and minimal-toxicity, which are well disclosed in Journal of
Pharmaceutical Science 85(11), pp 1142-1169 (1996).
[0006] Further, with regard to cyclodextrin inclusion compounds,
U.S. Pat. No. 4,371,673 discloses two types of water-soluble
cyclodextrin complexes with retinoid-polymers and complexes of
retinoids with ether type derivatives of cyclodextrins. U.S. Pat.
No. 4,596,795 discloses results for administering a complex of sex
hormone and cyclodextrin derivative via sublingual and buccal
route. And U.S. Pat. No. 4,727,064 describes results on the
conversion methods of drugs with ready crystallization and low
water-solubility into intrinsically amorphous complexes, which have
improved pharmaceutical properties by using cyclodextrin
derivatives. U.S. Pat. No. 5,134,127 discloses sulfoalkylether
cyclodextrin derivatives and their use as solubilizing agents for
poorly water-soluble drugs for oral, intranasal or parenteral
administration.
[0007] Recently, it has been proposed as a promising concept that
solid tumor growth beyond a certain size requires newly-formed
blood vessels for the transport of nutrients and oxygen, which is
called to be angiogenesis-dependent, and it is expected that the
inhibition of angiogenesis would provide a powerful and selective
therapy for a wide variety of tumors. In particular, fumagillol
derivative has been reported to exhibit pharmacological properties
as an effective inhibiting agent for tumor-induced
neovascularization by European Patent No. 415,294 and U.S. Pat. No.
6,063,812. However, further development of those compounds
applicable to the clinical use is hampered considerably by the fact
that they are poorly soluble in water and very unstable at room
temperature.
[0008] It is well known that low drug solubility causes low
absorption upon oral administration, precluding parenteral
formulations. Furthermore, low stability imposes short shelf-life
of products, low-temperature storage requirement and restrictions
on mechanical movement, resulting in economical inefficiency and
inconvenience.
[0009] Preparation studies of fumagillol derivatives are disclosed
in several literatures. Solubility improvement of fumagillol
derivatives was established in U.S. Pat. No. 5,196,406, but the
stabilization of resulting products was not disclosed in the art.
Other solubility improvement of fumagillol derivatives was
established in European patent No. 519,428 but organic solvents
such as ethanol, acetonitrile, isopropyl alcohol and acetone rather
than cyclodextrins contributed more to the improved solubility.
However, the use of organic solvent potentially may cause the side
effects of the therapy. That is, further dilution in a large volume
parenteral fluid such as saline or 5% dextrose solution on
intravenous or intramuscular administration could lead to
life-threatening precipitation followed by phlebitis due to the
limited solubility. Also, in the art, stability was not consistent
depending on the particular kind of cyclodextrin derivative. The
stability of the mixture with maltosyl-.beta.-cyclodextrin was
improved, whereas the stability of the mixture with
hydroxypropyl-.beta.-cyclodextrin was rather worse than the parent
compound alone. In addition, though the formulation with
maltosyl-.beta.-cyclodextrin seemed to be stable,
maltosyl-.beta.-cyclodextrin has not been yet guaranteed for a
parenteral use, and its cost has made its universal use for various
formulations economically unfavorable in contrast to
hydroxypropyl-.beta.-cyclodextrin that proved to be safe and
economical. Also, though a stable composition of fumagillol
derivatives was disclosed in U.S. Pat. No. 5,422,363, all
excipients, fatty acid esters of glycerin or polyglycerin, used in
the formulations are not suitable for the parenteral
application.
[0010] Therefore, there was a need to convert fumagillol
derivatives into a form, which is better soluble and stable, and
thus possesses improved pharmaceutical properties.
[0011] Concerning the above, the inventors of the present invention
developed novel fumagillol derivative and filed (U.S. Pat. No.
6,063,812), and the compounds used in the present invention are
identical to the compounds disclosed therein.
[0012] The present inventors continued studies to provide
fumagillol derivative preparations that can be applicable to
parenteral administration such as intravenous or intramuscular
injection, or oral administration by ensuring homogeneity, safety,
bioavailability and stability under storage at room temperature
through increasing solubility of fumagillol derivatives or their
salts which were found to be superior as an angiogenesis inhibitor
but unstable under storage at room temperature or in aqueous
solution. As a result, we discovered that the inclusion compound of
fumagillol derivative or its salt with
hydroxypropyl-.beta.-cyclodextrin or
sulfobutylether-7-.beta.-cyclodextrin is useful as an antitumor
agent or antimetastatic agent with superior water-solubility and
stability but low irritancy effect, and based on these, completed
the present invention.
[0013] Therefore, the object of the present invention is to provide
inclusion compound of fumagillol derivative or its salt with
hydroxypropyl-.beta.-cyclodextrin or
sulfobutylether-7-.beta.-cyclodextrin, and pharmaceutical
composition comprising the same.
DISCLOSURE OF INVENTION
[0014] The present invention relates to the inclusion compound of
fumagillol derivative of the following Formula 1 or its salt with
hydroxypropyl-.beta.-cyclodextrin or
sulfobutylether-7-.beta.-cyclodextrin, and pharmaceutical
compositions comprising the same:
##STR00001##
[0015] Wherein, [0016] X is hydroxy, Y is halogen, or X and Y
together forms oxirane ring [0017] B is O or H.sub.2; and [0018]
R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 independently
represent hydrogen, hydroxy, acetoxy, C.sub.1-C.sub.6 alkyl,
C.sub.1-6 alkoxy, amino, alkylamino, dialkylamino, aminoalkyl,
alkylaminoalkyl, dialkylaminoalkyl, aminoalkoxy, alkylaminoalkoxy,
dialkylaminoalkoxy, halogen, cyano, trifluoromethyl, nitro, formyl,
acetamido, methyleneoxycarboxy, methylenedioxy or ethylenedioxy
group, [0019] provided that R.sub.1, R.sub.2, R.sub.3, R.sub.4 and
R.sub.5 cannot be hydrogen at the same time.)
[0020] Specifically, the inclusion compound of the present
invention is characterized in that it includes angiogenesis
inhibitor as a main component, in particular fumagillol derivative
of said Formula 1 or its salt and as a solubilizing and stabilizing
agent, cyclodextrin derivative, in particular
hydroxypropyl-.beta.-cyclodextrin or
sulfobutylether-7-.beta.-cyclodextrin without addition of any
organic solvent.
[0021] Fumagillol derivative or its salt of the present invention
is preferred to be the following compounds: [0022]
O-(4-chlorocinnamoyl)fumagillol; [0023]
O-(4-aminocinnamoyl)fumagillol; [0024]
O-(4-dimethylaminoethoxycinnamoyl)fumagillol [0025]
O-(4-methoxycinnamoyl)fumagillol; [0026]
O-(4-dimethylaminocinnamoyl)fumagillol; [0027]
O-(4-hydroxycinnamoyl)fumagillol; [0028]
O-(3,4-dimethoxycinnamoyl)fumagillol; [0029]
O-(3,4-methylenedioxycinnamoyl)fumagillol; [0030]
O-(3,4,5-trimethoxycinnamoyl)fumagillol; [0031]
O-(4-nitrocinnamoyl)fumagillol; [0032]
O-(3,4-dimethoxy-6-aminocinnamoyl)fumagillol; [0033]
O-4-acetoxy-3,5-dimethoxycinnamoyl)fumagillol; [0034]
O-(4-ethylaminocinnamoyl)fumagillol; [0035]
O-(4-ethylaminoethoxycinnamoyl)fumagillol; [0036]
O-(3-dimethylaminomethyl-4-methoxycinnamoyl)fumagillol; [0037]
O-(4-trifluoromethylcinnamoyl)fumagillol; [0038]
O-(3,4-dimethoxy-6-nitrocinnamoyl)fumagillol; [0039]
O-(4-acetoxycinnamoyl)fumagillol; [0040]
O-(4-cyanocinnamyl)fumagillol; [0041]
4-(4-methoxycinnamoyl)oxy-2-(1,2-epoxy-1,5-dimethyl-4-hexenyl)-3-m-
ethoxy-1-chloromethyl-1-cyclohexanol; [0042]
O-(3,4,5-trimethoxycinnamyl)fumagillol; [0043]
O-(4-dimethylaminocinnamoyl)fumagillol; [0044]
O-(3,4,5-trimethoxycinnamoyl)oxy-2-(1,2-epoxy-1,5-dimethyl-4-hexenyl)-3-m-
ethoxy-1-chloromethyl-1-cyclohexanol; [0045]
O-(4-dimethylaminocinnamoyl)oxy-2-(1,2-epoxy-1,5-dimethyl-4-hexenyl)-3-me-
thoxy-1-chloromethyl-1-cyclohexanol; [0046]
O-(3,5-dimethoxy-4-hydroxycinnamoyl)fumagillol; or a salt
thereof.
[0047] More preferably, the fumagillol derivative or its salt
according to the present invention is the following compounds:
[0048]
4-(4-methoxycinnamoyl)oxy-2-(1,2-epoxy-1,5-dimethyl-4-hexenyl)-3-methoxy--
1-chloromethyl-1-cyclohexanol; [0049]
O-(4-methoxycinnamoyl)fumagillol; [0050]
O-(3,5-dimethyl-4-hydroxycinnamoyl)fumagillol; [0051]
O-(4-dimethylaminoethoxycinnamoyl)fumagillol; [0052]
O-(3,4,5-trimethoxycinnamoyl)fumagillol; [0053]
O-(3,4-dimethoxy-6-aminocinnamoyl)fumagillol; or a salt
thereof.
[0054] Further preferably, fumagillol derivative or its salt of the
present invention is O-(4-dimethylaminoethoxycinnamoyl)fumagillol
or O-(3,4,5-trimethoxycinnamoyl) fumagillol.
[0055] In addition, as the fumagillol derivative salt of the
present invention, it is preferable to select from a group
consisting of salts of fumagillol derivative with hydrochloric
acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid,
formic acid, acetic acid, trifluoroacetic acid, oxalic acid,
fumaric acid, tartaric acid, maleic acid, methanesulfonic acid,
benzenesulfonic acid or para-toluenesulfonic acid.
[0056] Fumagillol derivative represented with the formula 1 used in
the present invention were disclosed in U.S. Pat. No. 6,063,812,
and prepared according to the method disclosed therein.
[0057] The inclusion compound of the present invention can be
prepared by dissolving hydroxypropyl-.beta.-cyclodextrin or
sulfobutylether-7-.beta.-cyclodextrin in distilled water, and
adding fumagillol derivative or its salt under stirring, or can be
prepared by dissolving hydroxypropyl-.beta.-cyclodextrin or
sulfobutylether-7-.beta.-cyclodextrin in distilled water, and
adding fumagillol derivative or its salt under stirring after
adjusting the solution pH in a range of 6-8 with dilute
hydrochloric acid or sodium hydroxide solution.
[0058] In addition, the inclusion compound of the present invention
can be prepared by dissolving hydroxypropyl-.beta.-cyclodextrin or
sulfobutylether-7-.beta.-cyclodextrin in buffer solution which pH
was pre-adjusted in a range of 6-8 with phosphate buffer, and by
adding fumagillol derivative or its salt under stirring.
[0059] The produced inclusion compound can be provided as pure
product, i.e. in the form of solution or in a solid form via
lyophilization, and if necessary, before freeze drying, final
solution obtained after shaking can undergo pH adjustment step in
the range of 6-8.
[0060] The Inclusion compound obtainable according to the present
invention can be used as various forms, e.g. solid or solution.
[0061] In this invention, it is preferred that the molar ratio of
fumagillol derivative or its salt to
hydroxypropyl-.beta.-cyclodextrin or
sulfobutylether-7-.beta.-cyclodextrin is 1:1 to 1:10, and more
preferably, 1:1 to 1:6.
[0062] The inclusion compound of fumagillol derivative or its salt
according to the present invention has superior solubility and
stability compared to other preparations of fumagillol
derivative.
[0063] As result of solubility evaluation with cosolvents and
surfactants, O-(4-dimethylaminoethoxycinnanoyl)fumagillol exhibited
the solubility of about 5 mg/ml in aqueous formulations containing
a mixture of 10% ethanol and 10% Tween 80.RTM. or 10%
Cremophor-EL.RTM. alone. However, these formulations have a number
of disadvantages, which are the inability to guarantee the
long-term stability due to facile hydrolysis in solution state, the
inability to be buffered due to increased sensitivity to ions
resulting in precipitation and further, the toxicity of the
surfactants.
[0064] In contrast, the inclusion compound of the present invention
has been found to have improved solubility over other formulations
indicated above. For example, the solubility of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol is enhanced from 50
.mu.g/ml in water to about 7 mg/ml in 7 w/v %
hydroxypropyl-.beta.-cyclodextrin solution and about 30 mg/ml in
14% w/v hydroxypropyl-.beta.-cyclodextrin solution at pH 6.7,
respectively. The solubility of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol is about 5.5 mg/ml in
7 w/v % sulfobutylether-7-.beta.-cyclodextrin solution. Thus,
depending the pH and the concentration of
hydroxypropyl-.beta.-cyclodextrin or
sulfobutylether-7-.beta.-cyclodextrin present in solution, the
aqueous solubility of O-(4-dimethylaminoethoxycinnamoyl)fumagillol
is found to increase by 20-1000 fold.
[0065] In addition, the stability of the inclusion compound in the
present invention was surprisingly improved at room temperature.
The results showed that the degradation rate of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol included in
hydroxypropyl-.beta.-cyclodextrin was decreased greatly in solid
state at room temperature, compared with that of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone. The preferred
inclusion compound in the present invention also suppressed the
hydrolysis rate of O-(4-dimethylaminoethoxycinnamoyl)fumagillol in
solution.
[0066] Therefore, the inclusion compound in the present invention
can be applied to the parenteral or oral formulation, since it
eliminates the disadvantage of other formulations with cosolvents
and surfactants. The preferred inclusion compound in the present
invention also overcomes ionic strength effects, which permits the
use of buffers to control the pH of solution, and is fully
dilutable because of a linear increase in the solubility of the
fumagillol derivative as function of
hydroxypropyl-.beta.-cyclodextrin or
sulfobutylether-.beta.-cyclodextrin concentration. Therefore, it
can offer a wide choice of diluents such as electrolytes and
non-electrolytes.
[0067] The present invention is further characterized by providing
pharmaceutical composition comprising the inclusion compound
according to the present invention and pharmaceutically acceptable
additives.
[0068] The pharmaceutically acceptable additives include diluents
of pharmaceutically acceptable electrolytes or non-electrolytes,
buffers, flavoring agents, binders, thickeners, lubricants,
preservatives and the like, and the composition of the present
invention can include at least one selected from those
ingredients.
[0069] Herein, it is preferable that said buffer included in the
composition of the present invention is phosphate buffer.
[0070] The pharmaceutical formulation can be formulated into oral
or parenteral preparation. For parenteral preparation, injection,
eye drop, nasal formulation can be enumerated, and preferred
injection includes subcutaneous, intravenous, intramuscular,
intraarterial and infusion administrations.
[0071] Further, the pharmaceutical composition of the present
invention can be formulated into the sustained-release dosage
form.
[0072] The pharmaceutical composition according to the present
invention can be used as an anti-tumor agent or a tumor metastasis
inhibitor in human beings with tumor, and can also be used for the
treatment in warm-blooded animals such as rats, dogs, rabbits, cats
and chickens.
BRIEF DESCRIPTION OF DRAWINGS
[0073] FIG. 1 shows two-dimensional nuclear magnetic resonance
spectrum (NOSEY) for the inclusion compound of fumagillol
derivative in Example 12.
[0074] FIG. 2 represents respective plasma concentration-time curve
after intravenous administration of fumagillol derivative alone
(.circle-solid.) and the inclusion compound of fumagillol
derivative (.largecircle.) in Example 17.
BEST MODE FOR CARRYING OUT THE INVENTION
[0075] The following Examples represent preferred embodiments of
the present invention. However, the present invention is not
limited to the following Examples.
Example 1
[0076] O-(4-dimethylaminoethoxycinnamoyl)fumagillol (30 mg) was
added to 1:5, 3.5, 7.0, 14.0, 28.0 w/v %
hydroxypropyl-.beta.-cyclodextrin solution, respectively, and
stirred at 4.degree. C. After 72 hr, the mixture was filtered with
0.2 .mu.m membrane filter and
O-(4-dimethylaminoethoxycinnamoyl)fumagillol in the filtrate was
determined by high pressure liquid chromatography (HPLC). The
solubility of O-(4-dimethylamino ethoxycinnamoyl)fumagillol as a
function of the hydroxypropyl-.beta.-cyclodextrin concentration was
represented in Table 1.
TABLE-US-00001 TABLE 1 Solubility Concentration of
hydroxypropyl-.beta.-cyclodextrin (w/v %) (mg/ml) 0 0.05 1.5 1.03
3.5 2.93 7.0 6.95 14.0 11.54 28.0 22.64
[0077] As in apparent from Table 1, the solubility of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol was improved when
hydroxypropyl-.beta.-cyclodextrin was added to form a complex and
when the concentration of hydroxypropyl-.beta.-cyclodextrin was
increased.
Example 2
[0078] O-(4-dimethylaminoethoxycinnamoyl)fumagillol (30 mg) was
added to 1.5, 3.5, 7.0, 14.0, 28.0 w/v %
sulfobutylether-7-.beta.-cyclodextrin solution, respectively, and
stirred at 4.degree. C. After 72 hr, the mixture was filtered with
0.2 .mu.m membrane filter and
O-(4-dimethylaminoethoxycinnamoyl)fumagillol in the filtrate was
determined by high pressure liquid chromatography (HPLC). The
solubility of O-(4-dimethylaminoethoxycinnamoyl)fumagillol as a
function of the sulfobutylether-7-.beta.-cyclodextrin concentration
was represented in Table 2.
TABLE-US-00002 TABLE 2 Solubility Concentration of
sulfobutylether-7-.beta.-cyclodextrin (w/v %) (mg/ml) 0 0.05 1.5
0.87 3.5 2.24 7.0 5.42 14.0 10.43 28.0 21.30
[0079] As in apparent from Table 2, the solubility of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol was improved when
sulfobutylether-7-.beta.-cyclodextrin was added to form a complex
and when the concentration of sulfobutylether-7-.beta.-cyclodextrin
was increased.
Example 3
[0080] O-(3,4,5-trimethoxycinnamoyl)fumagillol (20 g) was added to
1.5, 3.5, 7.0, 14.0, 28.0 w/v % hydroxypropyl-.beta.-cyclodextrin
solution, respectively, and stirred at 4.degree. C. After 72 hr,
the mixture was filtered with 0.2 .mu.m membrane filter and
O-(3,4,5-trimethoxycinnamoyl)fumagillol in the filtrate was
determined by high pressure liquid chromatography (HPLC). The
solubility of O-(3,4,5-trimethoxycinnamoyl)fumagillol as a function
of the hydroxypropyl-.beta.-cyclodextrin concentration was shown in
Table 3.
TABLE-US-00003 TABLE 3 Solubility Concentration of
hydroxypropyl-.beta.-cyclodextrin (w/v %) (mg/ml) 0 0.002 1.5 0.43
3.5 1.52 7.0 3.54 14.0 6.72 28.0 12.02
[0081] As in apparent from Table 3, the solubility of
O-(3,4,5-trimethoxycinnamoyl)fumagillol was improved when
hydroxypropyl-.beta.-cyclodextrin was added to form a complex and
when the concentration of hydroxypropyl-.beta.-cyclodextrin was
increased.
Example 4
[0082] O-(3,4,5-trimethoxycinnamoyl)fumagillol (20 mg) was added to
1.5, 3.5, 7.0, 14.0, 28.0 w/v %
sulfobutylether-7-.beta.-cyclodextrin solution, respectively, and
stirred at 4.degree. C. After 72 hr, the mixture was filtered with
0.2 .mu.m membrane filter and
O-(3,4,5-trimethoxycinnamoyl)fumagillol in the filtrate was
determined by high pressure liquid chromatography (HPLC). The
solubility of O-(3,4,5-trimethoxycinnamoyl) fumagillol as a
function of the sulfobutylether-7-.beta.-cyclodextrin concentration
was shown in Table 4.
TABLE-US-00004 TABLE 4 Solubility Concentration of
sulfobutylether-7-.beta.-cyclodextrin (w/v %) (mg/ml) 0 0.002 1.5
0.58 3.5 1.38 7.0 2.75 14.0 5.69 28.0 11.67
[0083] As in apparent from Table 4, the solubility of
O-(3,4,5-trimethoxycinnamoyl) fumagillol was improved when
sulfobutylether-.beta.-cyclodextrin was added to form a complex and
when the concentration of sulfobutylether-.beta.-cyclodextrin was
increased.
Example 5
[0084] O-(4-dimethylaminoethoxycinnamoyl)fumagillol (50 mg) was
added to phosphate buffer (pH 6.7) containing 1.5, 3.5, 7.0 and
14.0 w/v % hydroxypropyl-.beta.-cyclodextrin, respectively, and
stirred at 4.degree. C. After 72 hr, the mixture was filtered with
0.2 .mu.m membrane filter and
O-(4-dimethylaminoethoxycinnamoyl)fumagillol in the filtrate was
determined by high pressure liquid chromatography (HPLC). The
solubility of O-(4-dimethylaminoethoxycinnamoyl)fumagillol as a
function of the hydroxypropyl-.beta.-cyclodextrin concentration was
shown in Table 5.
TABLE-US-00005 TABLE 5 Solubility Concentration of
hydroxypropyl-.beta.-cyclodextrin(w/v %) (mg/ml) 0 2.38 1.5 4.24
3.5 9.86 7.0 17.59 14.0 32.25
[0085] As in apparent from Table 5, the solubility of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol was improved when
hydroxypropyl-.beta.-cyclodextrin was added to form a complex and
when the concentration of hydroxypropyl-.beta.-cyclodextrin was
increased
Example 6
[0086] Hydroxypropyl-.beta.-cyclodextrin (13 g) was put to mass
flask and 60 ml of distilled water was added and stirred or
subjected to sonication at 4.degree. C. until clear solution was
obtained. The pH of the solution was adjusted in a range of 6-8
with dilute hydrochloric acid or sodium hydroxide.
O-(4-dimethylaminoethoxycinnamoyl)fumagillol (1 g) was added and
completely dissolved by stirring at 4.degree. C. If necessary, the
pH of the final solution was adjusted between 6-8 with dilute
hydrochloric acid or sodium hydroxide and filtered through 0.2
.mu.m membrane filter and the filtrate was lyophilized.
Example 7
[0087] Hydroxypropyl-.beta.-cyclodextrin (13 g) was put to mass
flask and 60 ml of distilled water was added and stirred or
subjected to sonication at 4.degree. C. until clear solution was
obtained. The pH of the solution was adjusted in the range of 6-8
with dilute hydrochloric acid or sodium hydroxide.
O-(3,4,5-trimethoxycimamoyl) fumagillol (1 g) was added and
completely dissolved by stirring at 4.degree. C. If necessary, the
pH of the final solution was adjusted in a range of 6-8 with dilute
hydrochloric acid or sodium hydroxide and filtered through 0.2 m
membrane filter and the filtrate was lyophilized.
Example 8
[0088] Potassium phosphate (6.8 g) and
hydroxypropyl-.beta.-cyclodextrin (21.67 g) were put to mass flask
and 100 ml of distilled water was added and stirred or subjected to
sonication at 4.degree. C. until clear solution was obtained.
O-(4-dimethylaminoethoxycinnamoyl)fumagillol (1.67 g) was added and
completely dissolved by stirring at 4.degree. C. The final solution
was filtered through 0.2 .mu.m membrane filter and the filtrate was
lyophilized.
Example 9
[0089] According to the same method as in Example 8 except using
sulfobutylether-7-.beta.-cyclodextrin instead of
hydroxypropyl-.beta.-cyclodextrin, inclusion compound of
sulfobutylether-7-.beta.-cyclodextrin with O-(4-dimethylaminoethoxy
cinnamoyl)fumagillol was prepared.
Example 10
[0090] Potassium phosphate (6.8 g) and
hydroxypropyl-.beta.-cyclodextrin (21.67 g) were put to mass flask
and 100 ml of distilled water was added and stirred or subjected to
sonication at 4.degree. C. until clear solution was obtained.
O-(4-dimethylaminoethoxycinnamoyl)fumagillol oxalate (1.84 g) was
added and completely dissolved by string at 4.degree. C. The final
solution was filtered through 0.2 .mu.m membrane filter and the
filtrate was lyophilized.
Example 11
[0091] According to the same method as in Example 10 except using
sulfobutylether-7-.beta.-cyclodextrin instead of
hydroxypropyl-f-cyclodextrin in Example 10, inclusion compound of
O-(4-dimethylamino ethoxycinnamoyl)fumagillol oxalate with
sulfobutylether-7-.beta.-cyclodextrin was prepared.
Example 12
[0092] The filtrate obtained in Example 6 was lyophilized. The
resulting lyophilized product was dissolved in heavy water
(D.sub.2O) and analyzed by using two-dimensional .sup.1H-NMR
(NOESY). The results are shown in FIG. 1. The cross peaks indicate
that there are interactions between four protons of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol and the protons of the
glucose skeleton of hydroxypropyl-4-cyclodextrin. These cross peaks
were not observed for the spectrum of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone. These results
indicate that O-(4-dimethylaminoethoxycinnamoyl)fumagillol forms an
inclusion compound with hydroxypropyl-.beta.-cyclodextrin.
Example 13
[0093] The stability 6f the lyophilized powders obtained in Example
6 was compared with that of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone under storage at
25.degree. C. The residual amount of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol was determined by
HPLC. The results are shown in Table 6.
TABLE-US-00006 TABLE 6 Residual amount of O-(4-dimethylaminoethoxy-
cinnamoyl)fumagillol (%) after after after after 1 month 3 month 6
month 12 month O-(4-dimethylaminoethoxy- 72.8 54.3 -- --
cinnamoyl)fumagillol Inclusion compound of 99.3 98.7 97.5 93.6
O-(4-dimethylaminoethoxy- cinnamoyl)fumagillol
[0094] As in apparent from Table 6, the stability of complex of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol with
hydroxypropyl-.beta.-cyclodextrin was improved as compared to that
of O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone.
Example 14
[0095] The stability of the lyophilized powders obtained in Example
7 was compared with that of O-(3,4,5-trimethoxycinnamoyl)fumagillol
alone at 25.degree. C. The residual amount of
O-(3,4,5-trimethoxycinnamoyl)fumagillol was determined by HPLC. The
results are shown in Table 7.
TABLE-US-00007 TABLE 7 Residual amount of O-(3,4,5-trimethoxy-
cinnamoyl)fumagillol (%) after after after after 1 month 3 month 6
month 12 month O-(3,4,5-trimethoxycinna- 65.3 -- -- --
moyl)fumagillol Inclusion compound of 99.5 97.7 94.4 93.2
O-(3,4,5-trimethoxycinna- moyl)fumagillol
[0096] As in apparent from Table 7, the stability of complex of
O-(3,4,5-trimethoxycinnamoyl)fumagillol with
hydroxypropyl-.beta.-cyclodextrin was improved as compared to that
of O-(3,4,5-trimethoxycinnamoyl)fumagillol alone.
Example 15
[0097] The stability of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol was investigated in
the presence of various concentrations of
hydroxypropyl-.beta.-cyclodextrin in acidic, neutral and basic
solutions at 50.degree. C. The residual amount of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol in each solution was
determined by HPLC. The results are shown in Table 8.
TABLE-US-00008 TABLE 8 Concentration of hydroxypropyl-.beta.-
Acidic (pH 3.2) Neutral (pH 7.2) Basic (pH 11.5) cyclodextrin
Inhibition Inhibition Inhibition (w/v %) k.sub.obs (h.sup.-1).sup.a
ratio (%) k.sub.obs (h.sup.-1) ratio (%) k.sub.obs (h.sup.-1),
ratio (%) 0 0.2042 -- 0.0162 -- 0.0918 -- 2 0.0976 52.20 0.0141
12.96 0.0816 5.00 5 0.0848 58.47 0.0137 15.43 0.0580 16.55 10
0.0754 63.08 0.0122 24.96 0.0453 22.77 20 0.0665 67.43 0.0115 29.01
0.0294 30.56 .sup.ak.sub.obs: Hydrolysis Rate Constant
[0098] As in apparent from Table 8, hydroxypropyl-f-cyclodextrin
suppressed the hydrolysis rate of O-(4-dimethylaminoethoxy
cinnamoyl)fumagillol significantly.
Example 16
[0099] Pre-prepared tumor mass (Lewis lung carcinoma) of 8 mm.sup.3
was subcutaneously implanted into the right axillary region of BDF1
mice (4 weeks). When the tumor size was 100-200 mm.sup.3, mice were
divided randomly into treatment group and control group. The
treatment group was administered subcutaneously with
O-(4-ethylaminoethoxycinnamoyl)fumagillol or the complex of
O-(4-ethylaminoethoxycinnamoyl)fumagillol with
hydroxypropyl-.beta.-cyclodextrin at a dose of 30 mg/kg or 120
mg/kg as O-(4-ethylaminoethoxycinnamoyl)fumagillol every other day
for 5 injections, while the control group was given injections of
0.2 ml of phosphate buffered saline. The tumors were weighed on the
final day, and tumor volume was calculated using the following
equation:
Tumor volume (mm.sup.3)=a.times.b.sup.2.times.0.5
(a: the longest diameter, b: the shortest diameter) Inhibition
ratio (IR %) of the treatment group relative to the untreated
control group was calculated using the following equation:
Inhibition Ratio %=(1-Tumor volume of Treatment group/Tumor volume
of Control group).times.100 Inhibition Ratio %
(1-Tumor weight of Treatment group/Tumor weight of Control
group).times.100
[0100] The results are shown in Table 9.
TABLE-US-00009 TABLE 9 Inhibition Ratio (%), Total Dose Tumor
Tissue Tumor Tissue (mg/kg) Volume Weight Control Group 150 0 0 600
0 0 Group administered 150 37.7 33.6 with O-(4-dimethyl- 600 63.3
71.4 aminoethoxycinna- moyl)fumagillol Group administered 150 32.2
34.7 with inclusion 600 60.4 70.8 compound of O-(4-
dimethylaminoethoxy- cinnamoyl)fumagillol
[0101] As in apparent from Table 9, the inclusion compound of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol exhibits comparable
antitumor activity with O-(4-dimethylaminoehoxycinnamoyl)fumagillol
alone.
Example 17
[0102] O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone and the
complex of O-(4-dimethylaminoethoxycinnamoyl)fumagillol with
hydroxypropyl-.beta.-cyclodextrin containing the same amount of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol were injected via
intravenous route, and blood level of the drug was determined. As
test animal, 5 male rats were used per 1 group.
[0103] Under light ether anesthesia, the femoral arteries and veins
of rats were cannulated with PB-50 polyethylene tubing. After
complete recovery from anesthesia,
O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone or the complex
of O-(4-dimethylaminoethoxycinnamoyl)fumagillol with
hydroxypropyl-.beta.-cyclodextrin dissolved in phosphate buffered
saline (pH 6.2) was administered intravenously to the femoral vein
through the catheter at a dose of 20 mg/kg as
O-(4-dimethylaminoethoxycinnamoyl)fumagillol, respectively. Blood
samples (0.15 ml) were collected via the femoral artery immediately
after the dose and at designated time intervals (15, 30, 45, 60,
120, 180 and 240 min). The blood samples were centrifuged
immediately and the concentrations of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol in the plasma were
determined by HPLC.
[0104] As apparent in FIG. 2, there were no significant differences
between the plasma concentrations of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone and those of the
complex of O-(4-dimethylaminoethoxycinnamoyl)fumagillol with
hydroxypropyl-.beta.-cyclodextrin. However, it was advantageous
that the administration of the complex of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol with
hydroxypropyl-.beta.-cyclodextrin causes little pain on the
injection site in contrast to that of
O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone.
INDUSTRIAL APPLICABILITY
[0105] The inclusion compound of fumagillol derivative or its salt
with hydroxypropyl-.beta.-cyclodextrin or
sulfobutylether-7-.beta.-cyclodextrin according to the present
invention shows improved water-solubility, superior long-term
stability at room temperature and reduced irritancy effect to
injection site with unaltered tumor growth inhibitory activity when
compared to fumagillol derivative alone, and thus may be useful for
the treatment of tumors as an angiogenesis inhibitor.
* * * * *