U.S. patent application number 14/310768 was filed with the patent office on 2014-10-09 for pharmaceutical composition for peroral administration of 3,3'-diindolylmethane.
The applicant listed for this patent is ZAKRYTOE AKTSIONERNOE OBSCHESTVO "VELES FARMA". Invention is credited to Vsevolod Ivanovich KISELEV, Irina Gennadievna VASSILIEVA.
Application Number | 20140303225 14/310768 |
Document ID | / |
Family ID | 43758874 |
Filed Date | 2014-10-09 |
United States Patent
Application |
20140303225 |
Kind Code |
A1 |
KISELEV; Vsevolod Ivanovich ;
et al. |
October 9, 2014 |
PHARMACEUTICAL COMPOSITION FOR PERORAL ADMINISTRATION OF
3,3'-DIINDOLYLMETHANE
Abstract
Block copolymer pharmaceutical compositions containing
3,3'-diindolylmethane (DIM). The pharmaceutical composition for
peroral administration comprises 3,3'-diindolylmethane as an active
component and a target additive, the target additive being a block
copolymer of oxyethylene and oxypropylene, in which the content of
the hydrophobic oxypropylene block is less than 50 mass % and the
molecular mass of the hydrophilic oxyethylene block is equal to
2,250 Da or more, at a ratio of the active component to the
selected block copolymer varying between 1:2 and 1:10. The
composition improves absorption of the active compound by the
bloodstream upon peroral delivery.
Inventors: |
KISELEV; Vsevolod Ivanovich;
(Moscow, RU) ; VASSILIEVA; Irina Gennadievna;
(Moscow, RU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ZAKRYTOE AKTSIONERNOE OBSCHESTVO "VELES FARMA" |
Moscow |
|
RU |
|
|
Family ID: |
43758874 |
Appl. No.: |
14/310768 |
Filed: |
June 20, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13377261 |
Dec 9, 2011 |
8791150 |
|
|
PCT/RU2010/000487 |
Sep 7, 2010 |
|
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14310768 |
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Current U.S.
Class: |
514/414 |
Current CPC
Class: |
A61K 47/34 20130101;
A61P 35/00 20180101; A61K 9/2031 20130101; A61K 31/404 20130101;
A61P 37/00 20180101; A61P 13/08 20180101 |
Class at
Publication: |
514/414 |
International
Class: |
A61K 31/404 20060101
A61K031/404; A61K 47/34 20060101 A61K047/34 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 18, 2009 |
RU |
2009-134872 |
Claims
1. A pharmaceutical composition for peroral administration
containing 3,3'-diindolylmethane as an active component and a
target additive, further comprising a block copolymer of
oxyethylene and oxypropylene as the target additive, wherein the
content of the hydrophobic block is less than 50 mass % and the
molecular mass of the hydrophilic block is 2,250 Da or more at a
ratio of the active component to the selected block copolymer
varying between 10:1 and 2:1.
2. The pharmaceutical composition as claimed in claim 1, further
containing Pluronic F127 as a block copolymer of oxyethylene and
oxypropylene.
3. The pharmaceutical composition as claimed in claim 2, further
containing Pluronic L10 as well.
4. The pharmaceutical composition as claimed in claim 1, further
containing a pharmaceutically acceptable carrier.
5. The pharmaceutical composition as claimed in claim 4, said
composition being in the form of a tablet, lyophilized powder,
suspension, or capsule.
Description
CROSS REFERENCE
[0001] The present application is a 37 C.F.R. .sctn.1.53(b)
continuation of, and claims priority to, U.S. application Ser. No.
13/377,261, filed Dec. 9, 2011. Application Ser. No. 13/377,261 is
the national phase under 35 U.S.C. .sctn.371 of International
Application No. PCT/RU2010/000487, filed on Sep. 7, 2010. Priority
is also claimed to Russian Application No. 2009-134872 filed on
Sep. 18, 2009. The entire contents of each of these applications is
hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to pharmacy, in particular, to new
pharmaceutical compositions for peroral administration of
3,3'-diindolylmethane (DIM) and to methods for treating diseases
with the help thereof.
BACKGROUND OF THE INVENTION
[0003] 3,3'-diindolylmethane (DIM), and its analogues and
derivatives have a broad spectrum of biological activities for
which reason DIM may be regarded as a pharmacologically active
compound of great promise. 3,3-diindolylmethane (DIM) is the main
oligomer product of indole-3-carbinol (I3C) proved to be highly
selective is respect of transformed cells of varied origin
(Aggarwal B. B., Ichikawa H. (2005), Molecular Targets and
Anticancer Potential of Indole-3-Carbinol and Its Derivatives, Cell
Cycle, 4(9), 1201-1215). Pharmacokinetic studies have shown that
perorally administered I3C is almost immediately transformed to DIM
in the acidic medium of the stomach (Arneson D. W., Hurwitz A.,
McMahon L. M., Robaugh D. (1999), Presence of 3,3'-Diindolylmethane
in Human Plasma after Oral Administration of Indole-3-Carbinol
(Abstr.), Proc. Am. Assoc. Cancer Res., 40, 2833). Many researchers
studying anticancer activity of I3C tend, therefore, to accept the
idea that a majority of clinical effects registered upon its
administration are actually produced by the dimer form of
indole-3-carbinol, or DIM.
[0004] It has been demonstrated experimentally that nearly all
multiple anticancer mechanisms induced by I3C in vitro and in vivo
are inherent in DIM as well (Chang X., Tou J. C., Hong C., et al.
(2005), 3,3'-Diindolylmethane Inhibits Angiogenesis and the Growth
of Transplantable Human Breast Carcinoma in Athymic Mice,
Carcinogenesis, 264(4), 771-778; Firestone G. L., Bjeldanes L. F.
(2003), Indole-3-Carbinol and 3,3-Diindolylmethane
Anti-Proliferative Signaling Pathways Control Cell Cycle Gene
Transcription in Human Breast Cancer Cells by Regulating
Promoter-Sp1 Transcription Factor Interactions, J. Nutr., 133,
2448S-2455S; Ge X., Yannai S., Rennert G., et al. (1996),
3,3'-Diindolylmethane Induces Apoptosis in Human Cancer Cells,
Biochem. Biophys. Res. Commun., 228, 153-158; Hong C., Kim H. A.,
Firestone G. L., et al. (2002), 3,3'-Diindolylmethane (DIM) Induces
a Cell Cycle Arrest in Human Breast Cancer Cells That Is
Accompanied by Sp-1-Mediated Activation of p21 WAF1/CIP1
Expression, Carcino-genesis, 23, 1297-1305; Leibelt D. A., Hedstrom
O. R, Fisher K. A. (2003), Evaluation of Chronic Dietary Exposure
to Indole-3-Carbinol and Absorption Enhanced 3,3'-Diindolylmethane
in Sprague-Dawley Rats, Toxicol. Sci., 74, 10-21; Li Y., Li X.,
Sarkar F. H. (2003), Gene Expression Profiles of 13C- and
DIM-Treated PC3 Human Prostate Cancer Cells Determined by cDNA
Microarray Analysis, J. Nutr., 133, 1011-1019; Nachshon-Kedmi M.,
Yannai S., Haj A., Fares F. A. (2003), Indole-3-Carbinol and
3,3'-Diindolylmethane Induces Apoptosis in Human Prostate Cancer
Cells, Food Chem. Toxicol., 41, 745-752). This conclusion applies
to prostate cancer as well. Like I3I, DIM in vitro and in vivo
inhibits growth of prostate cancer cells (Li Y., Li X., Sarkar F.
H. (2003), Gene Expression Profiles of I3C- and DIM-Treated PC3
Human Prostate Cancer Cells Determined by cDNA Microarray Analysis,
J. Nutr., 133, 1011-1019; Nachshon-Kedmi M., Fares F. A., Yannai S.
(2004), Therapeutic Activity of 3,3'-Diindolylmethane on Prostate
Cancer in an in vivo Model, Prostate, 61 (2), 153-160) and induces
their apoptosis (Li Y., Li X., Sarkar F. H. (2003), Gene Expression
Profiles of I3C- and DIM-Treated PC3 Human Prostate Cancer Cells
Determined by cDNA Microarray Analysis, J. Nutr., 133, 1011-1019;
Nachshon-Kedmi M., Yannai S., Fares F. A. (2004), Induction of
Apoptosis in Human Prostate Cancer Cell Line, PC3, by
3,3'-Diindolylmethane Through the Mitochondrial Pathway, Br. J.
Cancer, 91, 1358-1363), in which case it, similarly to I3C,
displays its activity at the submolecular level by regulating the
expression of genes responsible for proliferation, differentiation,
and survivability processes (Li Y., Li X., Sarkar F. H. (2003),
Gene Expression Profiles of I3C- and DIM-Treated PC3 Human Prostate
Cancer Cells Determined by cDNA Microarray Analysis, J. Nutr., 133,
1011-1019) and inhibiting multiple signaling pathways leading to
cellular hyperproliferation.
[0005] The hormone-sensitive prostate cells (culture LNCaP) have
been used to demonstrate that DIM can be bound concurrently to
androgen receptors to suppress in this way their translocation into
the nucleus and successive activation of gene transcription, and
also expression of the gene promoter encoding the prostate-specific
PSA antigen. The PSA protein (specific prostate protease) is a
classical marker of prostate cancer that is produced and secreted
in abundance by prostate cancel cells. The same paper established,
after structural studies undertaken, that DIM is very similar in
molecular geometry to the well-known synthetic anti-androgen
Casodex (Le H. T, Schaldach C. M., Bjeldanes L. F. (2003),
Plant-Derived 3,3'-Diindolylmethane Is a Strong Androgen Antagonist
in Human Prostate Cancer Cells, J. Biol. Chem., 278, 21136-21145)
that, in contrast to DIM, however, promotes translocation of
androgen receptors into the nucleus (Masiello D., Cheng S., Bubley
G. J., et al. (2002), Bicalutamide Functions as an Androgen
Receptor Antagonist by Assembly of a Transcriptionally Inactive
Receptor, J. Biol. Chem., 277, 26321-26326).
[0006] The capacity of DIM to display anti-angiogenic activity,
discovered only recently, is an extremely significant development.
Pathological growth of vessels almost always accompanies hyper- and
neoplastic processes. It is common knowledge that unless a network
of capillary vessels is formed to supply oxygen and nutrients to a
new tumor of 1 to 2 mm in diameter the tumor would not continue to
grow at all. It has been demonstrated that micromolar
concentrations of DIM in vitro suppress proliferation and migration
of endothelial cells and their capacity to form vessels
effectively. In vivo, DIM injected subcutaneously to experimental
animal (5 mg/kg daily) was 74% effective in suppressing
pathological neoangiogenesis (Chang X., Tou J. C., Hong C., et al.
(2005), 3,3'-Diindolylmethane Inhibits Angiogenesis and the Growth
of Transplantable Human Breast Carcinoma in Athymic Mice,
Carcinogenesis, 264(4), 771-778; McCarty M. F., Block K. I. (2005),
Multifocal Angiostatic Therapy: An Update, Integrative Cancer
Therapies, 4(4), 301-314).
[0007] The nuclear transcription factor NF-.kappa.B is the most
significant molecular target displaying an activity that modern
target preparations (directional preparations) developed and
adopted in clinical practice are intended to block. It has been
proved that this factor mediates the inflammatory response and has
a key role in regulating proliferative (anti-apoptotic),
angiogenic, migratory, and invasive cellular activities at the
final stage of signaling pathways induced by growth factors and
cytokines. Moreover, translocation of the active factor into the
nucleus and transcription activation of genes responsible for these
processes is a significant event. It has been found that, if used
in vitro, DIM (Rahman K. M., Ali S., Aboukameel A., et al. (2007),
Inactivation of NF-KappaB by 3,3'-Diindolylmethane Contributes to
Increased Apoptosis Induced by Chemotherapeutic Agent in Breast
Cancer Cells, Mol. Cancer Ther., 6(10), 2757-2765; Rahman K. M.,
Sarkar F. H. (2005), Inhibition of Nuclear Translocation of Nuclear
Factor-.kappa.B Contributes to 3,3'-Diindolylmethane-Induced
Apoptosis in Breast Cancer Cells, Cancer Res., 65, 364-371) and its
metabolic predecessor I3C are effective in suppressing nuclear
translocation and activity of NF-.kappa.B. This means that, in
addition to its anti-proliferative and anti-angiogenic effect, the
DIM-base preparation is capable of suppressing local inflammatory
reactions that frequently attend hyper- and neoplastic processes in
hormone-dependent organs and tissues.
[0008] A detailed study of patients with regression of cervical
dysplasias conducted within the framework of placebo-controlled
clinical research has helped trace a direct link between the
positive dynamics of the disease and the efficiency of I3C
conversion to DIM (Sepkovic D. W., Bradlow H. L., Bell M. (2001),
Quantitative Determination of 3,3'-Diindolylmethane in the Urine of
Individuals Receiving Indole-3-Carbinol, Natr. Cancer, 41, 57-63).
The high DIM concentration was determined in the urine of patients
receiving the preparation.
[0009] One of the most recent experimental studies demonstrated the
capacity of DIM to induce apoptosis of human cervical HPV-infected
keratinocytes in vitro. Moreover, in one of the three cellular
cervical cancer lines studied, DIM displayed a much higher
efficiency than I3I. The value of LD.sub.50 was 50 to 60 .mu.M for
DIM and 200 .mu.M for I3C, respectively, but, unlike its metabolic
predecessor (I3C), DIM did not induce any apoptotic changes in
normal (untransformed) keratinocytes (Chen D. Z., Qi M., Auborn K.,
Carter T. H. (2001), Indole-3-Carbinol and Diindolylmethane Induce
Apoptosis of Human Cervical Cancer Cells and in Murine
HPV16-Transgenic Preneoplastic Cervical Epithelium, J. Nutrit.,
I3I, 3294-3302).
[0010] To conclude, DIM has been discovered recently to have yet
another property, perhaps one of its most important advantages--its
immunomodulating activity. The researchers have shown that when
used in vitro DIM stimulates IFN.gamma.-dependent signaling
pathways in tumor cells by activating expression of IFN.gamma.
receptors, and also other IFN-responsive regulatory proteins.
[0011] The peroral method of dosing DIM-base preparations must be
given preference because it offers a series of advantages over
other dosing methods, in particular, patient comfort, flexible
treatment tactics, and treatment costs. Peroral dosing, however,
limits significantly the biological availability of DIM because of
its poor solubility and low absorption efficiency in the small
intestines. DIM typically shows poor solubility in physiological
salt solutions and has a limited capacity to pass through barrier
membranes. Furthermore, this compound is known to be bound to blood
plasma proteins and be involved in various unspecific reactions in
the bloodstream that reduce greatly the efficiency of its delivery
to the disease focus.
[0012] Several pharmaceutical compositions based on pegylated
vitamin E (TPGS) have been developed recently as a way to dispose
of the above-mentioned problems (Anderton M. J., Manson M. M., et
al. (2004), Physiological Modeling of Formulated and Crystalline
Diindolylmethane Pharmacokinetics Following Oral Administration in
Mice, Drug Metabolism and Disposition, 32(6), 632-638). Pegylated
vitamin E is known for its capacity to enhance solubility of
various compounds in water (Constantinides P. P., Tustian A.,
Kessler D. R. (2004), Tocol Emulsions for Drug Solubilization and
Parenteral Delivery, Adv. Drug Deliv. Rev., 56, 1243-1255) and
improve their biological availability when administered perorally
(Wu S. H. W., Hopkins W. K. (1999), Characteristics of
d-.alpha.-Tocopheryl PEG 1000 Succinate for Applications as an
Absorption Enhancer in Drug Delivery Systems, Pharm. Technol., 23,
52-68). TPGS-base compositions, however, can increase
insignificantly only (by 50% to 100% only) the biological
availability of DIM, and its analogues and derivatives (Zeligs, et
al., U.S. Pat. No. 6,416,793, Formulation and Use of
Controlled-Release Indole Alkaloids), for which reason the
therapeutic potential of these compounds cannot be utilized in
full.
SUMMARY OF THE INVENTION
[0013] It is an object of this invention, therefore, to improve DIM
delivery.
[0014] This object is achieved by a new pharmaceutical composition
for peroral delivery of DIM on the basis of block copolymers of
oxyethylene and oxypropylene.
[0015] The pharmaceutical composition for peroral administration
comprises 3,3'-diindolylmethane as an active component and a target
additive, which is a block copolymer of oxyethylene and
oxypropylene, in which the content of the hydrophobic block is less
than 50 mass %, and the hydrophilic block has a molecular mass of
2,250 Da or more, at a ratio of the active component to the
selected block copolymer equal to between 1:2 and 1:10.
[0016] The pharmaceutical composition preferably contains Pluronic
F127 copolymer as a block copolymer of oxyethylene and
oxypropylene.
[0017] The pharmaceutical composition may further contain Pluronic
L10.
[0018] The pharmaceutical composition may further contain a
pharmaceutically acceptable carrier.
[0019] The pharmaceutical composition may be in the form of a
tablet, lyophilized powder, suspension, or a capsule.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 illustrates dynamics of DIM dissolution (DIM
concentration is determined from changes in optical density).
[0021] Composition 1--DIM (control);
[0022] Composition 2--Pluronic F127 and DIM;
[0023] Composition 3--Pluronic F127, Pluronic L10, and DIM.
[0024] FIG. 2 illustrates pharmacokinetics of DIM in the plasma of
rats given the following compositions:
[0025] Composition 1--DIM (control);
[0026] Composition 2--Pluronic F127 and DIM;
[0027] Composition 3--lyophilically dried solution of Pluronic
F127, Pluronic L10, and DIM.
[0028] Composition 4--Pluronic F127 and DIM;
[0029] Composition 5--Pluronic L10 and DIM.
[0030] FIG. 3 shows the results of a morphological study of
patients with prostate gland adenoma (PGA) and prostate
intraepithelial neoplasia (PIN) before and after treatment:
[0031] Group I (18 patients) was given the DIM-base pharmaceutical
composition in accordance with the invention; and
[0032] Group II (16 patients) was given a pharmaceutical
composition containing crystalline DIM.
[0033] FIG. 4 shows the results of an immuno-histochemical
analysis, with IGF and EGF growth factors and TGF-13 regulatory
factor studied before and after administration of the
preparations:
[0034] Group I (4 patients) was given the claimed DIM-base
pharmaceutical composition; and
[0035] Group II (4 patients) was given a pharmaceutical composition
containing crystalline DIM.
DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
[0036] The block copolymers of oxyethylene and oxypropylene are
also known under the name of Pluronic and Poloxamer.
[0037] The hydrophobic-hydrophilic properties of Pluronics and
their capacity to solubilize water-insoluble compounds are
determined from the size and proportions of the
polyoxyethylene(hydrophilic) and polyoxypropylene(hydrophobic)
blocks. The following table (Table 1) shows the structural
properties of various Pluronics.
TABLE-US-00001 TABLE 1 Structural Properties of Various Pluronics
Mass of hydrophilic Content of hydrophobic Pluronic block, Da
block, mass % L10 3,200 60% L31 950 90% F35 950 50% L42 1,200 80%
L43 1,200 70% L44 1,200 60% L61 1,750 90% L62 1,750 80% L63 1,750
70% P65 1,750 50% F68 1,750 20% L72 2,050 80% P75 2,050 50% L81
2,250 90% P84 2,250 60% P85 2,250 50% F87 2,250 30% F88 2,250 20%
L92 2,750 80% F98 2,750 20% P103 3,250 70% P104 3,250 60% P105
3,250 50% F108 3,250 20% L121 4,000 90% L122 4,000 80% L123 4,000
70% F127 4,000 30% 10R5 1,000 50% 10R8 1,000 20% 12R3 1,200 70%
17R2 1,700 80% 17R2 1,700 80% 17R4 1,700 60% 17R8 1,700 20% 22R4
2,200 60% 25R1 2,500 90% 25R2 2,500 80% 25R4 2,500 60% 25R5 2,500
50% 25R8 2,500 50% 31R1 3,100 90% 31R2 3,100 80% 31R4 3,100 60%
[0038] Although the above block copolymers are used widely in
pharmaceutical and cosmetic compositions, for example, for
enhancing the solubility of hydrophobic water-insoluble compounds
(Foster B., Cosgrove T., Hammouda B. (2009), Pluronic Triblock
Copolymer Systems and Their Interactions with Ibuprofen, Langmuir,
25(12), 6760-6766), individualized approach to their use is needed
for each specific drug.
[0039] More than fifty ATP-dependent transporters capable of
influencing, in one way or another, the biological availability of
drugs are known today (Oostendorp R. L., Beijnen J. H., Schellens
J. H. (2009), The Biological and Clinical Role of Drug Transporters
at the Intestinal Barrier, Cancer Treat. Rev., 35(2), 137-147).
Moreover, the genetic polymorphism of these transporters also
contributes significantly to the variability of bioavailability of
different drugs (Nakamura T., Yamamori M., Sakaeda T. (2008),
Pharmacogenetics of Intestinal Absorption, Curr. Drug Deliv., 5(3),
153-169). The exact mechanisms of interaction between various
surface-active polymers and various transporters and their
combinations that restrict the bioavailability of different drugs
have not yet been established, and a composition having a positive
effect on the bioavailability of one active agent may be
ineffective for another agent, and conversely.
[0040] Oral bioavailability of compounds enhanced by block
copolymers of oxyethylene and oxypropylene is related to modulation
of P-glycoprotein activity and, accordingly, these block copolymers
are suggested for addition to compositions in which P-glycoprotein
substrates are the active agents (Kabanov A. V. et al., U.S. Pat.
No. 6,277,410, Copolymer Compositions for Oral Delivery), as are
several MRP formulations (Miller D. W., Batrakova E. V., Kabanov A.
V. (1999), Inhibition of Multidrug Resistance-Associated Protein
(MRP) Functional Activity with Pluronic Block Copolymers, Pharm.
Res., 16(3), 396-401). Similar properties were demonstrated for
other surface-active polymer compounds. In particular, in addition
to high solubilizing activity in respect of water-insoluble
compounds, the capacity of Solutol H15 to modulate activity of
P-glycoprotein was established (Coon J. S., Knudson W., Clodfelter
K., et al. (1991), Solutol HS 15, Nontoxic Polyoxyethylene Esters
of 12-Hydroxystearic Acid, Reverses Multidrug Resistance, Cancer
Res., 51, 897-902), making it potentially capable of being used as
an absorption enhancer of peroral drug formulations. Similar
properties were also revealed in Chremophor EL, Tween 80, TPGS
referred to above, and other similar compounds (Seelig A.,
Gerebtzoff G. (2006), Enhancement of Drug Absorption by Noncharged
Detergents Through Membrane and P-Glycoprotein Binding, Expert
Opinion on Drug Metabolism and Toxicology, 2(5), 733-752).
[0041] A direct experiment described below (Example 8) has shown
that DIM does not enhance the capture of rhodamine 123 by cells
expressing P-glycoprotein, which is an indication that DIM is not a
substrate of that transporter. This finding agrees well with the
results of the above-mentioned attempt to use TPGS as a highly
active P-glycoprotein modulator for enhancing DIM bioavailability,
an attempt that failed to enhance this parameter significantly. In
comparison, a number of surface-active polymers used in accordance
with this invention have led to a significant enhancement of oral
bioavailability of the drug.
[0042] An analysis of DIM solubility in Pluronics containing
different proportions of hydrophobic and hydrophilic blocks has
shown that polymers containing 50 mass % or more of the hydrophilic
block have a higher solubilizing capacity than polymers containing
less than 50 mass % of the hydrophilic block. Some Pluronics are
known to have a constant molecular mass of the hydrophilic element
and a variable content of oxyethylene groups.
[0043] It was discovered unexpectedly that DIM bioavailability also
depends on the molecular mass of the hydrophilic block. The higher
bioavailability is contributed by block copolymers of oxyethylene
and oxypropylene in which the content of the hydrophobic block is
under 50 mass % and the molecular mass of the hydrophilic block is
2,250 Da or more. Pluronic F127 displays the highest efficiency and
can be used to obtain stable water dispersions containing over 3
mg/ml of DIM. When this DIM composition was administered perorally
to rats its biological availability increased significantly (more
than fivefold). The proportions of the active component and
selected block copolymer may be varied depending on the desired
release time and averages between 1:2 and 1:10. The most optimal
ratio of DIM to Pluronic F127 is 1:6.
[0044] It was also found unexpectedly that Pluronic L10 (containing
about 40% of the hydrophilic block and about 3,200 in total
molecular mass), that had little effect on DIM solubility in water
and biological availability upon peroral administration to rats,
proved to be capable of enhancing significantly the effect of
Pluronic F 127 and increasing the bioavailability of the DIM
formulation more than 15-fold against control. The optimal ratio of
Pluronic F127 to Pluronic L10 was found to be within 8:1 to 1:1. It
was also a surprise that other Pluronics containing under 50% of
the hydrophilic block, for example, Pluronic P85 and Pluronic L61,
did not produce a similar effect.
[0045] Compositions of the invention may be produced, for example,
by joint or separate dissolution of components in suitable solvents
such as water, and alcohol or water-alcohol solutions, followed by
mixing the solutions in required proportions.
[0046] The resultant solutions may not necessarily be dried to
produce a solid drug formulation. Solutions are dried by any
technically suitable method or a combination of methods, including,
but not limited to, methods such as evaporation in a rotary
vaporizer or SpeedVac, lyophilic drying, or continuous flow
drying.
[0047] Ready drug formulations may be obtained by tabletizing dried
compositions with the use of necessary excipients, for example,
sodium stearate, lactose, cellulose derivatives, and so on.
[0048] Ready drug formulations may be obtained by packing dried
compositions into capsules, for example, gelatin capsules having
solid shells.
[0049] Pharmaceutical compositions containing an effective quantity
of DIM may be used to treat various diseases.
[0050] Considering the above-mentioned molecular targets of DIM, in
particular, its positive effect on the metabolism of estrogens,
restoration of apoptotic processes, and anti-proliferative,
antitumor, and anti-angiogenic activity, the compositions described
herein are suitable for treating proliferative diseases such as
uterine myoma, adenomyosis, and hyperplastic prostate diseases. We
have also demonstrated the high clinical efficiency of high
bioavailability DIM in treatment of infectious diseases of the
urogenital tract caused by intracellular infectious agents such as
Chlamydia trachomatis. Most probably, these effects are caused by
induction of programmed cellular death of epithelial cells infected
by Chlamydia trachomatis.
[0051] The effective quantity of 3,3'-diindolylmethane needed for
treatment and disease prevention may vary depending on the kind and
severity of disease and the patient's age and condition, and can be
determined by the doctor in charge on a case to case basis. The
doses used vary within 2 mg to 2,000 mg a day.
[0052] The invention is illustrated with the following
examples:
EXAMPLE 1
[0053] Determination of DIM Solubility in Aqueous Dispersions of
Various Pluronics
[0054] Preparation of Pluronic Solution
[0055] 400 mg of Pluronic, 9.7 ml of dehydrated ethyl alcohol, and
0.3 ml of distilled water were placed in a glass container. The
resultant mixture was stirred carefully in a magnetic mixer until a
transparent solution was obtained.
[0056] Preparation of DIM Solution
[0057] 10 mg of DIM was put in a glass container, and 1.0 ml of
dehydrated ethyl alcohol was added. The mixture was stirred
carefully in a magnetic mixer until a transparent solution was
obtained.
[0058] Preparation of Molecular Dispersions Containing Pluronic and
DIM
[0059] 0.5 ml of Pluronic solution (20 mg) and 0.2 ml of DIM
solution were placed in a 2 ml test tube. The resultant solution
was treated with ultrasound for 10 minutes and stirred for 1 hour.
Ethanol was removed from the resultant mixture in a rotary
vaporizer or SpeedVac, and evaporation continued in vacuum
overnight. As a result of these processes, the resultant mixture
was dissolved in 1.5 ml of distilled water and filtered, and DIM
concentration in the resultant solution was determined by the
spectrometric method. The results given in the following table
(Table 2) show that DIM had the highest solubility when Pluronic
F127 was used.
TABLE-US-00002 TABLE 2 DIM Solubility in Aqueous Dispersions of
Various Pluronics Mass of hydrophilic Content of DIM solubility in
Pluronic blocks hydrophobic blocks water, mg/ml L10 1,000 60%
>0.01 L31 950 90% >0.01 F35 950 50% 0.15 L42 1,200 80%
>0.01 L43 1,200 70% >0.01 L44 1,200 60% 0.1 L61 1,750 90%
>0.01 L62 1,750 80% >0.01 L63 1,750 70% >0.01 P65 1,750
50% 0.2 F68 1,750 20% 0.35 L72 2,050 80% >0.01 P75 2,050 50%
0.33 L81 2,250 90% >0.01 P84 2,250 60% 0.15 P85 2,250 50% 1.2
F87 2,250 30% 0.9 F88 2,250 20% 0.8 L92 2,750 80% >0.01 F98
2,750 20% 0.4 P103 3,250 70% >0.01 P104 3,250 60% 0.3 P105 3,250
50% 0.5 F108 3,250 20% 0.5 L121 4,000 90% >0.01 L122 4,000 80%
>0.01 L123 4,000 70% 0.02 F127 4,000 30% <3.0
EXAMPLE 2
[0060] Preparation of Molecular Suspensions Containing Pluronic
F127 and DIM
[0061] Preparation of Pluronic F 127 Solution
[0062] 400 mg of Pluronic F127, 9.7 ml of dehydrated ethyl alcohol,
and 0.3 ml of distilled water were placed in a glass container. The
resultant mixture was carefully stirred in a magnetic mixer until a
transparent solution was obtained.
[0063] Preparation of DIM Solution
[0064] 10 mg of DIM were placed in a glass container and 1.0 ml of
dehydrated ethyl alcohol was added. The content was carefully
stirred in a magnetic mixer until a transparent solution was
obtained.
[0065] Preparation of Molecular Suspensions Containing Pluronic
F127 and DIM
[0066] 0.5 ml of Pluronic F127 solution (20 mg) and 0.2 ml of DIM
solution were placed in a 2 ml test tube. The resultant solution
was treated with ultrasound for 10 minutes and stirred for 1 hour.
Ethanol was removed from the resultant mixture in a rotary
vaporizer or SpeedVac, and evaporation continued in vacuum
overnight. As a result of these processes, a wax-like mass was
obtained to be dissolved in distilled water to a target DIM
concentration of 3 mg in 1 ml of distilled water.
EXAMPLE 3
[0067] Preparation of Molecular Suspensions Containing Pluronic
F127, Pluronic L10, and DIM
[0068] Preparation of Pluronic L10 Solution
[0069] 250 mg of Pluronic L10 and 10 ml of dehydrated ethyl alcohol
were placed in a glass container. The resultant mixture was
carefully stirred in a magnetic mixer.
[0070] Preparation of Pluronic F 127 Solution
[0071] 400 mg of Pluronic F127, 9.7 ml of dehydrated ethyl alcohol,
and 0.3 ml of distilled water were placed in a glass container. The
resultant mixture was carefully stirred in a magnetic mixer until a
transparent solution was obtained.
[0072] Preparation of DIM Solution
[0073] 10 mg of DIM were placed in a glass container and 1.0 ml of
dehydrated ethyl alcohol was added. The content was carefully
stirred in a magnetic mixer until a transparent solution was
obtained.
[0074] Preparation of Molecular Suspensions Containing Pluronic
F127, Pluronic L10, and DIM
[0075] 0.5 ml of Pluronic F127 solution (20 mg), 0.2 ml of DIM
solution, and 0.1 ml of Pluronic L10 solution were placed in a 2 ml
test tube. The resultant solution was treated with ultrasound for
10 minutes and stirred for 1 hour. Ethanol was removed from the
resultant mixture in a rotary vaporizer or SpeedVac, and
evaporation continued in vacuum overnight. As a result of these
processes, a wax-like mass was obtained, to be dissolved in
distilled water to a target DIM concentration of 3 mg per 1 ml of
distilled water.
EXAMPLE 4
[0076] Preparation of Water-Soluble DIM Composition by
Lyophilization
[0077] 1 ml of distilled water was added to one of the DIM
solutions containing Pluronics described in Examples 1 and 2. The
mixture was stirred in a mixer until a transparent solution was
obtained. The solution was stable for 15 hours. The resultant
solution was frozen and placed in a lyophilic drier. Lyophilization
of the frozen solution produced colorless powder.
EXAMPLE 5
[0078] Preparation of DIM Composition by Spray Drying
[0079] 200 g of Pluronic F127, 300 ml of distilled water, and 10
liters of ethanol were placed in a 20-liter glass container. The
mixture was stirred until the Pluronic was dissolved completely and
a transparent solution was obtained. 25 g of Pluronic L10 and 20 g
of DIM were added to the resultant solution. The resultant mixture
was stirred until a transparent solution was obtained and was
filtered thereafter. The resultant solution was dried in a spray
drier at a temperature of 40.degree. C.
EXAMPLE 6
[0080] Preparation of a Composition by Dissolving DIM Directly in
Molten Pluronics
[0081] Pluronic F98 and Pluronic F 127, or a combination thereof in
an optimal proportion (F98 to F127 at approximately 1:4), were
mixed and melted (at 60.degree. C.), whereupon crystalline DIM was
added to the molten mass at vigorous stirring. After DIM was
dissolved, the solution was cooled rapidly to +5.degree. C. The
resultant solid mass was ground to powder.
EXAMPLE 7
[0082] Study of the Solubility of DIM-Containing Compositions
[0083] The study was done to determine the solubility of DIM
compositions obtained in Examples 2 and 3 in water. For this
purpose, 2 ml of 0.9% aqueous solution of sodium chloride was added
to each of the resultant compositions (buttons thereof contained 6
mg of DIM each) and placed in a horizontal shaker rotating at 200
revolutions per minute. 0.2 ml samples were taken periodically to
determined DIM concentration from changes in the optical density
thereof. The results of the experiments are shown in the drawing
(FIG. 1).
EXAMPLE 8
[0084] Study of the Inhibiting Activity of DIM Relative to Membrane
P-Glycoprotein
[0085] Absorption of rhodamine (Rhodamine 123 (R123) by MESSA/DX
cells expressing membrane P-glycoprotein (P-gp) was studied as a
model for experiments to be conducted. Negative P-gp MESSA/DX cells
were used as control. The cells were placed in a 96-well plate at a
concentration of 40,000 cells per well. After 24 hour of
incubation, R123 at a concentration of 3 .mu.M was added to the
cells and incubation continued for 1 hour at 37.degree. C. in the
presence of various concentrations of DIM and verapamil, a
well-known P-gp inhibitor. At the end of incubation, the solution
was removed and the cells were rinsed three times with a cooled
phosphate buffer. Rhodamine fluorescence was then measured in the
cell samples. All the experiments were run three times. As was
expected, MESSA/DX cells expressing membrane P-gp absorbed
rhodamine insignificantly in comparison with negative MESSA cells.
Verapamil, a well-known P-gp inhibitor, increases R123 accumulation
in MESSA/DX cells depending on dose, but does not affect R123
accumulation in any of the cell lines, which definitely means that
DIM is not a substrate for P-gp.
EXAMPLE 9
[0086] Pharmacokinetics of DIM in Experimental Animals given DIM
Compositions
[0087] DIM compositions prepared as in Examples 3 to 4 were used
for the purposes of this study. The results are given in the table
at the end of this example.
[0088] The compositions were administered to animals perorally in
the form of aqueous dispersions at a target DIM concentration of 3
mg in 1 ml of solvent. Crystalline DIM was administered as a
suspension of 15 mg of DIM in 5 ml of 0.5% methylcellulose in
distilled water.
[0089] Spraque-Dawley female rats weighing 250 to 350 g were used
in the experiment. All the experiments were conducted strictly in
accordance with the GLP rules. DIM formulations were administered
to the animals at 60 mg per 1 kg of body weight. Blood samples were
withdrawn at different time intervals (15, 30, 45 minutes and 1, 2,
4, 6, and 24 hours) after drug administration. The blood samples
were centrifuged immediately after withdrawal, and the plasma
separated from blood was frozen and stored at -80.degree. C.
[0090] Isofluran (Bimeta-MTC, Animal Health Inc., Cambridge, ON,
Canada). Blood was drawn from the jugular vein into
heparin-containing test tubes that were placed immediately on ice
for 5 to 10 minutes. The blood was then centrifuged to separate it
from plasma. Plasma samples were frozen and stored at -80.degree.
C.
[0091] Sample Extraction and Analysis
[0092] Plasma samples were unfrozen, centrifuged, and 100 .mu.l
aliquots were packed in plastics test tubes. The samples were then
extracted twice with 750 .mu.l of methyl-tert-butyl ester for 2
minutes while stirring at 180.degree. C. The samples were
centrifuged at 10,000 r.p.m. for 10 minutes. The supernatants were
separated and transferred to glass test tubes. The organic phase
was evaporated by nitrogen at 50.degree. C. until it was completely
dry. The dried samples were stored at -80.degree. C. The samples
studied were dissolved in 15 .mu.l of acetonitrile and 85 .mu.l of
mobile phase. The aliquots of a total volume of 20 .mu.l were then
analyzed by the HPLC method.
[0093] HPLC Performance:
[0094] C.sub.18 reverse-phase columns 50.times.4.6 mm,
Symmetry/shield 3.5 .mu.m (sorbent, grains in .mu.m), 30.degree.
C., flow speed 1.5 ml/min., injection volume 20 .mu.l, at 280
nm.
[0095] Mobile phase: Linear gradient of buffer B, from 0% to 100%,
buffer A: 5% acetonitrile, 0.1% trifluoroacetic acid, buffer B: 90%
acetonitrile, 0.1% trifluoroacetic acid, for 10 minutes.
[0096] DIM concentration was determined on a calibration curve, on
the basis of peak area (AUP). The area under the curves (AUC) was
determined from the trapezoidal rule (which is used to determine
specific integrals). The values of C. and AUC for control and
compositions are given in the following table (Table 3):
TABLE-US-00003 TABLE 3 Pharmacokinetics of DIM in various
compositions Ratio C.sub.max Ratio C.sub.max AUC.sub.0-24 h
AUC.sub.0-24 h Group Composition [.mu.g/mL] composition/control
[.mu.g h/mL] composition/control 1 Control 0.22 .+-. 0.02 3.88 .+-.
0.08 (0.5% methyl- cellulose) 2 L10/F127 (Example 4.47 .+-. 0.17
20.3 56.76 .+-. 6.25 14.6 3) 3 L10/F127 (lyo- 4.99 .+-. 0.64 22.7
58.56 .+-. 7.76 15.1 philically dried, Example 4) 4 F127 3.08 .+-.
0.17 14 21.55 .+-. 3.31 5.6 (Example 2) 5 L10 0.55 .+-. 0.04 2.5
7.75 .+-. 0.32 2.0 (Example2)
[0097] The data are presented graphically in the following drawing
(see: FIG. 2) illustrating pharmacokinetics of DIM in the plasma of
rats given the above compositions.
EXAMPLE 10
[0098] Study of Clinical Efficiency of Pharmaceutical DIM
Composition of Example 2 Purpose of the Study
[0099] The study was done to assess the clinical efficiency,
morphological effects, and safety of the new pharmaceutical DIM
composition (containing 50 mg of 3,3'-diindolylmethane in a
capsule) in comparison with a pharmaceutical composition containing
crystalline DIM (50 mg of crystalline DIM in a capsule).
[0100] The Objects of the Study were to: [0101] Assess the effect
of the preparations on the dynamics of dysfunction symptoms of the
lower urinary tracts and the quality of life of prostate adenoma
patients; [0102] Assess the effect of the preparations on the
principal urodynamic factors: maximum urine flow rate (Q.sub.max)
and residual urine volume (V.sub.res); [0103] Assess the effect of
the preparations on PSA dynamics; [0104] Assess the effect of the
preparations on prostate volume; [0105] Assess the nature of
morphological effects on prostate tissue in comparison with
placebo; and [0106] Assess the safety of the preparations on the
basis of an analysis of the frequency of undesirable events, side
effects, and dynamics of the principal biochemical parameters of
blood serum.
[0107] Tests were Conducted with:
[0108] A pharmaceutical composition containing DIM in accordance
with the invention (Group I), 2 capsules/twice a day.
[0109] A pharmaceutical composition containing crystalline DIM
(Group II), 2 capsules/twice a day.
[0110] Thirty-four patients with prostate gland adenoma (PGA) and
prostate intraepithelial neoplasia (PIN) were examined and treated
to assess the clinical efficiency, morphological effects, and
safety of the preparations. Group I (18 patients) were given 2
capsules of the DIM-containing pharmaceutical composition twice a
day, and Group II (16 patients) were given 2 capsule of the
crystalline DIM pharmaceutical composition twice a day.
[0111] The Patients were Selected for Treatment According to the
Following Criteria: [0112] outpatients and inpatients with
symptomatic and morphologically confirmed PGA and PIN; [0113] age
over 50 years; [0114] patients who gave written consent and
followed doctor's instructions on the treatment prescribed; [0115]
symptom manifestation over 7 on the I-PS S scale; [0116] Q.sub.max
over 5 and less than 15 ml/sec.; [0117] Residual urine not more
than 200 ml; [0118] Prostate volume over 25 cm.sup.3; and [0119]
PSA up to 10 ng/ml.
[0120] Assessment of the Effect of the Preparation on Clinical
Condition
[0121] At the starting point in time (V1), two morphological
characteristics, L-PIN and H-PIN, were recorded in both groups. A
patient's condition deteriorated, was unchanged, or improved under
the effect of the preparation. In all, there were seven possible
variants of clinical response within the time interval between V1
and V2. In this case, it is possible to make assessment of the
clinical change (between V1 and V2) against the ordinal scale. The
Mann-Whitney criterion is the most sensitive of all for comparing
clinical change in the group studied.
[0122] Changes in the morphological study data for all patients are
shown in the drawing (see: FIG. 3) that illustrates change in the
morphological characteristic in Groups I and II in the course of
the study.
[0123] On the basis of the dual-sided level of significance of the
therapeutic effect, Group I (that was given the DIM-containing
pharmaceutical composition according to the invention) and Group II
(given a pharmaceutical composition containing crystalline DIM)
differed significantly (p=0.002).
[0124] Comparison of Malignity Frequency
[0125] A separate study was conducted to explore the difference in
the malignity frequency in the groups studied. No instances of
malignity were observed in the core group (its 18 patients were
given the DIM-containing pharmaceutical composition in a new
formulation). Four instances of prostate cancer were recorded in
the control group (16 patients were given a pharmaceutical
composition containing crystalline DIM). Considering a probable
error of chi-square approximation, the more accurate Fischer
criterion was used.
[0126] Conclusion: The groups studied differed in the frequency of
malignity, and their differences were statistically significant
(p=0.039).
[0127] Assessment of the effect of the claimed DIM-containing
pharmaceutical composition on immuno-histochemical data:
[0128] An immuno-histochemical analysis was carried out in two
groups of four patients each from Group I given the DIM-containing
pharmaceutical composition in a new formulation and Group II that
received a pharmaceutical composition containing crystalline DIM,
respectively. Assessment was made of factors such as IGF and EGF
growth factors and TGF-.beta. regulation factor before and after
administration of the preparations studied. The starting values of
IGF, EGF and TGF-.beta. being statistically uniform, the following
differences in the level of these parameters were recorded upon
further measurement:
[0129] A statistically reliable decrease in the growth factors IGF
(p=0.004) and EGF (p=0.002) was recorded, as also was an increase
in the level of TGF-.beta. (p=0.047) in the group of patients
taking the DIM-containing pharmaceutical composition in a new
formulation. No reliable dynamics were registered in the control
group. The data obtained are shown graphically in the drawing below
(see: FIG. 4).
[0130] Conclusion
[0131] The data obtained for a reliable decrease in the IGF and EGF
growth factors and an increase in the TGF-.beta. level in the group
of patients taking the claimed DIM-containing pharmaceutical
composition point to the effect of the active agent of the
preparation on the principal signaling mechanisms of pathological
cellular proliferation, and also to the induction of apoptosis of
transformed cells.
[0132] No side effects or undesirable events were registered during
treatment.
[0133] The DIM-containing pharmaceutical composition in a new
formulation shows an anti-proliferative activity in patients with
prostate gland adenoma and prostate intraepithelial neoplasia.
[0134] The DIM-containing pharmaceutical composition in a new
formulation is a safe drug to treat PGA and PIN because it had no
side effects and undesirable events during the treatment
period.
* * * * *