U.S. patent application number 14/329923 was filed with the patent office on 2016-01-14 for formulations and compositions of vitamin d analogs for treating and preventing cancer and other diseases.
This patent application is currently assigned to Aphios Corporation. The applicant listed for this patent is Aphios Corporation. Invention is credited to Trevor P. Castor.
Application Number | 20160008377 14/329923 |
Document ID | / |
Family ID | 55066200 |
Filed Date | 2016-01-14 |
United States Patent
Application |
20160008377 |
Kind Code |
A1 |
Castor; Trevor P. |
January 14, 2016 |
FORMULATIONS AND COMPOSITIONS OF VITAMIN D ANALOGS FOR TREATING AND
PREVENTING CANCER AND OTHER DISEASES
Abstract
This invention is for formulations of analogs of the non-toxic
and inert Vitamin D3, its non-toxic and mostly inert pre-hormone
and its toxic and biologically active hormone, and for using these
formulations for preventing and treating certain cancers such as
breast, prostate, ovarian, kidney, renal and other cancers, Vitamin
D deficiency, autoimmune disease such as Multiple Sclerosis,
hypertension, osteoporosis, bone diseases, rickets, psoriasis and
infectious diseases. This invention also discloses compositions of
the analogs of the non-toxic and inert Vitamin D3 and the non-toxic
and mostly inert Vitamin D3 pre-hormone.
Inventors: |
Castor; Trevor P.;
(Arlington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Aphios Corporation |
Woburn |
MA |
US |
|
|
Assignee: |
Aphios Corporation
Woburn
MA
|
Family ID: |
55066200 |
Appl. No.: |
14/329923 |
Filed: |
July 12, 2014 |
Current U.S.
Class: |
424/450 ;
424/452; 514/167; 549/560; 552/653 |
Current CPC
Class: |
A61K 9/4866 20130101;
A61K 31/593 20130101; A61K 9/5153 20130101; A61K 9/127 20130101;
A61K 9/0019 20130101; A61K 9/4875 20130101; A61K 9/4858
20130101 |
International
Class: |
A61K 31/593 20060101
A61K031/593; A61K 9/48 20060101 A61K009/48; A61K 9/127 20060101
A61K009/127 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] Research leading to this invention was in part funded by the
National Cancer Institute, National Institutes of Health, Bethesda,
Md., USA.
Claims
1. A method of treating a disease responsive to Vitamin D3 or
Vitamin D3 analog comprising the steps of: a. Providing a
medicament having a plurality of nanosomes having at least one
bilayer of phospholipid in which a Vitamin D3 or Vitamin D3 analog
is distributed; and b. Administering an amount of medicament having
an effective amount of Vitamin D3 or Vitamin D3 analog to treat
such disease.
2. The method of claim 1 wherein said disease is selected from the
group consisting of cancer, Vitamin D deficiency, autoimmune
disease, hypertension, osteoporosis, bone disease, psoriasis and
infectious disease.
3. The method of claim 1 wherein said Vitamin D.sub.3 analog is
selected from the group consisting of the bromoacetate derivative
(1.alpha.,25-dihydroxyvitamin D.sub.3-3-bromoacetate
[1,25(OH).sub.2D.sub.3-3-BE]) of the active Vitamin D.sub.3 hormone
(AMPI-109), the bromoacetate derivative (25-hydroxyvitamin
D.sub.3-3-bromoacetate[25-OH-D.sub.3-3-BE]) of the non-toxic
pre-hormonal form of Vitamin D.sub.3 (AMPI-105), the epoxide
derivative (25-hydroxyvitamin D.sub.3-3-epoxide
[25-OH-D.sub.3-3-EPO]) of the non-toxic pre-hormonal form of
Vitamin D.sub.3 (AMPI-106), the epoxide derivative (Vitamin
D.sub.3-3-epoxide [D.sub.3-3-EPO]) of the non-toxic and inert
Vitamin D.sub.3 (AMPI-107) and the bromoacetate derivative (Vitamin
D.sub.3-3-bromoacetate [D.sub.3-3-BE]) of the non-toxic and inert
Vitamin D.sub.3 (AMPI-108).
4. A method of treating a disease responsive to Vitamin D3 or
Vitamin D3 analog comprising the steps of: a. Providing a gel
capsule having a gelatin outer layer defining an inner volume and
having an oil base contained in said inner volume, and further
comprising Vitamin D3 or Vitamin D3 analog dissolved in said oil
base; and b. Administering an effective amount of Vitamin D3 analog
by ingesting one or more gel capsules.
5. The method of claim 4 wherein said disease is selected from the
group consisting of cancer, Vitamin D deficiency, autoimmune
disease, hypertension, osteoporosis, bone disease, psoriasis and
infectious disease.
6. The method of claim 4 wherein said Vitamin D.sub.3 analog is
selected from the group consisting of the bromoacetate derivative
(1.alpha.,25-dihydroxyvitamin D.sub.3-3-bromoacetate
[1,25(OH).sub.2D.sub.3-3-BE]) of the active Vitamin D.sub.3 hormone
(AMPI-109), the bromoacetate derivative (25-hydroxyvitamin
D.sub.3-3-bromoacetate[25-OH-D.sub.3-3-BE]) of the non-toxic
pre-hormonal form of Vitamin D.sub.3 (AMPI-105), the epoxide
derivative (25-hydroxyvitamin D.sub.3-3-epoxide
[25-OH-D.sub.3-3-EPO]) of the non-toxic pre-hormonal form of
Vitamin D.sub.3 (AMPI-106), the epoxide derivative (Vitamin
D.sub.3-3-epoxide [D.sub.3-3-EPO]) of the non-toxic and inert
Vitamin D.sub.3 (AMPI-107) and the bromoacetate derivative (Vitamin
D.sub.3-3-bromoacetate [D.sub.3-3-BE]) of the non-toxic and inert
Vitamin D.sub.3 (AMPI-108).
7. As a composition of matter, a composition comprising an analog
of the non-toxic and inert Vitamin D.sub.3 selected from the group
consisting of Vitamin D.sub.3-3-epoxide [D.sub.3-3-EPO] and Vitamin
D.sub.3-3-bromoacetate [D.sub.3-3-BE].
8. A composition of claim 7 where such Vitamin D analog is Vitamin
D.sub.3-3-epoxide [D.sub.3-3-EPO].
9. A composition of claim 7 where such Vitamin D analog is the
Vitamin D.sub.3-3-bromoacetate [D.sub.3-3-BE].
Description
PRIORITY
[0001] This application claims priority to U.S. provisional
application Ser. No. 61/845,980.
FIELD OF THE INVENTION
[0003] This invention relates to formulations and compositions of
Vitamin D analogs for the prevention and treatment of cancers and
other diseases to minimize the toxic side-effects of the Vitamin D
hormone while improving therapeutic index. The formulation methods
feature supercritical, critical and near-critical fluids with and
without polar cosolvents. This invention also discloses
compositions of the analogs of the non-toxic and inert Vitamin D3
and the non-toxic and mostly inert Vitamin D3 pre-hormone.
BACKGROUND OF THE INVENTION
[0004] Vitamin D is the general name for a collection of natural
sterol-like substances including vitamin D2 and D3. As shown in
FIG. 1, Vitamin D3 is synthesized in the skin from
7-dehydrocholesterol, a cholesterol breakdown product, via
photochemical reactions using ultraviolet (UV) radiation from
sunlight. The inert vitamin D3 is first converted to a largely
inert intermediate by the liver to 25-HydroxyVitamin D3 (25-OH-D3)
and then converted by the kidney to the bioactive hormone
1-25-DihydoxyVitamin D.sub.3 (1,25(OH).sub.2D.sub.3) (FIG. 1). The
bioactive vitamin D hormone, 1,25(OH).sub.2D.sub.3, mediates its
action by binding to vitamin D receptor (VDR) that is principally
located in the nuclei of the target cell.
[0005] Vitamin D is a natural molecule that is biosynthesized by
the interaction of sunlight with 7-dehydrochoelsterol in the
epidermis. 1,25-dihydroxyvitamin D.sub.3 (1,25(OH).sub.2D.sub.3
named calcitriol), the dihydroxylated metabolite of vitamin D3 is
an essential nutrient for skeletal health. Calcitriol has profound
effects on the growth and maturation of normal and malignant cells.
Several epidemiological studies have demonstrated that people who
live in higher latitudes are at higher risk of developing and dying
of many cancers, including prostate cancer. It has also been
demonstrated that there is an inverse relationship between
latitude, sun-exposure and cutaneous synthesis of vitamin D (20).
In 1989, Garland et al. carried out an eight-year prospective study
among 26,520 healthy adults to demonstrate that if the initial
level of serum of calcifediol [25-hydroxyvitamin D.sub.3
(25-OH-D.sub.3)], the mono-hydroxylated pre-hormonal form of
calcitriol is at least 20 ng/ml, there is a 50% reduced risk of
developing colon cancer. Since this observation other investigators
have confirmed latitudinal impact and vitamin D intake on reducing
risk of various cancers, including breast, prostate, renal and
ovary. Vitamin D deficiency has also been correlated with
autoimmune disease such as Multiple Sclerosis, hypertension,
osteoporosis, bone diseases, rickets, psoriasis and infectious
diseases.
[0006] Prostate cancer (PCA) is the most prevalent cancer among
men; and the second leading cause of cancer death among men in the
US. More than 500,000 PCA cases are diagnosed each year, 1 in 6
American males will develop PCA and 30,000 die each year in the US.
Current clinical interventions for PCA include surgical removal of
prostate and radiation therapy, with adverse side effects such as
impotence, incontinence and alopecia. The mainstay of
hormone-sensitive prostate cancer (HSPCA) chemotherapy is
androgen-deprivation. After 9 to 30 months, HSPCA usually becomes
insensitive to hormonal therapy and rapidly leads to HRPCA for
which there are few interventions except for Sanofi-Aventis'
Taxotere.RTM. (docetaxel) that has problems of toxicity and other
adverse side-effects. New drugs have been recently approved for
castration-resistant, docetaxel-refractory prostate cancers that
extend life by 4.8 months.
[0007] Numerous studies have registered strong promise of
calcitriol as a therapeutic agent for prostate and other cancers.
However, its clinical use has been limited by risk of toxicity
related to hypercalcemia, hypercalciuria, and significant loss of
body weight. Attempts to address the toxicity-issue have taken two
paths. In the first, combinations of calcitriol with standard
chemotherapeutic agents are being investigated to harness synergy
between these compounds. For example, clinical and animal studies
have been carried out demonstrate that toxic effects of calcitriol
can be mitigated by a combination with dexamethasone or
paclitaxel.
[0008] Several attempts have been made to develop less/non-toxic
analogs of calcitriol with potent antiproliferative activities as
potential therapeutic agents. A Phase II clinical trial evaluated
Seocalcitol (EB-1089), a side-chain analog of the active vitamin D
hormone, in patients with inoperable pancreatic cancer. No
objective responses (anti-tumor) activity was observed; the most
frequent toxicity was dose-dependent hypercalcemia with most
patients tolerating a dose of 10-15 .mu.g/day in chronic
administration.
[0009] The nuclear vitamin D receptor (VDR) plays a central role in
the cell signaling process leading to anti-proliferation, and in
some cases apoptosis of cancer cells. In this respect calcitriol is
very similar to other steroidal and non-steroidal hormones such as
estrogen, androgens, retinoids, glucocorticoids etc. Furthermore,
VDR has high structural homology with nuclear receptors of other
hormones. It is well established that cellular regulation by
calcitriol and its analogs are initiated by highly specific binding
to VDR, which is translated into pro-differentiation and
concomitant antiproliferation of cells. Most human prostate cancer
cells contain VDR; and numerous studies have shown that several
prostate cancer cells respond to calcitriol. These findings
strongly support the use of vitamin D-based agents for first line
therapy and/or second line therapy when androgen deprivation
fails.
[0010] However, cancer-therapy with calcitriol is limited by its
rapid catabolic degradation by CYP-hydroxylases, which reduces its
potency. As a result high doses of calcitriol are required
clinically to harness its beneficial property; but such
pharmacological doses cause toxicity. A way of circumventing this
problem will be to covalently attach calcitriol into the
ligand-binding pocket of VDR as shown in FIG. 2, so that (i)
calcitriol is prevented from interacting with catabolic enzymes;
and (ii) VDR-mediated transcriptional process could be set in
motion since the ligand is inside the ligand-binding pocket of VDR
leading to conformational changes required for the transcriptional
process.
[0011] During the past decade hundreds of vitamin D analogs have
been synthesized with the goal of obtaining a better
antitumor/toxicity ratio and tumor-specific effect. Although a few
of these analogs have successfully completed preclinical studies
for several cancers; and at least one analog has recently failed
Phase II clinical trials for pancreatic carcinomas, the majority of
these compounds have been proved to be of limited therapeutic value
due to toxicity. As a result new strategies for developing such
analogs are required.
[0012] Aspects of the present invention employ materials known as
supercritical, critical or near-critical fluids. A material becomes
a critical fluid at conditions which equal its critical temperature
and critical pressure. A material becomes a supercritical fluid at
conditions which equal or exceed both its critical temperature and
critical pressure. The parameters of critical temperature and
critical pressure are intrinsic thermodynamic properties of all
sufficiently stable pure compounds and mixtures. Carbon dioxide,
for example, becomes a supercritical fluid at conditions which
equal or exceed its critical temperature of 31.1.degree. C. and its
critical pressure of 72.8 atm (1,070 psig). In the supercritical
fluid region, normally gaseous substances such as carbon dioxide
become dense phase fluids which have been observed to exhibit
greatly enhanced solvating power. At a pressure of 3,000 psig (204
atm) and a temperature of 40.degree. C., carbon dioxide has a
density of approximately 0.8 g/cc and behaves much like a nonpolar
organic solvent, having a dipole moment of zero Debyes.
[0013] A supercritical fluid displays a wide spectrum of solvation
power as its density is strongly dependent upon temperature and
pressure. Temperature changes of tens of degrees or pressure
changes by tens of atmospheres can change a compound solubility in
a supercritical fluid by an order of magnitude or more. This
feature allows for the fine-tuning of solvation power and the
fractionation of mixed solutes. The selectivity of nonpolar
supercritical fluid solvents can also be enhanced by addition of
compounds known as modifiers (also referred to as entrainers or
cosolvents). These modifiers are typically somewhat polar organic
solvents such as acetone, ethanol, methanol, methylene chloride or
ethyl acetate. Varying the proportion of modifier allows wide
latitude in the variation of solvent power.
[0014] In addition to their unique solubilization characteristics,
supercritical fluids possess other physicochemical properties which
add to their attractiveness as solvents. They can exhibit
liquid-like density yet still retain gas-like properties of high
diffusivity and low viscosity. The latter increases mass transfer
rates, significantly reducing processing times. Additionally, the
ultra-low surface tension of supercritical fluids allows facile
penetration into microporous materials, increasing extraction
efficiency and overall yields.
[0015] A material at conditions that border its supercritical state
will have properties that are similar to those of the substance in
the supercritical state. These so-called "near-critical" fluids are
also useful for the practice of this invention. For the purposes of
this invention, a near-critical fluid is defined as a fluid which
is (a) at a temperature between its critical temperature (T.sub.c)
and 75% of its critical temperature and at a pressure at least 75%
of its critical pressure, or (b) at a pressure between its critical
pressure (P.sub.c) and 75% of its critical pressure and at a
temperature at least 75% of its critical temperature. In this
definition, pressure and temperature are defined on absolute
scales, e.g., Kelvin and psia. To simplify the terminology,
materials which are utilized under conditions which are
supercritical, near-critical, or exactly at their critical point
with or without polar co-solvents such as ethanol will jointly be
referred to as "SuperFluids.TM." or referred to as "SFS."
SuperFluids.TM. were used for the nanoencapsulation of the Vitamin
D analog in the protective lipid layer of phospholipid
nanosomes.
SUMMARY OF THE INVENTION
[0016] Embodiments of the present invention are directed to the
composition, formulation and use of Vitamin D analogs, that bind
tightly into the Vitamin D receptor, and can be used
therapeutically at lower doses than their Vitamin D counterparts,
and are such less toxic than their Vitamin D counterparts.
[0017] AMPI-109 is a bromoacetate derivative
(1.alpha.,25-dihydroxyvitamin D.sub.3-3-bromoacetate
[1,25(OH).sub.2D.sub.3-3-BE]) the active Vitamin D3 hormone (FIG.
3).
[0018] AMPI-105 is a bromoacetate derivative (25-hydroxyvitamin
D.sub.3-3-bromoacetate[25-OH-D.sub.3-3-BE]) of the non-toxic
pre-hormonal form of Vitamin D.sub.3 (FIG. 4).
[0019] AMPI-106 is the epoxide derivative (25-hydroxyvitamin
D.sub.3-3-epoxide [25-OH-D.sub.3-3-EPO]) of the non-toxic
pre-hormonal form of Vitamin D.sub.3 (FIG. 5).
[0020] AMPI-107 is the epoxide derivative (D.sub.3-3-epoxide
[D.sub.3-3-EPO]) of the non-toxic and inert Vitamin D.sub.3 (FIG.
6).
[0021] Embodiments of the present invention are directed to
formulations of these Vitamin D analogs to prolong circulation time
while reducing systemic toxicity and enhancing therapeutic
index.
[0022] In order to prolong circulation time while reducing systemic
toxicity and enhancing therapeutic index, the less toxic Vitamin D
analogs are nanoencapsulated within the lipid bilayer of
phospholipid nanosomes. Nanoencapsulation also enhances serum
stability. Phospholipid nanosomes will also protect the ester bond
from hydrolysis increasing the half-life of Vitamin D analogs.
[0023] Nanoencapsulation allows Vitamin D analogs to kinetically
engage VDR to increase the half-life of calcitriol, thereby
potentially increasing its potency with less toxicity.
[0024] Using SCCNC fluids, AMPI-109 was encapsulated into
phospholipid nanosomes (APH-0701), which were .about.100 to 200 nm
in size, had high encapsulation efficiencies around 75%, with
passive in vitro release rates of .about.3 days.
[0025] APH-0701 was found to be stable in human serum and mouse
liver homogenates.
[0026] Both AMPI-109 and APH-0701 were effective in reducing
tumor-size in mouse xenograft models of DU-145
(androgen-insensitive) tumors. Compared to a nanosomal vehicle
control, AMPI-109 and APH-0701 reduced tumor size approximately 37%
and 49%.
[0027] Gross body-weights of AMPI-109 and APH-0701-treated animals
were not significantly different from control animals, indicating
lack of gross toxicity.
[0028] Collectively these results demonstrated that AMPI-109 and
APH-0701 have a strong translational potential as a therapeutic
agent in androgen-insensitive prostate cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 depicts Biosynthesis of inert Vitamin D.sub.3 in
skin, conversion into the largely inert pre-hormone in the liver
and the highly bioactive hormone in the kidney.
[0030] FIG. 2 depicts Cross-linking of 1,25(OH).sub.2D.sub.3-3-BE
(AMPI-109) into the VDR-ligand binding pocket via Cys.sub.288.
[0031] FIG. 3 depicts the bromoacetate derivative
(1.alpha.,25-dihydroxyvitamin D.sub.3-3-bromoacetate
[1,25(OH).sub.2D.sub.3-3-BE]) of the active Vitamin D.sub.3 hormone
(AMPI-109).
[0032] FIG. 4 depicts the bromoacetate derivative
(25-hydroxyvitamin D.sub.3-3-bromoacetate[25-OH-D.sub.3-3-BE]) of
the non-toxic pre-hormonal form of Vitamin D.sub.3 (AMPI-105).
[0033] FIG. 5 depicts the epoxide derivative (25-hydroxyvitamin
D.sub.3-3-epoxide [25-OH-D.sub.3-3-EPO]) of the non-toxic
pre-hormonal form of Vitamin D.sub.3 (AMPI-106).
[0034] FIG. 6 depicts the epoxide derivative (Vitamin
D.sub.3-3-epoxide [D.sub.3-3-EPO]) of the non-toxic and inert
Vitamin D.sub.3 (AMPI-107).
[0035] FIG. 7 depicts AMPI-109 Standard Curve.
[0036] FIG. 8 depicts SFS-CFN Apparatus.
[0037] FIG. 9 depicts Particle Size Analysis of APH-0701-27-02
Nanosomes.
[0038] FIG. 10 depicts Photomicrograph of APH-0701-27-02.
[0039] FIG. 11 depicts SEC Separation of APH-0701-27-02.
[0040] FIG. 12 depicts Release of AMPI-109 from APH-0701-27-02.
[0041] FIG. 13 depicts LNCaP cells: Antiproliferation assay with
AMPI-109 and APH-0701.
[0042] FIG. 14 depicts antiproliferative and cytotoxic activity of
AMPI-109 (Vitamin D.sub.3-3-epoxide [D.sub.3-3-EPO]) vs Calcitriol
(1,25(OH).sub.2D.sub.3) in normal kidney cells by .sup.3H-thymidine
incorporation assay.
[0043] FIG. 15 depicts antiproliferative and cytotoxic activity of
AMPI-109 (Vitamin D.sub.3-3-epoxide [D.sub.3-3-EPO]) vs Calcitriol
(1,25(OH).sub.2D.sub.3) in kidney cancer cells by .sup.3H-thymidine
incorporation assay.
[0044] FIG. 16 depicts DU-145 cells treated with various doses of
Calcitriol or AMPI-109 or ethanol (vehicle) for 20 hours followed
by .sup.3H-thymidine incorporation assay.
[0045] FIG. 17 depicts Effect of AMPI-109, Calcitriol or EB-1089 on
the growth of DU-145 cells.
[0046] FIG. 18 depicts Effect of 1,25(OH).sub.2D.sub.3-3-BE and
1,23(OH).sub.2D.sub.3 (Hormone) (q.o.d..times.10, i.p.) on tumor
volume in athymic DU-145 mice.
[0047] FIG. 19 depicts Effect of 1,25(OH).sub.2D.sub.3-3-BE and
1,23(OH).sub.2D.sub.3 (Hormone) (q.o.d..times.10, i.p.) on Body
Weight in athymic DU-145 mice.
[0048] FIG. 20 depicts Effect of 1,25(OH).sub.2D.sub.3-3-BE
(q.o.d..times.10, p.o.) on tumor volumes against tumor model DU-145
in athymic mice.
[0049] FIG. 21 depicts Effect of 1,25(OH).sub.2D.sub.3-3-BE
(q.o.d..times.10, p.o.) on body weights in tumor model DU-145 in
athymic mice.
[0050] FIG. 22 depicts Tumor Size of Androgen-Insensitive Tumor
(DU-145) Bearing Athymic Mice Treated with
1,25(OH).sub.2D.sub.3-3-BE, Liposomal 1,25(OH).sub.2D.sub.3-3-BE
and Vehicle Control.
[0051] FIG. 23 depicts Body Weight of Androgen-Insensitive Tumor
(DU-145) Bearing Athymic Mice Treated with
1,25(OH).sub.2D.sub.3-3-BE, Liposomal 1,25(OH).sub.2D.sub.3-3-BE
and Vehicle Control.
[0052] FIG. 24 depicts .sup.3H-Thymidine Incorporation Assays of
Keratinocytes, MCF-7, PZ-HPV-7, LNCaP and PC-3 Cells.
[0053] FIG. 25 depicts Cell Counting Assay of LAPC-4, LNCaP, MCF-7
and MC3T3 Cells Treated with 25-0H-D.sub.3-BE or
1,25(OH).sub.2D.sub.3.
[0054] FIG. 26 depicts Effect of 25-OH-D.sub.3-3-BE (every 3 days,
starting on day 11 and ending on day 31, p.o.) on tumor volumes
against tumor model DU-145 in athymic mice.
[0055] FIG. 27 depicts Effect of 25-OH-D.sub.3-3-BE (every 3 days,
starting on day 11 and ending on day 31, p.o.) on body weights of
tumor model DU-145 in athymic mice.
[0056] FIG. 28 depicts Effect of 24 h Treatment on WPMY-1 Cells
(Mean and SEM) by AMPI-107 and Calcitrol.
[0057] FIG. 29 depicts Antiproliferative evaluation of AMPI-107 vs.
Calcitrol in DU-145 Prostate Cancer Cells.
[0058] FIG. 30 depicts Antiproliferative evaluation of AMPI-107 vs.
Calcitrol in LNCaP Prostate Cancer Cells.
[0059] FIG. 31 depicts Antiproliferative evaluation of AMPI-107 vs.
Calcitrol in PC-3 Prostate Cancer Cells.
[0060] FIG. 32 depicts Effect of AMPI-109 (Vitamin
D.sub.3-3-epoxide [D.sub.3-3-EPO]) vs Calcitriol
(1,25(OH).sub.2D.sub.3) (q.o.d..times.10, i.p.) on tumor volumes
against tumor model DU-145 in athymic mice.
[0061] FIG. 33 depicts Effect of AMPI-109 (Vitamin
D.sub.3-3-epoxide [D.sub.3-3-EPO]) vs Calcitriol
(1,25(OH).sub.2D.sub.3) AMPI-017 (q.o.d..times.10, i.p.) on body
weights of tumor model DU-145 in athymic mice.
[0062] FIG. 34 depicts Effect of AMPI-109 (Vitamin
D.sub.3-3-epoxide [D.sub.3-3-EPO]) vs Calcitriol
(1,25(OH).sub.2D.sub.3) (q.o.d..times.10, p.o.) on tumor volumes
against tumor model DU-145 in athymic mice.
[0063] FIG. 35 depicts Effect of AMPI-109 (Vitamin
D.sub.3-3-epoxide [D.sub.3-3-EPO]) vs Calcitriol
(1,25(OH).sub.2D.sub.3) AMPI-017 (q.o.d..times.10, p.o.) on body
weights of tumor model DU-145 in athymic mice.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
AMPI-109 (1.alpha.,25-dihydroxyvitamin
D.sub.3-3-bromoacetate[1,25(OH).sub.2D.sub.3-3-BE])
[0064] The Vitamin D analog 1.alpha.,25-dihydroxyvitamin
D.sub.3-3-bromoacetate[1,25(OH).sub.2D.sub.3-3-BE]), AMPI-109, is a
derivative of calcitriol covalently links calcitriol inside the
ligand-binding pocket of VDR via a cysteine residue as shown in
FIG. 2. We also observed that such a process constitutively
activated VDR. Thus, AMPI-109 became a significantly stronger
anti-proliferative agent than calcitriol on a mole-per-mole basis
in LNCaP, PC-3 and DU-145 prostate cancer cells (6.5 more times for
DU-145 in hormone refractory prostate cancer (HRPCA) animal
models). AMPI-109 is a significantly stronger antiproliferative
agent than EB-1089, a side-chain analog of calcitriol that
underwent clinical trials, in DU-145 cancers. In addition, AMPI-109
induced apoptosis in these cells. Furthermore, in in vivo studies,
AMPI-109 produced strong anti prostate tumor effect without
inducing significant toxicity in athymic mice. Therefore, AMPI-109
demonstrates a strong translational potential as a therapeutic
agent for prostate cancer.
[0065] There may be concerns that AMPI-109 may: (i) behave like
protein/DNA alkylating compounds with significant side effects at
pharmacological doses; (ii) generate adverse immune responses; and
(iii) be prematurely hydrolyzed since it contains an ester bond.
Unlike protein/DNA alkylating compounds such as estramustine and
lomustine that are non target-specific and produce significant side
effects particularly at pharmacological doses, AMPI-109 will
interact with specific targets and cross-link to the
substrate/ligand-binding sites of enzymes and receptors and thus,
will have less side effects. Adverse immune response of the
alkylating agents, such as AMPI-109, is difficult to predict.
Calcitriol and its analogs are touted as potential drug-candidates
for immune-deficiency diseases, such as Type I diabetes. Therefore,
AMPI-109 is expected, if anything to show a positive immune
response. Since AMPI-109 contains an ester bond, hydrolysis would
produce calcitriol and bromoacetic acid; such a phenomenon might
limit bioavailability of the intact molecule. This phenomenon is
minimized by nanoencapsulation of the Vitamin D analog in the
protective lipid membrane of the nanoparticles. In a cellular
proliferation study, the antiproliferative property of AMPI-109 is
due solely to its un-hydrolyzed and intact form.
[0066] The covalent attachment of calcitriol into the
ligand-binding pocket of VDR prevents the catabolism of calcitriol
because it will be sitting deep inside the binding pocket, and will
be inaccessible to catabolizing CYP enzymes.
[0067] DU-145 is a highly aggressive androgen-insensitive human
prostate cancer cell line that does not respond well to calcitriol
due to increased expression of CYP 24-OHase and rapid catabolism.
AMPI-109 shows a strong and dose-dependent antiproliferative effect
in DU-145 cells, while calcitriol shows no effect. By decreasing
catabolism of calcitriol by first protecting its analog in the
lipid layer and then cross-linking it to the ligand-binding pocket
of VDR (via AMPI-109), the potency of the hormone is significantly
increased. AMPI-109 also modulate messages for human osteocalcin
and CYP 24-OHase (genes that are involved in the VDR-mediated
mechanism) in keratinocytes similar to clacitriol. The message for
24-OHase is up regulated by calcitriol and AMPI-109 in LNCaP cells,
and this message is obliterated by ZK 159222, a clacitriol
antagonist. These results strongly indicate that cellular effects
of AMPI-109 follow a mechanism similar to that of calcitriol.
Synthesis of AMPI-109
[0068] 1,25(OH).sub.2D.sub.3-3-BE (AMPI-109) shown in FIG. 3 was
synthesized. The structure of AMPI-109 was confirmed by proton and
.sup.13C NMR; the molecular weight was established by mass spectral
analysis to be 536.25; and the purity determined to be >98.3% at
265 nm by reversed phase HPLC.
Analysis of AMPI-109
[0069] AMPI-109 and the formulated product were analyzed by HPLC.
The HPLC method utilized a Phenomenex Luna C18(2), 100 A, 5 micron,
150.times.4.6 mm HPLC column (P/No.: OOF-4252-EO; S/No. 425310-16),
a mobile phase consisting of 95% acetonitrile:5% water, a flow rate
of 1.0 mL/min, a column temperature of 30.degree. C., an injection
volume of 20 .mu.L and a run time of 10 minutes with monitoring at
265 nm. AMPI-109's standard curve is shown in FIG. 7. A system
suitability requirement of Plates >4,000 was established for the
method. The limit of detection (LOD) was determined to be 0.013 ppm
and limit of quantification (LOQ) to be 0.04 ppm. Injection
replication was determined to have a root square difference (RSD)
of 0.21%.
Formulation of AMPI 109
[0070] We utilized the SuperFluids.TM. critical fluid nanosome
(CFN) process for the formation of small, uniform liposomes
(nanosomes) for encapsulating AMPI-109. Liposomal preparations are
identified as AMPI-109 (L) and APH-0701.
[0071] Twenty-seven encapsulation runs were performed in the
SFS-CFN apparatus shown schematically as FIG. 8. In a typical
SFS-CFN experiment, the solids chamber was charged with
dimyristoyl-phosphatidylcholine (DMPC) or phosphatidylcholine (PC)
and cholesterol and placed inline within the apparatus. The molar
ratio of lipid:cholesterol:drug was designed to be 20:1:1. The
system was then pressurized between 2,000 and 3,000 psig with a SFS
(Freon 23 or propane) and heated to the desired temperature (40 to
60.degree. C.). The lipids were dissolved into the SFS through
circulation of the SFS within the upper high pressure circulation
loop in the apparatus for .about.60 min, before adding AMPI-109
dissolved in ethanol (EtOH) via an injection port into the high
pressure circulation loop. After a specific residence time, the
resulting mixture was decompressed via a backpressure regulator
(valve) though a dip tube with a nozzle into a decompression
chamber (vessel B), which contained a buffer such as a 10% sucrose
solution. After decompression through the nozzle, the
SuperFluids.TM. were evaporated off leaving an aqueous solution of
nanosomes entrapping the hydrophobic AMPI-109 within the lipid
bilayers, forming APH-0701.
[0072] Three samples were typically taken: depressurization at
constant pressure, depressurization from operating pressure to 400
psig, and depressurization from 400 psig to atmospheric pressure.
Most of the AMPI-109 was contained in the second fraction. This
fraction was the sterile filtered through a 0.2 .mu.m polycarbonate
or a nitroplus cellulosic filter using compressed N2. The filtrates
were checked for AMPI-109 content as well as for particle size. In
a typical example, after filtration, the sterile samples were
dispensed with a sterile, disposable pipette in 5 mL aliquots into
sterile 20 mL vials and frozen at -80.degree. C. The samples were
then freeze-dried overnight and weighed. Lyophilized samples were
then reconstituted in 5 mL DI-H.sub.2O, sonicated for 20 seconds
three times, and analyzed for particle size and AMPI-109
content.
AMPI-105 and AMPI-106 (25-OH-D.sub.3 Analogs)
[0073] AMPI-105 and AMPI-106 are analogs of 25-HydroxyVitamin
D.sub.3 (25-OH-D.sub.3). AMPI-105 is a bromoacetate derivative
(25-hydroxyvitamin D.sub.3-3-bromoacetate[25-OH-D.sub.3-3-BE]) of
the non-toxic pre-hormonal form of Vitamin D.sub.3 (FIG. 4).
AMPI-106 is the epoxide derivative (25-hydroxyvitamin
D.sub.3-3-epoxide [25-OH-D.sub.3-3-EPO]) of the non-toxic
pre-hormonal form of Vitamin D.sub.3 (FIG. 5).
[0074] AMPI-105 and AMPI-106 are a class of novel, non-toxic VDR
affinity-binding analogs of 25-OH-D.sub.3. By covalently attaching
(alkylating) 25-OH-D.sub.3, a non-toxic and biologically inert
pre-hormonal form of 1,25(OH).sub.2D.sub.3, to the hormone-binding
pocket of VDR, 25-OH-D.sub.3 was to converted into a
transcriptionally active form. This makes 25-OH-D.sub.3
biologically active. Furthermore, it translates the non-toxic
nature of 25-OH-D.sub.3 into its VDR-alkylating analog. Thereby,
the 25-OH-D.sub.3 analogs now have the anti-cancer property of a
`1,25(OH).sub.2D.sub.3-like molecule` without systemic
toxicity.
[0075] As shown in the examples, the two (2) VDR-alkylating analogs
of 25-OH-D.sub.3 (AMPI-105 and AMPI-106) possess strong anti-tumor
activity in a mouse prostate tumor xenograft model.
AMPI-107 (Vitamin D.sub.3 Analog)
[0076] AMPI-107 is an epoxide analog of Vitamin D.sub.3 (FIG. 6).
Vitamin D.sub.3 is normally considered to be biologically
inert.
[0077] As shown in the examples, the very strong anti-growth
activity of AMPI-107, a vitamin D.sub.3 derivative, even at
10-times higher dose level is highly unexpected and
significant.
EXAMPLES
Example 1
APH-0701 Nanosomes Characterization
[0078] Experiment APH-0701-27 was conducted with SFS propane at
3,000 psig and 60.degree. C. Three depressurization fractions were
collected in 10% sucrose. The first was obtained by displacement
under constant pressure, the second by depressurization from 3,000
psig to .about.400 psig and the third by depressurization from
.about.400 psi to 0 psig. These were APH-0701-27-01, APH-0701-27-02
and APH-0701-27-03 respectively. The results of the analysis of
APH-0701-27 are summarized in Table 1.
Table 1: AMPI-109 Content and Size of APH-0701-27
TABLE-US-00001 [0079] TABLE 1 AMPI-109 Content and Size of
APH-0701-27 APH-0701-27 Amount of AMPI- Recovered Fractions 109
(mg) Size (nm) (%) APH-0701-27-01 0.013 4740 1.5 APH-0701-27-02
0.745 197 88.5 APH-0701-27-03 0.084 -- 10.0 Total 0.842 --
100.0
[0080] The particle size of APH-0701-27-02, which contained 88.5%
of AMPI-109, was determined to be 197 nm using a Coulter 4MD
particle size analyzer (FIG. 9). This particle size was confirmed
by photomicrography at a magnification of 1,000.times. (FIG.
10).
Example 2
Size Exclusion Chromatography
[0081] In order to determine the extent of encapsulation of
AMPI-109, a size exclusion separation of VDD-27-02 was conducted on
Sephadex LH-20. In this size exclusion separation, phospholipid
nanosomes should elute with the void volume with smaller molecules
retained onto the column and eluted after solvent wash. The
results, shown in FIG. 11, indicate that the majority (.about.90%)
of AMPI-109 elutes with the phospholipid nanosomes in the first
three fractions with particle sizes of 151 nm, 150 nm and 200 nm,
around those originally measured.
Example 3
In Vitro Release of AMPI-109 from Nanosomes
[0082] Release studies of VDD-27-02 into 10% Tween 80 solution in
FIG. 12 indicate that AMPI-109 is releasing from nanosomes slowly
over time until almost all has either diffused out or the nanosomes
have broken apart. Maximum release is observed after 3 days, after
which time, AMPI-109 is either precipitating or degrading.
Example 4
Antiproliferative and Cytotoxic Activity of APH-0701 and AMPI-109
in Prostate Cancer Cells by .sup.3H-Thymidine Incorporation
Assay
[0083] The activities of AMPI-109 in nanosomes (APH-0701) vs. naked
AMPI-109 were measured in androgen-sensitive LNCaP prostate cancer
cells. Anti-proliferative activity was compared with nanosomal
preparation of AMPI-109 (APH-0701) versus naked AMPI-109.
Antiproliferative activities in these cells were measured by
.sup.3H-thymidine incorporation assay.
.sup.3H-Thymidine-Incorporation Assay:
[0084] In a typical assay cells were grown to 50-60% confluence in
24-well plates in respective media containing 5% FBS, and serum
starved for 20 hours, followed by treatment with various agents (in
0.1% ethanolic solution) or ethanol (vehicle) in serum-containing
medium for 16 hours. After the treatment media was removed from the
wells and replaced with media containing .sup.3H-thymidine (Sigma,
0.1 .mu.Ci) per well, and the cells were incubated for 3 hours at
37.degree. C. After this period media was removed by aspiration and
the cells were washed thoroughly (3.times.0.5 ml) with PBS.
Ice-cold 5% perchloric acid solution (0.5 ml) was added to each
well and the cells were incubated on ice for 20 minutes. After this
incubation, perchloric acid was removed by aspiration, replaced
with 0.5 ml of fresh perchloric acid solution and the cells were
incubated at 70.degree. C. for 20 minutes. Solution from each well
was mixed with scintillation fluid and counted in a liquid
scintillation counter. There were eight (8) wells per sample;
statistics was carried out by Student's t test.
[0085] AMPI-109, both in naked and nanosomal forms has strong
antiproliferative effect in LNCaP prostate cancer cells (FIG. 13).
Both AMPI-109 and APH-0701 [aka AMPI-109 (L)] almost completely
inhibited the growth of LNCaP cells.
[0086] At 10.sup.-7M dose level of APH-0701 has significantly
stronger effect than an equivalent amount of AMPI-109.
Encapsulation of AMPI-109 prevents catabolic degradation of naked
AMPI-109; and as a result APH-0701 has a stronger
biological/cellular effect, as we have observed with LNCaP prostate
cancer cells.
Example 5
Antiproliferative and Cytotoxic Activity of AMPI-109 Vs
1,25(OH).sub.2D.sub.3 (Calcitriol) in Normal Kidney and Kidney
Cancer Cells by .sup.3H-Thymidine Incorporation Assay
[0087] The antiproliferative activities of AMPI-109 and Calcitriol
(1,25(OH).sub.2D.sub.3) are shown in FIG. 14 for normal kidney
cells and in FIG. 15 for RCC 54 kidney cancer cells.
[0088] At 10.sup.-6M, neither Calcitriol nor AMPI-109 had any
statistically different impact on normal kidney cells than the
control (FIG. 14).
[0089] Surprisingly, at doses ranging from 7.5.times.10.sup.-7 to
10.sup.-6M, Calcitriol did not have any statistically different
impact on RCC 54 kidney cancer cells over control whereas AMPI-109
had a >95% impact on the reduction of proliferation of RCC 54
kidney cancer cells (FIG. 15).
[0090] The APH-0701 nanosomal formulation of AMPI-109 will have a
similar impact to that shown in Example 4 of reducing its toxicity
to normal kidney cells and increasing its efficacy on kidney cancer
cells.
Example 6
Effect of 1,25(OH).sub.2D.sub.3-3-BE (AMPI-109) Vs
1,25(OH).sub.2D.sub.3 (Calcitriol) and EB-1089 on the Growth of
Androgen-Insensitive Human Prostate Cancer DU-145 Cells
[0091] DU-145 is a highly aggressive androgen-insensitive human
prostate cancer cell line that does not respond well to calcitriol
due to increased expression of CYP 24-OHase and subsequent rapid
catabolism. We hypothesized that covalent attachment of calcitriol
(via AMPI-109) into the ligand-binding pocket of VDR might make it
inaccessible to catabolic enzymes; and hence restore its activity.
To prove this point we treated DU-145 cells (grown to approximately
60% confluence) in DMEM media containing 5% FBS with
2.5.times.10.sup.-7M, 5.0.times.10.sup.-7M, 7.5.times.10.sup.-7M
and 10.0.times.10.sup.-7M (10.sup.-6M) of AMPI-109, calcitriol or
ethanol for 20 hours followed by antiproliferation analysis by
.sup.3H-thymidine-incorporation assay.
[0092] The data in FIG. 16 demonstrates that AMPI-109 showed a
dose-dependent antiproliferative effect in DU-145 cells with
maximum effect at 10.sup.-6M dose, while calcitriol showed no
effect. In a separate experiment (data shown in FIG. 17), we
treated DU-145 cells with 10.sup.-6M of calcitriol, AMPI-109 or
EB-1089 (a non-calcemic analog of calcitriol). AMPI-109 showed a
strong anti-proliferative effect at 0.sup.-6M, but both calcitriol
and EB-1089 failed to produce any discernible anti-proliferative
effect.
Example 7
Serum-Stability Study of AMPI-109 and APH-0701
[0093] One ml of pooled human serum was incubated at 37.degree. C.
for 60 minutes with 10 .mu.g of AMPI-109 or an equivalent amount of
APH-0701 followed by multiple (5 times) extraction with 0.5 ml of
ethyl acetate. The organic layer was dried in a stream of nitrogen
and the residue was analyzed by HPLC (5% H.sub.2O-- 95% methanol
1.5 ml/min, 265 nm detection, Agilent C18 column). Organic extracts
of both AMPI-109 and APH-0701 produced a peak at 6.68 min which
corresponds to the peak of a standard sample of AMPI-109.
[0094] These results demonstrate that APH-0701 is stable in human
serum.
Example 8
Stability Study of AMPI-109 and APH-0701 in Liver Homogenate
[0095] Pieces of liver, obtained from normal mice were minced and
homogenized in phosphated saline with a polytron. One mL of the
homogenate was incubated at 37.degree. C. for 60 minutes with 10
.mu.g of AMPI-109 or an equivalent amount of APH-0701 followed by
multiple (5 times) extraction with 0.5 mL of ethyl acetate. The
organic layer was dried in a stream of nitrogen and the residue was
analyzed by HPLC (as above). Organic extracts of both AMPI-109 and
APH-0701 produced a peak at 6.1-6.3 min which corresponds to the
peak of a standard sample of AMPI-109.
[0096] These results demonstrate that APH-0701 is stable in a mouse
liver homogenate.
Example 9
Maximum Tolerated Dose (MTD) of AMPI-109 and APH-0701 in SCID
Mice
[0097] DU-145 prostate cancer cells (ATCC, Manasas, Va.) were grown
in culture, and then approximately 5 million cells/animal was
injected under the skin in the flank area of SCID mice (Charles
River). Tumors grew in 2-3 weeks, and when they reached a size of
approximately 1 cm.sup.3, they were injected with 0.1, 0.5 and 1
.mu.g/kg dose of AMPI-109 (in 5% dimethylacetate in sesame oil)
intraperitoneally. Dosing levels were limited by concentrations and
volumes. Each group had six mice. Dosing was carried out every
third day and weight of each mouse was recorded. All mice in the 1
.mu.g/Kg dose died after three dosing.
[0098] Based on dosing, the relative MTD of AMPI-109 is estimated
to be .ltoreq.3 .mu.g/Kg.
[0099] Twenty (20) male nu/nu mice, 6 weeks old (Charles River
Laboratories, Wilmington, Mass.) were grouped in five (5) animals
each and injected (i.p.) with either vehicle (blank liposome) or
0.75 .mu.g/kg, 1.0 .mu.g/kg and 1.25 .mu.g/kg of APH-0701, the
liposomal preparation of AMPI-109 on every third day. Mice were
observed for sign of toxicity including lack of appetite, weight
loss, lethargy etc. After seven (7) injections three (3) mice (out
of a total of 5) receiving 1.25 .mu.g/kg of APH-0701 died, and the
experiment was stopped.
[0100] Based on dosing, the relative MTD of APH-0701 is estimated
to be >9 .mu.g/Kg.
[0101] Thus the maximum tolerated dose of APH-0701, the
nanoformulated Vitamin D analog, is at least 300% higher than that
of the naked Vitamin D analog, AMPI-109.
Example 10
In Vivo Efficacy of AMPI-109 and Calcitriol in Mouse Xenograft
Models of Androgen-Insensitive DU-145 Human Prostate Cancer Cells
(I.P.-Administration)
[0102] Male, athymic mice (average weight 20 gm) were fed normal
rat chow and water ad libitum. They were inoculated with DU 145
cells, grown in culture in their flanks under light anesthesia.
When the tumor size grew to approximately 100 mm.sup.3 the animals
were randomized into groups of ten (10) tumor-bearing animals, and
they were given AMPI-109 (0.1 .mu.g/kg), calcitriol (0.5 and 1
.mu.g/kg), and vehicle (5% DMA in sesame oil) by intraperitoneal
injection (i.p.) on every third day (when body weights were
determined); and one group was left untreated. Treatment started on
day 11 and stopped on day 30; and they were left untreated for two
(2) additional days when they were sacrificed.
[0103] AMPI-109 (0.1 .mu.g/kg) showed a strong anti-tumor effect
(solid purple triangle in FIG. 18). Effect of AMPI-109 (0.1
.mu.g/kg) was similar to calcitriol (0.5 .mu.g/kg). AMPI-109 was
approximately 5 times stronger in potency than calcitriol in
reducing tumor-size. However, molecular weights of calcitriol and
AMPI-109 are 416.65 and 537.8 respectively. Therefore, on a molar
basis AMPI-109 is approximately 6.5 times more potent than
calcitriol. Thus, covalently attaching calcitriol to VDR might
increase its potency (by decreasing catabolism).
[0104] As shown in FIG. 19 AMPI-109 (0.1 .mu.g/kg) showed some
reduction in body weight which was significantly less than with
calcitriol (0.5 .mu.g/kg and 1.0 .mu.g/kg). Another interesting
observation was that after the withdrawal of AMPI-109 (day 30)
weights of animals started increasing (similar to calcitriol). This
is an important finding because AMPI-109 is an alkylating compound,
and there may be concerns of sustained systemic toxicity.
Example 11
In Vivo Studies of AMPI-109 and Calcitriol in Nude Mice Inoculated
with DU-145 Human Prostate Cancer Cells (P.O.-Administration)
[0105] In the p.o. administration oral gavage mode AMPI-109 (0.5
.mu.g/kg) showed a strong anti-tumor effect (FIG. 20). This effect
was similar to calcitriol (0.5 .mu.g/kg). However, calcitriol (0.5
.mu.g/kg and 1.0 .mu.g/kg) caused significant loss in body weight
denoting toxicity, while AMPI-109 did not cause any significant
change in body weight of the animals (FIG. 19).
[0106] It is noteworthy that AMPI-109 was five (5) times less
potent in the p.o.-mode than in the i.p.-mode. This is to be
expected because in the i.p. mode the compound goes directly in the
blood stream, while in the p.o. mode a significant portion of
AMPI-109 is expected to undergo hydrolysis/metabolism before
reaching the blood stream. Therefore higher amounts would be
required to show any biological effect. Therapeutic agents
containing hydrolysable bonds are fairly common; for example
aspirin and acetaminophen contain hydrolysable ester and amine
bonds.
[0107] In summary, the results described above showed strong
anti-tumor activity and significant bioavailability of
AMPI-109.
Example 12
In Vivo Studies of AMPI-109 and APH-0701 in Nude Mice Inoculated
with DU-145 Human Prostate Cancer Cells (P.O.-Administration)
[0108] Male, athymic mice (average weight 20 gm) were fed normal
rat chow and water ad libitum. They were inoculated with DU-145
cells (5.times.10.sup.6 cells, dispersed in 100 .mu.l PBS) in the
flank. When the tumor size grew to approximately 100 mm.sup.3 the
animals were randomized into groups of eight (8), and they were
given AMPI-109 (0.5 .mu.g/Kg in 5% DMA in sesame oil, 5% DMA in
sesame oil (vehicle control), or APH-0701 (0.5 .mu.g/Kg, in 5% DMA
in sesame oil) by intraperitoneal injection (i.p.) on an average
every third day (body weights were determined at each dosing).
Treatment started on day 7 after tumor-implantation and was stopped
on day 42, when they were sacrificed.
[0109] Results of our in vivo efficacy and safety study are shown
in FIGS. 22 and 23. At the end of the experiment, average size of
vehicle-control, AMPI-109-treated and APH-0701-treated tumors were
approximately 750, 475 and 385 mm.sup.3 respectively, demonstrating
a strong reduction of tumor size by AMPI-109 (37% of control) and
APH-0701 (49% of control), with a 33% improvement of liposomal
versus naked drug (FIG. 22). On the other hand, gross body-weights
of AMPI-109- and APH-0701-treated animals were not significantly
different from control animals, indicating lack of toxicity (FIG.
23). Therefore, collectively these results demonstrated that
AMPI-109 and APH-0701 have a strong translational potential as a
therapeutic agent in androgen-insensitive prostate cancer.
Example 13
Antiproliferation Studies of Normal and Cancerous Cell Lines
Treated with AMPI-105 or Bioactive Vitamin D.sub.3 Hormone
[0110] Antiproliferation studies of keratinocytes (normal skin),
MCF-7 (breast cancer), PZ-HPV-7 (immortalized normal prostate),
LNcap (androgen-sensitive prostate cancer) and PC-3
(androgen-insensitive prostate cancer) cells, treated with
10.sup.-6M of 25-OH-D.sub.3-3-BE (AMPI-105) or
1,25(OH).sub.2D.sub.3 (bioactive Vitamin D.sub.3 hormone) were
performed with a .sup.3H-thymidine incorporation assay
[0111] Growth-inhibitory effect of 1,25(OH).sub.2D.sub.3 and its
analogs is known to vary among cell-lines and even among lines from
the same tissue. But, in general, strongest effect is observed at a
10.sup.-6 M concentration of the hormone or its analogs. Although
this concentration is considered to be physiologically irrelevant,
it produces optimal effect. Therefore, this dose was used for
screening of various cell lines.
[0112] PZ-HPV-7 cells were grown in MCDB media containing pituitary
extract, epidermal growth factor (EGF) and 1%
penicillin/streptomycin. Keratinocytes were also grown in the same
media with additional PG1 and insulin. PC-3, LNCaP, DU-145 cells
were grown in RPMI media containing 10% fetal bovine serum (FBS)
and antibiotics. MCF-7 cells were grown in DMEM media containing
10% FBS and antibiotics. LAPC-4 cells were maintained in IMEM media
containing antibiotics, 1% L-glutamine and 10 nM of R1881, a
synthetic progestin. MC3T3 mouse bone cells were grown in
.alpha.-MEM media containing 10% FBS and antibiotics. In general,
cells were grown in 35 mm dishes to 70-80% confluence and then
plated into 24-well plates in respective media. After the cells
grew to approximately 70% confluence, they were serum-starved for
20 hours (MCF-7, PC-3, LNCaP and DU-145 cells) followed by
incubation with steroid samples. Keratinocytes and PZ-HPV-7 cells,
after reaching 70% confluence, were kept in MCDB media without
additives for 20 hours before treatment with steroids. In general,
reagents were dissolved in ethanol (EtOH), and dilution with the
media was adjusted in such a way that the concentration of EtOH was
0.1% v/v.
[0113] Assays were carried out with six (6) replicates and
student's t-test was employed for statistical analysis. Results are
expressed relative to EtOH (100%) in FIG. 24.
[0114] As shown in FIG. 24-E, 10.sup.-6 M of 25-OH-D.sub.3-3-BE and
1,25(OH).sub.2D.sub.3 inhibited the growth of all the cells with
varying efficiency. However, the effect of 25-OH-D.sub.3-3-BE was
strongest in LNCaP and PC-3 prostate cancer cells.
[0115] For example, growth of LNCaP cells were inhibited by
approximately 60% and 98% with 1,25(OH).sub.2D.sub.3 and
25-OH-D.sub.3-3-BE, respectively (FIG. 24-D), while growth of PC-3
cells was reduced by 70% and 90% by 1,25(OH).sub.2D.sub.3 and
25-OH-D.sub.3-3-BE, respectively (FIG. 24-E). In contrast, growth
of normal immortalized prostate cells (PZ-HPV-7 cells) were
inhibited by approximately 50% and 65% by 10.sup.-6 M of
25-OH-D.sub.3-3-BE and 10.sup.-6 M of 1,25(OH).sub.2D.sub.3,
respectively (FIG. 24-C).
[0116] While growth inhibition by 25-OH-D.sub.3-3-BE was stronger
than an equivalent amount of 1,25 (OH).sub.2D.sub.3 in
keratinocytes (FIG. 24-A), the effect of 25-OH-D.sub.3-3-BE was
weaker than 1,25(OH).sub.2D.sub.3 in MCF-7 breast cancer cells
(FIG. 24-B). Furthermore, 10.sup.-6 M of 25-OH-D.sub.3 showed
marginal antiproliferative effect in PC-3 cells (FIG. 24-F). We
also observed that 10.sup.-6 M of 25-OH-D.sub.3-3-BE was cytotoxic
only to LNCaP and PC-3 cells, causing the cells to lift, float and
die, as seen under a phase contrast microscope.
[0117] In a cell counting assay, LNCaP and LAPC-4 cells had sharply
reduced number of cells with 10.sup.-6 M of 25-OH-D.sub.3-3-BE
after 24 hours incubation (FIG. 25) while MCF-7 and MC3T3 cells
(incubated for 48 hr) were affected to a much lesser extent than
for LNCaP and LAPC-4 cells, although the effect on MC3T3 cells was
significantly stronger than in MCF-7 cells. It should be noted that
in this assay cells were not serum-starved prior to addition of the
reagents, and 10.sup.-7M of 1,25(OH).sub.2D.sub.3 had little effect
on all the cells. 10.sup.-7 M of 1,25(OH).sub.2D.sub.3 was shown to
produce a significant effect in LNCaP cells after a longer period
(3-6 days) of incubation.
Example 13
Efficacy Study of 25-OH-D.sub.3-3-BE in Athymic Nude Mice
[0118] Male, athymic nude mice (average weight 20 gm) were fed
normal rat chow and water ad libitum. They were inoculated with DU
145 cells, grown in culture, in the flank under light anesthesia.
After the tumor size grew to approximately 100 mm.sup.3, the
animals were randomized into three (3) groups of ten (10)
tumor-bearing animals and they were given 25-OH-D.sub.3-3-BE (0.25
and 0.5 mg/kg, dissolved in 5% DMA in sesame oil) or left
untreated. Administration of the compound was done by oral gavage
on every third day (when weights were determined). Treatment
started on day 11 and stopped on day 31; and they were left
untreated for several additional days (as shown in FIG. 26) when
they were sacrificed and blood was collected.
[0119] 25-OH-D.sub.3-3-BE showed a dose-dependent anti-tumor effect
at 0.25 and 0.5 m.mu.g/kg doses (FIG. 26). For example, at the end
of treatment average size of the untreated tumor was approximately
900 mm.sup.3, while average tumor volumes were 650 mm.sup.3 and 500
mm.sup.3 with 0.25 and 0.5 .mu.g/kg of 25-OH-D.sub.3-3-BE
respectively. Toxicity was measured by following gross weight of
the animals during and after the treatment. As shown in FIG. 27
there was no significant difference between the
25-OH-D.sub.3-3-BE-treated and untreated animals. It is to be noted
that these results are preliminary in nature and we plan to carry
out more extensive dose-response studies both by oral gavage
administration mode as well as determination of serum-calcium
levels in the proposed studies.
Example 15
Viability Testing of Vitamin AMPI-107 and Calcitrol in Normal
Prostate Cells by MTT Reduction
[0120] Vitamin D epoxide (AMPI-109) was received at a concentration
of 10.sup.-2M (diluting 1000-fold to give 10.sup.-5M, and a later
10.times. dilution produced 10.sup.-6M. The hormone
[1,25(OH).sub.2D.sub.3] or Calcitrol positive control sample was
received at a concentration of 10.sup.-3M. A 1000-fold dilution
gave 10.sup.-6M.
[0121] Normal prostate WPMY cells were cultured to confluency in
10% fetal calf serum supplemented Dulbecco's modified Eagle's
medium plus 1% antibiotic/antimycotic in 96 well dishes and at
confluency, cells were exposed to 1 .mu.M Calcitrol, 1 and 10 .mu.M
AMPI-107 in complete medium for 24 hours.
[0122] Cells were then incubated with yellow tetrazolium MTT
(3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide) for
3 h and was then extracted in acid isopropanol (0.1 N HCl in
anhydrous isopropanol). MTT is reduced in metabolically active
cells, in part by the action of dehydrogenase enzymes, to generate
reducing equivalents such as NADH and NADPH. The resulting
intracellular purple formazan can be solubilized and quantified by
spectrophotometry. After 5 minutes at room temperature, absorbances
in plates were measured using a plate analyzer set for in dual
wavelength comparisons at wavelength of 540 nm with a reference
wavelength of 620 nm.
[0123] Compared to control cells, FIG. 28, AMPI-107 at 1 and 10
.mu.M did not significantly depress cell metabolism. By comparison,
1 .mu.M Calcitrol did slightly, but significantly reduce cell
metabolism (**p<0.01, one way ANOVA with Bonferroni
post-testing).
Example 16
Antiproliferative Evaluation of AMPI-107 Versus Calcitrol in
Prostate Cancer Cells
[0124] Prostate cancer cells (DU-145, PC-3 and LNCaP) were grown in
DMEM medium with 10% FBS, antibiotics, etc. When the confluence
reached approximately 50% they were dosed with ethanolic solutions
(0.1% in media) with various doses of either AMPI-107 or the
hormone [1,25(OH).sub.2D.sub.3] aka Calcitrol or ethanol (control)
on days 1,3 and 5, and cells were counted on a hemocytometer on the
7.sup.th day. Results are designated as percentage of ethanol
control.
[0125] As shown in FIG. 29, Calcitrol (10.sup.-6M) and AMPI-107
(10.sup.-5M) had approximately similar growth inhibitory activity
in DU-145 cells.
[0126] As shown in FIG. 30, Calcitrol (10.sup.-6M) and AMPI-107
(10.sup.-5M) strongly inhibited the growth of LNCaP cells, but
AMPI-107 had a significantly stronger effect at this dose.
[0127] As shown in FIG. 31, Calcitrol (10.sup.-6M) and AMPI-107
(10.sup.-5M) strongly inhibited the growth of PC-3 cells, but
AMPI-107 had a significantly stronger effect at this dose.
[0128] Cell-counting assay (dosing on 1.sup.st, 3.sup.rd, and
5.sup.th days, harvesting and counting on 7.sup.th day)
demonstrated that AMPI-107 is either equally effective (DU-145 and
PC-3 cells) or more (LNCaP cells) than Calcitrol. However,
concentration of AMPI-107 was 10-times higher than Calcitrol.
Example 17
In Vivo Studies of AMPI-107 in Nude Mice Inoculated with DU-145
Human Prostate Cancer Cells (P.O. and I.P. Administration)
[0129] Male, athymic mice (average weight 20 gm) were fed normal
rat chow and water ad libitum. They were inoculated with DU 145
human androgen-insensitive prostate cancer cells (10.sup.6), grown
in culture in the flank under light anesthesia. When the tumor size
grew to approximately 100 mm.sup.3 the animals were randomized into
groups of ten (10) tumor-bearing animals, and they were given
vitamin D.sub.3-3-epoxide, AMPI 107 (1 mg/kg),
1,25(OH).sub.2D.sub.3 hormone (0.5 and 1.0 .mu.g/kg), and vehicle
(5% DMA in sesame oil) by intraperitoneal injection (i.p.) or by
oral gavage (p.o.) on every third day (when body weights were
determined); and one group was left untreated. Treatment started on
day 11 and stopped on day 30; and they were left untreated for two
(2) additional days when they were sacrificed. The i.p. results are
respectively shown in FIGS. 32 and 33 for the effect of AMPI-107
and the 1,25(OH).sub.2D.sub.3 hormone on tumor growth and body
weight. The p.o. results are respectively shown in FIGS. 34 and 35
for the effect of AMPI-107 and the 1,25(OH).sub.2D.sub.3 hormone on
tumor growth and body weight. Note AMPI-107 is referred to as
MPI-107 in these figures.
[0130] In terms of efficacy vitamin D.sub.3-3-epoxide, MPI 107 (1
mg/kg) was similar to hormone (0.5 .mu.g/kg) in both i.p. and p.o.
administration modes (FIGS. 32 and 34). But 1,25(OH).sub.2D.sub.3,
hormone (0.5 and 1.0 .mu.g/kg) was clearly toxic as evidenced by
considerable loss of body weight in both cases, while vitamin
D.sub.3-3-epoxide, MPI 107 was completely non-toxic (FIGS. 33 and
35).
[0131] Surprisingly, the results of this study demonstrated that
vitamin D.sub.3-3-epoxide strongly reduced tumor size in this model
both in i.p. and p.o. administration modes without any significant
toxicities.
[0132] The APH-0701 nanosomal formulation of AMPI-105 will have a
similar impact to that shown in Example 4 of reducing its toxicity
and increasing its efficacy.
Example 18
Polymeric Nanoencapsulation of Vitamin D.sub.3 Analogs
[0133] Polymeric Spheres were formed which contained the Vitamin
D.sub.3 analogs in the following manner. A feed rate of 0.25 mg/ml
Vitamin D.sub.3 analog in an ethanolic buffer solution and a
supercritical, critical or near critical solution of solution of
poly(D, L-lactic acid), poly(glycolic acid) in propane was injected
into a decompression fluid of de-ionized water and produced a batch
of spheres having a mean particle diameter of 200 to 400
nanometers. The polymer solution was maintained prior to injection
at a pressure of 21 MPa and 30 degrees centigrade.
[0134] This suspension of spheres in a phosphate buffer was then
lyophilized. Dried spheres were stored at five degrees centigrade
until used or compressed into tablets. Prior to use, dried spheres
were re-constituted and formulated into a phosphate buffer
solution.
Example 19
Oil Capsule Formulation of Vitamin D.sub.3 Analogs
[0135] The Vitamin D.sub.3 analogs are formulated in different
doses ranging from 500 IU to 5,000 IU in gel capsules containing
the following. [0136] 500 IU to 5,000 IU of Vitamin D.sub.3 analogs
[0137] 30 mg of mixed tocopherol 90% as an antioxidant [10.7%]
[0138] 30 mg of Lecithin as an emulsifier to improve solubility and
bioavailability [10.7%] [0139] 15 mg of Medium Chain Triglyceride
(MCT) as a co-emulsifier [5.4%] [0140] 175 mg of Olive Oil as an
excipient with some nutritional value [62.5%]; Nitrogen head
Example 20
Water Capsule Formulation of Vitamin D.sub.3 Analogs
[0141] The Vitamin D.sub.3 analogs are formulated in different
doses ranging from 500 IU to 5,000 IU in gel capsules containing
the following: Medium Chain Triglyceride (MCT) as a co-emulsifier,
Lecithin Soy, Hydroxyl Propyl Methyl Cellulose (HPMC) and Purified
Water.
* * * * *