U.S. patent application number 17/499248 was filed with the patent office on 2022-03-31 for resiquimod topical and injectable compositions for the treatment of neoplastic skin conditions.
This patent application is currently assigned to THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. The applicant listed for this patent is THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. Invention is credited to Pankaj Karande, Shujing Liu, Alain Rook, Xiaowei Xu.
Application Number | 20220096459 17/499248 |
Document ID | / |
Family ID | 1000006025844 |
Filed Date | 2022-03-31 |
View All Diagrams
United States Patent
Application |
20220096459 |
Kind Code |
A1 |
Xu; Xiaowei ; et
al. |
March 31, 2022 |
RESIQUIMOD TOPICAL AND INJECTABLE COMPOSITIONS FOR THE TREATMENT OF
NEOPLASTIC SKIN CONDITIONS
Abstract
Compositions and methods for treatment of viral skin disease,
precancerous and cancerous skin disease, and other neoplasms are
disclosed.
Inventors: |
Xu; Xiaowei; (Monmouth
Drive, NJ) ; Liu; Shujing; (Philadelphia, PA)
; Rook; Alain; (Wynnewood, PA) ; Karande;
Pankaj; (Troy, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA |
Philadelphia |
PA |
US |
|
|
Assignee: |
THE TRUSTEES OF THE UNIVERSITY OF
PENNSYLVANIA
Philadelphia
PA
|
Family ID: |
1000006025844 |
Appl. No.: |
17/499248 |
Filed: |
October 12, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15740147 |
Dec 27, 2017 |
11179385 |
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PCT/US2016/040497 |
Jun 30, 2016 |
|
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17499248 |
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62186931 |
Jun 30, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 17/12 20180101;
A61K 9/0019 20130101; A61K 31/444 20130101; A61K 38/217 20130101;
A61K 2300/00 20130101; A61K 45/06 20130101; A61K 9/0014 20130101;
A61K 47/14 20130101; A61K 31/4745 20130101; A61P 35/04 20180101;
A61K 47/20 20130101; A61K 39/00 20130101; A61K 39/39558 20130101;
A61K 41/0057 20130101; A61K 47/12 20130101 |
International
Class: |
A61K 31/4745 20060101
A61K031/4745; A61K 45/06 20060101 A61K045/06; A61K 39/00 20060101
A61K039/00; A61K 31/444 20060101 A61K031/444; A61P 17/12 20060101
A61P017/12; A61P 35/04 20060101 A61P035/04; A61K 9/00 20060101
A61K009/00; A61K 38/21 20060101 A61K038/21; A61K 39/395 20060101
A61K039/395; A61K 41/00 20060101 A61K041/00; A61K 47/12 20060101
A61K047/12; A61K 47/14 20060101 A61K047/14; A61K 47/20 20060101
A61K047/20 |
Goverment Interests
GRANT SUPPORT STATEMENT
[0002] This invention was made with government support under Grant
Number AR054593 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1.-9. (canceled)
10. A method for the treatment of skin tumor lesion and virus
induced skin disease, comprising topical administration of the
composition comprising between 0.01% -1% resiquimod, said
composition being effective to reduce or eliminate said lesion or
disease while limiting adverse skin reactions selected from
erythema and inflammation, said composition reducing tumor lesion
penetration into surrounding tissue and metastasis to lymph
nodes.
11. The method of claim 10, wherein said skin tumor lesion is a
precancerous pigmented lesion selected from an atypical melanocytic
lesions, atypical melanocytic hyperplasia, atypical intraepidermal
melanocytic hyperplasia, atypical intraepidermal melanocytic
proliferation and actinic keratosis.
12. The method of claim 10 wherein said lesion is selected from
lentigo maligna, melanoma in situ, squamous cell carcinoma in situ,
said composition being effective to reduce local tissue
invasion.
13. The method of claim 10 wherein said lesion is indicative of
primary invasive melanoma, basal cell carcinoma, or a squamous cell
carcinoma, said composition being effective to reduce infiltration
through tissues and reduce metastasis to lymph nodes.
14. The method of claim 10 wherein said lesion is selected from
Bowen's disease, squamous cell carcinoma in situ, arsenical
keratosis, radiation keratosis, PUNA keratosis, bowenoid papulosis,
sebaceous carcinoma, porocarcinoma, extramammary Paget's disease,
Merkel cell carcinoma and cutaneous T cell lymphoma.
15. The method of claim 10 for the treatment of epidermal
keratinocyte proliferation, and seborrheic keratosis.
16. The method of claim 10, wherein said virus induced skin disease
is selected from molluscum contagiosum, fIPV infection, vulvar
intraepithelial neoplasia, vaginal intraepithelial neoplasia.,
common warts, and RSV infection.
17.-20. (canceled)
21. The method of claim 10, said treatment further comprising
systemic administration of at least one immune modulator selected
from anti-PD1, anti-PDL1, Anti-CTLA-4 antibody, anti-CD137
antibody, agonistic CD40 antibody, CD134 (Anti-OX40) agonist and
PLX3397.
22. The method of claim 10, further comprising systemic
administration of interferon gamma.
23. The method of claim 10, further comprising of administration of
local radiation with or without systemic anti-PD1 antibody.
24. The method of claim 10, further comprising of administration of
photodynamic therapy.
25. The method of claim 10, wherein said composition comprises a
filler.
26. The method of claim 25, wherein said filler is a dermal
filler.
27. The method of claim 25, wherein said dermal filler is at least
one of hydroxylapatite, sodium hyaluronate, poly-L-lactic acid,
collagen and hydrogel.
28. The method of claim 25 wherein said filler is purified oil.
29. The method of claim 28, wherein said purified oil is at least
one of soybean oil, triolein, castor oil, fractionated coconut oil,
miglyol 810, miglyol 812, Neobee MS or Captex 300.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. application Ser.
No. 15/740,147, filed Dec. 27, 2017, which is a .sctn. 371 of the
International Application No. PCT/US2016/040497, filed Jun. 30,
2016, which claims priority to U.S. Provisional application no.
62/186,931 filed Jun. 30, 2016. The entire disclosure of each of
the aforesaid applications being incorporated herein by reference
in the present application as though set forth in full.
FIELD OF THE INVENTION
[0003] This invention relates to the fields of dermatology and
oncology. More specifically, the invention provides resiquimod
containing compositions for treatment of non-cancerous skin
disorders, pre-cancerous skin disorders, primary and metastatic
neoplasms of the skin and other tissues.
BACKGROUND OF THE INVENTION
[0004] Several publications and patent documents are cited
throughout the specification in order to describe the state of the
art to which this invention pertains. Each of these citations is
incorporated herein by reference as though set forth in full.
[0005] Skin cancer is the most commonly diagnosed form of cancer of
which, the non-melanoma skin cancers, basal cell carcinoma and
squamous cell carcinoma, are the most common types. Non-melanoma
skin cancers are most often treated with surgical excision.
Non-surgical treatments are employed but only in a minority of
patients due to limited efficacy or cost. Melanoma results in the
greatest number of skin cancer-related deaths worldwide. Melanoma
is the 5.sup.th most common cancer in men and the 6.sup.th most
common cancer in women. It is the most common form of cancer for
young adults 25-29 years old, particularly in young women. In situ
melanoma is the very earliest stage of melanoma. There are cancer
cells in the top layer of skin (the epidermis) but they are all
contained in the epidermis where they start to develop. By curing
melanoma in situ, invasive melanoma can be prevented. Many
therapies have been investigated for the treatment of localized
cutaneous melanoma either as an adjuvant or in place of surgical
therapy.
[0006] Pre-cancerous skin lesions are more common than malignant
lesions. These precancerous lesions are indicative of changes in
skin that are not yet cancer but could become cancer over time.
Pre-cancerous pigmented lesions, including atypical melanocytic
lesions, atypical melanocytic hyperplasia, and atypical
intraepidermal melanocytic exhibit high proliferation levels. Some
of these lesions are thought to have the potential for developing
into melanoma. Classification of lesions is typically based on
visual or pathologic inspection of the lesion. In order to prevent
or inhibit progression to malignancy, new treatment protocols for
such pre-cancerous lesions are urgently needed.
[0007] Similar to skin, bladder also has a surface lining that has
cancerous transformation potential. Early bladder cancer is also
called superficial bladder cancer or nonmuscle invasive bladder
cancer. This means that the cancer cells are only in the inner
lining of the bladder. In CIS (carcinoma in situ) the cancer cells
are still only in the bladder lining of the bladder. They are in
flat sheets that look a bit like moss. CIS can occur in patches
throughout the bladder lining and the cells are very abnormal. This
cancer is associated with a high risk of spreading into the deeper
layers of the bladder. Early stage bladder cancer may be treated
with BCG vaccine administration into the bladder. By curing CIS,
invasive cancer can be prevented. However, current nonsurgical
therapy for early bladder cancer is very limited.
[0008] When cancerous lesions progress, they may become metastatic
and chemotherapy, radiation therapy and immunotherapy may be used
in addition to surgery. Resiquimod, a member of the
imidazoquinoline family related in structure to imiquimod, is an
immune response modifier which acts as a Toll-like receptor 7 and 8
agonist. However, it is distinctively different from Imiquimod
since Imiquimod signals through the toll-like receptor 7 (TLR7)
only. Imiquimod is FDA approved for the treatment of a number of
skin diseases. Compared with imiquimod, resiquimod is a more potent
inducer of IFN-.alpha., TNF-.alpha., IL-1, IL-6, IL-8, and IL-12.
Although resiquimod has been shown to be 10-100.times. more potent
than imiquimod in T cell activation, it has not yet been approved
for clinical use by the FDA. Resiquimod has been shown to promote
cross-presentation of exogenous antigens, resulting in the
efficient induction of antigen-specific CD8.sup.+ T-cell responses
in an animal model. Results from animal studies have confirmed the
ability of resiquimod to activate dendritic cells, including the
capacity to induce local activation of immune cells, stimulate the
production of proinflammatory cytokines, and enhance
antigen-presentation by dendritic cells leading to activation of
effective cellular responses. Systemic delivery of resiquimod, with
radiation, primes durable antitumor immune responses in a lymphoma
model (Dovedi S J 2013, Blood 121(2):251-9.). Resiquimod has been
used in clinical trials to treat actinic keratosis, cutaneous T
cell lymphoma and herpes simplex virus with mixed results. Other
prior art uses of resiquimod include administration as a vaccine
adjuvant agent to treat various diseases, including metastatic
melanoma with inconsistent results. Resiquimod has been used as a
vaccine adjuvant for NY-ESO-1 protein vaccine in treatment of
melanoma (Sabado R L Cancer Immunol Res. 2015).
[0009] A major advance in clinical immunotherapy has been the
blockade of inhibitory immune receptors and their ligands,
collectively termed immune checkpoints. Specifically, blockade of
CTLA4 and PD1 (or PDL1) has demonstrated clinical efficacy in a
number of different advanced malignancies. However, the response
rate is still relatively low in clinical trials and there is an
unmet clinical need to combine immune checkpoint blocks with other
reagents.
SUMMARY OF THE INVENTION
[0010] In accordance with the present invention, compositions and
formulations comprising an effective amount of resiquimod and
methods of use thereof for the treatment of non-cancerous,
pre-cancerous, and cancerous lesions are disclosed. In a preferred
embodiment of the invention, a composition comprising between 0.01%
-1% resiquimod for the treatment of various diseases is provided.
The inventive formulations are formulated for local (topical,
intracystic and intratumoral) administration, enhance permeation
and delivery of resiquimod, and facilitate beneficial local immune
reactions while limiting adverse reactions such as erythema,
inflammation or systemic cytokine release. Topical, intracystic and
intratumoral formulations are different as they are specifically
formulated to maximally enhance resiquimod's therapeutic effects
while also reducing unwanted side effects. In a particularly
preferred embodiment, the precancerous lesion is selected from
actinic keratosis, an atypical junctional melanocytic lesions,
atypical melanocytic hyperplasia, atypical intraepidermal
melanocytic hyperplasia, atypical intraepidermal melanocytic
proliferation. In alternative embodiments, the cancerous lesion is
basal cell carcinoma, squamous cell carcinoma, lentigo maligna,
melanoma in situ and melanoma, The neoplasm may also be selected
from Bowen's disease, squamous cell carcinoma in situ, arsenical
keratosis, radiation keratosis, PUVA keratosis, bowenoid papulosis,
sebaceous carcinoma, porocarcinoma, extramammary Paget's disease,
Merkel cell carcinoma, Kaposi's sarcoma and cutaneous T cell
lymphoma. The composition also has efficacy for the treatment of
abnormal epidermal keratinocyte proliferation, and seborrheic
keratosis. The composition also has efficacy for the treatment of
viral skin infections, such as, HPV infections including vulvar
intraepithelial neoplasia, vaginal intraepithelial neoplasia,
common warts that are difficult to treat, molluscum contagiosum and
HSV infection. In alternative embodiments, the cancerous lesion is
bladder carcinoma in situ and early bladder cancer (superficial
bladder cancer or nonmuscle invasive bladder cancer).
[0011] In another aspect, the resiquimod is administered in
combination with interferon gamma for the treatment of basal cell
carcinoma, squamous cell carcinoma, bladder superficial carcinoma,
lentigo maligna, melanoma in situ, primary invasive melanoma and
cutaneous T cell lymphoma. Use of resiquimod in combination with
radiation therapy for the treatment of basal cell carcinoma,
squamous cell carcinoma, bladder superficial carcinoma, lentigo
maligna, melanoma in situ, primary invasive melanoma and cutaneous
T cell lymphoma also forms an aspect of the invention. Use of
resiquimod in combination with photodynamic therapy for the
treatment of basal cell carcinoma, squamous cell carcinoma, bladder
superficial carcinoma, lentigo maligna, melanoma in situ, primary
invasive melanoma and cutaneous T cell lymphoma is also
disclosed.
[0012] In a particularly preferred embodiment of the invention, a
method for the treatment of non-cancerous, pre-cancerous, and
cancerous neoplasms of the skin and bladder, comprising topical or
intracystic administration of the composition(s) described above is
provided wherein the composition is effective to reduce or
eliminate said lesion.
[0013] When the method is for the treatment of deep cancer or
metastatic tumors including melanoma, intratumoral injection of
resiquimod or intratumoral resiquimod with systemic administration
of one or more immune modulators, including, for example, anti-PD1,
anti-PDL1, Anti-CTLA-4 antibodies, CD137 (Urelumab), CD40, CD134
(Anti-OX40) agonist and other immune-modulating small molecules
that can inhibit MDSCs (such as PLX3397) is preferred. The
intratumoral formulations and methods of use thereof can be used to
advantage to treat a wide variety of pathological conditions,
including without limitation, metastatic epithelial cancer,
squamous cell carcinoma, basal cell carcinoma, lung, bladder,
prostate, brain, breast, pancreas, ovary, liver, stomach, other
epithelial malignant tumor cells and mesenchymal malignant tumor
cells such as soft tissue sarcomas as well as hematopoietic
malignancy such as lymphoma and advanced cutaneous T cell
lymphoma.
[0014] In a particularly preferred embodiment of the invention, a
method for the treatment of deep or metastatic tumor, comprising
intratumoral administration of the composition (s) alone or in
combination of systemic administration of anti-PD1, or anti-PDL1,
or Anti-CTLA-4 antibodies or CD137 (Urelumab), CD40, CD134
(Anti-OX40) agonist and other immune-modulating small molecules
that can inhibit MDSCs (such as PLX3397) described above is
provided wherein the composition is effective to reduce or
eliminate said lesion.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A-1I. Resiquimod inhibits tumor growth in Tyr::creER,
BRAF.sup.ca mice. FIG. 1A) Treatment scheme. FIG. 1B) Large
pigmented lesion in a mouse treated with vehicle (n=5). FIG. 1C)
Residual dermal pigmentation in a mouse treated with resiquimod
(n=8). FIG. 1D) Pigmented lesion in a mouse before treatment. FIG.
1E) The same lesion 1 month after resiquimod treatment. FIG. 1F)
Histology of tumor treated with vehicle. There are many tumor cells
in the dermis. FIG. 1G) Histology of tumor treated with resiquimod.
Viable tumor cells are not seen and many macrophages with
pigmentation are seen in the dermis. FIG. 1H) Histology of the
lesion one month after resiquimod treatment. Viable tumor cells are
not seen and only macrophages with pigmentation are seen in the
dermis. FIG. 1I) Tumor growth curve of mice treated with vehicle or
resiquimod. * indicate p<0.05. Bars in b-e indicate 6 mm. Bars
in f-h indicate 200 .mu.m.
[0016] FIGS. 2A-2L. Resiquimod inhibits melanoma growth in
Tyr::creER, BRAFca, Phd2lox/lox mice. FIG. 2A) Treatment scheme.
FIG. 2B) Large tumor in a mouse treated with vehicle (n=10). FIG.
2C) Underside of skin filled with tumor. FIGS. 2D and 2E) Histology
of melanoma in a control mouse. The dermis is filled with tumor
cells. FIG. 2F) Resiquimod treated mouse with near complete
response (n=10). FIG. 2G) Underside of skin cleared of tumor. FIG.
2H) Histology of skin in a treated mouse, normal appearing. FIG.
2I) Macrophages with pigment are present in the dermis in a treated
mouse. Arrow points to macrophages. FIG. 2J)Tumor growth curve.
FIG. 2K) In situ melanoma in control mice. Arrows point to the
tumor cells in the epidermis and hair follicle. I) Normal appearing
epidermis in treated mice. *indicates p<0.05. Bars in b, c, f
and g indicate 6 mm. Bars in d, e, h and I, indicate 200 .mu.m.
Bars in k and 1 indicate 10 .mu.m.
[0017] FIGS. 3A-3I. Resiquimod inhibits melanoma growth in
Tyr::creER, BRAFca, Ptenlox/lox mice. FIG. 3A) Treatment scheme.
FIG. 3B) Representative image of a mouse with control treatment
(n=15). FIG. 3C) Representative image of a mouse with resiquimod
treatment (n=15). FIG. 3D) Histology of melanoma in a control
mouse. Arrow points to skeletal muscle. FIG. 3E) Histology of
melanoma in a treated mouse. Arrow points to skeletal muscle. FIG.
3F) Effect of resiquimod on tumor volume. FIG. 3G) Effect of
resiquimod on mouse survival. FIG. 3H) In situ melanoma in control
mice. Arrows point to the tumor cells in the epidermis. FIG. 3I)
Normal appearing epidermis in treated mice. *indicates p<0.05.
Bars indicate 6 mm in FIG. 3B and FIG. 3C, 400 .mu.m in FIG. 3D and
FIG. 3E and 50 .mu.m in FIG. 3H and FIG. 3I.
[0018] FIGS. 4A-4I. Reprogramming of tumor immune microenvironment
after resiquimod treatment. FIG. 4A) Treatment scheme. FIG. 4B)
Effect of resiquimod on positive lymph nodes (n=5). FIG. 4C) Effect
of resiquimod on tumor thickness. FIG. 4D) Effect of resiquimod on
intratumoral CD45.sup.+ cells. FIG. 4E) Effect of resiquimod on
intratumoral M-MDSCs. FIG. 4F) Effect of resiquimod on intratumoral
GramB MFI in CD8.sup.+ cells. FIG. 4G) Effect of resiquimod on
intratumoral GramB.sup.+CD8.sup.+ T cells . FIG. 4H) Effect of
resiquimod on intratumoral GramB.sup.+CD8.sup.+ki-67.sup.+ cells.
FIG. 4I) Effect of resiquimod on intratumoral PD1.sup.+CD8.sup.+
cells. All measurement was compared to the control. * indicates
p<0.05.
[0019] FIGS. 5A-5E. Resiquimod inhibits melanoma growth in B16
melanoma model. FIG. 5A) Treatment scheme. FIG. 5B) Effect of
resiquimod on melanoma growth (n=10). FIG. 5C) Effect of resiquimod
on mouse survival. FIG. 5D) Histology of melanoma from a control
(left) and resiquimod (right) treated mouse. There are more
inflammatory cells in the treated mice. Arrow points to the
inflammatory cells. FIG. 5E) Fold changes in cytokine and chemokine
mRNA expression in the tumor tissues from treated mice compared to
control mice. * indicates p<0.05. Bars in d indicate 25
.mu.m.
[0020] FIGS. 6A-6C. CD8+ T cells mediate the effect of resiquimod
on melanoma. FIG. 6A) Effect of resiquimod on Rag-1-decicient mice.
The survival curves showed that the effect of resiquimod was
abolished in these mice. FIG. 6B) Effect of resiquimod on
CD8-depleted mice.
[0021] The survival curves showed that the effect of resiquimod was
abolished in these mice. FIG. 6C) Effect resiquimod on cytokine
release by T cells after stimulation.
[0022] FIGS. 7A-7G. Combination of resiquimod and anti-PD1 therapy
dramatically reduced melanoma growth. FIG. 7A) Treatment scheme.
FIGS. 7B-7E) Representative images of mice treated with control
vehicle (FIG. 7B), anti-PD1 (FIG. 7C), resiquimod (FIG. 7D) and
resiquimod+anti-PD1 (FIG. 7E). FIG. 7F) Effect of therapies on
melanoma growth (n=5 each group). FIG. 7G) Effect of therapies on
lymph node metastasis. * indicates p<0.05. ** indicates
p<0.01. Bars indicate 6 mm.
[0023] FIGS. 8A-8D. Stability and solubility of resiquimod in a
topical formulation consisting of a combination on ethanol and
phosphate buffered saline (PBS). FIG. 8A-8C) HPLC chromatograms of
resiquimod formulations at different compositions in a
representative 50:50 ethanol:pbs topical formulation aged for 2
weeks. The chromatograms exhibit that resiquimod is stable and
soluble in the base formulation without degradation or formation of
by-products as noted by the presence of a single peak and
consistent retention times on a C18 column at various
concentrations in the base formulation. All data are collected
under the following gradient conditions: 0-5 mins:100% Water; 5-15
mins: 0 to 100% Acetonitrile gradient; 15-20 mins: 100%
Acetonitrile; 20-25 mins 100% to 0% Acetonitrile gradient; 25-30
mins: 100% Water in a 10 .mu.L injection containing (i) 16 nM; (ii)
12.7 nM; (iii) 9.5 nM; (iv) 6.4 nM; (v) 1.6 nM and (vi) 0.16 nM
resiquimod. FIG. 8D) A calibration curve showing area under the
curve of resiquimod as a function of the number of moles of
resiquimod present in the formulation. The strong statistical
correlation further indicates that resiquimod is stable and soluble
in a pbs:ethanol formulation.
[0024] FIGS. 9A-9D. Enhanced delivery of resiquimod from topical
formulations containing chemical permeation enhancers. FIG. 9A)
Human skin permeability of resiquimod from a formulation containing
50:50 PBS:ethanol (denoted as control), a formulation containing
10% (vol/vol) oleic acid as a chemical enhancer (denoted as
Formulation 1) and a formulation containing sodium lauryl sulfate
(5%) and oleic acid (2%) as chemical enhancers (denoted as
Formulation 2) at 6 hrs. These data demonstrate that chemical
permeation enhancers can significantly accelerate the topical
delivery of resiquimod across human skin as early as 6 hrs thereby
reducing the total topical dosage and/or reduction in lag time for
delivery of resiquimod to target for biological activity. FIG. 9B)
Amount of resiquimod delivered across the skin from three different
formulations in 6 hrs. FIG. 9C) Permeation of resiquimod across
human skin in three different formulations at 18 hrs is shown.
These data show enhanced permeation and enhanced delivery of
resiquimod across human skin in presence of permeation enhancers
compared to base formulation. FIG. 9D) Amount of resiquimod
delivered across the skin from three different formulations in 18
hrs.
[0025] FIG. 10. Adjuvancy potential of topical formulations. The
secretion of cytokine interleukin 1 alpha (IL-1.alpha.) from skin
tissue measured from 10 unique formulations that are also topical
permeation enhancers of varying efficacies. IL-1.alpha. is a
representative cytokine and marker of tissue inflammation
implicated in innate immune response.
DETAILED DESCRIPTION OF THE INVENTION
[0026] Skin cancer is usually superficially located. Melanoma in
situ is still confined to the epidermis and typically grows slowly.
Such lesions may take years to progress to invasive melanoma.
Invasive melanoma usually has an epidermal component but is no
longer confined to the epidermis as melanoma cells invade the
dermis. Metastatic cancer is usually deeply located. Because the
effectiveness of resiquimod requires the presence of functional
dendritic and T cells, it is currently impractical to test the
function of tumor killing effect of resiquimod in vitro. In
accordance with the present invention, we provide data
demonstrating that resiquimod functions in multiple in vivo
pigmented lesion transgenic mouse models and in a syngeneic
melanoma mouse model which have an intact immune system. Our data
showed that resiquimod was effective in inhibiting or killing
melanocytic tumor cells in nevus, atypical intraepidermal
melanocytic proliferation and melanoma. In addition, the mice
treated with resiquimod also had significantly reduced metastatic
melanoma in the lymph nodes, indicating that resiquimod can inhibit
melanoma cell metastasis. Additionally we showed that resiquimod in
combination with systemic anti-PD1 therapy significantly enhanced
the anti-PD-1 therapeutic effects in melanoma models. Importantly,
our data indicate that resiquimod induced an influx of CD45
positive leukocytes in the tumor and the effect of resiquimod is
mediated through both activation of CD8+ T cells and inhibition of
myeloid-derived suppressor cells. The implication of our discovery
is broad. Since the effect of resiquimod on tumors is mediated
through activation of CD8 T cells and inhibition of myeloid-derived
suppressor cells, the effect of resiquimod is not limited to
melanocytic tumors. It is also effective in treating other
epithelial cancers and cutaneous T-cell lymphoma.
[0027] Because metastatic cancer including melanoma is often in the
subcutis, or at other internal sites, topical application of
resiquimod is unlikely to reach these sites. To further improve the
treatment efficacy of resiquimod in late stage cancer/melanoma
patients, we developed an injectable form of resiquimod. We found
that even high doses of resiquimod injection did not cause
significant toxicity in mice, thus indicating that injectable
resiquimod is safe. Accordingly, the invention provides use of a
novel, injectable formulation of resiquimod alone or in
combination, with anti-PD1, or anti-PDL1, or Anti-CTLA-4 antibodies
or CD137 (Urelumab), CD40, CD134 (Anti-OX40) agonist and other
immune-modulating small molecules that can inhibit MDSCs (such as
PLX3397). This formulation, alone or in combination, and methods of
use thereof can be used advantageously to treat a wide variety of
pathological conditions, including without limitation, metastatic
epithelial cancer, squamous cell carcinoma, basal cell carcinoma,
lung, bladder, prostate, brain, breast, pancreas, liver, stomach,
other epithelial malignant tumor cells and mesenchymal malignant
tumor cells such as soft tissue sarcomas as well as hematopoietic
malignancy such as B cell lymphoma, leukemia and advanced cutaneous
T cell lymphoma.
[0028] Injectable formulations provide several advantages for
achieving enhanced penetration/permeation and/or immune adjuvancy
via the use of chemical moieties. US patent 2004/0265351 A1
(incorporated herein by reference) teaches methods for localizing
and retaining drugs like resiquimod within a tissue environment.
This is achieved by use of purified oils (soybean oil, safflower
oil, triolein, and castor oil, fractionated coconut oil,
miglyol810, 812, Neobee M S, Captex 300) and FDA approved dermal
fillers (hydroxylapatite, hyaluronic acid, Poly-L-Lactic Acid,
collagen, hydrogel) as components of injectable formulation. The
present invention includes novel applications of chemical moieties,
including for example, vehicles, gels and carriers for retarding
the diffusion of resiquimod from the site of injection using a
single component or a plurality of components towards this end. In
one approach, resiquimod is formulated in a solvent that ensures
stability and solubility for topical and injectable administration
(See FIG. 8). Notably, inclusion of chemical enhancers facilitates
the enhanced diffusion of the active ingredient, resiquimod,
locally across the tumor matrix (largely composed of cells and
extracellular matrix components) which is apparent from the data
presented in FIG. 9. Additionally, chemical enhancers have the
ability to cause local inflammation that is conducive to the
initiation and acceleration of an immune response by facilitating
the secretion of cytokines that are agents of inflammation and
mediators of the innate immune response. It is well-known that the
innate immune response synergizes with the adaptive immune response
and is beneficial in the overall activity against tumors and
malignant cells. This follows from the data presented in FIG. 10 on
the ability of chemicals to initiate the secretion of interleukin 1
alpha (IL-1.alpha.) in skin. IL-1.alpha. a is a known marker of
non-specific inflammation in tissues and provides a surrogate
measure of adjuvancy activity. Moieties capable of inducing
localized inflammation and tissue penetration enhancement include,
but are not limited to, members of a broad class of chemicals
including sulfoxides, alcohols, polyols, alkanes, fatty acids,
esters, amines and amides, terpenes, surface-active agents,
cyclodextrins, C.sub.2 or C.sub.3 alcohols and higher, a C.sub.3 or
C.sub.4 diol or higher, DMSO, DMF, DMA and related solvents,
1-n-dodecyl-cyclazacycloheptan-2-one, N-methyl-pyrrolidone and
N-(2-hydroxyethyl) pyrrolidone, and a broader class of azones, and
mixtures (binary, ternary or higher) thereof, that are otherwise
pharmacologically and chemically inert and chemically stable,
potent, nonsensitizing, nonphototoxic, noncomedogenic in ways other
than their claimed activities. Typical concentration ranges in
which such agents are employed depend on the specific agent
employed but are within a range of 0.01-50% wt/vol unless
constrained by their solubility in a base formulation that contains
0.5-50% vol/vol of ethanol in phosphate buffered saline.
[0029] The following definitions are provided to facilitate an
understanding of the present invention. They are not intended to
limit the invention in any way.
[0030] "Agonist" refers to a compound that can combine with a
receptor (e.g., a TLR) to induce a cellular activity. An agonist
may be a ligand that directly binds to the receptor. Alternatively,
an agonist may combine with a receptor indirectly by, for example,
(a) forming a complex with another molecule that directly binds to
the receptor, or (b) otherwise results in the modification of
another compound so that the other compound directly binds to the
receptor. An agonist may be referred to as an agonist of a
particular TLR (e.g., a TLR6 agonist) or a particular combination
of TLRs (e.g., a TLR 7/8 agonist--an agonist of both TLR7 and
TLR8).
[0031] "Ameliorate" refers to any reduction in the extent,
severity, frequency, and/or likelihood of a symptom or clinical
sign characteristic of a particular condition.
[0032] "Cell-mediated immune activity" refers to a biological
activity considered part of a cell-mediated immune response such
as, for example, an increase in the production of at least one
T.sub.H1 cytokine.
[0033] "Immune cell" refers to a cell of the immune system, i.e., a
cell directly or indirectly involved in the generation or
maintenance of an immune response, whether the immune response is
innate, acquired, humoral, or cell-mediated.
[0034] "Sign" or "clinical sign" refers to an objective physical
finding relating to a particular condition capable of being found
by one other than the patient.
[0035] "Symptom" refers to any subjective evidence of disease or of
a patient's condition.
[0036] "Treat" or variations thereof refer to reducing, limiting
progression, ameliorating, or resolving, to any extent, the
symptoms or signs related to a condition.
[0037] "Penetration enhancer" and "permeation enhancement" as used
herein relates to an agent and the increase in the permeability of
tissue to a drug respectively, i.e., so as to increase the rate and
extent at which the drug permeates through a tissue such as skin or
a tumor. The enhanced permeation effected through the use of such
enhancers can be observed, for example, by measuring the rate of
diffusion of drug through animal or human skin or tumor tissue
using a diffusion cell apparatus or in situ measurements. The
diffusion cell is described by Merritt et al. Diffusion Apparatus
for Skin Penetration, J. of Controlled Release, 1 (1984) pp.
161-162.
[0038] "Adjuvancy" as used here relates to the ability to influence
a non-specific inflammation or specific immune response caused by
an activator of the immune system.
[0039] As used herein, the term "anti-cancer response" to therapy
relates to any response of the cancer to therapy, preferably to a
change in tumor mass and/or volume after initiation of therapy.
Hyperproliferative disorder response may be assessed where the size
of a tumor after topical or systemic intervention is compared to
the initial size and dimensions as measured by CT, PET, mammogram,
ultrasound or palpation. Response may also be assessed by caliper
measurement or pathological examination of the tumor after biopsy
or surgical resection. Response may be recorded in a quantitative
fashion like percentage change in tumor volume or in a qualitative
fashion like "pathological complete response" (pCR), "clinical
complete remission" (cCR), "clinical partial remission" (cPR),
"clinical stable disease" (cSD), "clinical progressive disease"
(cPD) or other qualitative criteria. Assessment of
hyperproliferative disorder response may be done early after the
onset of therapy, e.g., after a few hours, days, weeks or
preferably after a few months. A typical endpoint for response
assessment is upon termination of chemotherapy or upon surgical
removal of residual tumor cells and/or the tumor bed. This is
typically three months after initiation of therapy.
[0040] As used herein "decreasing the size of a tumor" is defined
as a reduction in the size of a tumor. Such an effect can be
accomplished by reducing the number of proliferating tumor cells in
the tumor (e.g., by reducing cell division of the tumor cells)
and/or by inducing cytotoxicity or cell death (apoptosis) of
existing tumor cells. Accordingly, tumor growth is arrested or
prevented.
[0041] As used herein, the term "inhibiting cancer" or "inhibiting
cancer cell growth" is intended to include the inhibition of
undesirable or inappropriate cell growth. The inhibition is
intended to include inhibition of proliferation including rapid
proliferation. The term "inhibiting cancer cell growth" is also
intended to encompass inhibiting tumor growth which includes the
prevention of the growth of a tumor in a subject or a reduction in
the growth of a pre-existing tumor in a subject. The inhibition
also can be the inhibition of the metastasis of a tumor from one
site to another. A cancer is "inhibited" if at least one symptom of
the cancer is alleviated, terminated, slowed, or prevented. As used
herein, cancer is also "inhibited" if recurrence or metastasis of
the cancer is reduced, slowed, delayed, or prevented.
[0042] As used herein, "a," "an," "the," "at least one," and "one
or more" are used interchangeably. Thus, for example, a
pharmaceutical composition comprising "an" immune response
modulator (IRM) compound can be interpreted to mean that the
pharmaceutical composition includes at least one IRM compound.
[0043] As used herein, the term "subject" shall mean any animal
including, without limitation, a human, a mouse, a rat, a rabbit, a
non-human primate, or any other mammal. In one embodiment, the
subject is a primate. In another embodiment, the subject is a
human.
[0044] Also herein, the recitations of numerical ranges by
endpoints include all numbers subsumed within that range (e.g., 1
to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Additionally,
the inventive resiquimod containing compositions may contain
between 0.1-0.5%
[0045] As used herein, the term "synergistic" refers to a
combination of therapeutic agents described herein, which, when
taken together, is more effective than the additive effects of the
individual therapies. A synergistic effect of a combination of
therapies (e.g., a combination of therapeutic agents) permits the
use of lower dosages of one or more of the therapeutic agent(s)
and/or less frequent administration of the agent(s) to a subject
with a disease or disorder, e.g., a proliferative disorder. The
ability to utilize lower dosages of one or more therapeutic agent
and/or to administer the therapeutic agent less frequently reduces
the toxicity associated with the administration of the agent to a
subject without reducing the efficacy of the therapy in the
treatment of a disease or disorder. In addition, a synergistic
effect can result in improved efficacy of agents in the prevention,
management or treatment of a disease or disorder, e.g. a
proliferative disorder. Finally, a synergistic effect of a
combination of therapies may avoid or reduce adverse or unwanted
side effects associated with the use of either therapeutic agent
alone. As used herein, the term "in combination" refers to the use
of more than one therapeutic agent. The use of the term "in
combination" does not restrict the order in which the therapeutic
agents are administered to a subject with a disease or disorder,
e.g., a proliferative disorder. A first therapeutic agent, such as
a compound described herein, can be administered prior to (e.g., 5
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks before), concomitantly with, or subsequent to (e.g., 5
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks after) the administration of a second therapeutic agent, such
as an anti-cancer agent, to a subject with a disease or disorder,
e.g. a proliferative disorder, such as cancer.
[0046] Immune response modifiers ("IRMs") include compounds that
possess potent immunomodulating activity including but not limited
to antiviral and antitumor activity. Certain IRMs modulate the
production and secretion of cytokines. For example, certain IRM
compounds induce the production and secretion of cytokines such as,
e.g., Type I interferons, TNF-alpha, IL-1, IL-6, IL-8, IL-0, IL-12,
MIP-1, and/or MCP-1. As another example, certain IRM compounds can
inhibit production and secretion of certain TH2 cytokines, such as
IL-4 and IL-5. Additionally, some IRM compounds are said to
suppress IL-1 and TNF (U.S. Pat. No. 6,518,265).
[0047] Resiquimod,
(1-[4-amino-2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-
-ol) is an immune response modifier (IRM) that works by stimulating
cells through toll like receptors (TLR) 7 and 8.
[0048] As used herein, an "effective amount," when used with
respect to the combination of agents described herein includes,
without limitation, an amount of each agent in the combination that
provides a statistically significant desired effect on cancer
cells. In some embodiments, the effect amount can be narrowed to
further require clinical acceptability of the amount of toxicity to
non-cancer cells. Representative desired effects are described
herein. For example, the effect can be a decrease in the rate of
tumor growth, a cessation of tumor growth, or a reduction in the
size, mass, metabolic activity, or volume of the tumor, as measured
by standard criteria such as, but not limited to, the Response
Evaluation Criteria for Solid Tumors (RECIST), a statistically
significant increase in survival relative to treatment with
individual agents of the combination or sub-combinations of the
combination alone, and the like. The effective amount can vary
depending on such factors as the type of cell growth being treated
or inhibited, the type of therapeutic agent(s) employed, the
particular therapeutic agent, the size of the subject, or the
severity of the cancer cell growth or tumor. For example, the
choice of each of the individual agents which make up the
combination can affect what constitutes an "effective amount". One
of ordinary skill in the art would be able to study the
aforementioned factors and make the determination regarding the
effective amount of the combination of the therapeutic agents
without undue experimentation.
[0049] For example, an in vitro assay can be used to determine an
"effective amount" of the therapeutic agents. The ordinarily
skilled artisan would select an appropriate amount of each
individual agent in the combination for use in the aforementioned
in vitro assay. The cell survival fraction can be used to determine
whether the selected amounts were an "effective amount" for the
particular combination of therapeutic agents. For example, the
selected amounts used within the assay preferably should result in
a killing of at least 50% of the cells, more preferably 75%, and
most preferably at least 95%. In a preferred embodiment, the
effective dose of the therapeutic agent is a subtoxic dose. As used
herein, the term subtoxic dose refers to a dose which results in
the killing of less than about 10% of the cells.
[0050] The regimen (e.g., order) of administration can also affect
what constitutes an effective amount. Further, several divided
dosages, as well as staggered dosages, can be topically
administered daily or sequentially, or the dose can be continuously
infused. Further, the dosages can be proportionally increased or
decreased as indicated by the exigencies of the therapeutic
situation.
[0051] The phrase "pharmaceutically acceptable" is employed herein
to refer to those combinations of therapeutic agents, materials,
compositions, and/or dosage forms which are, within the scope of
sound medical judgment, suitable for use in contact with the
tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0052] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material, involved in carrying or
transporting the subject chemical from one organ, or portion of the
body, to another organ, or portion of the body. Each carrier must
be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0053] The term "pharmaceutically-acceptable salts" refers to the
relatively non-toxic, inorganic and organic acid addition salts of
the therapeutic agents encompassed by the invention. These salts
can be prepared in situ during the final isolation and purification
of the therapeutic agents, or by separately reacting a purified
therapeutic agent in its free base form with a suitable organic or
inorganic acid, and isolating the salt thus formed. Representative
salts include the hydrobromide, hydrochloride, sulfate, bisulfate,
phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate,
laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate,
fumarate, succinate, tartrate, napthylate, mesylate,
glucoheptonate, lactobionate, and laurylsulphonate salts and the
like. (See, for example, Berge et al. (1977) "Pharmaceutical
Salts", J. Pharm. Sci. 66:1-19)
[0054] Unless otherwise indicated, reference to a compound can
include the compound in any pharmaceutically acceptable form,
including any isomer (e.g., diastereomer or enantiomer), salt,
solvate, polymorph, and the like. In particular, if a compound is
optically active, reference to the compound can include each of the
compound's enantiomers as well as racemic mixtures of the
enantiomers.
Topical, Intracystic and Intratumoral Compositions Comprising
Resiquimod for the Treatment of Diseases
[0055] Chemical penetration enhancers are often used in topical and
transdermal formulations to enhance the absorption, uptake and
delivery of active pharmaceutical ingredients (drug or drug
substance) into the skin. Such formulations may also be employed to
complement further permeation within a localized compartment of the
tissue and/or to enhance an immune-related response in the tissue
in which they are delivered. Design of specific chemical enhancer
formulations is not trivial but requires the comprehensive
understanding of the effect of these agents on the skin
permeability. It is indeed feasible to match the specific chemical
enhancer formulation with the physicochemical properties of the
drug to specifically enhance the permeation and uptake of the drug
into the skin/tumor. Such formulations include, but are not limited
to, a combination of one or more chemical enhancers; one or more
solvents or vehicles that improve portioning of the drug and
chemical enhancers into the skin/tumor; and a gelling agent or
matrix for their incorporation as a topical or intratumoral
formulation. Several exemplars of chemicals under each category
have been previously disclosed in the literature. However, the
specific combination in which each ingredient is employed so that
they match the physicochemical properties of the drug and improve
its skin penetration are neither trivial nor singularly dependent
on the specific properties of the individual chemicals. It is
important to note that the effect of each ingredient on skin/tumor
is dependent of the concentration employed in the formulation and
there are synergistic effects to be anticipated from their
combination in a single formulation. These effects can be additive,
positively synergistic, or negatively synergistic. This then
implies that the kinetics of skin/tumor penetration and cumulative
effects of the drug delivered into and across the skin/tumor from
these formulations depends on the specific formulation employed. It
follows, therefore, that the physiological and biological and
therapeutic endpoints of the drug is determined to a large extent
by the formulation in addition to the drug properties. These
endpoints include, but are not limited to, the
pharmacokinetics/pharmacodynamics (PK/PD), cumulative absorption as
determined by the area under the curve (AUC), bioequivalence,
therapeutic index (TI), to highlight a few. Furthermore, the
specific formulation of the drug affects not only its potency but
also the tolerance/safety on the skin on application. This can
include, but is not limited to, adverse effects such as irritation,
skin toxicity, erythema amongst others.
[0056] A beneficial effect of chemical permeation enhancers in
addition to enhancing the permeation of a drug across a tissue, or
within a localized tissue compartment such as a tumor, is to
provide an inflammatory effect that is influential in directing the
immune response important in tumor therapy. Incorporation of agents
in topical, intracystic and/or injectable formulations can
therefore achieve additive or more than additive effects towards
the therapeutic endpoint of tumor treatment.
[0057] Formulations which are suitable for administration include
creams, ointments, solutions, gels, lotions, pastes, patches, foams
or spray formulations containing such carriers as are known in the
art to be appropriate. Dosage forms for the topical, intracystic or
intratumoral administration of resiquimod and other agents include
powders, sprays, ointments, pastes, creams, ointments lotions,
gels, solutions, and patches. The active component may be mixed
under sterile conditions with a pharmaceutically-acceptable
carrier, and with any preservatives, buffers, or propellants which
may be required. The ointments, pastes, creams, lotions, solutions,
foams and gels may contain, in addition to a therapeutic agent,
excipients, such as animal and vegetable fats, oils, waxes,
paraffins, starch, tragacanth, cellulose derivatives, polyethylene
glycols, silicones, bentonites, silicic acid, talc and zinc oxide,
or mixtures thereof. Injectable formulations may also be achieved
by use of purified oils (soybean oil, safflower oil, triolein, and
castor oil, fractionated coconut oil, miglyol810, 812, Neobee MS,
Captex 300) and FDA approved dermal fillers (hydroxylapatite,
hyaluronic acid, Poly-L-Lactic Acid, collagen, hydrogel) as
components of injectable formulation.
[0058] Topical, intracystic and intratumoral formulations can be
designed that are optimized by selecting the specific ingredients
of the formulation including the enhancer, vehicle and the matrix
(Karande 2004 Nature Biotechnology, Karande 2005 PNAS, and related
references within and citing these works). Specific formulation can
have a profound effect on efficacy of the drug and the target
indication. We have established the physicochemical properties of
resiquimod from its structure and chemical composition
(http://pubchem.ncbi.nlm.nih.gov/compound/Resiquimod#section=Depositor-Su-
pplied-Synonyms). We have identified chemical descriptors for
permeation enhancers that allow us to balance their potency and
safety. Based on these we have identified combinations of chemical
permeation enhancers that contain chemicals from the list below in
the total concentration range of 0-2% wt/vol in weight fractions
ranging between 0 and 100%. The formulation may contain 2 or 3
individual chemicals in a solvent to make the formulation.
Resiquimod will be combined in such formulations.
[0059] Sodium octyl sulfate, Sodium decyl sulfate, Sodium dodecyl
sulfate, Sodium tetradecyl sulfate, Sodium heptadecyl sulfate,
Sodium eicosyl sulfate, Sodium laureth sulfate, Nicotine sulfate,
Sodium taurocholic sulfate, Dimethyl sulfoxide, Sodium tridecyl
phosphate, ChemBetaine CAS, ChemBetaine Oleyl, ChemBetaine C,
Hexadecyldimethyl ammonio propane sulfonate, Decyldimethyl ammonio
propane sulfonate, Dodecyldimethyl ammonio propane sulfonate,
Myristyldimethyl ammonio propane sulfonate, Benzyl pyridinium
chloride, Dodecyl pyridinium chloride, Cetyl pyridinium chloride,
Benzyldimethyl dodecyl ammonium chloride, Benzyldimethyl myristyl
ammonium chloride, Benzyldimethyl stearyl ammonium chloride
Octyltrimethyl ammonium bromide, Decyltrimethyl ammonium bromide,
Dodecyltrimethyl ammonium bromide, Myristyltrimethyl ammonium
chloride, Cetyltrimethyl ammonium bromide Sorbitan monolaurate,
Sorbitan monopalmitate, Sorbitan monostearate, Sorbitan monooleate
Polyoxyethylene sorbitan monolaurate, Polyoxyethylene sorbitan
monopalmitate, Brij 97 Brij 30, Brij 56, Triton X-100, Hexanoic
acid, Octanoic acid, Decanoic acid, Undecanoic acid Dodecanoic
acid, Tridecanoic acid, Myristic acid, Palmitic acid, Stearic acid,
Oleic acid, Elaidic acid, Linoleic acid, Linolenic acid, Cholic
acid, Methyl hexanoate, Ethyl undecanoate, Methyl laurate, Methyl
tridecanoate, Methyl myristate, Isopropyl myristate, Isopropyl
palmitate, Palmityl palmitate, Diethyl sebaccate, Tetracaine,
Glyceryl monolaurate, Glyceryl monooleate Ethylpiperazine
carboxylate, N-Lauryl sarcosinate, Sodium caprylate, Sodium
decanoate, Sodium palmitate, Sodium oleate, Octyl amine, Decyl
amine, Dodecyl amine, Tetradecyl amine, Oleyl amine, Urea, Methyl
pyrrolidone, Cyclohexyl pyrrolidone, Octyl pyrrolidone, Decyl
pyrrolidone, Decyl methyl pyrrolidone, Methyl piperazine, Phenyl
piperazine, Octanamide Hexadecanamide, Caprolactam, Carveol, Pinene
oxide, Limonene, Menthol, Pulegone, Carvacrol Pinene, Menthone,
Terpineol, Cineole, Fenchone, Triacetin, Trimethoxy propylene
methyl benzene, Linalool, Geraniol, and Octyl dodecanol. Others
include sulfoxides, alcohols, polyols, alkanes, fatty acids,
esters, amines and amides, terpenes, surface-active agents,
cyclodextrins, C.sub.2 or C.sub.3 alcohols and higher, a C.sub.3 or
C.sub.4 diol or higher, DMSO, DMF, DMA and related solvents,
1-n-dodecyl-cyclazacycloheptan-2-one, N-methyl-pyrrolidone and
N-(2-hydroxyethyl)pyrrolidone, and a broader class of azones, and
mixtures (binary, ternary or higher)
[0060] The physicochemical and structural properties of resiquimod
indicate that it is a small molecule with reasonable hydrophobicity
attributed by its aromatic nature. Chemical enhancers that improve
transport of such molecules generally, but not exclusively, tend to
be long chain hydrocarbons with a polar head group such as
surfactants, fatty acids and fatty esters. Unsaturation (single or
double) in the hydrocarbon chain can additionally aid the
fluidization of skin lipid bilayers and permeation of resiquimod.
Oleic acid, linoleic acid, linolinic acid, palmitic acid, myristic
acid, etc. are exemplars of fatty acids. Esters of fatty acids such
as palmitate, myristate, oleate, linoleate, etc. with a small alkyl
group such as methyl, ethyl, propy or butyl are examples of fatty
esters. Salts of fatty esters such as sodium lauryl sufate, sodium
oleate, etc, are examples of surfactants. Short chain alcohols
including ethanol, isopropanol, butanol, hexanol, etc. are examples
of solvents. Combinations of chemicals and solvent can
significantly enhance the permeation of resiquimod across skin. All
potential combinations of such exemplars are viable formulations
with varying potency and safety with varying PK/PD profiles for the
API resiquimod.
[0061] Examples of formulations that embody the chemicals noted
above and their combinations are as follows: Resiquimod
concentration is 0.01, 0.02, 0.03, 0.04 to 1%, and can be combined
with the components listed in i, ii, iii, iv, v, vi, vii, viii, ix,
x, xi, xii or xiii.
i. Oleic acid (50%) and isopropyl myristate (50%) in a solution
containing 50 ml phosphate buffered saline and 50 ml ethanol at a
total concentration of 1% wt/vol. ii. Isopropyl myristate (50%) and
sodium lauryl sulfate (50%) in a solution containing 50 ml
phosphate buffered saline and 50 ml ethanol at a total
concentration of 0.5% wt/vol. iii. Oleic acid (50%) and sodium
lauryl sulfate (50%) in a solution containing 50 ml phosphate
buffered saline and 50 ml ethanol at a total concentration of 0.5%
wt/vol. iv. Oleic acid (33%), isopropyl myristate (33%) and sodium
lauryl sulfate (33%) in a solution containing 50 ml phosphate
buffered saline and 50 ml ethanol at a total concentration of 0.5%
wt/vol. v. Isopropyl palmitate (50%) and sodium oleate (50%) in a
solution containing 50 ml phosphate buffered saline and 50 ml
ethanol at a total concentration of 2% wt/vol. vi. Sodium lauryl
sulfate (25%) and linoleic acid (75%) in a solution containing 50
ml phosphate buffered saline and 50 ml ethanol at a total
concentration of 1.0% wt/vol. vii. Palmitic acid (50%) and
isopropyl laurate (50%) in a solution containing 50 ml phosphate
buffered saline and 50 ml ethanol at a total concentration of 2.0%
wt/vol. viii. Oleic acid (50%) and linoleic acid (50%) in a
solution containing 50 ml phosphate buffered saline and 50 ml
ethanol at a total concentration of 1.5% wt/vol. ix. Linoleic acid
(25%), oleic acid (25%) and isopropyl linoleate (50%) in a solution
containing 50 ml phosphate buffered saline and 50 ml ethanol at a
total concentration of 0.5% wt/vol. x. Sodium oleate (33%), oleic
acid (33%) and methyl palmitate (33%) in a solution containing 50
ml phosphate buffered saline and 50 ml ethanol at a total
concentration of 2.0% wt/vol. xi. A solution containing 50 ml
phosphate buffered saline and 50 ml ethanol. xii. Oleic acid (10%)
in a solution containing 50 ml phosphate buffered saline and 50 ml
ethanol. xiii. Oleic acid (2%) and sodium lauryl sulfate (5%) in a
solution containing 50 ml phosphate buffered saline and 50 ml
ethanol.
[0062] The following example is provided to illustrate certain
embodiments of the invention. It is not intended to limit the
invention in any way.
EXAMPLE I
Topically Applied Resiquimod Inhibits The Progression of
Precancerous and Cancerous Lesions
[0063] A majority of nevi and melanomas contain BRAF mutations,
particularly the BRAFV600E mutation. To test whether resiquimod can
be used to treat cutaneous diseases that have the potential to
become malignant tumors and early stage malignant tumors, we tested
the treatment efficacy of resiquimod on skin tumors using four
different mouse models: 1) a Tyr:creER, BrafCa nevus model in which
the mice develop nevus like lesions including atypical
proliferation of melanocytes in the epidermis (atypical
intraepidermal melanocytic proliferation), 2) a Tyr::CreER,
Phd2lox/lox, BRafCA melanoma model in which these mice will develop
primary melanoma were selected 3) Tyr::creER, BRAF.sup.ca,
.sup.Ptenlox/lox a melanoma model where the tumors develop very
quickly, and 4) the B16 melanoma model.
[0064] In the genetically engineered mouse (GEM) models described
above TLR activation induced changes were assessed. In the GEM
models, tumors arise de novo in the context of a normal immune
system and co-evolve with surrounding stroma. In addition, in situ
melanoma can only be observed and studied in GEM models but not in
the xenograft tumor models.
Effect of Resiquimod on Pigmented Tumor Progression in Tyr::creER,
BRAF.sup.ca Mice
[0065] We first tested treatment efficacy of resiquimod in
Tyr::creER, BRAT.sup.ca mice. This formulation comprised 0.2%
resiquimod, propylene glycol, colloidal silicon dioxide, and
triacetin. These mice develop nevus-like pigmented tumors after
4-HT induction, but lymph node metastasis does not occur. Mice
treated with blank topical gel were used as controls. When
pigmented lesions became palpable in Tyr::creER, BRAT.sup.ca mice,
we treated these mice with topical resiquimod or blank gel twice a
week for six weeks (FIG. 1A). Tumor diameters were measured using
calipers every three days. The pigmented tumors in the control
group continued to grow (FIG. 1B and 1I), while the pigmented
lesions treated with resiquimod regressed (FIG. 1C and 1I).
Histological examination showed that pigmented tumor cells occupied
the dermis in the control mice (FIG. 1F), while the tumor cells in
mice treated with resiquimod disappeared and only many macrophages
with pigment can be seen in the dermis (FIG. 1G). Pigmented tumor
cells in the epidermis also regressed completely (data not shown).
In a different experiment, we waited until the pigmented lesions
became palpable (FIG. 1D), then we treated these mice with topical
resiquimod for 1 month. The same pigmented lesion disappeared after
treatment (FIG. 1E). These mice were sacrificed and histology
showed only pigment containing macrophages in the dermis and viable
tumor cells were not seen.
Effect of Resiquimod on Melanoma Progression in Tyr::creER,
BRAE.sup.ca, Phd2.sup.lox/lox Mice
[0066] The Tyr:creER, BRAT.sup.ca, phd2.sup.lox/lox mouse melanoma
model was developed in Dr. Xu's lab. Melanoma can be induced with
100% penetrance using topical 4-HT. Lymph node metastasis is
frequent in these mice. However, unlike the Tyr::creER,
BRAF.sup.ca, Pten.sup.lox/lox mice, the latency of melanoma
development is significantly longer and the median survival after
Cre activation is about 180 days instead of 60 days in Tyr::creER,
BRAF.sup.ca, Pten.sup.lox/lox mice. Once these mice developed
palpable tumor (140 days after induction), we started topical
resiquimod treatment (0.2%) twice a week for 6 weeks (FIG. 2A). The
melanoma in control mice grew and the tumor became very big at day
180 (FIG. 2B). These mice were sacrificed. The underside of skin
was occupied completely by tumor (FIG. 2C). Histologically, the
dermis was filled entirely by melanoma cells (FIG. 2D and FIG. 2E).
In contrast, melanoma growth was inhibited after resiquimod
treatment (FIG. 2F). The underside of skin had significantly less
tumors (FIG. 2G). Histologically, dermal melanoma had regressed
(FIG. 2H) and numerous macrophages with pigment were present in the
dermis (FIG. 2I), indicating melanoma has regressed. None of the
treated mice developed lymph node metastasis. In the periphery of
melanoma in the control mice, melanoma cells were seen in the
epidermis (FIG. 2K) and they disappeared after treatment (FIG.
2I).
Effect of Resiquimod on Melanoma Progression in Tyr::creER,
BRAF.sup.ca, Pten.sup.lox/lox Mice
[0067] Because melanoma in Tyr::creER, BRAT.sup.ca,
Phd2.sup.lox/lox mice has long latency and melanoma growth is
relatively slow, we next tested resiquimod gel in Tyr::creER,
BRAT.sup.ca, Pten.sup.lox/lox mice, in which melanoma grows much
faster. Once these mice developed palpable tumors, we treated the
mice with resiquimod twice a week for 6 weeks (FIG. 3A). The mice
treated with control vehicle developed bulky tumors (FIG. 3B and
3F). Resiquimod treatment significantly slowed melanoma growth
(FIG. 3C and 3F). Furthermore, resiquimod treatment prolonged the
lifespan of the treated mice (FIG. 3G). Histologically, melanoma
infiltrated through the panniculus carnosus muscle in the control
mice (FIG. 3D); while melanoma was thinner and did not infiltrate
through the skeletal muscle in the resiquimod treated mice (FIG.
3E). In the periphery of melanoma in the control mice, in situ
melanoma was seen in the epidermis (FIG. 3H) and they disappeared
after treatment (FIG. 3I). In another experiment, mice were treated
with control vehicle or resiquimod for 3 weeks and then sacrificed
while they were still on the treatment (FIG. 4A). All 5 mice in the
control group developed lymph node metastasis and the number of
positive lymph nodes ranged from 2-4 per mouse (FIG. 4B), while in
the resiquimod treated group 2 of 5 mice developed lymph node
metastasis and 1 positive node each was identified in these mice
(FIG. 4B). The treated mice had significantly thinner tumors (FIG.
4C). Tumor and spleen tissues were processed and multicolor flow
cytometry analysis was performed. Our analysis detected a
significant increase of CD45.sup.+ inflammatory cells (FIG. 4D) and
decrease of CD45.sup.+CD11B.sup.+F4/80.sup.-Ly6g.sup.high MDSCs in
the tumor after treatment (FIG. 4E). The expression of granzyme B
increased in CD8.sup.+ T cells (FIG. 4F); the number of
GranB.sup.+CD8.sup.+ and GranB.sup.+CD8.sup.+Ki-67.sup.+ T cells
were increased after resiquimod treatment (FIG. 4G and 4H)
indicating effector T cell activation. Interestingly,
PD1.sup.+CD8.sup.+ T cells were also significantly increased,
suggesting an expanded tumor recognizing T cell repertoire. Data
from spleen are similar to these in the tumor (data not shown).
Effect of Resiquimod on Melanoma Progression in the B16 Melanoma
Model
[0068] To study whether the effect of resiquimod depends on BRAF
mutation, we tested the 0.2% resiquimod gel in C57BL/6J mice with
the B16-F10 melanoma cells. Once these mice developed palpable
tumors, we started resiquimod treatment twice a week (FIG. 5A).
Resiquimod treatment significantly slowed the melanoma growth (FIG.
5B) and prolonged the lifespan of the treated mice (FIG. 5C).
Histology showed more inflammatory cells in the tumor after
resiquimod treatment (FIG. 5D). Tumor tissues were processed and we
measured cytokine and chemokine mRNA expression in the tumor using
qRT-PCR. The analysis showed increased TNF-.alpha., INF-.gamma. and
CCL2 expression after resiquimod treatment compared to levels in
control mice (FIG. 5E).
Effect of Resiquimod on Melanoma Progression is Mediated Through
CD8.sup.+ T Cells
[0069] To study whether the effect of resiquimod depends on T
cells, the effects of resiquimod gel in RAG-1-deficient mice using
B16-F10 melanoma cells were tested. RAG-1-deficient mice do not
have mature B and T lymphocytes. Once these mice developed palpable
tumors, resiquimod treatment was administered twice a week.
Resiquimod treatment had little effect on melanoma growth (FIG.
6A). To study whether the effect of resiquimod depends on CD8+ T
cells, we tested the effect of resiquimod gel in CD8 depleted C57
mice using B16-F10 melanoma cells. CD8 depletion was achieved using
anti-CD8 antibody. Once these mice developed palpable tumors,
resiquimod was administered twice a week. Resiquimod treatment had
little effect on melanoma in CD8 T cell depleted mice, indicating
that the effect is mediated through CD8 T cells (FIG. 6B).
Resiquimod also has effects on cytokine release from T cells.
Resiquimod treated T cells from spleen showed increased expression
of IFN-.gamma. after stimulation (FIG. 6C).
Combination of Resiquimod with Anti-PD1 Therapy
[0070] The efficacy of 0.2% resiquimod in combination with anti-PD1
in Tyr::creER, BRAT.sup.ca, Pten.sup.lox/lox mice was assessed.
Once these mice developed palpable tumors, they were treated the
mice with resiquimod (twice a week for 6 weeks) and anti-PD1
therapy (200 .mu.g, i.p., twice a week for 5 weeks, FIG. 7A). The
mice treated with control vehicle developed bulky tumor (FIG. 7B)
and the mice treated with anti-PD antibody also developed bulky
tumors (FIG. 7C). Resiquimod treatment significantly slowed
melanoma growth (FIG. 7D) and the combination therapy showed the
most significant efficacy (FIG. 7E). The tumor volume was measured
and shown in FIG. 7F. Anti-PD1 therapy alone had no effect on lymph
node metastasis, while resiquimod significantly reduced the number
of positive lymph nodes and the combination further decreased
positive lymph nodes (FIG. 7G)
Enhanced Delivery of Resiquimod in Presence of Chemical Permeation
Enhancers
[0071] 0.2% resiquimod permeation was determined for three
different formulations across human skin. FIG. 8 shows the
solubility and stability of resiquimod in the base formulation on
50:50 ethanol: PBS. Additionally, data in FIG. 9 show the enhanced
permeation of resiquimod from formulations containing skin
permeation enhancers. Chemical permeation enhancers are capable of
reducing the lag time of permeation of resiquimod across human skin
as well as increase the total amount of resiquimod delivered across
human skin.
Adjuvancy Potency of Chemical Enhancer Formulations
[0072] Immune stimulating capacity of test formulations was
evaluated in terms of release of interleukin 1.alpha. (IL-1.alpha.)
in an in vitro skin model EpiDerm.TM., a 3D cell culture of human
normal epidermal keratinocytes after exposure to the formulations.
To study the adjuvancy potential of test formulations on skin, cell
cultures were exposed to 10 .mu.L of the test formulation for 4 h.
Each test formulation was analyzed in duplicate. Post exposure, the
incubation medium was removed and stored at -20.degree. C. Cytokine
content, specifically 1L-1.alpha., was measured using standard
human colorimetric ELISA kits for IL-1.alpha.. The optical
absorbance data from the extracted samples were then used, along
with the standards, to determine the IL-1.alpha. released (ng/ml).
Triton X100 (1% wt./vol.) was used as the positive control and 1:1
PBS:EtOH was used as the negative control. Data in FIG. 10 shows
adjuvancy potential of 10 unique formulations tested. Formulations
were designed as described previously.
[0073] While certain of the preferred embodiments of the present
invention have been described and specifically exemplified above,
it is not intended that the invention be limited to such
embodiments. Various modifications may be made thereto without
departing from the scope and spirit of the present invention, as
set forth in the following claims.
* * * * *
References