U.S. patent application number 10/643141 was filed with the patent office on 2005-04-07 for immune stimulation by phosphorothioate oligonucleotide analogs.
This patent application is currently assigned to Coley Pharmaceutical Group, Inc.. Invention is credited to Glover, Josephine M., Hutcherson, Stephen L..
Application Number | 20050075302 10/643141 |
Document ID | / |
Family ID | 32109766 |
Filed Date | 2005-04-07 |
United States Patent
Application |
20050075302 |
Kind Code |
A1 |
Hutcherson, Stephen L. ; et
al. |
April 7, 2005 |
Immune stimulation by phosphorothioate oligonucleotide analogs
Abstract
Methods of stimulating a local immune response in selected cells
or tissues employing immunopotentiating oligonucleotide analogs
having at least one phosphorothioate internucleotide bond are
provided. Methods of enhancing the efficacy of a therapeutic
treatment by stimulating a local immune response in selected cells
or tissues employing oligonucleotide analogs having at least one
phosphorothioate bond are also provided. The oligonucleotide
analogs may have antisense efficacy in addition to
immunopotentiating activity. Methods of modulating cytokine release
in skin cells and immunopotentiators which include oligonucleotide
analogs having at least one phosphorothioate bond capable of
eliciting a local inflammatory response are also provided.
Inventors: |
Hutcherson, Stephen L.;
(Richmond, VA) ; Glover, Josephine M.; (Surrey,
GB) |
Correspondence
Address: |
Helen C. Lockhart
Wolf, Greenfield & Sacks, P.C.
Federal Reserve Plaza
600 Atlantic Avenue
Boston
MA
02210
US
|
Assignee: |
Coley Pharmaceutical Group,
Inc.
Wellesley
MA
|
Family ID: |
32109766 |
Appl. No.: |
10/643141 |
Filed: |
August 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10643141 |
Aug 18, 2003 |
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09009634 |
Jan 20, 1998 |
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6727230 |
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09009634 |
Jan 20, 1998 |
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08712135 |
Sep 11, 1996 |
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5723335 |
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08712135 |
Sep 11, 1996 |
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08217988 |
Mar 25, 1994 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
C12N 15/1133 20130101;
A61K 31/7105 20130101; C12N 2310/18 20130101; C12N 2310/321
20130101; C12N 15/1131 20130101; A61K 38/00 20130101; C12N 2310/321
20130101; C12N 2310/315 20130101; C12N 2310/321 20130101; A61K
31/7125 20130101; C12N 15/117 20130101; C12N 15/1135 20130101; C12N
2310/3521 20130101; C12N 2310/3527 20130101 |
Class at
Publication: |
514/044 |
International
Class: |
A61K 048/00 |
Claims
1. (canceled).
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25. (canceled).
26. A method for stimulating an immune response in a human
comprising: administering by a route selected from the group
consisting of inhalation, ophthalmic, intranasal, parenteral, oral
and intradermal to the human as an immunopotentiator an amount of a
phosphorothioate oligonucleotide analog effective to stimulate an
immune response, wherein the phosphorothioate oligonucleotide
analog is not antisense.
27. The method of claim 26, wherein the phosphorothioate
oligonucleotide analog is an immunopotentiator of an antibody
response.
28. The method of claim 26, wherein the human has cancer.
29. The method of claim 26, wherein the human has an infection.
30. The method of claim 26, wherein the human is having
surgery.
31. The method of claim 26, wherein the phosphorothioate
oligonucleotide analog is formulated in a vehicle selected from the
group consisting of liposomes and cationic lipids.
32. The method of claim 26, wherein all of the internucleotide
linkages of the phosphorothioate oligonucleotide analog are
phosphorothioate linkages.
33. The method of claim 26, wherein the phosphorothioate
oligonucleotide analog includes at least one 2'-O-alkyl
modification.
34. The method of claim 26, wherein the 2'-O-alkyl modification is
a 2'-O-methyl modification.
35. The method of claim 26, wherein the 2'-O-alkyl modification is
a 2'-O-propyl modification.
36. The method of claim 26, further comprising administering a
therapeutic modality, before, after or simultaneously with the
phosphorothioate oligonucleotide analog.
37. The method of claim 26, wherein the therapeutic modality is a
drug.
38. A method for stimulating a systemic or humoral immune response
in a human comprising: administering to the human as an
immunopotentiator an amount of a phosphorothioate oligonucleotide
analog formulated in a vehicle selected from the group consisting
of liposomes and cationic lipids effective to stimulate the
systemic or humoral immune response, wherein the phosphorothioate
oligonucleotide analog is not antisense.
39. The method of claim 38, wherein the phosphorothioate
oligonucleotide analog is an immunopotentiator of an antibody
response.
40. The method of claim 38, wherein the human has cancer.
41. The method of claim 38, wherein the human has an infection.
42. The method of claim 38, wherein the human is having
surgery.
43. The method of claim 38, wherein all of the internucleotide
linkages of the phosphorothioate oligonucleotide analog are
phosphorothioate linkages.
44. The method of claim 38, wherein the phosphorothioate
oligonucleotide analog includes at least one 2'-O-alkyl
modification.
45. The method of claim 38, wherein the 2'-O-alkyl modification is
a 2'-O-methyl modification.
46. The method of claim 38, wherein the 2'-O-alkyl modification is
a 2'-O-propyl modification.
47. The method of claim 38, further comprising administering a
therapeutic modality, before, after or simultaneously with the
phosphorothioate oligonucleotide analog.
48. The method of claim 38, wherein the therapeutic modality is a
drug.
Description
FIELD OF THE INVENTION
[0001] This invention is directed towards methods for stimulating a
localized immune response and for enhancing the efficacy of
antiinfective and anticancer agents through local immune
stimulation. This invention is further directed toward
immunopotentiators comprising phosphorothioate oligonucleotide
analogs which produce the desired immune stimulation.
BACKGROUND OF THE INVENTION
[0002] Developments in recombinant DNA technology and peptide
synthesis have made possible the creation of a new generation of
drugs. However, small peptides and other agents do not always
invoke the immune response necessary for a therapeutic effect.
Substances which increase cell-mediated and/or humoral response may
be required in the formulation for efficacy. The potency of a
variety of agents, particularly antiinfective and antitumor drugs,
may be enhanced by stimulation of an immune response.
[0003] The cell-mediated immune response ("local immune response")
is produced by T-cells or thymus derived lymphocytes. T-cells are
able to detect the presence of invading pathogens through a
recognition system referred to as the T-cell antigen receptor. Upon
detection of an antigen, T-cells direct the release of multiple
T-cell lymphokines including the interleukin-2 family (IL-2). IL-2
is a T-cell growth factor which promotes the production of many
more T-cells sensitive to the particular antigen. This production
constitutes a clone of T-cells. The sensitized T-cells attach to
cells containing the antigen. T-cells carry out a variety of
regulatory and defense functions and play a central role in
immunologic responses. When stimulated to produce a cell-mediated
immune response, some T-cells respond by acting as killer cells,
killing the host's own cells when these have become infected with
virus and possibly when they become cancerous and therefore
foreign. Some T-cells respond by stimulating B cells while other
T-cells respond by suppressing immune responses.
[0004] The antibody or humoral immune response ("systemic immune
response") depends on the ability of B-cells, or bone
marrow-derived lymphocytes, to recognize specific antigens. The
mechanism by which B-cells recognize antigens and react to them is
as follows. Each B cell has receptor sites for specific antigens on
its surface. When an antigen attaches to the receptor site of a
B-cell, the B-cell is stimulated to divide. The daughter cells
become plasma cells which manufacture antibodies complementary to
the attached antigen. Each plasma cell produces thousands of
antibody molecules per minute which are released into the
bloodstream. As the plasma cells die, others are produced, so that,
once the body is exposed to a particular antigen, antibodies are
produced against that antigen as long as the antigen is present in
the body. Many B-cells appear to be regulated by the helper T-cells
and suppressor T-cells. Helper T-cells appear to stimulate B-cells
to produce antibodies against antigens, while suppressor T-cells
inhibit antibody production by either preventing the B-cells from
functioning or preventing the helper T-cells from stimulating the
B-cells. Some B-cells, however, are T-cell independent and require
no stimulation by the T-cells.
[0005] Immunopotentiators, such as adjuvants, are substances which
are added to therapeutic or prophylactic agents, for example
vaccines or antigens used for immunization, to stimulate the immune
response. Adjuvants cause an accumulation of mononuclear cells,
especially macrophages, at the site of injection. Macrophages
involved in this first stage of the immune response take in the
protein antigens and break them down into peptide fragments which
are then exposed on the cell surface where they form a physical
association with class II histocompatibility antigens. The T helper
cells recognize only protein fragments associated with class II
histocompatibility antigen, and not the free undegraded protein.
Nonprotein antigens are similarly processed by macrophages or other
antigen-presenting cells. The macrophages release monokines from
the interleukin-1 family (IL-1) which stimulate the T helper cells
to secrete IL-2. The actions of IL-1 and IL-2 result in the clonal
expansion of T helper cells. The clonal expansion of T helper cells
is followed by their interaction with B-cells, which in turn
secrete antibody.
[0006] Administration of an adjuvant resulting in stimulation of
IL-1 and other cytokines results in a complex spectrum of
biological activities. In addition to being a primary
immunostimulatory signal, IL-1 proteins have been linked with
prostaglandin production, inflammation and induction of fever. IL-1
proteins have been shown to have multiple effects on cells involved
in inflammation and wound healing and are known to stimulate
proliferation of fibroblasts and attract cells involved in the
inflammatory response.
[0007] Adjuvants encompass several broad classes including aluminum
salts, surface-active agents, polyanions, bacterial derivatives,
vehicles and slow-release materials. At present, most adjuvants
have been found to stimulate macrophages at the site of action;
however, certain adjuvants have been found to act as T-cell
replacers enabling B-cells to respond to antigen in the absence of
T-cells. An example of such an adjuvant is endotoxin, a B-cell
mitogen.
[0008] Polynucleotides and other polyanions have been shown to
cause release of cytokines. Also, bacterial DNA species have been
reported to be mitogenic for lymphocytes in vitro. Furthermore,
deoxyoligonucleotides (30-45 nucleotides in length) have been
reported to induce interferons and enhance natural killer (NK) cell
activity. Kuramoto et al. (1992) Jpn. J. Cancer Res. 83:1128-1131.
Oligonucleotides that displayed NK-stimulating activity contained
specific palindromic sequences and tended to be guanosine rich.
Immune stimulation has also been reported for antisense oligomers
that are complementary to the initiation sequence of HIV rev and to
the mink cell focus-forming (MCF) envelope gene initiation region.
Krieg et al. (1989) J. Immunol. 143:2448-2451; Branda et al. (1993)
Biochemical Pharmacology 45:2037-2043. The MCF sequence is an
endogenous retroviral sequence found in mice. In a study designed
to determine whether expression of these endogenous viral sequences
suppresses lymphocyte activation (as expressed infectious
retroviral sequences can), antisense oligonucleotides and analogs
complementary to the MCF env gene AUG region were used to inhibit
expression of MCF mRNA. This resulted in increased lymphocyte
activation. However, this was believed to be a specific effect
resulting from inhibition of the target gene, rather than an effect
of oligonucleotides per se. In this case both phosphodiester and
phosphorothioate oligonucleotides complementary to this target had
the same effect, whereas antisense oligonucleotides to other
retroviral targets and phosphorothioate control oligonucleotides
had no effect. Krieg et al. (1989) J. Immunol. 143:2448-2451;
Branda et al. (1993) Biochemical Pharmacology 45:2037-2043. Branda
et al. showed that an anti-rev phosphorothioate oligonucleotide
analog is mitogenic in both mononuclear cells from murine spleens
and human peripheral blood mononuclear cells. A
concentration-dependent stimulation of immunoglobulin production
was also observed in vitro and in vivo. This mitogenic effect was
specific for B-cells. These effects on B-cells were believed to be
specific to this anti-rev oligomer as oligonucleotides
complementary to the gag-pol initiation site and the 3' splice site
of endogenous retroviral sequences were known not to be stimulatory
(Krieg et al. (1989) J. Immunol. 143:2448-2451) and because another
phosphorothioate oligonucleotide analog of similar size, targeted
to the human p53 protein, did not exhibit the same effect. The data
suggested that endogenous retroviruses may suppress lymphocyte
activation and that antisense oligonucleotides specific for these
inhibitory retroviruses may reverse this suppression and stimulate
B-lymphocytes. Though Branda et al. speculated about the
possibility that the immune stimulation associated with this
oligomer may be independent of its antisense activity, for example,
contamination with endotoxin, no evidence for this could be found.
Furthermore, the lymphocyte stimulation seen was to an extent not
usually seen with exposure to double-stranded RNAs, which stimulate
lymphokines. Immune stimulation was concluded not to be a general
property of oligodeoxynucleotides, as they have been used by others
to inhibit T-cell function. Branda et al. (1993) Biochemical
Pharmacology 45:2037-2043.
[0009] The ability to reverse transforming growth factor-.beta.
(TGF-.beta.)-mediated cellular immunosuppression in malignant
glioma by addition of TGF-.beta.2-specific
phosphorothioate-antisense oligonucleotide analogs
(TGF-.beta.2-S-ODN's) has also been reported. Jachimaczak et al.
(1993) J. Neurosurg 78:944-951. TGF-.beta., an immunosuppressive
factor produced by malignant gliomas, is characterized by a wide
range of immunoregulatory properties including depression of T-cell
mediated tumor cytotoxicity, inhibition of IL-1- or IL-2-dependent
T-cell proliferation, lymphokine-activated killer and natural
killer cell activation, generation of cytotoxic macrophages and
B-cell function. The oligonucleotide analogs in these experiments
were used to block TGF-.beta. protein synthesis at the translation
level. In in vitro studies, preincubation of tumor cell cultures
with TGF-.beta.2-S-ODN's enhanced lymphocyte proliferation up to
2.5 fold and autologous tumor cytotoxicity up to 60%. Jachimaczak
et al. suggested these observations may have implications for in
vivo and in vitro activation of a cellular immune response against
autologous malignant glioma cells by inhibiting TGF-.beta.
synthesis.
[0010] Thus, as illustrated by the above-described studies,
antisense oligonucleotides and analogs have been used to
specifically inhibit expression of genes implicated in
immunosuppression, thus reversing the immunosuppressive
effects.
[0011] An antisense oligonucleotide targeted to the cellular
proto-oncogene c-myb has been demonstrated to block T-cell
proliferation in peripheral blood mononuclear cells. Gewirtz et al.
(1989) Science 245:180-183. Antisense oligonucleotides targeted to
interleukin-2 (IL-2) have been shown to specifically inhibit T-cell
functions, i.e., proliferation in response to allo-antigen or PHA
and IL-2 production. Kloc et al. (1991) FASEB J. 5:A973.
[0012] Thus, antisense oligonucleotides have been used to
specifically inhibit the expression of genes involved in T-cell
proliferation, thus blocking proliferation and resulting in an
immunosuppressive effect. Phosphorothioate monomers and congeners
thereof also have been demonstrated to affect humoral and
cell-mediated immune responses. It was shown that mice treated with
O,O,S-trimethyl phosphorothioate (OOS-TMP), a contaminant of
malathion and other organophosphate pesticides, developed
immunosuppression characterized by a decreased ability to make
either humoral or cell-mediated immune responses to subsequent
immunizations. Rodgers et al. (1987) Toxicol. Appl. Pharmacol. 88:
270-281. On the contrary, O,S,S-trimethylphosphorodithioate
(OSS-TMP) enhanced the generation of humoral and cell-mediated
immune responses in mice. Rodgers et al. (1988) Toxicol.
51:241-253.
[0013] Bacterial DNA and certain synthetic polynucleotides, both
single- and double-stranded, can stimulate proliferation of
lymphocytes in mice. One such example is AMPLIGEN.RTM.
[polyI:poly(C.sub.12U), HEM Research Inc., Rockville, Md.], a
double-stranded RNA (dsRNA) which acts as a lymphokine to mediate
cellular immune activity. This includes killer cell modulation,
macrophage modulation, B-lymphocyte modulation, tumor necrosis
factor modulation, interferon modulation and modulation of
interferon-induced intracellular enzymes. AMPLIGEN.RTM. has been
reported to stabilize T4 cell counts in patients with AIDS-related
complex and to have antineoplastic effects. AMPLIGEN.RTM. is a
specific form of mismatched dsRNA which has a uridine substituted
for every twelfth cytosine in the poly(C) strand. Poly(I):poly(C)
without this mismatching was highly immunogenic but proved to be
severely toxic and was abandoned as a clinical candidate in the
1970s. U.S. Pat. No. 5,194,245.
[0014] Certain synthetic oligonucleotides and analogs have been
shown to be mitogenic in vitro. These oligonucleotides were
polydeoxyguanosine, polydeoxycytosine or a mixture of the two.
Phosphorothioates were found to be more active than the
corresponding phosphodiesters. Pisetsky et al., (1993) Life
Sciences 54:101-107. In addition, a 21-mer phosphorothioate
oligonucleotide analog, ISIS 1082 (SEQ ID NO: 2), was also shown to
stimulate proliferation and antibody production by murine B cells.
This oligonucleotide is complementary to the translation initiation
codon of the herpes simplex virus UL13 gene. It was concluded that
the mitogenic effects of this and certain other oligonucleotides on
B cells may be due to preferential uptake of phosphorothioates and
other mitogenic oligonucleotides by B cells, and that the enhanced
penetration promotes a high intracellular concentration of these
compounds, leading to non-specific activation.
[0015] Oligonucleotides having a sequence identical to a portion of
the sense strand of the mRNA encoding the p65 subunit of NF-kB, a
DNA binding protein, were found to stimulate splenic cell
proliferation both in vitro and in vivo. The proliferating spleen
cells were shown to be B cells. Immunoglobulin secretion and NF-kB
activity in these cell lines was also increased by the sense
oligonucleotide. Both phosphodiester and phosphorothioate sense
oligonucleotides stimulated the splenocyte proliferation. The
antisense phosphorothioate oligonucleotide complementary to the
same region of p65 did not have this effect, and the stimulatory
effect was abolished by mixing the sense and antisense
oligonucleotides. Sense oligonucleotides having two mismatches from
the target sense sequence also failed to elicit the proliferative
effect. It was concluded that this was a sequence-specific effect
which may involve direct binding of the sense sequence to specific
proteins. McIntyre et al. (1993) Antisense Res. and Devel.
3:309-322.
[0016] It has now been found, surprisingly, that oligonucleotide
analogs having at least one phosphorothioate bond can induce
stimulation of a local immune response. This immunostimulation does
not appear to be related to any antisense effect which these
oligonucleotide analogs may or may not possess. These
oligonucleotide analogs are useful as immunopotentiators, either
alone or in combination with other therapeutic modalities, such as
drugs, particularly antiinfective and anticancer drugs, and
surgical procedures to increase efficacy. In addition, the
antiinfective and anticancer effects already possessed by certain
antisense oligonucleotide analogs are enhanced through such immune
stimulation.
[0017] It has also been found that oligonucleotide analogs having
at least one phosphorothioate bond can be used to induce
stimulation of a systemic or humoral immune response. Thus, these
oligonucleotides are also useful as immunopotentiators of an
antibody response, either alone or in combination with other
therapeutic modalities.
SUMMARY OF THE INVENTION
[0018] The present invention provides methods of stimulating a
local immune response in selected cells or tissues by administering
an oligonucleotide analog having at least one phosphorothioate bond
to the cells or tissues. Phosphorothioate oligonucleotide analogs
have been shown to stimulate a local immune response in animals and
humans. These methods are believed to be useful for enhancing the
efficacy of a therapeutic treatment, particularly an antiinfective
or anticancer treatment.
[0019] The present invention also provides oligonucleotide
immunopotentiators having at least one phosphorothioate bond which
are capable of eliciting a local inflammatory response. These
oligonucleotide immunopotentiators may also possess a therapeutic
activity, for example antisense activity. Several embodiments of
these immunopotentiators are provided which have been shown to
stimulate a local immune response in animals and humans.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Oligonucleotides and oligonucleotide analogs have recently
become accepted as therapeutic moieties in the treatment of disease
states in animals and man. For example, workers in the field have
now identified antisense, triplex, decoy and other oligonucleotide
therapeutic compositions which are capable of modulating expression
of genes implicated in viral, fungal and metabolic functions and
diseases. Oligonucleotide drugs have been safely administered to
humans and several clinical trials of antisense oligonucleotide
analog drugs are presently underway. It is, thus, established that
oligonucleotides and analogs can be useful therapeutic
instrumentalities and that the same can be configured to be useful
in regimes for treatment of cells, tissues and animals, especially
humans.
[0021] The present invention provides a method for stimulating a
local immune response in selected cells or tissues. The method
comprises administering to selected cells or tissues an effective
amount, preferably the amount needed to elicit a local inflammatory
response, of an oligonucleotide analog having at least one
phosphorothioate bond. It is preferred that selected cells or
tissues be infected by a fungus bacterium or virus. In one
embodiment, the cells are skin cells infected with a virus, such as
Herpes Simplex Virus Type-1 (HSV-1), Herpes Simplex Virus Type-2
(HSV-2) or Human Papilloma Virus. In one embodiment, the tissues
are condyloma acuminata (genital warts).
[0022] The present invention also provides a method for enhancing
the efficacy of a therapeutic treatment, preferably treatment with
an antiinfective or anticancer drug or a surgical treatment, by
administering to cells or tissues an effective amount, preferably
the amount needed to elicit a local inflammatory response, of an
oligonucleotide analog having at least one phosphorothioate bond.
In one embodiment, the cells are skin cells infected with a virus,
such as Herpes Simplex Virus Type-1 (HSV-1), Herpes Simplex Virus
Type-2 (HSV-2) or Human Papilloma Virus, and the therapeutic
treatment is treatment with an antiviral drug or surgical excision.
In one embodiment, the tissues are condyloma acuminata (genital
warts).
[0023] The present invention employs phosphorothioate antisense
oligonucleotide analogs which elicit a local inflammatory response.
These oligonucleotide analogs can be used alone to stimulate a
local immune response or can be administered in combination with
another therapeutic modality, either a drug or a surgical
procedure. These oligonucleotide analogs can modulate cytokine
release in skin cells upon contacting skin cells with an effective
amount of oligonucleotide analog. By an "effective amount" it is
meant an amount sufficient to elicit an immune response resulting
in the release of cytokines. In one embodiment of the invention,
oligonucleotide analogs are provided which have both therapeutic
efficacy (through antisense or other means) and immunopotentiating
activity. In one embodiment, the therapeutic activity is antisense
activity against a foreign nucleic acid (bacterial, fungal, viral
or oncogene-derived) in a host. Examples of several
phosphorothioate oligonucleotide analog sequences useful in the
present invention are provided in SEQ ID NO: 1, SEQ ID NO: 2 and
SEQ ID NO: 3.
[0024] In the context of this invention, the term
"immunopotentiator" refers to a material which produces
non-specific immune stimulation. Immune stimulation can be assayed
by measuring various immune parameters, for example
antibody-forming capacity, number of lymphocyte subpopulations,
mixed leukocyte response assay or lymphocyte proliferation assay.
Immune stimulation may result in increased resistance to infection
or resistance to tumor growth upon administration.
[0025] The term "oligonucleotide" refers to a plurality of joined
nucleotide units formed from naturally-occurring bases and
cyclofuranosyl groups joined by native phosphodiester bonds.
[0026] "Oligonucleotide analog," as that term is used in connection
with this invention, refers to moieties which function similarly to
oligonucleotides but which have non naturally-occurring portions.
Thus, oligonucleotide analogs may have altered sugar moieties or
inter-sugar linkages. Exemplary among these are the
phosphorothioate and other sulfur containing species which are
known for use in the art. They may also comprise altered base units
or other modifications consistent with the spirit of this
invention. In accordance with this invention, at least one of the
phosphodiester bonds of the oligonucleotide is replaced by a
phosphorothioate bond. The oligonucleotide analog may have
additional modifications to enhance the uptake, stability, affinity
or other features of the oligonucleotide. Some examples of such
modifications are modifications at the 2' position of the sugar
such as 2'-O-alkyl modifications, preferably lower alkyl such as
2'-O-methyl and 2'-O-propyl. All such analogs are comprehended by
this invention so long as they function effectively to produce an
immune response. The oligonucleotide analogs in accordance with
this invention preferably comprise from about 15 to about 50
subunits. As will be appreciated, a subunit is a base and sugar
combination suitably bound to adjacent subunits through
phosphodiester or other bonds.
[0027] Certain oligonucleotide analogs of this invention are
designed to be specifically hybridizable with messenger RNA of a
virus or oncogene, for example HSV-1, HSV-2, HPV or ras. This
relationship between an oligonucleotide and its complementary RNA
target is referred to as "antisense". These antisense
oligonucleotide analogs, which also stimulate an immune response in
keeping with the nature of the invention, thus can be said to have
a "combination" or "multimodal" mechanism of action. Several
embodiments of this type are phosphorothioate oligonucleotide
analogs of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
[0028] "Hybridization," in the context of this invention, means
hydrogen bonding, also known as Watson-Crick base pairing, between
complementary bases, usually on opposite nucleic acid strands or
two regions of a nucleic acid strand. Guanine and cytosine are
examples of complementary bases which are known to form three
hydrogen bonds between them. Adenine and thymine are examples of
complementary bases which form two hydrogen bonds between them.
[0029] "Specifically hybridizable" and "substantially
complementary" are terms which indicate a sufficient degree of
complementarity such that stable and specific binding occurs
between the target and the oligonucleotide or analog. It is
understood that an oligonucleotide need not be 100% complementary
to its target nucleic acid sequence to be specifically
hybridizable. An oligonucleotide is specifically hybridizable when
binding of the oligonucleotide to the target interferes with the
normal function of the messenger RNA to cause a loss of utility,
and there is a sufficient degree of complementarity to avoid
non-specific binding of the oligonucleotide to non-target sequences
under conditions in which specific binding is desired, i.e., under
physiological conditions in the case of in vivo assays or
therapeutic treatment, or, in the case of in vitro assays, under
conditions in which the assays are conducted.
[0030] The functions of messenger RNA to be interfered with include
all vital functions such as translocation of the RNA to the site
for protein translation, actual translation of protein from the
RNA, maturation of the RNA and possibly even independent catalytic
activity which may be engaged in by the RNA. The overall effect of
such interference with the RNA function is to cause interference
with expression of the targeted nucleic acid.
[0031] The oligonucleotide analogs of this invention are used as
immunopotentiators. For therapeutic or prophylactic treatment,
oligonucleotide analogs are administered to animals, especially
humans, in accordance with this invention. Oligonucleotides may be
formulated in a pharmaceutical composition, which may include
carriers, thickeners, diluents, buffers, preservatives, surface
active agents and the like in addition to the oligonucleotide.
Pharmaceutical compositions may also include one or more active
ingredients such as antimicrobial agents, antiinflammatory agents,
anesthetics, and the like in addition to oligonucleotides.
[0032] The pharmaceutical composition may be administered in a
number of ways depending on whether local or systemic treatment is
desired, and on the area to be treated. Administration may be done
topically (including ophthalmically, vaginally, rectally,
intranasally), intralesionally, orally, by inhalation, or
parenterally, for example by intravenous drip or subcutaneous,
intraperitoneal, intradermal or intramuscular injection. It is
generally preferred to apply the oligonucleotide analogs in
accordance with this invention topically, intralesionally or
parenterally. Formulations for topical administration may include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous,
powder or oily bases, thickeners and the like may be necessary or
desirable.
[0033] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders may be desirable.
[0034] Formulations for parenteral administration may include
sterile aqueous solutions which may also contain buffers, diluents
and other suitable additives.
[0035] In certain embodiments, the oligonucleotide analog is
administered in conjunction with a therapeutic agent, for example
an antiinfective or anticancer drug, or a surgical procedure. When
oligonucleotide analog is administered in conjunction with another
such therapeutic modality, the oligonucleotide analog may be
administered before, after and/or simultaneously with the
alternative treatment. In one embodiment of the invention, the
oligonucleotide analog is administered by intradermal injection to
the wound area upon excision of genital warts. In another
embodiment of the invention, the oligonucleotide analog is
administered by intradermal injection into genital warts.
[0036] Dosing is dependent on severity and responsiveness of the
condition to be treated, but will normally be one or more doses per
day, with course of treatment lasting from several days to several
months or until a cure is effected or a diminution of disease state
is achieved. Persons of ordinary skill can easily determine optimum
dosages, dosing methodologies and repetition rates.
[0037] In accordance with certain embodiments of the invention, a
number of antisense oligonucleotides which are targeted to selected
mRNAs were made. Natural oligonucleotides containing a
phosphodiester backbone were screened for anti-viral activity in an
infectious yield assay. The sequences showing the best activity in
this assay were synthesized as phosphorothioate analogs, the
phosphorothioate backbone modification greatly enhancing the
antiviral activity of the oligonucleotides through stimulation of a
local immune response.
[0038] Phosphorothioate oligonucleotide analogs include at least
one modified or unnatural internucleotide linkage which, in
addition to its enhancement of immune stimulation, can confer
stability and enhance uptake of oligonucleotide into cells. An O
(oxygen) of the phosphate diester group linking nucleotides is
modified to S (sulfur). Phosphorothioates often have in vivo
half-lives over 24 hours and have been shown to be stable in cells,
tissues, and drug formulations. Phosphorothioate oligonucleotide
analogs are believed to enter cells by receptor-mediated
endocytosis, and cellular uptake is often dependent on length and
size, specific sequences, protein binding, and pendant
modifications. Liposomes and cationic lipids can significantly
enhance the uptake and fate of oligonucleotides and analogs.
[0039] ISIS 1082 (SEQ ID NO: 2), a phosphorothioate oligonucleotide
analog 21 nucleotides in length targeted to the translation
initiation codon for the UL13 gene of Herpes Simplex Virus (HSV)
type 1 and 2, has been shown to inhibit HSV-1 replication in vitro.
Synthesis of the UL13 protein in vitro by translational arrest with
an IC.sub.50 of 200-400 nm has been observed. In vitro assessment
of the cellular toxicity of ISIS 1082 demonstrated that the
predicted therapeutic index for the compound is equivalent to or
better than that predicted for ACV in parallel assays. The
demonstration that ISIS 1082 shows antiviral activity in
ACV-resistant strains of virus and the favorable therapeutic index
observed with the compound underscore the potential clinical value
of this class of antiviral compounds. Studies have shown that the
compound is minimally toxic at therapeutically relevant
concentrations in vitro. The safety profile of this and other
related phosphorothioates has also been evaluated in animal models.
It has been observed that the compound causes an immune cell
activation in rodents at the site of injection. Specifically,
repeated intradermal administrations to rats elicited an infiltrate
of mononuclear cells. This was believed to be a consequence of the
interaction between the oligonucleotide analog and keratinocytes of
the skin, and the resulting release of cytokines.
[0040] To better understand the mechanism of the local
immunostimulatory response, the effects of ISIS 1082 on IL-1.alpha.
release and viability in a 3-dimensional in vitro human skin model
consisting of neonatal keratinocytes and fibroblasts were examined.
This system was chosen because epidermal cytokines play an
important role in mediating inflammatory and immune responses in
the skin. Keratinocytes are the principal source of cytokines in
the epidermis. This in vitro skin model displays many of the
functional and metabolic properties of a differentiated epidermis
and has been induced to specifically release IL-1.alpha. in
response to a mixture of lipopolysaccharide/phorbol myristate
acetate. Incubation of the skin model with ISIS 1082 resulted in a
concentration dependent increase of cytokine release with
essentially no effect on cellular viability, as measured by the
Neutral Red assay. These data indicate that IL-1.alpha., and
possibly other cytokines, are released from keratinocytes in
response to ISIS 1082 (SEQ ID NO: 2) may contribute to the immune
cell responses seen in vivo. It was subsequently determined that an
oligonucleotide (ISIS 1049, SEQ ID NO: 2) having the same sequence
as ISIS 1082 but with a phosphodiester backbone did not induce
IL-1.alpha. release in the skin model. To further elucidate the
relationship between oligonucleotide structure and IL-1.alpha.
release, a series of oligonucleotides and analogs having SEQ ID NO:
2 and either phosphorothioate (P.dbd.S) or phosphodiester (P.dbd.O)
backbones were prepared. These oligonucleotide analogs were further
modified at the 2' position. Table 1 shows these oligonucleotides
and their ability to induce IL-1.alpha. induction from the skin
model.
1TABLE 1 Oligonucleotide induction of IL-1.alpha. (all are SEQ ID
NO: 2) ISIS # Backbone 2' group Induce IL-1.alpha.? ISIS 1049
P.dbd.O deoxy no ISIS 1082 P.dbd.S deoxy yes ISIS 7374 P.dbd.O
O-methyl no ISIS 2007 P.dbd.S O-methyl yes ISIS 7389 P.dbd.O
O-propyl no ISIS 7337 P.dbd.S O-propyl yes
[0041] The ability to induce IL-1.alpha. in this assay is
correlated with the presence of the phosphorothioate backbone. It
is likely that a uniformly phosphorothioate backbone is not
necessary for cytokine induction, i.e., gapped, alternating or
otherwise mixed backbones containing at least one phosphorothioate
linkage may also induce IL-1.alpha.. These results also demonstrate
that other modifications, such as the sugar modifications in this
example, can also be present as long as at least one
phosphorothioate is present.
[0042] Antisense oligonucleotides and analogs have been used to
inhibit the replication of virus in cell culture. Studies have also
shown the effectiveness of antisense oligonucleotides in animal
models of viral infection. Animal models of HSV-induced keratitis
are well suited for such studies. Such ocular HSV infections are
usually treated topically and thus provide a relatively simple way
to test the effectiveness of antisense oligonucleotides in vivo;
The drugs can be applied topically in aqueous solution and several
parameters of the infection can be monitored. In one experiment
using a murine model, the effectiveness of the phosphorothioate
antisense oligonucleotide analog ISIS 1082 (SEQ ID NO: 2) made in
accordance with the teachings of the invention was tested for
treatment of herpetic keratitis. It was found that topical
treatment with this anti-UL13 oligonucleotide analog significantly
reduced the severity of HSV-induced stromal keratitis.
[0043] Three different concentrations of the oligonucleotide analog
as well as a buffer control (50 mM sodium acetate, pH 5.8, 0.15 M
NaCl) and untreated animals infected with HSV-1 were tested. All
animals were infected with 1.times.10.sup.5 plaque forming units
(pfu) following scratching of the cornea. It was found that
treatment with 0.3% and 1.0% ISIS 1082 did not affect the severity
of blepharitis, but treated mice healed slightly faster. Treatment
with ISIS 1082 reduced stromal disease and vascularization on days
11, 13, and 15 post-infection. This reduction in disease was
statistically significant on some days but not on others, probably
because of small sample size and variability in the disease. These
results indicate that antisense oligonucleotide analogs of the
invention may be useful in treating HSV keratitis.
[0044] ISIS 2105 (SEQ ID NO: 1) is a phosphorothioate 20 mer
complementary to the translation initiation of both HPV types 6 and
11 mRNA encoded by the HPV E2 open-reading frame. HPV-6 and HPV-11
are associated with genital warts. ISIS 2105 has been shown to
inhibit E2-dependent transactivation by HPV-11 E2 expressed from a
surrogate promoter. ISIS 2105 is among the first compounds to have
specific antiviral effect on papillomavirus, as demonstrated by
inhibition of focus formation.
[0045] The effects of ISIS 2105 on IL-1.alpha. release and
viability in the 3-dimensional in vitro human skin model was
examined. Incubation of the skin model with ISIS 2105 resulted in a
concentration dependent increase of cytokine release similar to
that seen with ISIS 1082. There was essentially no effect on
cellular viability, as measured by the Neutral Red assay. These
data suggest that IL-1.alpha. (and possibly other cytokines) is
released from keratinocytes in response to ISIS 2105 (SEQ ID NO:
1).
[0046] Intradermal administration of ISIS 2105 in rabbits has
resulted in no local or systemic toxicity. Phosphorothioate
oligonucleotide analogs, both as single doses and as daily doses
over a several-week period, can be administered to mice, rats and
rabbits without significant acute or subacute toxicity. ISIS 2105
has also been administered to cynomolgus monkeys by intradermal
injection at doses up to 10 mg/kg every other day for four weeks,
and was found to be well tolerated. No antibodies to ISIS 2105 were
detectable in monkey plasma at the end of the study, indicating
that ISIS 2105 is not intrinsically antigenic, i.e., while it
stimulates an immune response, it is not itself an antigen.
[0047] Intradermal administration of ISIS 2105 does produce a local
inflammatory response, however, in all species examined, including
rats, mice, rabbits, guinea pigs, monkeys and humans. This response
appears to be a class effect of all phosphorothioate
oligonucleotide analogs, as similar responses were produced in rat
skin by both ISIS 2105 and ISIS 1082 in 14-day studies. This
response is not a delayed-type hypersensitivity involving memory
T-lymphocytes but rather a result of the immunostimulation caused
by these oligonucleotide analogs acting as adjuvants or
immunopotentiators. Thus, while the phosphorothioate
oligonucleotide analogs do not appear to be intrinsically
antigenic, they are immunostimulatory. Immune stimulation is also
indicated by an increased humoral immune response in rats and
B-cell proliferation in the spleens of mice. Lymphoid hyperplasia
in the spleen of both rats and mice, and in the lymph nodes of
mice, was seen after ISIS 2105 treatment.
[0048] Mice and rats given repeated intradermal injections of ISIS
1082 (SEQ ID NO: 2) or repeated intravenous or subcutaneous
injections of several other phosphorothioate oligonucleotide
analogs [ISIS 2105 (SEQ ID NO: 1), ISIS 2503 (SEQ ID NO: 3,
targeted to the ras oncogene)] developed, on a subacute basis,
splenomegaly characterized by lymphoid hyperplasia. Lymphoid
hyperplasia was also observed in lymph nodes under many
experimental conditions. In addition, a predominantly mononuclear
inflammatory infiltrate has been observed in other organs/tissues
following repeated parenteral administration of phosphorothioate
oligonucleotide analogs. These effects were not associated with any
organ damage or dysfunction, and were reversible upon cessation of
oligonucleotide administration.
[0049] Studies in rats to determine the association of this
hyperplasia and the humoral component of the immune response to a
T-cell dependent antigen demonstrated that the IgM antibody-forming
cell response to the antigen was increased by 72% in rats dosed
daily with ISIS 2105 at 3.3 mg/kg/day, compared with rats dosed
with vehicle only. This was considered significant.
[0050] In clinical trials, 21 human subjects completed the trial
with seven different dosing regimens. All subjects showed some
degree of inflammation at the injection site, the extent of which
was related to size and frequency of dose. Biopsies were taken from
the injection sites of two of the three men in the dosing group
receiving ISIS 2105 injections in the forearm twice weekly (1.02
mg/injection at 3 sites) for three weeks. Both subjects had a dense
inflammatory reaction at the injection sites. This was detected by
histological examination of biopsies from injection sites. There
was both T- and B-cell involvement which is indicative of a local
immunological response to ISIS 2105.
[0051] Blood samples taken from three subjects at least two months
after completion of the trial showed no evidence of circulating
antibodies to ISIS 2105. This indicates that, as was found in
monkeys, ISIS 2105 is not intrinsically antigenic in humans.
[0052] Radiolabelled ISIS 2105 has been injected intradermally into
each of four genital warts (condyloma acuminata) in five male
patients. Systemic absorption of radiolabelled compound was
monitored by blood sampling at intervals postinjection. Warts were
removed at 1, 24, 48, 72, 96, 120 and 144 hours postinjection.
After injection, ISIS 2105 was localized at the site of injection
with rapid absorption (70% in 4 hours). Appreciable amounts of
intact drug (4 .mu.M) still remained in the wart tissue at 72
hours. Current estimates from in vitro studies indicate that
concentrations of approximately 1 .mu.M (and perhaps lower) are
therapeutically effective. The prolonged retention time at the site
of injection indicates that twice-weekly intralesional injections
should be sufficient for therapeutic effect.
[0053] The invention is further illustrated by the following
examples which are meant to be illustrations only and are not
intended to limit the present invention to specific
embodiments.
EXAMPLES
Example 1
Preparation of Oligonucleotides and Analogs
[0054] Oligonucleotides and analogs were synthesized at ISIS
Pharmaceuticals on an automated DNA synthesizer using standard
phosphoramidite chemistry with oxidation by iodine.
.beta.-cyanoethyldiisopropyl-phosphoramidites were purchased from
Applied Biosystems (Foster City, Calif.). For phosphorothioate
oligonucleotide analogs, the standard oxidation bottle was replaced
by a 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in
acetonitrile for the stepwise thiation of the phosphite linkages.
The thiation cycle wait step was increased to 68 seconds and was
followed by the capping step.
[0055] 2'-O-methyl phosphorothioate oligonucleotide analogs were
synthesized according to the procedures set forth above
substituting 2'-O-methyl .beta.-cyanoethyldiisopropyl
phosphoramidites (Chemgenes, Needham, Mass.) for standard
phosphoramidites and increasing the wait cycle after the pulse
delivery of tetrazole and base to 360 seconds. 2'-O-propyl
phosphorothioate oligonucleotide analogs were prepared by slight
modifications of this procedure.
[0056] Prior to use in various assays, oligonucleotides and analogs
were prepared by first incubating stock solutions at 37.degree. C.
for 1 hour and diluting prewarmed drug in tissue culture medium to
specified concentrations. Diluted compounds were filter sterilized
by centrifugation through 0.2 .mu.m pore size Centrex filters.
Example 2
Cell Line Maintenance
[0057] HeLa (ATCC CCL2) cells were maintained as monolayer cultures
in low glucose Dulbecco's Modified Eagles Medium (DME) supplemented
with 10% heat inactivated fetal bovine serum (FCS) while normal
human dermal fibroblasts (NHDF) [Clonetics #CC2010] were grown in
Fibroblast Basal Medium (Clonetics #CC-3130) with 0.2% FCS) in a 5%
CO.sub.2-humidified incubator at 37.degree. C.
Example 3
In Vitro Cellular Proliferation Assay
[0058] Asynchronous, logarithmically growing HeLa cells (10.sup.4)
were plated in 24 well tissue culture plates in 2.0 ml of 10% DME
and allowed to attach to plate surfaces overnight. The next day,
medium was aspirated and 2.0 ml of medium containing increasing
concentrations of ISIS 1082 or medium alone was added to each well
and placed in the incubator for 5 days. At the end of the
incubation period, the cells were harvested and counted in the
presence of trypan blue.
Example 4
In Vitro Skin Model
[0059] The in vitro model of skin (Full thickness model ZK1200) was
obtained from Advanced Tissue Sciences (La Jolla, Calif.). Nylon
mesh squares of tissue derived from neonatal keratinocytes and
fibroblasts were removed from storage wells containing agarose and
transferred to sterile, 24 well tissue culture plates containing
low glucose DME supplemented with 10% FCS and allowed to
equilibrate in a 37.degree. C. incubator overnight. The next day,
the growth medium was removed and replaced with assay medium (DME,
2% FCS) containing oligonucleotide and incubated with the tissue
for 24 hours.
Example 5
Neutral Red Assay
[0060] The keratinocyte tissue substrates were incubated for 24
hours at 37.degree. C., 5% CO.sub.2, 90% humidity in the presence
of oligonucleotide or LPS/PMA in assay media. The test agents were
removed, replaced with neutral red solution (50 .mu.g/ml), and
incubated for 3 hours. The neutral red was removed and tissue
substrates were washed with PBS. After a brief exposure to 0.5%
formaldehyde/lk calcium chloride solution, incorporated dye was
extracted using 1% acetic acid in 50% aqueous ethanol. The color
intensity of the solution, measured at 540 nm, was proportional to
viability of cells after drug exposure.
Example 6
Human IL-1.alpha. Immunoassay
[0061] A murine monoclonal antibody specific for IL-1.alpha. was
applied to microtiter plates. A 200 .mu.l aliquot of sample
supernatant was pipetted into the wells and incubated at room
temperature for 2 hours. After washing away any unbound proteins, a
polyclonal antibody against IL-1.alpha. conjugated to horseradish
peroxidase was added to the wells to sandwich any immobilized
IL-1.alpha. and incubated for 1 hour at room temperature. Following
a wash to remove any unbound antibody-enzyme, a substrate solution
of hydrogen peroxide and tetramethylbenzidine was added to the
wells and color developed in proportion to the amount of bound
IL-1.alpha.. The color development was terminated by the addition
of 2N sulfuric acid and the intensity of the color was measured at
450 nm.
Example 7
Immunological Evaluation of ISIS 2105 in Rats
[0062] The effects of repeated administration of ISIS 2105 to rats
on the humoral component of the immune response to a T-cell
dependent antigen were determined. Lymphoid hyperplasia in the
spleen and lymph nodes of rats dosed with ISIS 2105 had previously
been observed. Histomorphologic changes were found to be associated
with increased antibody production capacity in the spleen. Doses of
0.033, 0.18, 0.33 or 3.3 mg/kg/day were administered intradermally
to groups of 5 female Sprague-Dawley rats daily for 14 days. The
control group was given vehicle alone. A positive control group
received cyclophosphamide (25 mg/kg/day) by intraperitoneal
injection on days 11-14 of the study. All animals were sensitized
to sheep RBCs on day 11 by intravenous injection. At the end of the
14-day dosing period, the rats were euthanized and terminal body
weights, spleen and thymus weights were recorded. The IgM
antibody-forming cell response of the spleen was determined ex vivo
in spleen homogenates by quantifying plaque formation after
addition of sheep RBCs. High-dose animals had increased spleen
weights, both absolute (55%) and percent of body weight (48%), and
an increased spleen cellularity (27%) compared to vehicle-treated
animals. The IgM antibody-forming cell response to the T-dependent
sheep erythrocyte antigen, when evaluated as total spleen activity,
was increased by 72% in the 3.3 mg/kg/day group compared to
vehicle-treated animals. This was considered to be significant. The
positive control, cyclophosphamide, produced anticipated decreases
in immune parameters. In conclusion, ISIS 2105 appeared to enhance
the humoral response in rats receiving 3.3 mg/kg/day.
Example 8
Immunological Evaluation of ISIS 2105 in Mice
[0063] The effects of ISIS 2105 on various immune parameters in
female B6C3F1 mice when administered by intradermal injection daily
for 14 days were determined. Lymphoid hyperplasia in the spleen of
mice dosed with ISIS 2105 had previously been observed. Groups of 5
females each received doses of 0 (vehicle control), 0.066, 0.33,
0.66 or 6.6 mg/kg/day. On the day after the last injection (day
15), the animals were sacrificed, spleens were removed and weighed,
and a spleen cell homogenate was prepared for determination of
immunologic parameters, including enumeration of lymphocyte
subpopulations using specific antibodies, the mixed leukocyte
response (MLR) assay, and the lymphocyte proliferation assay. No
animals died during the study, and there were no treatment-related
effects on body weight or weight gain. Spleen weight (both absolute
and relative to body weight) was increased by approximately 50-60%
in the high-dose group (6.6 mg/kg/day) and this was associated with
increases in total spleen cell number (35%) and in the fraction of
Ig.sup.+ cells (45%) which is a marker for B-lymphocytes. Results
at the lower doses were inconsistent. The MLR, an indicator of
T-cell-dependent immune function, was decreased at the two highest
doses, but there was no effect on the spleen cell proliferative
response to the T-cell mitogen, Con A, at any dose level, which
indicates that the proliferative capacity of T-lymphocytes was not
altered. These results are somewhat inconsistent and must be
considered preliminary; however, it was concluded that the high
doses of ISIS 2105 may cause a form of immunostimulation.
Example 9
Intradermal Injection of ISIS 2105 in Humans
[0064] ISIS 2105 for clinical trials was formulated as sterile
phosphate-buffered solution for intradermal injection of volumes of
0.1 ml to 0.15 ml per injection. The concentration of ISIS 2105
varied depending on desired dose. Intradermal injections of ISIS
2105 were given into the ventral surface of the forearm of healthy
male volunteers.
Example 10
Immunostimulatory Response in Humans
[0065] Skin biopsies were performed in two human subjects following
administration of 5 doses of 1.02 mg of ISIS 2105. A skin ellipse
measuring 1.2.times.0.5 cm having a central pigmented area of 0.2
cm was removed from the forearm injection site. This ellipse was
bisected and processed for microscopic histological analysis. The
histological analysis revealed a moderately dense, inflammatory
infiltrate in all layers of the dermis from both subjects.
Immunohistochemistry revealed a mixture of cell types present.
T-cells were predominant; however, B-cells were also present
suggesting the immunological response was both T-cell and B-cell in
nature.
Example 11
Injection of ISIS 2105 into Genital Warts in Human Subjects
[0066] To evaluate its pharmacokinetics, the phosphorothioate
oligonucleotide analog ISIS 2105 (SEQ ID NO: 1) was .sup.14C
labeled in the 2-position of thymine. Approximately 1 mg (3.5
.mu.Ci/mg) was injected intradermally in each of four genital warts
(condyloma acuminata) in five male patients. Systemic absorption of
radiolabelled compound was monitored by blood sampling 1, 4, 8, 12,
24, 48, 72 and 144 hours postinjection. Warts were removed at 1,
24, 48, 72, 96, 120 and 144 hours postinjection. Urine and CO.sub.2
samples for .sup.14C analysis were taken at intervals
postinjection. Safety monitoring of these patients revealed no
clinically significant abnormalities. After injection, ISIS 2105
was rapidly absorbed (70% in 4 hours). However, appreciable amounts
of intact drug (4 .mu.M) remained in the wart tissue at 72 hours.
Current estimates indicate that concentrations of approximately 1
.mu.M are therapeutically effective. Peak plasma concentrations
were achieved within 1 hour following the absorption of labeled
ISIS 2105 from the injection site. Drug was cleared from plasma
with a rapid distribution and prolonged elimination phase. The
total body elimination half-life was estimated at 156 hours. The
oligonucleotide was slowly metabolized and the radiolabel was
eliminated, principally as CO.sub.2 in expired air and in urine. In
summary, following a single dose, intact ISIS 2105 was localized at
the site of injection with rapid absorption but prolonged retention
time in wart tissue. This indicates that twice-weekly intralesional
injections should be sufficient for therapeutic effect.
Example 12
Evaluation of ISIS 2105 as Surgical Adjuvant Therapy
[0067] Condyloma acuminata (genital warts) measuring at least
1.times.1 mm.sup.2 are surgically removed. Upon cessation of
bleeding with electrocautery, skin surrounding the ablated area is
injected with 0.1 cc of ISIS 2105 drug formulation containing 0.3
mg or 1 mg of ISIS 2105. Up to 4 warts are treated.
Sequence CWU 1
1
3 1 20 DNA Artificial sequence Oligonucleotide analog 1 ttgcttccat
cttcctcgtc 20 2 21 DNA Artificial sequence Oligonucleotide analog 2
gccgaggtcc atgtcgtacg c 21 3 20 DNA Artificial sequence
Oligonucleotide analog 3 tccgtcatcg ctcctcaggg 20
* * * * *