U.S. patent application number 10/742451 was filed with the patent office on 2004-09-30 for antiviral agent for use in treatment of cancer.
This patent application is currently assigned to INSTITUT GUSTAVE ROUSSY. Invention is credited to Abdulkarim, Bassam, Bourhis, Jean, Deutsch, Eric.
Application Number | 20040192637 10/742451 |
Document ID | / |
Family ID | 8242168 |
Filed Date | 2004-09-30 |
United States Patent
Application |
20040192637 |
Kind Code |
A1 |
Bourhis, Jean ; et
al. |
September 30, 2004 |
Antiviral agent for use in treatment of cancer
Abstract
The invention relates to an antiviral agent for use in
combination with an anticancer agent, for the treatment of cancer.
Especially, the invention provides means for the treatment of
non-virus-associated cancer.
Inventors: |
Bourhis, Jean; (Sceaux,
FR) ; Abdulkarim, Bassam; (Vanves, FR) ;
Deutsch, Eric; (Paris, FR) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
INSTITUT GUSTAVE ROUSSY
Villejuif Cedex
FR
UNIVERSITE PARIS-SUD XI
Orsay Cedex
FR
|
Family ID: |
8242168 |
Appl. No.: |
10/742451 |
Filed: |
December 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10742451 |
Dec 19, 2003 |
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10138999 |
May 2, 2002 |
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6723712 |
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10138999 |
May 2, 2002 |
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PCT/EP00/11246 |
Nov 3, 2000 |
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Current U.S.
Class: |
514/47 ;
514/50 |
Current CPC
Class: |
A61K 33/243 20190101;
A61P 35/00 20180101; A61K 31/675 20130101; Y02A 50/30 20180101;
A61K 45/06 20130101; A61K 33/24 20130101; A61K 31/675 20130101;
A61K 31/675 20130101; A61K 31/28 20130101; A61K 31/675 20130101;
A61K 2300/00 20130101; A61K 33/24 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
514/047 ;
514/050 |
International
Class: |
A61K 031/7076; A61K
031/7072; A61K 031/551 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 4, 1999 |
EP |
99402748.0 |
Claims
1. An antiviral agent for use in combination with an anticancer
agent, for the treatment of cancer.
2. Use of an antiviral agent of the manufacture of a drug for the
treatment of cancer, wherein said drug is used in a combined
treatment with an anticancer agent.
3. Use of an antiviral agent for the manufacture of a drug suitable
for the treatment of cancer by systemic route.
4. Antiviral agent or its use according to anyone of claim[[s]] 1
to 3, wherein the cancer is a virus-associated cancer.
5. Antiviral agent or its use according to anyone ef claim[[s]] 1
to 3, wherein the cancer is a non-virus-associated cancer.
6. Kit of parts comprising an antiviral agent and an anticancer
agent, for combined or separate use.
7. An antiviral agent, its use or a kit comprising an antiviral
agent, according to anyone of claims[[s]] 1 to 6, wherein the
antiviral agent is virus non-specific.
8. An antiviral agent, its use or a kit comprising an antiviral
agent, according to anyone of claim[[s]] 1 to 7, wherein the
antiviral agent has a broad-spectrum antiviral activity.
9. An antiviral agent, its use or a kit comprising an antiviral
agent, according to anyone of claim[[s]] 1 to 8, wherein the
antiviral agent has a cytotoxic activity.
10. An antiviral agent, its use or a kit comprising an antiviral
agent, according to anyone of claim[[s]] 1 to 8, wherein the
antiviral agent has an inhibition activity against viral and/or
cellular polymerases.
11. An antiviral agent, its use or a kit comprising an antiviral
agent according to anyone of claim[[s]] 1 to 8, wherein the
antiviral agent is a nucleoside phosphonate analogue.
12. An antiviral agent, its use or a kit comprising an antiviral
agent, according to anyone of claim[[s]] 1 to 11, wherein the
antiviral agent is an acyclic nucleoside phosphonate analogue.
13. An antiviral agent, its use or a kit comprising an antiviral
agent, according to claim 11, wherein the antiviral agent is HPMPC
[(S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine].
14. An antiviral agent, its use or a kit comprising an antiviral
agent, according to anyone of claim[[s]] 1 to 9, wherein said
antiviral agent has an activity against cell cycle regulation,
especially through cyclins.
15. An antiviral agent, its use or a kit comprising an antiviral
agent according to anyone of claim[[s]] 1 to 14, wherein the
antiviral agent is active against cyclins.
16. An antiviral agent, its use or a kit comprising an antiviral
agent according to anyone of claim[[s]] 1 to 14, for use in
combination with anticancer radiotherapy.
17. An antiviral agent, its use or a kit comprising an antiviral
agent according to anyone of claim[[s]] 1 to 14, for use in
combination with an anticancer chemotherapeutic agent.
18. An antiviral agent, its use or a kit comprising an antiviral
agent according to anyone of claim[[s]] 1 to 14, for use in
combination with an anticancer immunotherapeutic agent.
19. An antiviral agent, its use or a kit comprising antiviral agent
according to claim 18, wherein the immunotherapeutic agent is
associated with a cytokin.
20. An antiviral agent, its use or a kit comprising an antiviral
agent according to anyone of claim[[s]] 1 to 14, for use in
combination with at least two anticancer agents chosen among
radiation, chemotherapeutic and immunotherapeutic agents.
21. An antiviral agent, its use or a kit comprising an antiviral
agent according to anyone of claim[[s]] 1-20 for the manufacture of
a drug for systemic treatment of virus-associated cancer in
human.
22. An antiviral agent, it use or a kit comprising an antiviral
agent according to anyone of claim[[s ]]1 to 20, for the
manufacture of a drug for systemic treatment of
non-virus-associated cancer in human.
23. An antiviral agent, its use or a kit comprising an antiviral
agent according to anyone of claim[[s]] 1 to 20, for the
manufacture of a drug for intratumoral treatment of cancer in
human.
24. An antiviral agent, its use or a kit comprising an antiviral
agent according to claim 23, for the systemic treatment of cancer
in human.
25. Use according to anyone of claim[[s]] 1 to 20 or composition
according to anyone of claims 21 to 23, wherein the antiviral agent
is HPMPC and the other anticancer therapeutic agent is
radiotherapy.
26. An antiviral agent, a composition or a use according to anyone
of claims 23-25, for the treatment of a virus-associated
cancer.
27. An antiviral agent, a composition or a use according to anyone
of claims 23 to 25, for the treatment of a non virus-associated
cancer.
28. Antiviral agent or its use according to anye ne of claim[[s]]
4, 6 to 20 or composition according to anyone of claims 21-24
wherein the viral-associated cancer is associated with an infection
by a virus chosen among herpes-, adeno-, polyoma-, papilloma-,
Epstein-Barr or Hepatitis DNA viruses.
29. Antiviral agent or its use according to anyone of claim[[s]] 4,
6 to 20, wherein the cancer is a virus-associated cancer involving
infection by EBV or HPV virus.
Description
[0001] The present invention relates to the field of the treatment
of cancer and especially involves the use of antiviral agents for
the treatment of cancer. The invention relates to the treatment of
virus-associated cancer, or non-virus-associated cancer.
[0002] In studying various human tumor cell lines derived from
virus-associated cancers, the inventors have shown that antiviral
agents used in combination with other therapeutic agents, may
provide a new way for the treatment of cancer, with improved sucess
in controlling the development of the tumor. The inventors have
also shown that said combination of an antiviral agent with another
therapeutic agent may also be used advantageously in the treatment
of non virus-associated cancers.
[0003] Although there has been no direct relationship established
between a detected viral infection and the occurrence of cancer in
human, studies have shown in the past that virus infection can be a
co-factor frequently associated with carcigonenesis in infected
cells and as a consequence can be linked to the development of
malignant lesion and in general related with the development of
cancer.
[0004] In these situations where an infection by a virus can be
correlated with the development of malignancy especially in the
human body, it is believed that other contributing factors may also
be involved.
[0005] To date, it appears that human cancers, associated with
virus infection are mainly represented by lymphomas and carcinomas.
For example, infection by the Epstein-Barr virus (EBV) has been
detected in nasopharyngeal carcinomas, Burkitt and other lymphomas,
papillomavirus infection (HPV) has been shown to be involved in
some head and neck carcinomas, and uterine cervix carcinomas,
infections by Hepatitis B or C viruses have been associated with
the occurrence of hepatocarcinomas.
[0006] These virus-associated cancers, where viral infection is a
co-factor involved in the carcinogenesis of human cancer represent
15 to 20% of the whole number of cancers in the world (26,27).
[0007] From a general point of view, cancers, including
virus-associated cancers, are treated through different ways. It is
especially well-known that cancer treatment comprises surgery,
radiation and chemotherapy. More recently immunotherapy has been
introduced as a further available treatment regimen. It is also
noted that cancers may be treated, if appropriate, with a
combination of several of these available treatments. Therefore,
the above-cited treatment regimens can be viewed as constituting a
primary therapy or depending upon the specific cases, as an
adjudant therapy.
[0008] As far as virus-associated cancers are concerned, it is
noted that conventional treatments of the type of the above-cited
treatment, have shown a relatively high level of failure to cure or
improve the situation of the patients, especially in locally
advanced disease (40-60% failure in stage III-IV nasopharyngeal
carcinoma and in stage III carcinoma of the uterine cervix
(27)).
[0009] Therefore, new approaches for therapeutic treatment of
cancers are d sirable. Such an alternative or complementary
possibility of treatment of cancers is provided by the inventors
through the definition of means involving the use of antiviral
agents.
[0010] Interestingly, the efficiency which has been observed by the
inventors on the control of tumors associated with viral infection,
when using antiviral agents, has also been shown unexpectedly on
non virus-associated cancers.
[0011] In a publication (1), Andrei G. et al (<< Inhibiting
Effect of Cydofovir (HPMPC) on the Growth of the Human Cervical
Carcinoma (SiHa) Xenografts in Athymic Nude Mice >>), have
disclosed that in view of a strong association noticed between
infection with specific genital viruses (HPV viruses) and the
development of cervical cancer, an assay was made, to treat cell
lines derived from human cervical carcinoma with HPMPC
([(S)-1-[3-hydroxy-2-(phosphonomatoxy)propy]cytosine,
(phosphonomatoxy)propyl]cytosine, Cidofovir) which is known to be
an antiviral agent.
[0012] As a result of this experimental work, Andrei et al (1) have
shown that cell proliferation of these cell lines was inhibited in
a concentration-dependent and in a time-dependent fashion. They
further report that effects of HPMPC on the growth of cervical
carcinoma xenografts in athymic nude mice has been observed,
allowing to condude that animals that were injected intratumorally
with HPMPC at a certain dose, have shown statistically significant
reduction in tumor size compared to a placebo group or to a group
of animals treated with another specific antiviral agent. They
further state that, -when HPMPC was administered topically or
systemically, no reduction of tumor growth was observed when
nontoxic concentrations of the compound were used.
[0013] Within the frame of the present invention, the inventors
have observed that contrary to what has been concluded by Andrei et
al in the above-cited publication, HPMPC, among other antiviral
agents, can be used for the treatment of cancer and especially by
using non toxic systemic concentrations. Both virus-associated
cancers and non virus-associated cancers may be treated by the use
of antiviral agent in appropriate conditions defined in the present
invention.
[0014] The inventors provide means for the treatment of cancer,
that comprise the us of antiviral agents in combination with known
groups of anticancer agents, said combination enabling a synergic
effect to occur between the antiviral agent and the anticancer
agent. It is stated that anticancer agents implicated in the
production of this synergic effect indude conventional anticancer
agents among those used for the anticancer conventional therapy
cited hereabove.
[0015] The present invention therefore relates to a method of
treatment of cancer, which comprises the steps of:
[0016] administering to a patient in need thereof an anticancer
agent and
[0017] administering to said patient an antiviral agent.
[0018] Each of the features described hereafter for the definition
of the type of cancer to be treated or in relation to the nature or
use of the anticancer agent or of the antiviral agent is applicable
for the implementation of said method of treatment.
[0019] The sequences of administration of said anticancer and said
antiviral agents are defined by the skilled person.
[0020] According to the invention, the expression << synergic
effect >> signifies that the effect obtained with the
combination of several agents within the scope of the invention is
higher than the effect which is obtained with only one of these
agents or, advantageously the effect which is obtained with the
combination of the above said agents is higher than the addition of
the effects obtained with each of these agents used separately.
[0021] Accordingly, the inventors have shown that antiviral agents
can be used in combination with other groups of molecules,
compositions, or irradiation treatments used as anti-cancer agents
for the treatment of cancer, and especially for the treatment of
virus-associated cancers, thereby producing an improved effect on
the tumor development.
[0022] In the present invention, the expression << antiviral
agents >> relates to agents having an interaction effect and
for instance an inhibitory effect on the infection of cells by a
virus. Within the possible effects of said antiviral agents, one
may include the capacity of the antiviral agent to inhibit the
infection of the host cells by the virus and/or to inhibit the
replication of the virus or the proliferation of the virus in host
cells. Additionally or alternatively, the antiviral agents of the
invention are agents which can have a direct effect on the infected
host cells including for instance against their transformation
towards a malignant state.
[0023] For the purpose of the invention, an antiviral agent which
appears to produce a result in the treatment of cancer, when
combined with an anticancer agent as defined hereafter, is
designated as an antiviral agent.
[0024] By the expression << anticancer agent >> is
meant according to the present invention, any known agent or ag nt
to be developed which has, in proper conditions, an activity on th
formation of a malignant I sion and/or n th growth or on the
spreading of the formed malignant lesion towards the formation,
growth and spreading of the tumor.
[0025] In other words, it is pointed out that, according to the
invention, an anticancer agent can interfere with the process of
malignant transformation of a normal cell and/or with the
development or spreading of the tumor. In some cancers, anticancer
agents can interfere with abnormal cell differenciation or
metastases.
[0026] It is also emphasized that, the definition of the anticancer
agent applies to agents having effects on the control of the
biologic and/or biochemical basis for cancer disease, or on the
control of the clinical progress of the disease or recurrence
thereof. In a particular embodiment, the anticancer agent is able
to cure the cancer disease.
[0027] The expression << treatment of cancer >>
according to the invention, can be construed as encompassing the
effect that is normally sought with an anticancer agent, as defined
above. Advantageously, it encompasses the effect which can be
obtained on malignant cells or on developed tumors, following
administration of the combination of the antiviral-agent and the
anticancer agent. Especially it ncompasses reduction in tumor size,
which can be measured in accordance with the assays provided in the
examples of this patent application.
[0028] The word << combination >> which is used
according to the invention, designates the use of the antiviral
agent and of the anticancer agent in the treatment of a detected
cancer either a virus-associated cancer or a non virus-associated
cancer. In a particular embodiment of the invention, it encompasses
the associated use of both agents if they can be used together, for
instance in the same composition. Alternatively, it designates the
separated administration of th se agents. Said << separated
>> administration includes the simultaneous, concomitant or
sequential administration in time, either as a consequence of the
difference in physical or chemical nature of the agents or as a
result of the regimen or schedul of treatment requiring that the
agent be used separately in time, or be used through separated rout
s of administration.
[0029] Antiviral agents or anticancer agents are as a consequence
proposed for use in treatment of cancer, when they are capable, in
combination, to produce an int raction effect on the occurrence or
on the development of a malignant lesion and/or on the occurrence
or on the development of the resulting tumor.
[0030] According to a particular embodiment, the invention relates
to the use of an antiviral agent replying to one or several of the
various definitions provided in the present application, for the
manufacture of a drug for the treatment of a cancer either of a
virus-associated cancer or of a non-virus-associated cancer,
wherein said drug is used in combination with an anti-cancer
agent.
[0031] The invention also relates to the use of an antiviral agent
for the manufacture of a drug suitable for the treatment by
systemic route of a cancer, in accordance with the above-given
definitions.
[0032] Accordingly, in a particular embodiment, the invention
relates to a method for the treatment of cancer comprising the
steps of
[0033] administering an antiviral agent through the systemic route,
to a patient in need thereof,
[0034] administering to said patient, an anticancer agent.
[0035] Depending on its nature and properties, the anticancer agent
can also be administered through the systemic route. Alternatively,
it can be provided to the patient through another route, especially
locally.
[0036] Among the antiviral agents which can be used according to
the invention, antiviral agents which are non-specific for a
particular virus or for a determined group of viruses, are of
particular interest.
[0037] In a particular embodiment, the invention relates to an
antiviral agent as defined according to the invention, for use in
appropriate conditions, wherein this agent is chosen among
compounds or compositions having a broad spectrum antiviral
activity.
[0038] Antiviral agents may be classified in several groups which
may sometimes ov rlap, depending on the param t rs which are used
for the classification.
[0039] The specificity of the antiviral agent with respect to a
particular type of virus, or to the contrary with regard to its
activity against a broad spectrum of viruses, may be one of the
possibilities of classification of these agents.
[0040] It is also noted in accordance with the invention, that the
antiviral agents can be chosen with respect to their capacity to
interact with the targeted virus or with the host cells, especially
when the treated cancer is associated with viral infection.
[0041] In a particular embodiment, the invention relates to an
antiviral agent replying to one or several aspects of the
definitions given above, for use in the treatment of cancer,
wherein this antiviral agent has a cytotoxic activity on the cells
infected by the virus.
[0042] In addition or alternatively, the antiviral agent used in
accordance with the present invention is an antiviral agent capable
of inhibiting viral polymerases and/or cellular polymerases.
[0043] Advantageously, the invention proposes the use of antiviral
agents for the treatment of cancer, wherein the agent has an
activity on the cell cycle regulation of tumor cells. For instance,
this activity is observed as an action against the pathway
involving cyclins; preferably the antiviral agent interferes with
cyclin A in th tumor cells. The antiviral agents capable of
interfering with the cyclins' pathway are advantageously selected
among those which reply to one or several of any of the
characteristics which are disclosed in the present patent
application.
[0044] In a preferred embodiment of the invention, the antiviral
agent which is used is a nucleoside analogue and in a particular
embodiment, it is an acyclic nucleoside phosphonate analogue.
[0045] According to a preferred embodiment, the acyclic analogues
of nucleoside are substituted-N-alkyl derivatives of heterocyclic
basis, in which the nucleoside sugar moiety is replaced by a
substituted carbon chain bearing hydroxy groups. Once administered
to an organism, the biologically active nucleoside analogues
usually modify and give rise to production of 5' monophosphates,
active in vivo.
[0046] Preferred antiviral ag nts concerned by the invention are
acydic nucleoside phosphonate analogues. It is pointed out that
said acyclic nucleosid phophonate analogues, are characterized in
that their predominant activity is due to DNA polymerase
inhibition. Advantageously, in accordance with the invention, they
are not dependent upon the presence of a viral tyrosine kinase for
their activity. A number of these nucleotide analogues have been
synthesized and evaluated both in vitro and in vivo. Examples of
these are the [3-hydroxy-2-phosphonylmethoxyprop- yl] derivatives
of adenine (HPMPA) or cytosine (HPMPC, cidofovir), cyclic HPMPC
(cHPMPC), 9-(2-[phosphonylmethoxyethyl) derivatives of adenine
(PMEA, adefovir) or guanine (PMEG), 2-6 diaminopurine (PMEDAP),
cyclo-propyl PMEDAP (cPr-PMEDAP) and related compounds with similar
activities (29).
[0047] The inventors have obtained particularly interesting results
in using HPMPC
[(S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine] (designated
Cidofovir.RTM.). This antiviral agent has been extensively
disclosed in European patent 0 253 412.
[0048] These anti-viral agents have a predominant mode of action
that is targeted at viral and cellular DNA. Their activity is
mainly directed to the viral DNA, although not selectively since
they have also cellular effects leading to cytotoxicity, specially
at concentrations much higher than those needed for the viral
inhibition.
[0049] One of the predominant mechanisms involved in the anti-viral
effect of the nucleoside phosphonate analogues is the inhibition of
the viral DNA polymerase, at a concentration generally 10 to 1000
lower than that needed to inhibit the cellular DNA polymerases
alpha, beta, delta and hence cellular proliferation (28,29).
[0050] The inventors have also shown that the enhanced tumor
radiosensitivity observed when combining the administration of
radiotherapy with a treatment with an antiviral agent can be
associated in several human virus-related cancers, with a down
regulation of some viral oncoproteins and with an increase of
radiation-induced apoptosis.
[0051] The above disclosed elements relating to the biological
pathways explaining the antiviral activity, shall not be construed
as providing limitati n regarding the activity which is required
especially to enable the interaction of the antiviral agent, with
the virus or with the host cells. To the contrary, any antiviral
agent showing an activity in the treatment of cancer through
another biological pathway could be used, provided a result is
obtained in cancer treatment.
[0052] In a particular and preferred embodiment, the antiviral
agent of the invention is used in combination with an anticancer
agent comprising administration of radiotherapy. The antiviral
agent is advantageously Cidofovir used in combination with a
treatment of radiotherapy.
[0053] The inventors have indeed observed that the association of
an antiviral agent and of radiation therapy against tumors
including against virus-associated tumors or non virus-associated
tumors, enables a synergic effect to occur thereby remarkably
improving the likelyhood of success of treatment and moreover
nabling the antiviral agent to be used in accordance with treatment
modalities which were presented in the prior art as unacceptable
for the systemic route of cancer treatment.
[0054] As a matter of fact, the inventors have shown that doses of
antiviral agents which are lower than doses which were assayed in
the prior art to try to obtain a therapeutic effect, can be used in
accordance with the invention, enabling to obtain an unexpected
effect with respect to the result which would have been obtained,
in cumulating the effects of the individual administration of the
antiviral agent with the same dose on the one hand or of the
anticancer agent on the other hand.
[0055] Therefore, antiviral agents which would have been
disregarded for the treatment of cancer especially by the systemic
route of treatment, because the doses which would have been
required to obtain a therapeutic effect was not admissible in terms
of toxicity, in view of the results disclosed in the prior art have
been shown to present an interest in accordance with the present
invention, when us d in combination with another anticancer
agent.
[0056] Especially the combin d us of th se antiviral agents with
anticancer ag nts such as radiation, provid s an ffect on tumors or
on malignant cells, resulting from cooperation of both agents and
in the absence of occurrence of toxic effects, thereby becoming
suitable agents for the treatment of cancer, either of
virus-associated cancer or of virus-associated cancer.
[0057] The synergic effect has especially been shown on tumors
which presented poor reactions when treated by radiation only, or
by the antiviral agent alone, including when intratumoral
administration of the antiviral agents had no effect or a poor
effect on the tumor growth.
[0058] The inventors have shown to the contrary that the
combination of the antiviral agent and of radiation induces or
enables a significant effect on the growth of the tumor, even
enabling the complete remission of the tumor for a period of time
over fourty days.
[0059] This effect has been shown in the context of the invention,
either after intratumoral administration of the antiviral agent or
after subcutaneous administration of said agent in this latter
case, the doses which can be administered were lower than doses
which were disclosed in the prior art, as toxic for the
organism.
[0060] In order to illustrate the possible conditions for the
treatment, it is indicated that for an anticancer agent that would
be radiotherapy doses comprised within the range of 40 to 70 Gy can
be used and for an antiviral agent that would be Cidofovir doses of
the order of 1 to 100 mg/kg may be envisaged in human.
[0061] According to another embodiment of the present invention,
the antiviral agent is proposed for use in the treatment of cancer,
either for the treatment of virus-associated cancer, or for the
treatment of non-virus-associated cancer, in combination with an
anti-cancer chemotherapeutic agent. This anticancer agent can be
chosen in the group of well-known chemotherapeutic agents used in
the treatment of cancer, independently of the association of the
treated cancer with any virus infection. As an example, cisplatine
and etoposide are for instance cited. Treatments involving the use
of cytokines are also concerned.
[0062] According to another embodiment, the invention provides for
an antiviral agent and its use in the treatment of virus-associated
cancer wherein the antiviral agent is used in combination with an
anticancer immunotherapeutic agent.
[0063] It is noted that the conventional treatment available for
cancer, independently of presence or absence of an associated-virus
can be also combined, and especially both radiation,
chemotherapeutic and/or immunotherapeutic agents can be used in
addition to the treatment by the antiviral agent.
[0064] The antiviral agent which is used according to the invention
for the treatment of virus-associated cancer or of
non-virus-associated cancer or for the manufacture of a drug for
said treatment can be used either through systemic, intratumoral or
topical routes, and therefore can be formulated according to the
appropriate way depending upon the administration route.
[0065] Parenteral administration is preferred including
intravenous, intradermal, intramuscular, intrathecal, and other
parenteral administration routes.
[0066] The invention also relates to compositions comprising an
antiviral agent which are suitable for administration to the human
body comprises the use of antiviral agent at doses which are not
toxic for the organism when administered by systemic route and
especially which are capable of producing the effect sought. These
doses are determined in accordance with the usual practice in this
field.
[0067] Such a composition of the invention is appropriate for the
treatment of cancer, in particular for the treatment of cancer in a
human patient, and in a particular embodiment when combined with
the use of another treatment protocol including radiation,
chemotherapy and immunotherapy.
[0068] Based on the above given definitions, the invention provides
compositions comprising an antiviral agent, said compositions being
adapted for use in a treatment of cancer, in combination with
anticancer agents.
[0069] Where the antiviral and anticancer agents are both
chemotherapeutic substances, th y may be associated in kits, if
appropriate.
[0070] Other features of the invention and advantages of the use of
antiviral agents in accordance with the invention are provided in
the following examples.
[0071] When a virus is associated with the occurrence of the cancer
requiring therapeutic treatment, it can be in particular a target
of the treatment as DNA virus.
[0072] Within the group of cancers associated with infection by DNA
viruses, the invention relates to the treatment of virus-associated
cancers wherein the occurrence of the cancer is linked with the
infection by a virus chosen among Herpes viruses, Adenoviruses
(21), Polyoma viruses, Papillomaviruses (HPV)(2,3, 4, 9, 10, 20,
22), Epstein-Barr viruses (5, 15,23), Hepatitis DNA viruses (HBV or
HCV).
[0073] In the above paragraphs, some specific cancers have been
cited which are known to be associated with infection by particular
viruses or virus strains.
[0074] The invention especially concerns the use of antiviral
agents in the above and following described conditions in the
treatment of HPV-associated cancers, EBV-associated cancers or
HBV-associated cancers, HCV-associated cancers.
[0075] It is emphasized that the effect which is sought in using
antiviral agents for the treatment of virus-associated cancers is
not dependent upon the cellular type of the malignant cells or
dependent upon the tumor and unexpectedly is efficient v n in the
absence or virus.
[0076] The invention also relates to the association, for example
in a kit, of an antiviral agent and of an anticancer agent.
[0077] Said antiviral agent and said anticancer agent can be used,
depending upon their nature, either together, including in the same
composition for administration to the patient or can be physically
separated for simultaneous or concomitant use. Alternatively, the
antiviral agent and the anticancer agent can be used sequentially
during the administration of the treatment.
LEGEND TO FIGURES
[0078] FIG. 1 shows the effect of combining th nucl sid phosphonate
HPMPC (Cidofovir), and irradiation on clonog nic cell survival in 3
human cancer cell lines. The effect of the cidofovir alone was
substracted for each point measurement. The increased cell kill
obtained by the combined treatment was observed both for the
virus-related (Raji and HTB33) and for the non-virus-related cancer
cells (HTB31).
[0079] FIG. 2 shows the effect of the combination of intra-tumor
injection of cidofovir and irradiation on tumor growth of several
human xenograft cancers in nude mice. The effect is expressed as
the % of the initial tumor volume as a function time after the
treatment (days). For each cell line, a control group was used as
well as a group of irradiation alone (IR), irradiation+cidofovir
(IR+VIT), and cidofovir alone (VIT). The results show in all cases
a major effect on tumor growth in the group combining the 2 agents
(HEP2, HTB33, C15, Raji). For the C15 experiments is also shown the
effect of sub-cutaneous injection of 50 mg/kg of cidofovir in
combination with irradiation (VSC+IR), showing that intra-tumor and
subcutaneous administrations of cidofovir induced, in combination
with irradiation, an anti-tumor effect of the same magnitude.
[0080] FIG. 3: Proportion of apoptopic cells and LMP-1/Bcl2
expression in EBV+cells (C15 and RAJI) with and without
Cidofovir.
[0081] a, RAJI cells were cultured in the presence of Cidofovir (5
and 10 .mu.g/ml) and irradiated 48 hours later with 3 Gy and then
assayed for apoptosis by FACS analysis. C15 tumors were injected
intra-tumorally for 5 days with Cidofovir (50 mg/kg/day) and
received 7 Gy on day 3 and 5. Animals were sacrified on day 7 and
tumors were dissected and prepared for FACS analysis of
propidium-iodide nuclei staining as described above.
[0082] b, RAJI cells were cultured in the presence or absence of
Ciodofovir (10 .mu.g/ml). At 48 hrs, cells were harvested and
analyzed for the expression of LMP1, bcl2 and bax by Western-blot.
For C15, at day 7 portions of tumors where lysed in RIPA buffer and
protein extracts were immunoblotted with anti-LMP-1 antibody. The
blot from b was stripped and re-probed with a monoclonal
.beta.-actin antibody. Comparable results were obtained in 3
independent experiments.
[0083] FIG. 4 :Viral oncoproteins (E6/E7) and cellular proteins
(p53) expression in HPV+HTB33 and HEP2 cells, with and without
Cidofovir.
[0084] a.sub.2b.sub.2 HEP2 (a) and HTB33 (b) cells were cultured in
the presence or absence of Cidofovir (10 .mu.g/ml) for 3 and 6
days, and cells were collected in exponential phase, lysed and
total protein were immunoblotted with the Ab-1 anti-E7, C1P5
anti-B6, and DO7 anti-p53. The blots were stripped and re-probed
with a monoclonal .beta.-actin antibody.
[0085] Densitometric analysis of E6 and p53 bands
[0086] HTB33: E6 band was scored 1 without Cidofovir, 0.67 and
0.32, respectively for 3 and 6 days of Cidofovir exposure. P53
density was scored 1 without Cidofovir, 2.1 and 3.7 respectively
for 3 and 6 days of Cidofovir exposure.
[0087] HEP2: E6 band was scored 1 without Cidofovir, 0.7 and 0.25,
respectively for 3 and 6 days of Cidofovir exposure. P53 density
was scored 1 without Cidofovir, 1.2 and 1.65 respectively for 3 and
6 days of Cidofovir exposure. Comparable results were obtained in 3
independent experiments.
EXAMPLES
Cell Lines
[0088] A typical EBV+ human nasopharyngeal carcinoma cell line like
C15 (30) was used in vivo. An EBV+ lymphoma cell lines (Raji) EBV+
and 2 HPV+ squamous cell carcinoma lines, 1 of which originated
from the uterine cervix (HTB33) and 1 from the head and neck (HEP2,
HPV18+) were used.
[0089] A panel of 3 human cancer cell lines from the same tissue
origin, but lacking the viral infection were also used; namely 2
HPV- squamous carcinoma cells SCC97 (h ad and neck), HTB31 (cervix)
and the EBV- Ramos lymphoma cells.
[0090] Cell lines Raji (CCL86), Ramos (CRL-1596), Hep2 (CCL-23),
C33A (HTB-31) and Me-180 (HTB-33) are available in the ATCC
(American Type Culture Collection) catalogue.
[0091] Cells were grown in MEM medium supplemented with 15% fetal
calf serum, penicillin/streptomycin and 2 mM glutamine at 5%
CO2.
Apoptosis Assay
[0092] The cell cycle distribution was estimated by staining
ethanol-fixed cells with propidium iodide and monitoring by FACScan
flow cytometer (Becton Dickinson) using cellQuest software.
Briefly, 1.times.10.sup.6 cells were cultured without or with 5 and
10 .mu.g/mIl of Cidofovir, 24 hours later cells were irradiated
with 3 and 6 Gy and collected 12 and 24 hours after irradiation.
The percentage of apoptotic cells was determined by sub-G1
peak.
Histological Sections
[0093] At 10 day after treatment, mice with HPV+ tumors from all
groups were sacrified and tumors were excised and fixed in 10%
neutral buffered formalin (NBF). The sections from each group were
heamatoxylin and eosin (H&E)-stained for histological
analysis.
Immunoblot Analysis
[0094] To prepare total proteins, cells lysates were extracted with
lysis buffer 50 mM tris, pH 8, 120 mM NaCl, 0,1% SDS, and 0,5%
NP-40. The protein concentration in the soluble fraction was
detemnined by using a BioRad protein assay reagent. The viral
oncoprotein LMP1 was detected by immunoblotting with a monodonal
anti-LMP1 antibody (done CS1,CS2,CS3 & CS4 cocktail, RDI). The
following antibodies were used: anti-Bcl2 (clone 100 Santa Cruz
Biotechnology), anti-Bax (clone B-9, Santa Cruz, anti-E6 (clone
CIP5, abcam), anti-E7 (clone Ab-1, oncog ne), anti-p53 (clon DO7,
Dako). Anti .beta.-actin (clon AC-40, Sigma) was used to control
protein loading. Autoradiograms of the W st m blot were scanned
with the Gil doc 1000 image scanner (Bio-rad, Hercules, Calif.) and
densitometric analysis of the bands was performed using the
molecular analyst software program (Bio-rad, Hercules, Calif.).
Statistical Analysis
[0095] In vivo data are reported as the percentage of original (day
0) tumor volume and pl tted as fractional tumor volume.+-.S.E.
Statistical significance was determined by Kruskal/Wallis and
Mann-Withney U tests.
In vivo Experiments
[0096] Female swiss nu/nu mice were housed throughout experiments
in sterile isolators and fed ad libitum with irradiated food (UAR,
Villemoisson/Orge, France) and filtered water. Experiments were
performed according to the regulation n.sup.o86/609/CEE of the
European Community. Cell lines were established in vivo in Swiss
athymic mice by subcutaneous injection in the right flank of
5.times.10.sup.6 cells per animal and subsequently maintained in
vivo by sequential passages in animals aged 6 to 8 weeks. Nude mice
bearing 500-1000 mm3 tumors were used for in vivo experiments. Two
types of control were used including both injection of PBS
(intra-tumor and intravenous (IV)), irradiation atone, or
chemotherapy alone.
Irradiation and Clonogenic Survival Assay.
[0097] Irradiation of cells was performed using a .sup.137Cs
.gamma.-rays source at a dose rate of 1.45 Gy.min.sup.-1. Briefly
survival curves were obtained by irradiating cells (0 to 6 Gy). The
linear-quadratic model was used for fitting survival curves.
Quantification of radiosensitivity was obtained by the surviving
fraction at 2 Gy (SF2). Cell survival was also assessed by using
proliferation tests (incorporation of tritiated thymidine and MTT).
Irradiation of animals was performed using a 200 kv apparatus.
Mol cular Basis of the Invention
[0098] This aspect was mainly studied using western immunoblots and
flow cytometry (FACS) for protein expression analysis and protein
co-precipitation analysis (immunoprecipitation).
The Combination of Cidofovir and Ionizing Radiation Increases
Apoptosis and Tumor Necrosis.
[0099] Cidofovir is a potent inhibitor of the replication of EBV
and HPV in cell culture. In addition, the incorporation into the
cellular DNA of the infected cell may disrupt the g nomic integrity
and enhance susceptibility to apoptosis or necrosis. In C15 and
RAJI cell lines, Cidofovir (10 .mu.g/ml) combined with irradiation
(3 and 6 Gy) induced a marked increase of apoptosis (FIG. 3a). The
histological sections showed that increased tumor necrosis was
predominantly seen in the 2 HPV+ models (data not shown). Both in
EBV and HPV cancer cells, no significant change in
radiation-induced DNA repair was observed, as measured at the
chromosomal level by Fluorescence in Situ Hybridization (FISH)
(data not shown).
Cidofovir Induces a Modulation of some Viral Oncoproteins.
[0100] The effect of the combined treatment was major in the EBV+
and HPV+ cancer cells, whereas it was relatively marginal in the
corresponding virus negative models, suggesting a potential role
for some viral oncoproteins in this process (FIG. 1). Indeed, the
transforming properties of the EBV and HPVs are attributed to the
interaction of viral oncoproteins with critical cellular proteins
that control cell proliferation and apoptosis cell death (31,32).
Gene-transfer experiments have shown that the expression of LMP-1
specifically inhibits p53-mediated apoptosis, by inducing the
antiapoptotic cellular genes, Bcl-2 and A20.
[0101] Cidofovir exposure was able to induce a marked decrease of
LMP-1 expression both in RAJI and C15 cells (FIG. 3b). We observed
significant inhibition of LMP-1 expression as arly as 24 hours
(data not shown) and more pronounced inhibition 48 hours after
xposure to Cidofovir (FIG. 3b). The result might b related to th
inhibition of the viral replication and it is compatible with the
short halflife of LMP-1 protein (33). Importantly, the inhibition
of LMP-1 was associated with a downregulation of the LMP-1
inducible gene Bcl2, an up-regulation of the pro-apoptotic Bax
expression (FIG. 3b). In latent EBV infection as it occurs in C15
an RAJI cells, the induction of antiapoptotic genes by LMP-1
presumably contributes to protect cells from apoptosis (31). Such a
down regulation of LMP-1 and Bcl2 by Cidofovir could explain the
enhancement of the sensitity to readiation-induced apoptosis
observed in the FBV+ cell lines (RAJI and C15). Downregulation of
LMP-1 is reported here for the first time in cancer cells using a
pharmacological approach.
[0102] E6 and E7 oncoproteins are involved in cellular
transformation by interacting with the tumor suppressor proteins Rb
and p53, respectively (34, 35). P53 functional assay in yeast (36)
showed that p53 gene was wild type in cervical carcinoma HTB33 and
head and neck HEP2 cells (data not shown). However, the basal level
of p53 expression was relatively low as shown in FIG. 4a, and a
previously reported (35) which is compatible with a
proteasome-mediated degradation of p53 by E6 oncoprotein expressed
in these two cell lines. Unlike other cancers in which p53 is
mutated, the notion has arisen that the effect of E6 with respect
to p53 is equivalent to an inactivating mutation of p53 (32). This
underlines the importance of targeting E6 for therapeutic
intervention since blocking E6-mediated degradation of p53 may be
efficient to restore a normnal p53 expression in HPV1 cells (32).
In this study, we showed that Cidofovir exposure was able to
down-regulate E6 xpression with subsequent increase of p53
expression (FIG. 4a, 4b). This phenomenon was likely to enhance the
sensitivity to ionising radiation since the restoration of a normal
wtp53 expression has been shown to increase the radiosensitivity in
many human carcinoma models (37,38). In addition, a decreased
expression of the viral oncoprotein E7 was observed when these HPV+
cells were exposed to Cidofovir (FIG. 4b). It is important to point
out that Cidofovir was able to influ nce both E6 and E7 xpressi n
which may be more efficient for growth suppression than targ ting
ach of th s proteins s parately (39). Conversely, recent data have
shown that E6/E7 viral oncoproteins overexpression was associated
to p53 downregulation and radiation induced apoptosis inhibition in
cervical carcinoma cell line (40).
S me Examples of Interaction between the Acyclic Nucleoside
Phosphonate Analogues and Cytotoxic Agents are Presented
herewith:
Example N.sup.o1
Combination of an Acyclic Nucleoside Phosphonate Analogue with
irradiation in Human Cancer Cells in vitro
[0103] The example presented herewith relates to the combination of
cidofovir with irradiation, which was evaluated in vitro in various
virus-related and non virus-related human cancers.
[0104] It was studied in EBV+ human carcinoma C15 cells, EBV+
lymphoma Raji cells, 2 HPV+ carcinoma HTB33 and HEP2 cells. The
effect of the combination was also studied in 3 human cell lines
lacking the viral infection namely Ramos (lymphoma) HTB31 and SCC97
(carcinoma) cells.
[0105] For each cell line, several concentrations of cidofovir were
used in vitro in combination with irradiation (between 1 and 10
.mu.g/ml). The effect of the combined treatment was evaluated using
both a proliferation test and a donogenic assay for cell survival.
These two methods gave convergent results showing in all the EBV+
and HPV+ cell lines, that the addition of cidofovir to irradiation
produced a pronounced radiosensitization as shown by the dramatic
decrease of the SF2 (surviving fraction at 2 Gy). This marked
radiosensitizing effect was not restricted to the cell lines
exhibiting a viral infection since non virus-containing cells
Ramos, HTB31, and SCC97 were also markedly radiosensitized by the
cidofovir (see examples in FIG. 1).
Example N.sup.o2
Combination of an Acyclic Nucleoside Phosphonate Analogue with
Irradiati n in Human Canc r X nografts in vivo
[0106] The xample pres nted herewith also relates to th combination
of cidofovir with irradiation which was evaluated in vivo in
various human viral-associated and also in non viral associated
human cancers. The effect of cidofovir alone or combined with
irradiation was studied in vivo in nude mice bearing 500-1000 mm3
tumor x nografts. The treatment consisted of 60 mg/kg daily of
cidofovir intra-tumor injection for 5 days. In the combined group,
irradiation (7 Gy) was performed on day 3 and 5 of cidofovir. As
shown in FIG. 2; both irradiation alone and cidofovir alone induced
a weak growth delay, whereas the concomitant association of both
agents dramatically reduced the growth delay for the virus-positive
tumors (C15, Raji, HEP2, HTB33 . . . ), as well as for the
non-virally induced tumors tested. In all cases, in the group
receiving the combined treatment, nearly all the tumors were found
to be in complete remission, 30 to 40 days after the treatment,
suggesting th existence of a major interaction between the 2
agents.
Effect of the Route of Administration in vivo
[0107] In vivo, the effect of non toxic doses 25 to 75 mg/kg from
day 1 to day 5 of the nucleoside phosphonate analogues administered
intra-eritoneally or subcutaneously (C15, HTB33 etc . . . ) was
found to be as efficient for tumor inhibition (FIG. 2), as compared
to the intra-tumor mode of administration. The doses used
intra-peritoneally and subcutaneously were in the range of 0.5 to
1.66 times the doses used for intra-tumor injections.
M lecular Basis of the Observed Effect
[0108] The molecular basis of the interaction is not fully
understood. In the non-virus related cancer types, nucleoside
phosphonate analogue were found to interfere with DNA repair,
apoptosis and cell cycle regulation (cyclin D1, E and A), which
could explain the observed radiosensitization. In addition to these
mechanisms, in the virus-associated cancer types, an inhibition of
the viral oncoproteins could be also observed. For example, in the
EBV+ raji cells, nucleoside phosphonate analogues induced a down
regulati n of the viral oncoprotein LMP1 and consequ ntly th
anti-apoptotic Bcl2 g n xpression was down regulated, contributing
to increase radio-induced tumor cell kill.
[0109] In conclusion, the combination of an anti-viral nucleoside
phosphate analogue and irradiation described in this example
represents a totally new approach for the treatment of
virus-associated and non-virus-associated human cancers. Indeed, th
results obtained showed a major effect of combining this type of
anti-viral agent with irradiation.
Example N.sup.o3
Combination of an Acyclic Nucleoside Phosphonate Analogue with
Chemotherapeutic Agents in Human Cancer Cells.
[0110] Similar experiments combining a nucleoside analogue with
cytotoxic agents other than irradiation were performed
(chemotherapeutic drugs, cytokines). The example presented herewith
relates to the combination of cidofovir with chemotherapeutic drugs
which was evaluated in Wimr and in vivo in various virus-associated
and non virus-associated human cancers. Two drugs were tested,
showing a synergic inhibitory effect on tumor cell growth using a
combination of a nucleoside phosphonate analogue with cis platinum
(2.5 .mu.g/ml, in vitro) and VP16 (5 .mu.g/ml, vitro).
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