U.S. patent application number 10/840238 was filed with the patent office on 2005-02-24 for inhibition of viruses.
Invention is credited to Arnold, Jamie, Balzarini, Jan, Brown, Daniel M., Cameron, Craig, Castro, Christian, Graci, Jason, Korneeva, Victoria, Loakes, David, Moriyama, Kei, Negishi, Kazuo.
Application Number | 20050043268 10/840238 |
Document ID | / |
Family ID | 26246745 |
Filed Date | 2005-02-24 |
United States Patent
Application |
20050043268 |
Kind Code |
A1 |
Loakes, David ; et
al. |
February 24, 2005 |
Inhibition of viruses
Abstract
Disclosed is a pharmaceutical composition comprising a
ribonucleoside analogue in accordance with general formula (I) or
(II) as herein defined in admixture with a physiologically
acceptable excipient diluent or carrier.
Inventors: |
Loakes, David; (Cambridge,
GB) ; Brown, Daniel M.; (Cambridge, GB) ;
Negishi, Kazuo; (Okayama, JP) ; Moriyama, Kei;
(Okayama, JP) ; Balzarini, Jan; (Leuven, BE)
; Cameron, Craig; (State College, PA) ; Arnold,
Jamie; (State College, PA) ; Castro, Christian;
(State College, PA) ; Korneeva, Victoria; (State
College, PA) ; Graci, Jason; (State College,
PA) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Family ID: |
26246745 |
Appl. No.: |
10/840238 |
Filed: |
May 7, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10840238 |
May 7, 2004 |
|
|
|
10207005 |
Jul 30, 2002 |
|
|
|
Current U.S.
Class: |
514/46 ;
514/263.2; 514/263.4; 514/267; 514/47 |
Current CPC
Class: |
C07H 19/16 20130101;
A61P 31/14 20180101 |
Class at
Publication: |
514/046 ;
514/263.2; 514/263.4; 514/267; 514/047 |
International
Class: |
A61K 031/52; A61K
031/7076; A61K 031/519 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 7, 2001 |
GB |
0126701.2 |
Claims
1. A pharmaceutical composition comprising a ribonucleoside
analogue in accordance with general formula I or II 3where: n=1-4,
preferably 2-4, X.sup.1=N or CH or CR.sup.5 X.sup.2=N or S or
CR.sup.5 X.sup.3=NR.sup.6 or O or S, or R.sup.6 when X.sup.2=N, or
X.sup.3=NR.sup.6 or R.sup.6 when X.sup.2=S, and X.sup.3 is absent
when X.sup.2=CR.sup.5 R.sup.1=H or alkyl or aryl or alkaryl or acyl
R.sup.2=H or aryl or alkaryl or acyl; when X.sup.2=S, R.sup.2 is
absent; R.sup.3=H or NR.sup.5R.sup.6 or NR.sup.5NR.sup.5R.sup.6 or
NR.sup.5OR.sup.5 R.sup.5=H or alkyl or alkenyl or alkynyl or aryl
or alkaryl or acyl R.sup.6=H or alkyl or alkenyl or alkynyl or aryl
or alkaryl or acyl and R.sup.4=H or 4wherein Z=O or S or CH.sub.2
or CHF or CF.sub.2 or NR.sup.5 X.sup.4=OH or F R.sup.7=H or
PO.sub.3.sup.2- or P.sub.2O.sub.6.sup.3- or P.sub.3O.sub.9.sup.4-
or a masked phosphate derivative, in admixture with a
physiologically acceptable excipient, diluent or carrier.
2. A pharmaceutical composition according to claim 1, wherein the
ribonucleoside analogue is provided as the base analogue or the
ribonucleotide analogue.
3. A pharmaceutical composition according to claim 1 or claim 2,
wherein the ribonucleoside analogue comprises a purine
analogue.
4. A pharmaceutical composition according to claim 1 which,
following administration to a human or animal subject, gives rise
to a chemical entity which, inside a cell of the subject, is
incorporated into a RNA molecule by a cellular, or preferably
viral, RNA polymerase present in the cell.
5. A pharmaceutical composition according to claim 4, wherein the
cell is infected by an RNA virus, the RNA molecule is an RNA copy
of at least part of the viral genomic nucleic acid molecule.
6. A pharmaceutical composition according to claim 1, wherein the
ribonucleoside analogue is such that Z is O.
7. A pharmaceutical composition according to claim 1, wherein
X.sup.2 is N.
8. A pharmaceutical composition according to claim 1, wherein
X.sup.3 is O or comprises N.
9. A pharmaceutical composition according to claim 1, wherein
X.sup.4 is OH.
10. A pharmaceutical composition according to claim 1, wherein
X.sup.2 is N and X.sup.3 is NH.sub.2.
11. A pharmaceutical composition according to claim 10, comprising
a ribonucleoside analogue having the structure shown in FIG. 3 or
FIG. 7.
12. A pharmaceutical composition according to claim 1, wherein
X.sup.2 is N, X.sup.3 is O and R.sup.1 is alkyl.
13. A pharmaceutical composition according to claim 12, wherein
R.sup.1 is methyl or substituted methyl.
14. A pharmaceutical composition according to claim 13, comprising
a ribonucleoside analogue having the structure shown in FIG. 11, or
the corresponding ribonucleotide analogue.
15. A method of making a pharmaceutical composition suitable for
preventing and/or treating an RNA virus infection in a human or
animal subject, the method comprising the step of mixing a
ribonucleoside analogue in accordance with general formula I or II
of claim 1 with a physiologically acceptable excipient, diluent or
carrier.
16. (canceled)
17. A method according to claim 15, comprising the step of
combining a plurality of different ribonucleoside analogues, each
analogue being in accordance with general formula I or II.
18. A method according to claim 15, comprising the step of
including in the pharmaceutical composition a further antiviral
agent.
19. A method according to claim 18, wherein the further antiviral
agent is an inhibitor of reverse transcriptase.
20. A method according to claim 18, wherein the further antiviral
agent is active against HIV or other retrovirus.
21. A method according to claim 15, further comprising the step of
packaging the composition in unitary dose form.
22.-23. (canceled)
24. A method of treating an RNA virus infection in a human or
animal subject, the method comprising the step of administering to
a subject infected with an RNA virus an effective amount of a
ribonucleoside analogue in accordance with general formula I or II
as defined in claim 1.
25. A method according to claim 24, comprising administering to the
subject a pharmaceutical composition in accordance with claim
1.
26. (canceled)
27. A method according to claim 37, wherein the ribonucleoside
analogue has the structure shown in FIG. 2 or is the corresponding
ribonucleoside analogue.
28. (canceled)
29. A pharmaceutical composition according to claim 1 which, when
administered to a human or animal subject infected with an RNA
virus, inhibits replication of the virus and/or causes an increase
in the mutation frequency of the virus.
30. A pharmaceutical composition according to claim 1 which, when
administered to a human or animal subject infected with an RNA
virus, causes inhibition of LTR-mediated transcription of viral
nucleic acid.
31. (canceled)
32. A composition suitable for application to a plant, for the
purpose of preventing and/or treating an RNA virus infection of the
plant, the composition comprising an RNA nucleoside analogue
conforming to general formula I or II of claim 1.
33. A composition according to claim 32, further comprising a
surfactant and/or a plant penetration enhancer.
34. A method of preventing and/or treating an RNA virus infection
in a susceptible plant, the method comprising the step of applying
to the plant an efefctive amount of a composition according to
claim 32 or 33.
35. and 36. (canceled)
37. A method of treating or preventing an RNA virus infection in a
human or animal subject which comprises administering thereto an
amount of a composition according to claim 1 sufficient to inhibit
LTR-mediated transcription of viral nucleic acid.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method of inducing
mutations in viruses, a method of inhibiting the replication of
viruses, pharmaceutical compositions for use in inhibiting the
replication of viruses, and the use of various compounds in the
preparation of medicaments to inhibit viral replication. The
invention specifically applies to RNA viruses, that is, viruses
which have an RNA genome or which replicate via an essential RNA
intermediate.
BACKGROUND OF THE INVENTION
[0002] RNA viruses are responsible for many diseases of man and
animals. Examples of RNA viruses which are human pathogens include
influenza virus, poliovirus, rhinovirus and HIV. A specific example
of a pathogenic DNA virus which replicates via an essential RNA
intermediate is hepatitis B virus (HBV).
[0003] Very few effective antiviral agents are currently available.
Certain compounds which are moderately effective against HIV are
deoxynucleoside analogues. These act by inhibiting HIV replication
by acting as "chain terminators" i.e. causing termination of HIV
reverse transcriptase-mediated DNA synthesis. However the efficacy
of such drugs is limited because of the emergence of resistant
strains of viruses. RNA viruses in general, and HIV in particular,
have a very high mutation rate during replication, and this high
mutation frequency enhances the likelihood of resistant strains
emerging.
[0004] Recently the idea has developed that RNA viruses may be
close to the "edge of viability". That is, the mutation frequency
of such viruses is so high that a comparatively modest increase in
mutation frequency may be sufficient to render the great majority
of the viral population non-viable, due to the presence of
deleterious mutations at essential loci in the viral genome. This
well-known concept is known as "error catastrophe" and results with
the wide spectrum antiviral ribavirin in the context of poliovirus
strongly suggest that the concept is well-founded (Crotty et al,
2000 Nature Medicine 6, 1375-1379; Crotty et al, 2001 Proc. Natl.
Acad. Sci. USA 98, 6895-6900; Sierra et al, J. Virol., 2000, 74,
8316-8323).
[0005] Loeb et al, (WO 98/18324 and U.S. Pat. No. 6,063,628)
disclose the use of ribonucleoside analogues to increase the
mutation rate in (and thereby inhibit the replication of) RNA
viruses such as HIV or HCV. Loeb et al state that the
ribonucleoside analogue may typically be an analogue of cytidine,
uridine, adenosine or guanosine, but that analogues of cytidine or
uridine (i.e. pyrimidine analogues) are preferred (U.S. Pat. No.
6,063,628; column 3 lines 44-45). Loeb et al do not specifically
refer to many purine nucleoside analogues, but adenosine analogues
specifically mentioned include: 1,N.sup.6-ethenoadenosine,
3-methyladenosine and N.sup.6-methyladenosine. Guanosine analogues
specifically mentioned include 8-hydroxyguanosine,
O.sup.6-methylguanosine, O.sup.6-ethylguanosine,
O.sup.6-isopropylguanosi- ne, 3,N.sup.2-ethenoguanosine,
O.sup.6-alkylguanosine, 8-oxo-guanosine, 2,N.sup.3-ethenoguanosine,
and 8-aminoguanosine.
[0006] Interestingly, neither WO 98/18324 nor U.S. Pat. No. 6 063
628 contain any data from experiments performed by the inventors to
support the claims made therein. Only one experiment is described
in which HIV is passaged in vitro in the presence of either
5-hydroxyuridine or 5-bromocytidine. The results after 4 passages
are shown in FIG. 3 of U.S. Pat. No. 6 063 628: no decline in viral
titer is apparent in the Figures.
[0007] The content of all documents mentioned in this specification
is incorporated herein by reference.
SUMMARY OF THE INVENTION
[0008] The present invention relates to certain nucleoside
analogues which the present inventors, in contrast to the data
presented by Loeb et al, have found to be effective in inhibiting
RNA virus replication, even within 4 passages in vitro.
[0009] In a first aspect the invention provides a method of
inhibiting the replication and/or increasing the mutation rate of
an RNA virus, the method comprising administering an RNA nucleoside
analogue to a cell infected by an RNA virus (as herein defined),
the analogue being incorporated by a polymerase into an RNA copy of
the viral genomic nucleic acid molecule, wherein the nucleoside
analogue conforms to the general formula I or II below: 1
[0010] where:
[0011] n=1-4, preferably 2-4,
[0012] X.sup.1=N or CH or CR.sup.5
[0013] X.sup.2=N or S or CR.sup.5
[0014] X.sup.3=NR.sup.6 or O or S or R.sup.6 when X.sup.2=N, or
X.sup.3=NR.sup.6 or R.sup.6 when X.sup.2=S, and X.sup.3 is absent
when X.sup.2=CR.sup.5
[0015] R.sup.1=H or alkyl or aryl or alkaryl or acyl
[0016] R.sup.2=H or alkyl or aryl or alkaryl or acyl; when
X.sup.2=S, R.sup.2 is absent;
[0017] R.sup.3=H or NR.sup.5R.sup.6 or NR.sup.5NR.sup.5R.sup.6 or
NR.sup.5OR.sup.5
[0018] R.sup.5=H or alkyl or alkenyl or alkynyl or aryl or alkaryl
or acyl
[0019] R.sup.6=H or alkyl or alkenyl or alkynyl or aryl or alkaryl
or acyl and
[0020] R.sup.4=H or 2
[0021] wherein
[0022] Z=O or S or CH.sub.2 or CHF or CF.sub.2 or NR.sup.5
[0023] X.sup.4=OH or F
[0024] R.sup.7=H or PO.sub.3.sup.2- or P.sub.2O.sub.6.sup.3- or
P.sub.3O.sub.9.sup.4- or a masked phosphate derivative.
[0025] Alkyl groups, if present, are preferably methyl groups
(desirably unsubstituted). Aryl groups, if present, are preferably
phenyl groups, substituted or unsubstituted. Desirably no more than
one aryl or alkaryl group is present in a molecule according to the
general formulae. Conveniently at least one of R.sup.1-R.sup.6 is H
and preferably at least two of R.sup.1-R.sup.6 are H.
[0026] A masked phosphate derivative is a modified phosphate group
in which the negative charge(s) which would normally be present in
an unmodified phosphate group are reduced or (more preferably)
entirely neutralized by additional moieties. This has the benefit
of facilitating transport of compounds comprising the modified
phosphate group across a lipid membrane (e.g. across a cell
membrane). Examples of masked phosphate derivatives are
bis-POM/bis-POM PMEA (see Delaney et al, 2001 Antiviral Chemistry
and Chemotherapy 12, 1-35), cycloSal (Meier et al, Eur. J. Org.
Chem. 1998, 837) and SATE (Lefebvre et al, J. Med. Chem., 1995, 38,
3941-3950). (SATE is an abbreviation of S-acyl thioethyl).
[0027] For present purposes an "RNA virus" is considered to include
all viruses with an RNA genome (encompassing both "conventional"
RNA viruses and retroviruses) and any virus which requires a
genomic RNA intermediate for the purposes of replication. Examples
of relevant viruses include ortho- and paramyxoviruses, poliovirus,
rhinovirus, retroviruses (especially HIV-1 and HIV-2), hepatitis B
and C viruses (HBV and HCV respectively), rotaviruses, flaviviruses
(e.g. West Nile virus) and certain arboviruses (e.g. Dengue Fever
virus).
[0028] "Conventional" RNA viruses include both negative and
positive stranded ss (single stranded) RNA viruses and ds (double
stranded RNA viruses). In particular, a "conventional" RNA virus
may be defined as a virus having a single or double stranded RNA
genome which encodes a viral RNA polymerase. An extensive, but not
necessarily exhaustive, list of RNA viruses is shown in Table 1 in
the Appendix. Those whose hosts are vertebrates, (especially
mammalian vertebrates, and in particular man or domesticated
mammals) are particularly suitable for inhibition by a
pharmaceutical composition in accordance with the invention.
[0029] The invention encompasses the administration of a
ribonucleoside analogue (that is, a base analogue covalently joined
to a ribosyl residue) to an infected cell. The administered
ribonucleoside analogues may be converted to the corresponding
ribonucleotide analogues intracellularly by known enzymes. However
it is also possible to perform the invention by administering the
base analogue (without an attached ribosyl residue), which base
analogue is then converted by phosphoribosylation (in vivo if
administered to a living multicellular organism, or intracellularly
if administered to a cell in vitro) into a ribonucleotide analogue.
Equally the invention encompasses within its scope the
administration of a ribonucleotide analogue (that is, a
ribonucleoside analogue esterified to a phosphate group, or a di-
or tri-phosphate). For the purposes of economy, the compounds of
use in the invention are referred to as ribonucleoside analogues,
although those skilled in the art will appreciate that the general
formulae presented above encompass both base analogues and
ribonucleotide analogues, and unless the context dictates
otherwise, the term "ribonucleoside" analogue is intended to
embrace both base analogue and ribonucleotide analogue. It is
generally preferred that the base analogue incorporated in the
ribonucleoside analogue is a purine base analogue, which term
specifically includes 7-deaza purine analogues.
[0030] In some instances it may be preferred to perform the
invention by use of base analogues, especially in preference to
ribonucleoside analogues, since these may be better absorbed by
mammalian subjects following administration in vivo.
[0031] Compounds for use in the invention and in accordance with
the general formulae presented above are commercially available
and/or are readily capable of being synthesised by those skilled in
the art using published protocols. Other compounds may be obtained
by following the detailed teaching provided in the present
specification.
[0032] In preferred embodiments Z is O. In the same or other
preferred embodiments X.sup.2 is N. In the same or other preferred
embodiments X.sup.3 is O or comprises N. In the same or other
preferred embodiments X.sup.4 is OH. Desirably, in one embodiment,
Z is O, X.sup.2 is N, X.sup.3 is N or O and X.sup.4 is OH. In an
especially preferred embodiment Z is O, X.sup.2 is N, X.sup.3 is O,
X.sup.4 is OH and R.sub.1 is alkyl, especially methyl.
[0033] Generally preferred are ribonucleotide analogues which have
low cytotoxicity but high viral mutagenicity and/or high viral
inhibitory activity. Particular examples of preferred
ribonucleoside analogues include those illustrated in FIGS. 3, 7
and 11, and the corresponding base analogues and ribonucleotide
analogues. Cytotoxicity can be readily assayed in vitro, by those
skilled in the art using for example, tissue cultures of relevant
tissues (e.g. cells, HeLa cells, CEM/O cells and the like).
Typically an IC.sub.50 value can be determined (that is, the
concentration of the agent under investigation which causes 50%
inhibition of growth of the tissue culture cells relative to
untreated control cultures). The amount of inhibition of growth may
be estimated by any suitable means e.g. incorporation of tritiated
thymidine; time taken for cultures to reach confluency, etc.
[0034] Anti-viral activity may be measured in vivo or in vitro.
Suitable assay methods will be apparent to those skilled in the art
with the benefit of the present disclosure. In particular, for
example, viral inhibition may be measured in vitro by using plaque
reduction assays, and determining for example the IC.sub.50
concentration of the compound under test (i.e. the concentration
which reduces by 50% the number of viral plaques formed in a
monolayer of susceptible cells after a fixed length incubation,
relative to virus grown in the absence of the test compound).
[0035] The ribonucleoside analogue having the structure shown in
FIG. 11, which compound has the full name
2-amino-6-methoxyamino-9-.beta.-D-ribofu- ranosylpurine,
abbreviated for simplicity as rK, and the corresponding base
analogue K and ribonucleotide analogue rKP (which expression
incorporates in particular mono-, di- and triphosphates) may be
particularly useful. The di- and triphosphates may be referred to
as rKDP and rKTP. The inventors have found that rK is active in
reducing viral titer, especially the titer of HIV-1 when the virus
is grown in vitro in tissue culture.
[0036] In order to be effective, the ribonucleoside analogues of
the invention need to be incorporated into the RNA copy of the
viral genomic nucleic acid with reasonable efficiency and must
therefore be recognisable as a suitable substrate by the relevant
RNA polymerase inside the host cell. For "conventional" RNA viruses
this is an RNA polymerase encoded by the virus. For retroviruses,
the relevant RNA polymerase is the RNA polymerase encoded by the
host cell. Generally speaking, viral RNA polymerases are less
faithful and less discriminating than host cell RNA polymerases and
will be more likely to utilise the ribonucleoside analogues as
substrates after in vivo conversion (when required) to
triphosphates. Accordingly the pharmaceutical composition, method,
and other associated aspects of the invention are preferably
intended and adapted for use in the prevention and/or treatment of
infections by conventional RNA viruses which encode a viral RNA
polymerase.
[0037] The inventors have additionally made the surprising
discovery that certain ribonucleoside analogues, preferably but not
necessarily in accordance with general formulae I or II above, can
inhibit retroviral transcription, which finding has not previously
been suggested or in any way disclosed in the prior art. Without
wishing to be bound by any particular theory, the inventors believe
that this may be due to an inhibitory effect of the ribonucleoside
analogue on transcription promoted by a 5' long terminal repeat
("LTR"), although the mechanism by which this inhibition might be
mediated is unknown. Accordingly, preferred ribonucleoside
analogues in accordance with the invention are those which exhibit
the property of inhibiting viral production, e.g. by inhibition of
transcription or by an error catastrophe mechanism. Methods of
assaying compounds for such a property are disclosed herein and may
be employed by those skilled in the art to identify ribonucleoside
analogues possessing this desirable characteristic. The effect of
inhibiting retroviral transcription is that there are fewer RNA
copies of the viral genome present in an infected cell:
accordingly, at a given concentration of ribonucleoside analogue
there are fewer RNA copies of the viral genome which are likely to
escape incorporation of the mutagenic ribonucleoside analogue. A
preferred compound in this regard is that denoted by the structure
shown in FIG. 2 referred to as rP, for simplicity (Moriyama et al,
Nucleic Acids Research, 1998, 26, 2105-2111), and the corresponding
base analogue (P) and the corresponding ribonucleotide analogue rPP
(especially the triphosphate, rPTP).
[0038] It will be appreciated that increasing the mutation rate in
the manner of the first aspect of the invention can, in accordance
with the concept of error catastrophe, cause a significant increase
in the number of non-viable viral particles produced, especially
when the ribonucleoside analogue is present at an effective
concentration for a plurality of cycles of viral replication, since
mutations will accumulate in the viral genome over time. In
contrast, although the ribonucleoside analogue will probably be
incorporated into messenger RNA in the host cell (resulting in
production of mutant polypeptides), mRNA is rapidly turned over and
degraded and therefore will not accumulate mutations over time.
Equally, the ribonucleoside analogue will generally not be
incorporated into the DNA genome of the host cell or, if
incorporated, will be removed by the "house-keeping" enzymes which
are responsible for maintaining the integrity of the host cell
genome. Accordingly, the method of the invention finds therapeutic
application in the treatment of RNA virus infections.
[0039] Thus, in a second aspect the invention provides a method of
treating an RNA virus infection in a human or animal subject, the
method comprising administering to a subject infected with an RNA
virus, an effective amount of a ribonucleoside analogue in
accordance with general formula I or II.
[0040] In a third aspect the invention provides a pharmaceutical
composition comprising an effective amount of a ribonucleoside
analogue in accordance with general formula I or II in admixture
with a physiologically acceptable excipient, diluent or
carrier.
[0041] In a fourth aspect the invention provides a method of making
a pharmaceutical composition, the method comprising mixing a
ribonucleoside analogue in accordance with general formula I or II
with a physiologically acceptable excipient, diluent or carrier.
The method optionally includes the further step of packaging the
composition in unitary dose form.
[0042] In a fifth aspect the invention provides for use of a
ribonucleoside analogue according to general formula I or II in the
preparation of a medicament to treat an RNA viral infection in a
human or animal subject.
[0043] The ribonucleoside analogues of use in one or more of the
various aspects of the invention will preferably be substantially
soluble in water and be readily capable of entering
virally-infected cells. Where the compound consists of a base
analogue, the compound may generally be ribosylated and
phosphorylated in vivo, or at least intracellularly. Where the
compound is a ribonucleoside analogue it may typically be
phosphorylated to form a ribonucleotide analogue. Possibly it is
the ribonucleotide analogue which is integrated into the RNA genome
of the RNA virus (or DNA virus which replicates via an essential
genomic RNA intermediate), although it is important to note that
the inventors make no assumption as to mode of action. Thus the
active compound may be the base analogue and/or the ribonucleoside
analogue and/or the ribonucleotide analogue. Specifically in
respect of integrating retroviruses, such as HIV, the presence of
the active compound incorporated by a cellular polymerase probably
leads to mutation by the viral reverse transcriptase during DNA
synthesis prior to integration into the host genome, which
mutations are not recognisable by repair enzymes; over several
cycles such mutations will accumulate.
[0044] Pharmaceutical compositions in accordance with the invention
may be administered by any conventional route known to those
skilled in the art. The preferred route is oral administration, but
the composition may alternatively be administered, for example,
intravenously, subcutaneously, transdermally, or via a rectal or
intranasal route.
[0045] The composition may be administered as a solid (e.g. in the
form of a tablet, pill, capsule, powder or the like) or may be a
liquid (e.g. solution, suspension), semi-solid (e.g. a gel),
aerosol or spray.
[0046] Physiologically acceptable excipients, diluents and carriers
are well known to those skilled in the art of medical formulations
and include, for example: saline, Ringer's solution, distilled
water, dextrose solution, calcium carbonate, silicates, starches
and modified starches and plant-derived polysaccharide gums and
gels (e.g. xanthan gum; carrageenans and the like).
[0047] An "effective amount" of a ribonucleoside analogue or
pharmaceutical composition comprising the same is understood to
mean, for present purposes, an amount sufficient to cause a
measurable decrease in the viral titer in suitable samples (e.g.
blood, saliva, or tissue biopsy specimens) taken from the subject,
or a measurable decrease in the amount of viral antigen detected in
such samples, or a discernible amelioration in the symptoms of the
viral infection experienced by the subject. Methods of obtaining
suitable samples from a subject, and of analysing same to measure
viral titer or viral antigen (e.g. by ELISA or other immunoassay)
are well known to those skilled in the art.
[0048] The appropriate dose of the ribonucleoside analogue will
depend on several factors, such as the body mass of the subject,
level of toxicity (if any) of the analogue, the age of the subject
and the severity of the viral infection (and/or any additional
condition afflicting the subject). Guidance is given in U.S. Pat.
No. 6,063,628. Conveniently the dose of ribonucleoside analogue
will be in the range 1 mg/Kg body weight to 500 mg/Kg per day,
preferably in the range 5 mg/Kg-250 mg/Kg, more preferably 10
mg-100 mg/Kg.
[0049] Typically a dose at the lower end of the acceptable range is
administered to the subject and, if there is no discernible
improvement in the subject's condition, the dose may be increased
if there are no contra-indications, until an effective dose is
achieved. By such trial and error clinicians will readily be able
to find an appropriate dose for any particular subject.
[0050] Advantageously the pharmaceutical composition in accordance
with the invention may comprise more than one anti-viral agent. For
instance, the composition may comprise a plurality of different
ribonucleoside analogues, each being in accordance with general
formula I or II defined above.
[0051] Additionally, or alternatively, the composition may comprise
one or more antiviral agents which do not conform to general
formula I or II. Examples include conventional antiviral agents
such as ribavirin, AZT, HIV protease inhibitors, and compounds of
the sort explicitly disclosed in U.S. Pat. No. 6,063,628. The other
aspects of the invention may conveniently reflect such
embodiments.
[0052] Alternatively, the method of treating the subject may
comprise separate administration of a further pharmaceutical
composition comprising an additional anti-viral agent, such as
those aforementioned, or a substance that reduces the
intra-cellular concentration of the naturally-occurring
ribonucleotide(s) with which the ribonucleoside analogue must
compete for incorporation into the viral RNA genome.
[0053] In a further aspect the invention provides a composition
suitable for application to a plant, for the purpose of preventing
or treating an RNA virus infection of the plant, the composition
comprising an RNA nucleoside analogue conforming to general formula
I or II as defined elsewhere, the term "nucleoside analogue" also
incorporating reference to a nucleotide analogue and a base
analogue.
[0054] The composition will typically be applied to a plant by
means of spraying a solution or suspension of the active antiviral
agent (typically an aqueous solution or suspension). Conveniently
the composition is supplied to a user in concentrated form and is
diluted with water prior to application. Conveniently the
composition will further comprise other substances conventional in
the field of plant protection, to assist adherence of the
composition to the plant to which the composition is applied and
uptake of the active agent by the plant. Such substances include,
for example, surfactants and penetration enhancers, which are well
known to those skilled in the pertinent art.
[0055] The list of viruses in the Appendix shows many RNA viruses
which infect plants. In principle, any of these could be inhibited
by the composition defined above. The invention thus also further
provides a method of preventing and/or treating an RNA virus
infection in a susceptible plant, the method comprising the step of
applying to the plant an effective amount of a composition
comprising an RNA nucleoside analogue conforming to general formula
I or II defined above, and a method of making a plant protection
composition for preventing and/or treating an RNA virus infection
in a plant.
[0056] The invention will now be further described by way of
illustrative example and with reference to the accompanying
drawings, in which:
[0057] FIG. 1 shows the structural formula of a deoxyribonucleoside
analogue, dP;
[0058] FIG. 2 shows the structural formula of a ribonucleoside
analogue rP, the `ribo` equivalent of the compound shown in FIG.
1;
[0059] FIGS. 3-20 show the structural formula of various
ribonucleoside analogues in accordance with general formula I or II
identified above;
[0060] FIGS. 21 and 22 are graphs of p24 antigen (ng/ml) against
time (in days);
[0061] FIG. 23 is a schematic representation of a transcription
system of use in screening ribonucleoside analogues for use in the
present invention;
[0062] FIG. 24 is a bar chart showing the amount of RNA transcript
produced (in femtomoles) by a transcription system of the sort
illustrated in FIG. 23, in the presence or absence of a
ribonucleotide analogue rPTP;
[0063] FIGS. 25A and 26A are images of PAGE analysis of nucleic
acid extension assays performed using certain ribonucleotide
analogues in accordance with the invention and FIGS. 25B and 26B
are representations of the same results in bar chart form.
[0064] FIGS. 27 and 28 show the general structural formulae of
certain masked phosphate derivatives of use in the invention.
EXAMPLES
Example 1
Synthesis of Purine Ribonucleoside Analogues
[0065] The inventors synthesised several ribonucleoside analogues
in accordance with general formula I or II, and also a
ribonucleoside (N.sup.4-hydroxycytidine) specifically mentioned by
Loeb et al in U.S. Pat. No. 6,063,628. For brevity the synthesised
compounds are referred to herein as JA22-JA31. An additional
compound, JA21 (Hill et al, Proc. Natl. Acad. Sci. USA, 1998, 95,
4258-4263), was synthesised and used as a control. JA21 is the
deoxyribonucleoside equivalent of the ribonucleoside analogue JA22.
JA29 is the compound indicated by Loeb et al as being useful in
increasing the mutation frequency of RNA viruses (although no data
are presented by Loeb et al in support of that assertion). The
inventors also prepared a number of different base analogues
(JA32-JA39). The table below (Table 1) indicates the systematic
name of each of the compounds referred to as JA21-JA39, and also
any trivial name if such a name has been used previously.
1TABLE 1 Compound Trivial Name Number Systematic Name (if any) JA21
6-(2-deoxy-.beta.-D-ribofura- nosyl)- dP
3,4-dihydro-8H-pyrimido[4,5-c] [1,2] oxazin-7-one JA22
6-(.beta.-D-ribofuranosyl)-3,4- rP dihydro-8H-pyrimido[4,5-c] [1,2]
oxazin-7-one JA23 2-amino-N.sup.6-methyladenosine -- JA24
N.sup.6-amino-9-.beta.-D-r- ibofuranosyl- -- 2,6-diaminopurine JA25
N.sup.6-aminoadenosine -- JA26 N.sup.6-methoxyadenosine -- JA27
N.sup.6-amino-N.sup.6-methyladenosine -- JA28 N.sup.6
hydroxyadenosine -- JA29 N.sup.4-hydroxycytidine -- JA30
2-amino-N.sup.6-hydroxyadenosine -- JA31 2-amino-6-methoxyamino-9--
.beta.- rK D-ribofuranosylpurine JA32 N.sup.6-hydroxyadenine --
JA33 N.sup.6-aminoadenine -- JA34 N.sup.6-amino-2,6-diaminopurine
-- JA35 N.sup.6-amino-N.sup.6-meth- yladenine -- JA36
N.sup.6-methyl-N.sup.6-diaminopurine -- JA37
N.sup.6-methyl-N.sup.6-methoxyadenine -- JA38
N.sup.6-methoxy-2,6-diaminopurine K JA39 N.sup.6-methoxyadenine
Z
[0066] The structures of compounds JA21-JA39 are shown in FIGS.
1-19 respectively. One of the main problems associated with
nucleoside analogues as therapeutic agents is that they are often
either only poorly phosphorylated by kinases to their
monophosphate, or they are not substrates for the kinase at all.
There are methods to generate nucleoside monophosphates in cells.
One is to use the nucleobases, for example analogues JA32-JA39.
These nucleobases may then be converted in vivo or in vitro to
their nucleoside monophosphates by nucleoside phosphorylases (for a
review see Pugmire and Ealick, Biochem. J., 2002, 361, 1-25). An
alternative method requires the use of masked monophosphates, such
as SATE-, cyclosal- or PMEA-derivatives. Nucleoside mono-, di- or
tri-phosphates may also be delivered into cells using transfection
agents, such as liposomes (e.g. any commercially available liposome
should, in principle, suffice--the inventors have used DMRIE-C,
from Gibco BRL, as a specific example).
[0067] As examples of compounds of use in accordance with the
present invention and in accordance with general formula I or II,
JA23-JA31 (except JA29) were synthesised from
6-chloro-9-.beta.-D-ribofuranosylpuri- ne or
2-amino-6-chloro-9-.beta.-D-ribofuranosylpurine (Aldrich). These
were treated with the following available reagents: hydroxylamine
hydrochloride, methoxyamine hydrochloride, N,O-dimethyl
hydroxylamine hydrochloride, anhydrous hydrazine and
N-methylhydrazine.
Example of General Method
2-Amino-6-methoxyamino-9-.beta.-D-ribofuranosylpurine-(JA31)
[0068] Synthesis of this compound has been described previously
(Ueda, et al. Chem. Pharm. Bull., 1978, 26, 2122).
[0069] The 2-amino-6-chloropurine derivative (302 mg; 1 mMol),
methoxyamine hydrochloride (160 mg; 4 equiv.) and triethylamine
(0.2 ml) in ethanol (9 ml) were heated overnight at 85.degree. C.
in a sealed bottle shielded from light. Complete reaction was
judged by thin layer chromatography (tlc.) in 20%
MeOH--CH.sub.2Cl.sub.2. Evaporation in vacuo then trituration with
ethanol of the residue gave the product as a white powder (90%)
which gave needles on crystallisation from dioxan-water.
[0070] In the synthesis of compounds from
6-chloro-9-.beta.-D-ribofuranosy- lpurine the reaction conditions
required lower temperatures and shorter reaction times.
[0071] The synthesis of compounds in accordance with general
formula I or II has been described in a number of other
publications:
[0072] JA23, 24, 27 and 30, see Taito et al, (1964, Chem. Pharm.
Bull. 12, 951);
[0073] JA25, see Johnson et al, (1958, J. Amer. Chem. Soc. 80,
699);
[0074] JA26, see Fuji et al, (1991, Chem. Pharm. Bull. 39, 39);
[0075] JA28, see Giner-Sorolla et al, (1966, J. Med. Chem. 9,
143).
[0076] JA32, see Baker et al, (1969, J. Med. Chem., 12,
684,687).
[0077] JA33, JA34, See Montgomery et al, (1957, J. Am. Chem. Soc.,
74, 2185).
[0078] JA37, see Fujii et al, (1983, Chem. Pharm. Bull., 31,
3149-3159).
[0079] JA39, see Fujii et al, (1971, Chem. Pharm. Bull., 19,
1731).
[0080] The analogue 7-deaza-N.sup.6-aminoadenosine (FIG. 20) has
been previously prepared and shown to have slight activity against
cytomegalovirus (HCMV) (Pudlo et al, J. Med. Chem., 1988, 31,
2086-2092). HCMV is a DNA virus, and the mode of action of this
compound is suggested to be as an adenosine kinase inhibitor. As
such its mode of action is different from that described
herein.
[0081] The particular compound disclosed by Pudlo et al is, in
terms of the general formula I used herein, that in which
X.sup.1=CH, X.sup.2=N, X.sup.3=NR.sup.6, R.sup.1=H, R.sup.2=H and
R.sup.6=H. In preferred embodiments, the present invention thus
excludes from its scope a pharmaceutical composition in which the
sole active anti-viral compound is that defined immediately above.
Since Pudlo et al were concerned only with acyclovir analogues
(acyclovir being a "selective inhibitor of the virus-encoded DNA
polymerase") the prior art does not suggest in any way that the
compound disclosed therein might be useful in inhibiting RNA
viruses. Moreover the compound in question (1d in Table 1 of Pudlo
et al) was shown to have an IC.sub.50 for human foreskin
fibroblasts (HFF) of 2 .mu.M, whereas the IC.sub.50 in the plaque
reduction assay for cytomegalovirus was 4 .mu.M i.e. the compound
was more inhibitory for HFF cells than for viral replication, and
so of little or no therapeutic usefulness.
[0082] Thus in an alternative preferred embodiment the
pharmaceutical composition of the present invention preferably
comprises an anti-viral active agent which has an IC.sub.50 in
respect of a conventional RNA virus (especially, for example, polio
virus) e.g. as judged by a plaque reduction assay, which is lower
than its IC.sub.50 for HFF cells, according to the assay method
disclosed by Pudlo et al.
[0083] all of the compounds synthesised were recrystallized,
characterised by nmr and shown to be substantially pure.
Example 2
[0084] Following synthesis, the various compounds were tested in
vitro for toxicity, by measuring the IC.sub.50 (i.e. the
concentration which caused 50% inhibition) in respect of the
inhibitory effects of the compounds on the proliferation of human
T-lymphocytes (CEM/O cells). The results are shown below in Table
2.
2 TABLE 2 Compound IC.sub.50.sup.a (.mu.M) JA21 690 .+-. 14 JA22
698 .+-. 11 JA23 622 .+-. 8 JA24 62 .+-. 6 JA25 12 .+-. 3 JA26 44
.+-. 2 JA27 17 .+-. 2 JA28 156 .+-. 15 JA29 16 .+-. 1 JA30 78 .+-.
3 JA31 377 .+-. 62 .sup.a50% inhibitory concentration.
Example 3
[0085] Having established an indication of the toxicity of the
various compounds, the ribonucleoside analogues were then tested to
determine whether they exhibited any effect on the replication of
RNA viruses in in vitro cell cultures.
[0086] HIV-1 infected CEM cells were subcultured every 4-5 days in
the presence of sub-toxic concentrations (in the range of 10-20% of
their respective IC.sub.50 value) of the compounds under test. At
each sub-culture, cell-free supernatant (10-20 .mu.l) was
transferred to fresh 1 ml cell cultures. At regular intervals the
cultures were inspected microscopically to assess the extent of any
cytopathic effect (giant cell formation). As an alternative, it is
also possible to perform an immunoassay to quantify viral p24
production.
[0087] The preliminary results for up to 7 passages are shown below
in Table 3.
3 TABLE 3 Concentration Passage number.sup.a,b Drug (.mu.M) 1 2 3 4
5 6 7 JA-21 (dP) 400 100 100 25 50 37 12 6 JA-22 (rP) 400 100 100
100 100 100 100 100 JA-23 400 100 100 12 25 3 0 0 JA-24 10 100 100
25 100 100 100 25 4 100 100 19 100 100 100 12 JA-25 2 100 100 100
100 100 100 100 0.8 100 100 87 100 100 100 100 JA-26 10 100 100 25
100 100 12 3 4 100 100 25 100 100 12 3 JA-27 4 100 100 6 25 25 0 0
JA-28 40 100 100 50 100 100 75 6 20 100 100 19 100 100 100 100
JA-29 2 100 100 25 100 100 100 100 0.8 100 100 12 100 100 100 100
JA-30 10 100 100 25 100 100 100 50 JA-31 (rK) 50 100 100 0 0 0 0 0
20 100 100 3 19 12 0 0 Control (no -- 100 100 25 100 100 100 100
drug) .sup.aSubcultivation of the drug-treated HIV-1(III.sub.B)
exposed CEM cell cultures was performed every 5 days. .sup.bData
represent the percentage of cytopathic effect (giant cell
formation) as recorded microscopically.
[0088] The results show that JA31 (rK) in particular is effective
at inhibiting the replication of RNA viruses as exemplified by HIV.
Other compounds also appear to be moderately effective: JA23 and
JA27 in particular. JA29, mentioned by Loeb et al, does not
demonstrate any antiviral activity in this assay.
[0089] In order to demonstrate that the reduction in viral titer,
as evidenced by the decline in observed cytopathic effect, is due
to induction of accumulated mutations in the viral genome, proviral
DNA will be isolated from the cultures and the sequence of the
reverse transcriptase gene determined by routine DNA sequencing
reactions. The determined sequence can be compared with the known
sequence of the original input virus and the number of mutations
calculated relative to those in the virus in the control
culture.
[0090] Further Studies
[0091] Mechanism of action studies will be undertaken to study the
effect of the 5'-triphosphate derivatives of the ribonucleotide
analogues on human and viral RNA polymerase-catalysed RNA synthesis
as well as HIV-1 reverse transcriptase-catalysed conversion of
nucleotide analogue-containing RNA to DNA. Also, the substrate
affinity of recombinantly produced ribonucleoside kinases for the
ribonucleoside analogues and their efficacy of conversion of the
ribonucleoside analogues to their 5'-monophosphates will be
determined. Insights in the above-mentioned characteristics of the
ribonucleos(t)ide analogues should allow optimisation of the viral
mutagenicity of the compounds whilst ideally minimising toxicity,
so as to enhance the therapeutic usefulness of the compounds.
Masked phosphate derivatives of the ribonucleoside analogues will
also be investigated.
Example 4
[0092] Other experiments were performed using ribonucleoside
analogues present as the phosphorylated ribonucleotide in the
presence of transfection agents. For example, the triphosphate of
rK, referred to as rKTP, was synthesised as described by Moriyama
et al, (1998 Nucl. Acids Res. 26, 2105). The triphosphate of rP,
rPTP, was prepared in an analogous manner.
[0093] These two compounds were then investigated for an inhibitory
effect on the replication of HIV in persistently infected
Molt4/IIIB cells, or acutely infected MT4/IIIB cells. The compounds
were compared with equivalent concentrations of dideoxycytidine
(ddC) or dideoxycytosine triphosphate (ddCTP), or a negative
control (no drug).
[0094] Effect on Persistently-Infected Cells
[0095] 2 nmol of the relevant drug (final concentration 1 .mu.M)
was mixed with 4 .mu.l of liposome DMRIE-C (Gibco BRL) in 800 .mu.l
of serum-free RPMI 1640 medium (Sigma). After incubating for 45
minutes at room temperature, 10.sup.5 Molt4/IIIB cells in 200 .mu.l
of serum-free RPMI 1640 medium were added and held at 37.degree. C.
for 4 hours. At the end of this interval 1 ml of RPMI 1640 medium
supplemented with 20% serum was added and the mixture cultured at
37.degree. C. at 24 hrs, 72 hrs and 5 days, aliquots of supernatant
were collected and the amount of p24 antigen present was quantified
using the Lumipuls.TM. system (Fuji Rebio). The results are shown
in FIG. 21.
[0096] Effect on Acutely-Infected Cells
[0097] 10.sup.3 pfu of HIV.sub.IIIB were added to 10.sup.5 MT4
cells in 1 ml of serum-free RPMI 1640 medium and incubated for 90
minutes at 37.degree. C. The cells were washed three times in
serum-free medium and resuspended in 200 .mu.l of serum-free
medium. Drug administration (100 nM final concentration), culture
and p24 assay were then performed as above. The results are shown
in FIG. 22.
[0098] FIG. 21 is a graph of viral titer (as measured by amount of
p24 antigen in ng/ml) against time (in days), showing the results
for cultures of persistently-infected Molt4/IIIB cells with no drug
("Control", triangles), or 1 .mu.M final concentration of ddC (open
circles), ddCTP (open squares), PTP (filled circles) or rKTP
(filled squares). FIG. 22 is a graph of p24 antigen (in ng/ml)
against time (in days) for cultures of acutely-infected MT4/IIIB
cells in the presence of drugs at a final concentration of 100 nM,
the legend is as for FIG. 21.
[0099] The results illustrated in FIGS. 21 and 22 show that both
rKTP and rPTP significantly inhibit viral replication compared to
controls, and reduce viral titers to levels comparable with known
dideoxy chain-terminating compounds which inhibit reverse
transcriptase. The ribonucleotide analogues of the invention are
believed, however, to be less vulnerable to the evolution of
resistant virus strains.
Example 5
Mutations Induced on HIV-1 Pol Gene of MT4/IIIB by PTP or KTP
[0100] Genomic DNA of MT4/IIIB was collected 3 days after drug
administration (final concentration was 100 nM) by DNeasy Tissue
Kit (QIAGEN). A part of the pol gene (873 bp) was amplified by
2-step polymerase chain reaction (2-step PCR). A first PCR reaction
mixture contained 50 pmol of forward primer-1
(5'-GGTACAGTATTAGTAGGACC-3'), 50 pmol of reverse primer-1
(5'-TGTGTCAGTTAGGGTGACAA-3'), 200 .mu.M each dNTP, 5 .mu.l of
collected genomic DNA, 3 U of Pfu DNA polymerase (Promega), 20 mM
Tris-HCl pH 8.8 10 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.- 4, 2 mM
MgSO.sub.4, 0.1% Triton X-100, and 0.1 .mu.g/.mu.l BSA in 50 .mu.l
and was divided into five tubes. Each mixture was incubated for 2
min at 95.degree. C. Then it was applied to a thermal cycle
reaction comprising 95.degree. C., 1 min; 52.degree. C., 30 sec;
and 72.degree. C., 2 min for 45 cycles, followed by incubation for
5 min at 72.degree. C., the cycling controlled by Mastercycler
gradient apparatus (Eppendorf).
[0101] A second PCR reaction mixture contained 50 pmol of forward
primer-2 (5'CAGGGATTAGATATCAGTAC-3'), 50 pmol of reverse primer-2
(5'-TCTCTAACTGGTACCATAAT-3'), 200 .mu.M each dNTP, 1 .mu.l of 1st
PCR product from each tube, 1.5 U of Pfu DNA polymerase (Promega),
20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 2
mM MgSO.sub.4, 0.1% Triton X-100, and 0.1 .mu.g/.mu.l BSA in 50
.mu.l and was similarly divided into five tubes. Each mixture was
incubated for 2 min at 95.degree. C. Then it was applied to a
thermal cycle reaction comprising 95.degree. C., 1 min; 52.degree.
C.; 30 sec; and 72.degree. C., 2 min. for 30 cycles, followed by
incubation for 5 min at 72.degree. C.
[0102] Divided 2nd PCR products (total twenty-five tubes for one
sample) were collected into one tube, ethanol precipitated, and
digested by EcoRV and KpnI. After ligation with pBluescriptIISK(+),
the constructed plasmid was introduced into Escherichia coli
DH5.alpha. by electroporation. Cloned PCR product was then applied
to standard DNA sequencing reaction using forward sequencing primer
(5'-AAAGCTGGAGCTCCACCGCG-3') or reverse sequencing primer
(5'-AGTGAGCGCGCGTAATACGACTCACTA-TAGGGCGAATTGG-3') and the Thermo
Sequenase II dye terminator cycle sequencing kit (Amersham
Pharmacia Biotech). Electrophoresis and analysis was carried out by
DNA sequencer 378A (Applied Biosystems).
[0103] The sequencing revealed that the presence of either rPTP or
rKTP increased the mutation frequency, according to the results
presented in Table 4 below.
4TABLE 4 Transition Transversion Sequenced Frequency G-to-A T-to-A
Total (nucleotides) (.times.10.sup.-3) Control 1 2 3 3,113 0.96 PTP
3 6 9 4,809 1.9 KTP -- 6 6 4,642 1.3
Example 6
[0104] The inventors constructed an in vitro transcription system
promoted by HIV 5'-long terminal repeat (LTR) using HeLa nuclear
extract supplemented with HIV Tat protein. A 668 bp PCR product
from pLTR-luc plasmid, which includes HIV 5'-LTR promoter and
luciferase gene, was used as a DNA template for a transcription
reaction. From this template, 310-mer run-off transcripts were
produced. The system is illustrated schematically in FIG. 23.
[0105] The effect of incorporation of rPTP, at 200 .mu.M, in
transcription reactions was investigated. The reaction mixture
contained conventional nucleotide triphosphates (ATP, GTP, CTP and
UTP) at 50 .mu.M (the GTP being .alpha..sup.32P radio labelled with
10 .mu.Ci of radioactivity), +/-200 .mu.M PTP, 100 ng of template
DNA, 40 Units of RNase inhibitor (Wako), 1 .mu.l of diluted (1:20)
Tat protein and 8 units of HeLa cell nuclear extract in 1.times.
transcription buffer (10 mM HEPES pH 7.9, 2 mM DTT, 6.25 .mu.M
ZnSO.sub.4, 100 mM KCl, 20% glycerol, 4 mM MgCl.sub.2). The
reaction mixture was incubated for 10 minutes at 30.degree. C. and
the reaction terminated by adding 7 volumes of stop solution (300
mM Tris. HCl pH 7.4, 300 mM sodium acetate, 0.5% SDS, 2 mM EDTA, 3
.mu.g/ml tRNA). Transcripts were then purified by phenol/chloroform
extraction and ethanol precipitation. Whole samples were loaded on
a 5% polyacrylamide gel and subjected to electrophoresis (40W, for
2 hours). The intensity of the bands corresponding to the 310 mer
transcripts was measured by a BAS-2000 image analyser (Fujifilm).
The intensity of the band in the control reaction (no PTP) was
considered to be 100%. The results of the control reaction and the
rPTP reaction are shown in FIG. 24 below. This shows that the
presence of rPTP at 200 .mu.M reduced the amount of transcript
produced by nearly 50%.
Example 7
[0106] The foregoing examples are primarily concerned with
demonstrating an inhibitory effect of various ribonucleoside
analogues on the replication of HIV. However, as explained above,
the compositions of the present invention should also find
particular use in combatting infections caused by "conventional"
RNA viruses.
[0107] In general terms, those skilled in the art can readily
ascertain the likely efficacy of various ribonucleoside analogues,
by incubating an RNA virus of interest with suitable susceptible
host cells in the presence or absence of various concentrations of
the ribonucleoside analogue(s) under test, and using an appropriate
parameter to measure the amount of viral replication. Suitable
parameters might include, for example, an assay of numbers of pfu
of virus after a certain length of incubation, or an assay of viral
antigen, or amount of cytopathic effect.
[0108] A specific example of a suitable screening assay, to
identify compounds effective in inhibiting replication of
poliovirus, is set forth below. Essentially similar protocols,
suitably modified, could be employed to screen for compounds active
against other "conventional" RNA viruses.
[0109] HeLa cells are propagated in D-MEM/F-12 media (Invitrogen)
supplemented with dialyzed fetal bovine serum (2%, Invitrogen). For
poliovirus infection assays, cells are plated in 24-well dishes
(1.times.10.sup.5 cells/well) 48 h before the experiment, test
compounds are preloaded 24 hours before the experiment, and cells
are infected with 2000 pfu poliovirus per well. Upon reaching 100%
cytopathic effect (CPE), virus is harvested by freeze-thaw and
serial dilutions are plaqued on 6-well dishes of confluent HeLa S3
cells. After 72 hours, cells are stained with Crystal Violet (0.2%
in 20% ethanol) to visualize plaques. Time to 100% CPE is recorded
as the number of days required for poliovirus (2000 pfu) to cause
visibly complete cell death.
Example 8
General Synthesis of Triphosphates
[0110] To a solution of 50 mg of either 6-chloropurine riboside
triphosphate or 2-amino-6-chloropurine riboside triphosphate in 0.5
cm.sup.3 of water was added the hydroxylamine or hydrazine
derivative (5 equivalents) and the reaction stirred at room
temperature for 4 hours. The synthesis of the 2-amino derivatives
required reaction at 40.degree. C. for 4 hours. The solution was
lyophilised, dissolved in water, and purified by HPLC. HPLC
(Phenomenex Luna (10 .mu.m diameter particle size) C-18 reverse
phase column, buffer A, 0.1 M TEAB; buffer B, 0.1 M TEAB, 50% MeCN)
% to 40% buffer B over 40 minutes at 8 ml/min.). Samples were
evaporated and converted to their sodium salts by passage through a
Dowex 50WX4-200 resin (Na.sup.+ form).
[0111] JA23 5'-triphosphate Yield 49 mg. .delta..sub.P (D.sub.2O)
-4.40 (d, .gamma.-P), -9.73 (d, .alpha.-P), -20.22 (t,
.beta.-P).
[0112] JA24 5'-triphosphate Yield 20 mg. .delta..sub.P (D.sub.2O)
-4.56 (d, .gamma.-P), -9.80 (d, .alpha.-P), -20.43 (t,
.beta.-P).
[0113] JA26 5'-triphosphate Yield 30 mg. .delta..sub.P (D.sub.2O)
-4.71 (d, .gamma.-P), -9.79 (d, .alpha.-P), -20.36 (t,
.beta.-P).
[0114] JA27 5'-triphosphate Yield 27 mg. .delta..sub.P (D.sub.2O)
-4.41 (d, .gamma.-P), -9.74 (d, .alpha.-P), -20.22 (t,
.beta.-P).
[0115] JA28 5'-triphosphate Yield 24 mg. .delta..sub.P (D.sub.2O)
-4.49 (d, .gamma.-P), -9.77 (d, .alpha.-P), -20.35 (t,
.beta.-P).
[0116] JA31 triphosphate Yield 24 mg. .delta..sub.P (D.sub.2O)
-4.51 (d, .gamma.-P), -9.77 (d, .alpha.-P), -20.36 (t,
.beta.-P).
Example 8A
Incorporation of Nucleoside Analogues into Sym/Sub-U and Sym/Sub-C
by Polio Virus Strain 3D (PV3D)
[0117] This was carried out generally as previously described
(Crotty et al, Proc. Natl. Acad. Sci. USA, 2001, 98, 6895-6900;
Crotty et al, Nature Medicine, 2000, 6, 1375-1379). All nucleotide
and nucleoside incorporation experiments were carried out in a
reaction buffer containing 50 mM HEPES pH=7.5, 5 mM MgCl.sub.2, 10
mM beta-mercaptoethanol, and 60 .mu.M ZnCl.sub.2 and using short
synthetic primer/template systems comprising either uracil or
cytidine as the first base opposite which incorporation is to take
place (referred to as sym/sub-U or sym/sub-C respectively). The
assay was performed by incubating PV 3D polymerase (2 .mu.M) with
either sym/sub-U or sym/sub-C (1 .mu.M) for 90 seconds at
30.degree. C. and the reaction was initiated by the addition of the
nucleotide or analogue (500 .mu.M). Reactions were stopped by
quenching with EDTA (50 mM final concentration) 2 minutes after
initiation.
[0118] Analysis of the products formed in the reaction was done
using a 23% PAGE denaturing gel. Gels were visualized by using a
PhosphorImager and quantitated using the ImageQuant software.
[0119] Sequence of sym/sub-U primer/template:
5 5'-GCAUGGGCCC CCCGGGUACG-5'
[0120] Sequence of sym/sub-C primer/template:
6 5'-GAUCGGGCCC CCCGGGCUAG-5'
[0121] The results are shown in FIGS. 25 A/B and 26 AB. FIGS. 25A
and 26A are images of PAGE analysis of the products obtained using
the sym/sub-U and sym/sub-C systems respectively. FIGS. 25B and 26B
are pictorial representations of the results in bar chart form. The
height of the bars indicates the amount of product obtained (in
arbitary units) relative to control reaction mixtures comprising
ATP nucleotide. The JA numbers refer to the respective
5'-triphosphates.
[0122] FIGS. 25A/B show that incorporation of all compounds into
sym/sub-U by PV 3D.sup.pol takes place to a similar extent as the
correct nucleotide, ATP. FIG. 25A shows a 23% PAGE denaturing gel
with the products formed in the reaction. The template is the major
band at the bottom of the gel. The extended product(s) is
represented by the fainter band(s) higher up the gel. FIG. 25B
shows the amount of product being formed when the inhibitors were
used in comparison with the incorporation of AMP.
[0123] JA numbers refer to their 5'-triphosphates.
[0124] The results with sym/sub-C are shown in FIGS. 26 A/B. In the
case of sym/sub-C, incorporation of the analogues occurs in a
similar scale as the incorporation of GMP with the exception of
JA-26, for which the efficiency of incorporation is nearly half
that of GMP. The reaction with sym/sub-C shows the formation of a
product 12-nucleotides long as the major product. This is the
result of incorporation of the analogues opposite both cytidine and
uracil located next to each other. In the case of sym/sub-U, above,
uracil is followed by adenosine, which appears not to be a good
`template` for the analogues. JA-28 shows products of incorporation
12- and 13-nucleotides long. This indicates that JA-28 may be both
a purine and a pyrimidine analogue.
Example 9
General Synthesis of Nucleobases
[0125] To a solution of either 2-chloropurine or
2-chloro-6-aminopurine in water was added 10 equivalents of reagent
(e.g. methoxyamine, methyl hydrazine, in the case of hydrochloride
salts, e.g. hydroxylamine hydrochloride, 10 equivalents of
triethylamine are also added) and the solution heated at 60.degree.
C. for 1-48 hours (2-chloropurine reacts faster than
2-chloro-6-aminopurine). On cooling the product precipitates and is
isolated by filtration.
Example 10
General Synthesis of Masked Phosphate Derivatives
[0126] 10.1 The general structure of cyclosal derivatives is shown
in FIG. 27, wherein X is H or CH.sub.3 and Y is Cl or H.
[0127] Using a mixture of DMF and acetonitrile as solvent, DIPEA as
base and the 3-methylchlorophosphane or 5-chlorochlorophosphane,
the nucleosides were prepared according to Meier et al (Eur. J.
Org. Chem., 1998, 837-846) and then oxidised with t-BuOOH.
[0128] 10.2 The general structure of SATE derivatives is shown in
FIG. 28.
[0129] Bis(S-pivaloyl-2-thioethyl) N,N-diisopropylphosphoramidite
was prepared as described by Lefebvre et al, (J. Med. Chem., 1995,
38, 3941-3950). Turbo-Tet (ethylthiotetrazole) (3 equivalents) was
added to a stirred solution of the ribonucleoside analogue and
bis(S-pivaloyl-2-thioethyl) N,N-diisopropylphosphoramidite (1.2
equivalents) in a mixture of DMF and THF. The solution was stirred
at room temperature for 30 mins. The solution was then either
cooled to -40.degree. C. and a solution of 3-chloroperbenzoic acid
(1.3 equivalents) in dichloromethane added, and the solution
allowed to warm to room temperature over 1 hour. Sodium sulfite was
then added to neutralise the 3-chloroperbenzoic acid.
[0130] Alternatively the reaction mixture was treated with iodine
oxidation solution (DNA synthesis grade). The reaction mixture was
concentrated, dissolved in ethyl acetate, washed with aqueous
sodium bicarbonate and evaporated. The product was purified by
column chromatography.
Appendix
[0131]
7TABLE 1 Order Family Subfamily Genus Type Species Host The
Negative Stranded ssRNA Viruses Mononegavirales Bornaviridae
Bornavirus Borna disease virus Vertebrates Filoviridae "Ebola-like
viruses" Ebola virus Vertebrates "Marburg-like viruses" Marburg
virus Vertebrates Paramyxoviridae Paramyxovirinae Respirovirus
Human parainfluenza virus 1 Vertebrates Morbillivirus Measles virus
& rinde pest virus Vertebrates Rubulavirus Mumps virus
Vertebrates Pneumovirinae Pneumovirus Human respiratory syncytial
virus Vertebrates Metapneumovirus Turkey rhinotracheitis virus
Vertebrates Rhabdoviridae Vesiculovirus Vesicular stomatitis
Indiana virus Vertebrates Lyssavirus Rabies virus Vertebrates
Ephemerovirus Bovine ephemeral fever virus Vertebrates
Novirhabdovirus Infectious hematopoietic necrosis virus Vertebrates
Cytorhabdovirus Lettuce necrotic yellow virus Plants
Nucleorhabdovirus Potato yellow dwarf virus Plants Orthomyxoviridae
Influenzavirus A Influenza A virus Vertebrates Influenzavirus B
Influenza B virus Vertebrates Influenzavirus C Influenza C virus
Vertebrates Thogotovirus Thogoto virus Vertebrates Bunyaviridae
Bunyavirus Bunyamwera virus Vertebrates Hantavirus Hantaan virus
Vertebrates Nairovirus Nairobi sheep disease virus Vertebrates
Phlebovirus Sandfly fever Sicilian virus Vertebrates Tospovirus
Tomato spotted wilt virus Plants Tenuivirus Rice stripe virus
Plants Ophiovirus Citrus psorosis virus Plants Arenaviridae
Arenavirus Lymphocytic choriomeningitis virus Vertebrates
Deltavirus Hepatitis delta virus Vertebrates The Positive Stranded
ssRNA Viruses Narnaviridae Narnavirus Saccharomyces cerevisiae 20S
narnavirus Yeast Mitovirus Cryphonectria parasitica NB631 virus
Yeast Leviviridae Levivirus Enterobacteria phage MS2 Bacteria
Allolevivirus Enterobacteria phage Q.beta. Bacteria Picornaviridae
Enterovirus Poliovirus 1 Vertebrates Rhinovirus Human rhinovirus 1A
Vertebrates Hepatovirus Hepatitis A virus Vertebrates Cardiovirus
Encephalomyocarditis virus Vertebrates Aphthovirus 1AH
Food-and-mouth disease virus O Vertebrates Parechovirus Human
echovirus 22 Vertebrates "Cricket paralysis-like viruses" Cricket
paralysis virus Invertebrates Sequiviridae Sequivirus Parsnip
yellow fleck virus Plants Wakavirus Rice tungro spherical virus
Plants Comoviridae Comovirus Cowpea mosaic virus Plants Fabavirus
Broad bean wilt virus 1 Plants Nepovirus Tobacco ringspot virus
Plants Potyviridae Potyvirus Potato virus Y Plants Rymovirus
Ryegrass mosaic virus Plants Macluravirus Maclura mosaic virus
Plants Ipomovirus Sweet potato mild mottle virus Plants Bymovirus
Barley yellow mosaic virus Plants Tritimovirus Wheat streak mosaic
virus Plants Caliciviridae Vesiculovirus Swine vesicular exanthema
virus Vertebrates Lagovirus Rabbit hemorrhagic disease virus
Vertebrates "Norwalk-like viruses" Norwalk virus Vertebrates
"Sapporo-like viruses" Sapporo virus Vertebrates "Hepatitis E-like
Hepatitis E virus Vertebrates viruses" Astroviridae Astrovirus
Human astrovirus 1 Vertebrates Nodaviridae Alphanodavirus Nodamura
virus Invertebrates Betanodavirus Striped jack nervous necrosis
virus Vertebrates Tetraviridae Betatetravirus Nudaurelia capensis
.beta. virus Invertebrates Omegatetravirus Nudaurelia capensis
.omega. virus Invertebrates Sobemovirus Southern bean mosaic virus
Plants Luteoviridae Luteovirus Barley yellow dwarf virus Plants
Polerovirus Potato leafroll virus Plants Enamovirus Pea enation
mosaic virus 1 Plants Umbravirus Carrot mottle virus Plants
Tombusviridae Tombusvirus Tomato bushy stunt virus Plants
Carmovirus Carnation mottle virus Plants Avenavirus Oat chlorotic
stunt virus Plants Aureusvirus Pothos latent virus Plants
Necrovirus Tobacco necrosis virus Plants Dianthovirus Carnation
ringspot virus Plants Machlomovirus Maize chlorotic mottle virus
Plants Panicovirus Panicum mosaic virus Plants Nidovirales
Coronaviridae Coronavirus Avian infectious bronchitis virus
Vertebrates Torovirus Berne virus Vertebrates Arteriviridae
Arterivirus Equine arteritis virus Vertebrates Flaviviridae
Flavirus Yellow fever virus Vertebrates Pestivirus Bovine diarrhea
virus Vertebrates Hepacivirus Hepatitis C virus Vertebrates
Togaviridae Alphavirus Sindbis virus Vertebrates Rubivirus Rubella
virus Vertebrates Tobamovirus Tobacco mosaic virus Plants
Tobravirus Tobacco rattle virus Plants Hordeivirus Barley stripe
mosaic virus Plants Furovirus Soil-borne wheat mosaic virus Plants
Pomovirus Potato mop-top virus Plants Pecluvirus Peanut clump virus
Plants Benyvirus Beet necrotic yellow vein virus Plants
Bromoviridae Alfamovirus Alfalfa mosaic virus Plants Ilarvirus
Tobacco streak virus Plants Bromovirus Brome mosaic virus Plants
Cucumovirus Cucumber mosaic virus Plants Oleavirus Olive latent
virus 2 Plants Ourmiavirus Ourmia melon virus Plants Idaeovirus
Raspberry bushy dwarf virus Plants Closteroviridae Closterovirus
Beet yellows virus Plants Crinivirus Lettuce infectious yellows
virus Plants Capillovirus Apple stem grooving virus Plants
Trichovirus Apple chlorotic leaf spot virus Plants Vitivirus
Grapevine virus A Plants Tymovirus Turnip yellow mosaic virus
Plants Carlavirus Carnation latent virus Plants Potexvirus Potato
virus X Plants Allexivirus Shallot virus X Plants Foveavirus Apple
stem pitting virus Plants Barnaviridae Barnavirus Mushroom
bacilliform virus Fungi Marafivirus Maize rayado fino virus Plants
The dsRNA Viruses Cystoviridae Cystovirus Pseudomonas phage .PHI.6
Bacteria Reoviridae Orthoreovirus Reovirus 3 Vertebrates Orbirirus
Bluetongue virus 1 Vertebrates Rotavirus Simian rotavirus SA11
Vertebrates Coltivirus Colorado tick fever virus Vertebrates
Aquareovirus Golden shiner virus Vertebrates Cypovirus Bombyx mori
cypovirus 1 Invertebrates Fijivirus Fiji disease virus Plants
Phytoreovirus Wound tumor virus Plants Oryzavirus Rice rattged
stunt virus Plants Birnaviridae Aquabirnavirus Infectious
pancreatic necrosis virus Vertebrates Avibirnavirus Infectious
bursal disease virus Vertebrates Entomobirnavirus Drosphila X virus
Invertebrates Totiviridae Totivirus Saccharomyces cerevisiae virus
L-A Fungi Giardiavirus Giardia lamblia virus Protozoa
Leishmaniavirus Leishmania RNA virus 1-1 Protozoa Partitviridae
Partitvirus Gaeumannomyces graminis virus 019/6-A Fungi Chrysovirus
Penicillium chrysogenum virus Fungi Alphacryptovirus White clover
cryptic virus 1 Plants Betacryptovirus White clover cryptic virus 2
Plants Hypoviridae Hypovirus Cryphonectria hypovirus 1-EP713 Fungi
Varicosavirus Lettuce big-vein virus Plants
[0132]
Sequence CWU 1
1
8 1 20 DNA Artificial Sequence Description of Artificial
Sequencesynthetic oligonucleotide 1 ggtacagtat tagtaggacc 20 2 20
DNA Artificial Sequence Description of Artificial Sequencesynthetic
oligonucleotide 2 tgtgtcagtt agggtgacaa 20 3 20 DNA Artificial
Sequence Description of Artificial Sequencesynthetic
oligonucleotide 3 cagggattag atatcagtac 20 4 20 DNA Artificial
Sequence Description of Artificial Sequencesynthetic
oligonucleotide 4 tctctaactg gtaccataat 20 5 20 DNA Artificial
Sequence Description of Artificial Sequencesynthetic
oligonucleotide 5 aaagctggag ctccaccgcg 20 6 40 DNA Artificial
Sequence Description of Artificial Sequencesynthetic
oligonucleotide 6 agtgagcgcg cgtaatacga ctcactatag ggcgaattgg 40 7
10 DNA Artificial Sequence Description of Combined DNA/RNA Molecule
synthetic oligonucleotide 7 gcaugggccc 10 8 10 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule synthetic
oligonucleotide 8 gaucgggccc 10
* * * * *