Patent Application
20090012046
This invention relates to methods of treating patients infected with Hunan Immunodeficiency Virus (HIV) that has a K65R mutation in the viral genome encoding for reverse transcriptase.
Drugs that are currently on the market or under development to combat HIV viral infection belong to classes such as reverse transcriptase inhibitors (RTIs), protease inhibitors (PIs) and the more recent fusion inhibitors. RTIs prevent viral replication by intervening in the reverse transcription mechanism while PIs intervene in the viral assembly. RT inhibitors interact with the RT enzyme in a number of ways to inhibit its functioning so that viral replication becomes blocked. PIs bind to the active site of the viral protease enzyme, thereby inhibiting the cleavage of precursor poly proteins necessary to produce the structural and enzymatic components of infectious virons.
Nucleoside Reverse Transcriptase Inhibitors (NRTIs) belong to a class of RT inhibitors that are intracellularly converted to nucleoside triphosphates and compete with the natural nucleoside triphosphates for incorporation into elongating viral DNA by reverse transcriptase. Chemical modifications that distinguish these compounds from natural nucleosides result in DNA chain termination events.
Currently available NRTIs include zidovudine (ZDV or AZT), didanosine (ddl), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), abacavir (ABC), emtricitabine (FTC). A nucleotide reverse transcriptase inhibitor (N(t)RTI) is tenofovir disoproxil fumarate (TDF). For example, AZT, one of the first HIV RT inhibitors identified, is converted to the triphosphate (TP) by cellular kinases. HIV-1 RT is subsequently able to use AZT-TP as an efficient alternative substrate in the building of the viral DNA.
However, AZT-TP lacks a 3'OH necessary for further DNA elongation, thereby causing termination of the growing DNA chain following incorporation.
Another class of RT inhibitors are the Non-Nucleoside RT Inhibitors (NNRTIs): delavirdine, efavirenz (EFV), and nevirapine (NVP)
Although effective in suppressing HIV, each of these drugs, when used alone, is confronted with the emergence of resistant mutants. This led to the introduction of combination therapy of several anti-HIV agents usually having a different activity profile. In particular the introduction of “HAART” (Highly Active Anti-Retroviral Therapy) resulted in a remarkable improvement in anti-HIV therapy, leading to a large reduction in HIV-associated morbity and mortality. HAART involves various combinations of nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs). Current guidelines for antiretroviral therapy recommend such triple combination therapy regimen even for initial treatment. However, none of the currently available drug therapies is capable of completely eradicating HIV. Even HAART can face the emergence of resistance, often due to non-adherence and non-persistence with antiretroviral therapy. In these cases HAART can be made effective again by replacing one of its components by one of another class. If applied correctly, treatment with HAART combinations can suppress the virus for many years, up to decades, to a level where it no longer can cause the outbreak of AIDS.
NRTIs are a basic component of HAART combinations. An NRTI that is frequently used in current combinations is tenofovir or its derivative tenofovir disoproxil fumarate (TDF), usually in combination with 3TC or with FTC. The use of tenofovir disoproxil fumarate, or of drug combinations containing tenofovir disoproxil fumarate, results in the selection of HIV that has a K65R mutation in reverse transcriptase.
A series of compounds that belong to a novel class of HIV RT inhibitors has been described1,2. They differ from N(t)RTIs by chemical structure, absence of chain terminating properties, and lack of phosphorylation requirement, and from NNRTIs in terms of mechanism of action and binding pocket. Since they reversibly bind to the RT active site in competition with natural dNTP substrates, this class is referred to as Nucleotide-competing RT Inhibitors (NcRTIs).
It now has been found that NcRTIs show hypersusceptibilty towards the K65R mutation and therefore can be used to treat patients that are infected with HIV having this mutation. The present invention is aimed at using NcRTIs for treating patients that are infected with HIV having a K65R mutation in the viral genome.
The present invention relates to a method for treating patients infected with HIV that has a K65R mutation in the viral genome encoding for reverse transcriptase, said method comprising administering an effective amount to said patients of an NcRTI. In another aspect there is provided a method for treating patients infected with HIV that has a K65R mutation in the viral genome encoding for reverse transcriptase, said method comprising administering an effective amount to said patients of a combination of HIV inhibitors, at least one of which is an NcRTI.
Or, in another aspect, the invention provides the use of an NcRTI for the manufacture of a medicament for treating patients infected with HIV that has that has a K65R mutation in the viral genome encoding for reverse transcriptase. In another aspect, the invention provides the use of a combination of HIV inhibitors, at least one of which is an NcRTI for the manufacture of a medicament for treating patients infected with HIV that has that has a K65R mutation in the viral genome encoding for reverse transcriptase.
Nucleotide-competing reverse transcriptase inhibitors (NcRTI) bind to the active site of HIV reverse transcriptase (RT) in competition with the next incoming nucleotide. NcRTIs have been found to be ribonucleotide or pyrophosphate sensitive RT inhibitors and can be identified by running a test compound through an enzymatic RT inhibitory test with or without a nucleoside phosphate or a pyrophosphate being present and selecting those compounds that show an increase in inhibition of reverse transcriptase.
NcRTIs therefore can be found by a method comprising the steps:
NcRTs can also be identified as follows:
NcRTIs in particular are those compounds that are as well competitive towards an incorporated nucleotide and that may be identified as described in the previous paragraph, as being ribonucleotide or pyrophosphate sensitive RT inhibitors, which may be identified by the methodology mentioned above.
NcRTIs have been described, for example, in WO 04/046163, WO 05/111034, WO 05/111035, WO 05/111047 and WO 05/111044. Combinations of the compounds of WO 04/046163 with certain HIV inhibitors have been described in WO 05/110411.
Interesting NcRTIs for use in the invention are those compounds having the formula:
wherein
Of interest are the compounds of formula (I) wherein R2 is hydrogen, C1-6alkyl optionally substituted with NR4aR4b, pyrrolidinyl, piperidinyl, homopiperidinyl, piperazinyl, 4-(C1-4alkyl)-piperazinyl, morpholinyl, aryl, furanyl.
Of particular interest are the compounds of formula (I) wherein R2 is C1-6alkyl optionally substituted with NR4aR4b, pyrrolidinyl, piperidinyl, morpholinyl; and wherein R4a and R4b independently from each other are hydrogen, C1-4alkyl.
Also interesting for use in the present invention are compounds of formula (II)
wherein R1 is as R1, R3 is as R3 and
wherein
—CpH2p—CH(OR14)—CqH2q—R15 (b-3);
—CH2—CH2—(O—CH2—CH2)m—OR14 (b-4);
—CH2—CH2—(O—CH2—CH2)m—NR17aR17b (b-5);
wherein in radical (b-3) one of the hydrogen atoms in —CpH2p— and one of the hydrogen atoms in —CH(OR14)—CqH2q—, that is not part of R14, may be replaced by a direct bond or a C1-4alkanediyl group;
p is 1, 2 or 3;
q is 0, 1, 2 or 3;
each m independently is 1 to 10;
each R14 independently is hydrogen, C1-4alkyl, aryl C1-4alkyl, aryl, C1-4alkylcarbonyl, —SO3H, —PO3H2;
R15 is cyano, NR16aR16b, pyrrolidinyl, piperidinyl, homopiperidinyl, piperazinyl, 4-(C1-4alkyl)-piperazinyl, 4-(C1-4alkylcarbonyl)-piperazinyl, 4-(C1-4alkyloxycarbonyl)-piperazinyl, morpholinyl, thiomorpholinyl, aryl, furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, pyrazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, hydroxy-carbonyl, C1-4alkylcarbonyl, N(R16aR16b)carbonyl, C1-4alkyloxycarbonyl, pyrrolidin-1-ylcarbonyl, piperidin-1-ylcarbonyl, homopiperidin-1-ylcarbonyl, piperazin-1-ylcarbonyl, 4-(C1-4alkyl)-piperazin-1-ylcarbonyl, morpholin-1-yl-carbonyl, thiomorpholin-1-yl-carbonyl, 1-oxothiomorpholin-1-ylcarbonyl and 1,1-dioxo-thiomorpholin-1-ylcarbonyl; or R15 may additionally be aryl substituted with a radical —COOR4; or a radical selected from —NR5a—C(═NR5b)—NR5cR5d, —NR5a—C(═NR5e)—R5f, —O—NR5a—C(═NR5b)—NR5cR5d, —O—NR5a—C(═NR5e)—R5f, -sulfonyl-R6, —NR7R8, —NR9R10, a radical (a-1), (a-2), (a-3), (a-4) or (a-5); wherein R4, R5a, R5b, R5c, R5d, R6, R7, R8, R9, R10, and the radicals (a-1), (a-2), (a-3), (a-4), (a-5), independently are as defined above;
Also interesting for use in the invention are the following compounds:
wherein R1, R3 and R2 are as specified above for (I) or for (II) and −a1=a2−a3=a4-represents a bivalent radical of formula
—CH═CH—CH═CH— (c-1);
wherein one, two, three or four of the hydrogen atoms in (c-1) is replaced by a radical C1-6alkyl, C1-4alkoxy, halo, hydroxy, (R5g)(R5h)N—(C1-4alkanediyl)-O—, (R7)(R8)N—(C1-4alkanediyl)-O—, trifluoromethyl, cyano, a radical —COOR4, (R5a)(R5b)N-carbonyl, formyl, C1-6alkylcarbonyl, nitro, hydroxyC1-6alkyl, C1-4alkoxyC1-6alkyl, (R4OOC)—C1-6alkyl, a radical —N(R5a)(R5b), a radical
morpholinyl, thiomorpholinyl, (R5g)(R5h)N—(C1-4alkanediyl)-N(R5c)—, (R7)(R8)N—(C1-4alkanediyl)-N(R5c)—, C1-6alkyl-carbonylamino, C1-6alkyloxycarbonylamino, trifluoroacetylamino, C1-6alkylsulfonyl-amino, (R5a)(R5b)N—C1-4alkyl; aryl;
R5g and R5h independently are hydrogen or C1-4alkyl; R5a and R5c independently are hydrogen or C1-4alkyl; Q1 and R11 are as defined above; as well as the pharmaceutically acceptable addition salts thereof.
An interesting subgroup of the compounds of formula (I), (II) or (III) comprises those compounds, wherein R3 or R3 is nitro.
In any of the above compounds represented by (I), (II) or (III) the phenyl moiety substituted with R3 or R3 may be replaced by furyl, thienyl, pyridyl, pyrimidinyl, benzofuryl, benzo-thienyl, indolyl, imidazopyridyl, purinyl, optionally substituted with one or two substituents selected from halo, cyano, C1-6alkyl, CF3, —COOR4, (R5a)(R5b)N-carbonyl, hydroxy, C1-6alkyloxy, C1-6alkylthio, C1-6alkylsulfonyl; or the phenyl moiety substituted with R3 or R3 may be replaced by furyl, thienyl, pyridyl, indolyl, imidazopyridyl, optionally substituted with one or two substituents selected from halo, cyano, C1-6alkyl, CF3, —COOR4, (R5a)(R5b)N-carbonyl, C1-6alkyloxy, C1-6alkylthio, C1-6alkylsulfonyl; or the phenyl moiety substituted with R3 or R3 may be replaced by halopyridyl, in particular by 6-chloro-4-pyridyl.
The term “C1-4alkyl” as a group or part of a group defines straight and branched chained saturated hydrocarbon radicals having from 1 to 4 carbon atoms, such as, for example, methyl, ethyl, propyl, butyl, 2-methyl-propyl and the like. The term “C1-6alkyl” as a group or part of a group defines straight and branched chained saturated hydrocarbon radicals having from 1 to 6 carbon atoms such as, for example, the groups defined for C1-4alkyl and pentyl, hexyl, 2-methylbutyl, 3-methylpentyl and the like.
The term “C2-6alkyl” as a group or part of a group defines straight and branched chained saturated hydrocarbon radicals having from 2 to 6 carbon atoms such as for example, ethyl, propyl, butyl, 2-methyl-propyl, pentyl, hexyl, 2-methylbutyl, 3-methylpentyl and the like.
The term “C1-10alkyl” as a group or part of a group defines straight and branched chained saturated hydrocarbon radicals having from 1 to 10 carbon atoms such as, for example, the groups defined for C1-6alkyl and heptyl, octyl, nonyl, decyl and the like. The term C2-6alkenyl as a group or part of a group defines straight and branched chained hydrocarbon radicals having saturated carbon-carbon bonds and at least one double bond, and having from 2 to 6 carbon atoms, such as, for example, ethenyl, prop-1-enyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-2-enyl, hex-1-enyl, hex-2-enyl, hex-3-enyl, 1-methyl-pent-2-enyl and the like.
The term “C2-10alkenyl” as a group or part of a group defines straight and branched chained hydrocarbon radicals having saturated carbon-carbon bonds and at least one double bond, and having from 2 to 10 carbon atoms, such as, for example, the groups of C2-6alkenyl and hept-1-enyl, hept-2-enyl, hept-3-enyl, oct-1-enyl, oct-2-enyl, oct-3-enyl, non-1-enyl, non-2-enyl, non-3-enyl, non-4-enyl, dec-1-enyl, dec-2-enyl, dec-3-enyl, dec-4-enyl, 1-methyl-pent-2-enyl and the like. The term C3-7cycloalkyl is generic to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
The term “halo” is generic to fluoro, chloro, bromo or iodo.
Aryl is phenyl optionally substituted with one, two or three substituents independently selected from C1-6alkyl, C1-4alkoxy, halo, hydroxy, amino, trifluoromethyl, mono- and di-C1-6alkylamino, nitro, cyano, carboxyl, C1-4alkoxycarbonyl; in particular aryl is phenyl optionally substituted with one, two or three substituents independently selected from C1-6alkyl, C1-4alkoxy, halo, hydroxy, amino, nitro, cyano.
It should be noted that different isomers of the various heterocycles may exist within the definitions as used throughout the specification. For example, oxadiazolyl may be 1,2,4-oxadiazolyl or 1,3,4-oxadiazolyl or 1,2,3-oxadiazolyl; likewise for thiadiazolyl which may be 1,2,4-thiadiazolyl or 1,3,4-thiadiazolyl or 1,2,3-thiadiazolyl; pyrrolyl may be 1H-pyrrolyl or 2H-pyrrolyl.
It should also be noted that the radical positions on any molecular moiety used in the definitions may be anywhere on such moiety as long as it is chemically stable. For instance pyridyl includes 2-pyridyl, 3-pyridyl and 4-pyridyl; pentyl includes 1-pentyl, 2-pentyl and 3-pentyl.
Examples of compounds for use in the invention are:
A compound of particular interest is:
5-Methyl-1-(4-nitro-phenyl)-2-oxo-2,5-dihydro-1H-pyrido[3,2-b]indole-3-carbonitrile; hereafter referred to as “NcRTI-1”, which compound is represented by the following chemical structure:
Further compounds of interest are:
Further compounds of interest are:
Further compounds of interest are:
Compounds of particular interest are:
The above-mentioned compounds may be used in free form or as a pharmaceutically acceptable addition salt form wherein the salts may be derived from acids such as, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butandioic acid), maleic, fumaric, malic (i.e. hydroxyl-butandioic acid), tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids. A particular group of compounds for use in this invention are the pharmaceutically acceptable addition salts of the compounds listed above by their chemical name.
Where applicable, the above compounds or their salts may be used in the form of racemates, stereoisomers or stereoisomeric mixtures.
The NcRTI that is administered, either alone or in a combination with one or more other HIV inhibitory agents, preferably is formulated into a suitable pharmaceutical formulation. Suitable formulations have been described in the references cited in the previous paragraph. The NcRTI can be administered as such, or in salt form; it can further be administered in the form of a stereoisomer or stereoisomeric mixture. It can also be administered as a pro-drug. Salts, stereoisomers and stereoisomeric mixtures and pro-drugs have also been described in the references cited in the previous paragraph.
In a further aspect, the present invention provides a method for treating patients infected with HIV that have been treated with tenofovir (or a derivative such as TDF) and/or 3TC, said method comprising administering an effective amount to said patients of an NcRTI. Or alternatively, the invention provides the use of an NcRTI for the manufacture of a medicament for treating patients infected with HIV that have been treated with tenofovir (or a derivative such as TDF) and/or 3TC.
The susceptibility of over 6000 recent clinical HIV-1 isolates for NcRTI-1 and different N(t)RTIs and NNRTIs was assessed.
82% of the profiled clinical isolates remained susceptible for NcRTI-1 (fold change in EC50 (FC)<4), 15.7% of the population showed a reduced susceptibility (FC4-10) while only 2.3% showed resistance (FC>10). No cross-resistance was observed between NcRTI-1 and the NNRTIs EFV or NVP, neither with the N(t)RTIs ZDV, TDF, and ABC. Only with the N(t)RTIs 3TC and FTC, limited cross-resistance could be detected (Pearson Correlation Coefficient=0.56) (Table 1).
Analysis of the genotype of a random set of more than 1700 of the 6000 tested clinical isolates (
Analysis also indicated that the K65R mutation not only is associated with hypersusceptibility to NcRTI-1 but also reverses M184V-induced resistance of HIV-1 for NcRTI-1. These findings were confirmed in site-directed mutant (SDM) strains.
This reciprocity between the K65R and M184V mutation is unparalleled among RT inhibitors. When replicating wild-type HIV-1 in the presence of NcRTI-1, M184V+Y115F were selected. In the presence of both NcRTI-1 and tenofovir, NcRTI-1 prevents the selection of K65R. NcRTI-1 activity is not affected by the presence of NNRTI resistance mutations, nor by the presence of the major N(t)RTI induced, multi-drug resistant mutation patterns: the thymidine-associated mutations (TAMs), the T69 insertion complex, and the Q151M complex.
Findings with clinical isolates were confirmed in site-directed mutant (SDM) strains (Table 2):
Recombinant HIV-1 viruses, derived from clinical samples, were constructed by cotransfection of MT4 cells with sample derived viral protease and RT coding sequences and an HIV-1 HXB2-derived proviral clone deleted in the protease and RT coding region2.
Site-directed mutant RT coding sequences were generated from a pGEM vector containing the HIV-1 clone HXB2 protease and RT coding sequence by using a QuikChange™ site-directed mutagenesis kit, and HPLC-purified primers. Plasmids were sequenced to confirm that they contained the desired mutations. Mutant viruses were created by recombination of the mutant protease-RT sequence with a protease-RT deleted HIV-1 HXB2 proviral clone2.
To in vitro select viral strains resistant to RT inhibitors, MT4-LTR-EGFP cells were infected in the presence of the inhibitor (at 2 or 3 times EC50). Cultures were passaged every 3 to 4 days at the same concentration of inhibitor, until full virus breakthrough. At that time, virus was harvested and used for a new round of selection at a higher compound concentration. At each breakthrough, the harvested virus was genotyped to identify acquired mutations. For several RT inhibitors, alone or in combination, the mutations selected when replicating wild type HIV-1 (IIIB) in their presence, were determined (see Table 3).
These results demonstrate that while the single mutations M184V and Y115F showed a moderate fold change of 5.0 and 7.9, respectively, the combination resulted in a FC of 75. When NcRTI-1 was present as sole inhibitor, wild type HIV-1 IIIB acquired mutations M184V and Y115F, in line with the results obtained with clinical isolates and SDMs (
The presence of the K65R point mutation causes an increased susceptibility of HIV-1 for NcRTI-1 (FC=0.46), and reverses M184V-induced reduction of NcRTI sensitivity (FC from 5.0 to 0.89).
NcRTI-1 remained active on a wide variety of strains containing NNRTI resistance associated mutations. The major N(t)RTI induced, multi-drug resistant mutation patterns (TAMs, T69 insertion complex and Q151M complex) do not affect NcRTI-1 activity.
When 3TC or FTC was combined with TDF, the K65R mutation was selected, in accordance with previous findings3. Combining NcRTI-1 with TDF did not result in selection of the K65R mutation, even after prolonged exposure (190 days). Instead, the virus acquired the K70E or K70N mutation.
From the above experiments, the following conclusions can be drawn. Mutational patterns associated with reduced and increased susceptibility to NcRTIs are different from patterns associated with currently used RT inhibitors. Only limited cross-resistance with 3TC was detected.
The combination of RT-active site mutations M184V+Y115F correlated most significantly with resistance to NcRTI-1 (FC=75). This combination is also selected in vitro when replicating HIV-1 IIIB in the presence of increasing concentrations of NcRTI-1.
Unlike 3TC or FTC, where M184V causes complete resistance (FC>100), the effect of M184V on NcRTI-1 susceptibility is limited (FC=5.0).
The K65R mutation causes hypersusceptibility to NcRTI-1. In addition, presence of K65R reverses the M184V-induced reduction of NcRTI sensitivity. This reciprocity between the K65R and M184V mutation is unparalleled among RT inhibitors.
When combined with TDF, NcRTI-1 prevents the selection of K65R by TDF. These experiments showed that the viruses acquired K70E, a mutation previously suggested as an alternative pathway of TDF resistance4.
The present invention also relates to a method for treating patients infected with HIV that has developed resistance towards an NcRTI, said method comprising administering an effective amount to said patients of tenofovir or of a tenofovir derivative, in particular tenofovir disoproxil fumarate (TDF). In particular said HIV that has developed resistance towards an NcRTI shows a fold change of at least 10, or at least 20, or at least about 50, or at least about 75. More in particular said HIV that has developed resistance towards an NcRTI has a M184V or Y115F, or a double M184V or Y115F mutation in the viral genome encoding for reverse transcriptase. This method is useful in treating patients that have been pre-treated with an NcRTI or NcRTI containing combination and have developed resistance towards the NcRTI, as indicated by the fold change. These patients can be treated with an effective amount to said patients of tenofovir or of a tenofovir derivative, in particular tenofovir disoproxil fumarate (TDF). In an alternative aspect, this invention concerns the use of tenofovir or of a tenofovir derivative, in particular tenofovir disoproxil fumarate (TDF) for the manufacture of a medicament for treating infected with HIV that has developed resistance towards an NcRTI, in particular with HIV that has developed resistance towards an NcRTI showing a fold change of at least 10, or at least 20, or at least about 50, or at least about 75; or more in particular with HIV that has developed resistance towards an NcRTI has a M184V or Y115F, or a double M184V or Y115F mutation in the viral genome encoding for reverse transcriptase.
All references mentioned in this specification are incorporated herein in their entirety.
Lloyd, R., Huong, J., Rouse, E., et al. HIV-1 RT Mutations K70E and K65R are Not Present on the Same Viral Genome when Both Mutations are Detected in Plasma. 45th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC): Dec. 16-19, 2005; Washington, D.C., USA. Poster H-1066.
| Number | Date | Country | Kind |
|---|---|---|---|
| 06101297.7 | Feb 2006 | EP | regional |
| 06114705.4 | May 2006 | EP | regional |
This application claims priority of the benefits of the filing of PCT Application No. PCT/EP2007/051087 filed Feb. 5, 2007, European Patent Application No. 06101297.7 filed Feb. 3, 2006 and European Patent Application No. 06114705.4 filed May 30, 2006. The complete disclosures of the aforementioned related patent applications are hereby incorporated herein by reference for all purposes.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP07/51087 | 2/5/2007 | WO | 00 | 7/7/2008 |
