EA040090B1 - COMBINATION FOR TREATMENT AND REDUCTION OF RSV INFECTION AND PHARMACEUTICAL COMPOSITION BASED ON IT - Google Patents

Description

The invention is directed to a combination for treating or attenuating an infection caused by a respiratory syncytial virus (RSV) containing 3-({5-chloro-1-[3-(methylsulfonyl)propyl]-1 H-indol-2-yl} methyl)-1-(2,2,2-trifluoroethyl)-1,3-dihydro-2H-imidazo[4,5-c]pyridin-2-one or a pharmaceutically acceptable salt thereof as compound A and N-(2 -fluoro-6-methylphenyl)-6-(4-(5methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinamido)benzoyl)-5,6-dihydro-4H-benzo[ b]thieno [2,3d]azepine-2-carboxamide or a pharmaceutically acceptable salt thereof as compound B, as well as a pharmaceutical composition based on it.

RSV, or human respiratory syncytial virus, is a large RNA virus that belongs to the Pneumoviridae family of the genus Orthopneumovirus together with bovine RSV. Human RSV causes a number of respiratory diseases in people of all ages throughout the world. It is the leading cause of lower respiratory tract disease in infancy and childhood. More than half of all babies experience RSV in their first year of life and nearly all during their first two years. Infection in young children can lead to lung damage that persists for years and may contribute to the development of chronic lung disease later in life (chronic bronchopulmonary obstruction, asthma). Adolescents and adults often suffer from a (bad) cold with RSV infection. Susceptibility increases again in old age, and RSV has been implicated in many outbreaks of pneumonia in the elderly, resulting in significant mortality.

So far, only three drugs have been approved for use against RSV infection. The first is ribavirin, a nucleoside analog that provides an aerosolized agent for the treatment of severe RSV infection in hospitalized children. The other two drugs, RespiGam® (RSV-IG) and Synagis® (palivizumab), immunostimulants based on polyclonal and monoclonal antibodies, are intended for prophylactic use. Both are very expensive and require parenteral administration. A number of compounds for the treatment of RSV infection are in clinical trials. One such compound is 3-({5-chloro-1-[3-(methylsulfonyl)propyl]-1H-indol-2-yl}methyl)-1-(2,2,2trifluoroethyl)-1,3- dihydro-2H-imidazo[4,5-c]pyridin-2-one disclosed in WO 2012/080447 as compound P55.

It has now been found that the combination of 3-({5-chloro-1-[3-(methylsulfonyl)propyl]1H-indol-2-yl}methyl)-1-(2,2,2-trifluoroethyl)-1 ,3-dihydro-2H-imidazo[4,5-c]pyridin-2-one (hereinafter referred to as compound A) and N-(2-fluoro-6-methylphenyl)-6-(4-(5- methyl 2-(7-oxa-2azaspiro[3.5]nonan-2-yl)nicotinamido)benzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepin-2-carboxamide ( hereinafter referred to as Compound B), wherein Compound B has RSV replication inhibiting properties, provides improved therapy in the treatment of RSV infection.

Thus, an object of the present invention is a combination for the treatment or amelioration of respiratory syncytial virus (RSV) infection containing 3-({5-chloro-1-[3-(methylsulfonyl)propyl]-1H-indole-2 -yl}methyl)-1-(2,2,2-trifluoroethyl)-1,3-dihydro-2Nimidazo[4,5-c]pyridin-2-one or a pharmaceutically acceptable salt thereof as Compound A and N-( 2fluoro-6-methylphenyl)-6-(4-(5-methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinamido)benzoyl)-5,6-dihydro-4H-benzo [b]thieno[2,3-d]azepine-2-carboxamide or a pharmaceutically acceptable salt thereof as compound B, wherein the amount of compound A corresponds to a concentration value of 0.19 to 1250 nM and the amount of compound B corresponds to a concentration value of 0.058 up to 42 nM.

Another object of the present invention is a pharmaceutical composition with activity against RSV, containing a therapeutically effective amount of the above combination and a pharmaceutically acceptable carrier.

Compound A is 3-({5-chloro-1-[3-(methylsulfonyl)propyl]-1H-indol-2-yl}methyl)-1-(2,2,2-trifluoroethyl)-1,3- dihydro-2H-imidazo[4,5-c]pyridin-2-one disclosed in WO 2012/080447 as compound P55 having the following structure:

Compound B is N-(2-fluoro-6-methylphenyl)-6-(4-(5-methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinamido)benzoyl)- 5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide

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As used hereinafter, the terms compound A, compound B, or similar expressions are intended to include both compound A and/or compound B in its free base form and their pharmaceutically acceptable salt forms.

Compound A or Compound B can be used in pharmaceutically acceptable salt forms or in free (ie, non-salt) form. Salt forms can be obtained by treating the free form with an acid or base. Of interest are pharmaceutically acceptable acid and base addition salts, which are intended to include the therapeutically active non-toxic acid and base addition salt forms that these compounds are capable of forming. Pharmaceutically acceptable acid addition salts of Compound A or Compounds B can be readily obtained by treating the free form with such appropriate acid. Suitable acids include, for example, inorganic acids such as hydrohalic acids such as hydrobromic acid or in particular hydrochloric acid; or sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic, maleic, fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, etc. -toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids. Compound A or Compound B can also be converted into pharmaceutically acceptable metal or amine addition salt forms by treatment with appropriate organic or inorganic bases. Suitable basic salt forms include, for example, ammonium salts, alkali and alkaline earth metal salts, eg lithium, sodium or potassium salts; or salts of magnesium or calcium; salts of organic bases, for example salts of benzathine, N-methyl-D-glucamine, hydrabamine, and salts of amino acids such as, for example, arginine, lysine and the like. The term addition salt form is also intended to include any solvates that Compound A or Compound B may form, as well as their salts. Such solvates are, for example, hydrates, alcoholates, for example ethanolates and the like.

The amount of Compound A and the amount of Compound B in the combinations of the present invention are such that a synergistic antiviral effect is provided against RSV.

All quantities mentioned in this and the following paragraphs refer to the free form (ie non-salt form). The above values represent free-form equivalents, i.e. values corresponding to the intended introduction of the free form. When introducing salts, the amounts must be calculated depending on the ratio of the molecular weights of the salt form and the free form.

The above daily doses are based on an average body weight of approximately 70 kg and should be recalculated for pediatric use or for use in patients with significant body weight variance.

Dosages may be presented as one, two, three or four or more dose portions administered at regular intervals throughout the day. The dosage used preferably corresponds to the daily amount of Compound A and Compound B above, or a fraction of the dose, such as 1/2, 1/3 or 1/4 of it. The dosage form may contain compound A and compound B in an amount equivalent to the ranges or values specified in the previous paragraphs, either in separate formulations or in a combined formulation. Such a combination composition is preferred.

Where Compound A and Compound B are to be administered once a day, this can be done by administering a combined fixed dose combination containing Compound A and Compound B. Dosage forms that can be administered are described hereinafter, wherein dosage forms for oral administration, in particular solutions for pe

- 2 040090 oral administration are preferred.

The active ingredients may be formulated into pharmaceutical compositions either singly or as a combined pharmaceutical composition. In the latter case, a pharmaceutical composition is provided comprising a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof and Compound B or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. A therapeutically effective amount in this context is an amount sufficient to stabilize or reduce the intensity of RSV infection in infected subjects. Therapeutically effective amounts may in particular correspond to the amounts indicated above for daily administration or portions thereof for the convenience of multiple daily administrations.

In a further aspect, the present invention relates to a process for the preparation of a pharmaceutical composition as defined herein, which comprises intimately mixing a pharmaceutically acceptable carrier with a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof, and an effective amount of Compound B, or a pharmaceutically acceptable salt thereof.

The combinations provided herein can also be formulated as a combined preparation for simultaneous or sequential use in anti-RSV therapy. In such a case, Compound A is formulated in a pharmaceutical composition containing other pharmaceutically acceptable excipients, and Compound B is formulated separately in a pharmaceutical composition containing other pharmaceutically acceptable excipients. Relatively speaking, these individual pharmaceutical compositions may be part of a kit for simultaneous or sequential use.

The individual components of the combination of the present invention may be administered simultaneously or separately at different points in time during therapy or in parallel in separate or single combined forms.

Therefore, Compound A and Compound B, alone or in combination, can be formulated into various pharmaceutical compositions that are suitable for administration purposes. In such compositions, a therapeutically effective amount of each of the specific Compounds A and B is combined with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. Pharmaceutical compositions may be formulated for oral, parenteral (including subcutaneous, intramuscular and intravenous), rectal, buccal or intranasal administration. Suitable compositions for oral administration include powders, granulates, aggregates, tablets, compressed or coated pills, dragees, sachets, hard or gelatine capsules, syrups and suspensions. Suitable compositions for parenteral administration include aqueous or non-aqueous solutions or emulsions, while suitable compositions for rectal administration include suppositories with a hydrophilic or hydrophobic carrier medium. For intranasal delivery, suitable aerosol delivery systems can be used.

For example, in the preparation of compositions for oral administration, any conventional pharmaceutical medium may be used, such as, for example, water, glycols, oils, alcohols, and the like, in the case of liquid compositions for oral administration, such as suspensions, syrups, elixirs, emulsions, and solutions. ; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrants and the like, in the case of solid compositions. In the case of compositions for parenteral administration, the carrier will generally contain sterile water, at least in large part, although other ingredients such as solubilizers, emulsifiers or additional auxiliaries may be added to the carrier. Injectable solutions can be prepared in which the carrier contains saline, glucose solution, or a mixture of both. Injectable suspensions can also be prepared, in which case suitable liquid carriers, suspending agents, and the like can be used. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations such as powders for dissolution.

Pharmaceutical compositions may conveniently be presented in unit dosage form for ease of administration and dose uniformity. Examples include tablets (including divisible or coated tablets), capsules, pills, suppositories, powder packs, wafers, injection solutions or suspensions, and the like, as well as multiples thereof.

The combination of Compound A and Compound B as described herein is suitable for the treatment of warm-blooded animals, in particular humans, infected with RSV.

As used herein, the expressions treat, treating, treating, therapeutic, and therapy do not necessarily mean the complete cure or elimination of a disease or condition. Any reduction in viral load value or alleviation of any undesirable signs or symptoms of a disease or condition can be considered to some extent as a treatment and/or therapy.

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The terms "therapeutically effective amount" and "effective amount" are used to refer to the amount of active compound or pharmaceutical agent that produces an observable biological or medical response. For example, a therapeutically effective amount of a compound may be the amount necessary to prevent, alleviate, or lessen the symptoms of a disease, lower a viral load value, or prolong the lifespan of a subject being treated. This response may occur in a tissue, system, animal, or human, and includes a decrease in viral load value, alleviation of the signs or symptoms of the disease being treated. Determination of an effective amount is well within the skill of those skilled in the art in view of the disclosure provided herein. The therapeutically effective amount of the compounds disclosed herein required as a dose will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the particular animal in question. The dose may be modified to achieve the desired effect, but will depend on factors such as weight, diet, concurrent drug use and other factors that will be understood by specialists in the field of medicine.

Various metrics for determining the effectiveness of a method for treating a respiratory syncytial virus (RSV) infection are known to those skilled in the art. Examples of suitable measures include, without limitation, a reduction in viral load value, a reduction in viral replication, a reduction in time to seroconversion (a virus that cannot be detected in a patient's serum), a reduction in morbidity or mortality in clinical outcomes, and/or another measure of disease response.

The combination of Compound A and Compound B, or pharmaceutically acceptable salts of the above compounds, can reduce viral titers to undetectable levels, such as less than 1.7 log ₁₀ plaque forming units (PFUe)/ml or less than 0.3 log ₁₀ plaque forming units (PFUe) /ml The combination of compounds described herein can provide a reduction in viral load value compared to the value of the viral load before the introduction of the combination (for example, 60 hours after receiving the initial dose of the combination). The combination of compounds described herein can reduce the viral load value to less than 1.7 log ₁₀ (PFUe)/ml or less than 0.3 log ₁₀ (PFUe)/ml. The combination of compounds described herein can provide a reduction in viral titer in a subject's serum ranging from about 1.5-fold log to about 2.5-fold log reduction, from about 3-fold log to about 4-fold log reduction, or greater than approximately 5-fold logarithmic reduction compared to the value of the viral load before the introduction of the combination. For example, the viral load value is measured before the administration of the combination and several hours after receiving the initial dose of the combination (eg, 60 hours after receiving the initial dose of the combination).

The combination of compound A and compound B or pharmaceutically acceptable salts of the above compounds may provide at least 1-, 2-, 3-, 4-, 5-, 10-, 15-, 20-, 25-, 50-, 75-, A 100-fold or greater reduction in RSV replication relative to pre-treatment levels in a subject, as determined several hours after receiving the initial dose of the combination (eg, 60 hours after receiving the initial dose of the combination). The combination described herein can provide a reduction in RSV replication relative to pre-treatment levels ranging from about 2- to about 5-fold, from about 10- to about 20-fold, from about 15- to about 40-fold, or from approximately 50 to approximately 100-fold reduction. In some embodiments, the combination described herein may provide a reduction in RSV replication ranging from 1 to 1.5 times log, from 1.5 times log to 2 times log, from 2 times log to 2.5 times log. , 2.5- to 3-fold logarithmic, 3-fold logarithmic to 3.5-fold logarithmic, or 3.5- to 4-fold logarithmic or greater reduction in RSV replication compared to the reduction in RSV achieved through the use of one an anti-RSV agent administered as monotherapy, or the same reduction may be achieved in a shorter period of time.

Over time, infectious agents may become resistant to one or more therapeutic agents. The term resistance as used herein refers to a viral strain exhibiting a delayed, reduced and/or nil response to a therapeutic agent(s). For example, after treatment with an antiviral agent, the viral load value of a subject infected with a resistant virus may be reduced to a lesser extent than that exhibited by a decrease in the viral load value of a subject infected with a resistant strain. A combination of Compound A and Compound B, or pharmaceutically acceptable salts of the above compounds, can be administered to an RSV-infected subject who is resistant to one or more different anti-RSV agents (eg, ribavirin). In some embodiments, the development of sustainable

- 4 040090 strains of RSV may be delayed in the treatment of subjects with the combination of compounds described herein, compared with the development of strains of RSV resistant to other anti-RSV agents administered as monotherapy.

The combination of Compound A and Compound B, or pharmaceutically acceptable salts of the above compounds, can reduce the percentage of subjects who experience complications from an RSV viral infection compared to the percentage of subjects who experience complications from treatment with an anti-RSV agent alone. For example, the percentage of subjects treated with the combination of compounds described herein that experience complications may be 5%, 10%, 25%, 40%, 50%, 60%, 70%, 80%, and 90% less than the value for subjects treated with only one anti-RSV agent administered as monotherapy.

A potential advantage of using a combination of Compound A and Compound B or pharmaceutically acceptable salts of the above compounds may be a reduction in the required amount(s) of Compound A or its pharmaceutically acceptable salt and/or Compound B or their pharmaceutically acceptable salts, which(which) is (are) effective(s) in the treatment of RSV infection compared to the amount required to achieve the same therapeutic result when using compound (B) or its pharmaceutically acceptable salt and/or compound A or its pharmaceutically acceptable salt. For example, the amount of Compound A or a pharmaceutically acceptable salt thereof and/or Compound B or a pharmaceutically acceptable salt thereof may be less than the amount of the above compounds required to achieve the same viral load reduction when administered as monotherapy. Another potential advantage of using the combination described herein is that the use of two or more compounds having different modes of action may create a higher barrier to the development of resistant viral strains than when the compound is administered alone. Additional benefits of using the combination described herein may include little to no cross-resistance to the compounds of the combination; various routes of excretion of the compounds of the combination; overlapping toxicity profiles of compounds of the combination - from a small degree to its absence; effects on cytochrome P450 are small to negligible; and/or pharmacokinetic interactions of compounds of the combination - from small to none.

Examples

The following examples are intended to illustrate the invention and not to limit it.

Example 1 Analysis for RSV.

Black 384-well clear bottom microtiter plates (Corning, Amsterdam, The Netherlands) were filled by sonic drop popping using an Echo liquid handling device (Labcyte, Sunnyvale, CA). 200 nl of compound stock solutions (100% DMSO) were transferred to assay plates. Compound (A) or a pharmaceutically acceptable salt thereof was serially diluted (1:3) to give 12 different concentrations horizontally in a 384-well plate, and compound (B) or a pharmaceutically acceptable salt thereof was serially diluted (1:3) to give 12 different concentrations in the vertical direction in a 384-well plate. Space was also allocated to titrate each of the compounds separately for use as reference controls. The assay was initiated by adding 10 µl of culture medium to each well (RPMI medium without phenol red, 10% heat-inactivated FBS, 0.04% gentamicin (50 mg/ml). All additional steps were performed using a multichannel dispenser (Thermo Scientific, Erembodegem, Belgium) The rgRSV224 virus (MOI=1) diluted in culture medium was then plated.The rgRSV224 virus is an engineered virus that includes an additional GFP gene (Hallak LK, Spillmann D., Collins PL, Peeples ME Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection; Journal of virology (2000), 74(22), 10508-13) and licensed from NIH (Bethesda, MD, USA) Finally, 20 µl was dispensed HeLa cell suspension (3000 cells/well) Media, virus-infected controls, and sham-infection controls were included in each test.The wells contained 0.05% DMSO per vol. and incubated at 37°C in an atmosphere of 5% CO ₂ . Three days after exposure to the virus, viral replication was quantified by measuring GFP expression in cells using an in-house developed MSM laser microscope (Tibotec, Berset, Belgium). EC ₅₀ was defined as 50% inhibitory concentration for GFP expression.

Cell viability analysis.

Compounds were incubated in parallel for three days in a set of white 384-well microtiter plates (Corning) and the cytotoxicity of the compounds in HeLa cells was determined by measuring the ATP content of the cells using the ATPLite kit (Perkin Elmer, Zaventem, Belgium) according to the manufacturer's instructions. CC ₅₀ was defined as 50% concentration for cytotoxicity.

Data analysis.

Each experiment was performed at N=3 for both anti-RSV and anti-RSV activity.

- 5 040090 carrying cell viability. The mean percent inhibition of replicon values from 3 experiments was obtained and the additivity index (CI) was calculated for activity against RSV using the program from the CalCuSyn software package from Biosoft (Biosoft, UK):

CI=[(D) 1 /(Dx) 1 ]+[(D)2/(Dx)2].

The combination was defined as: CI<1: synergy; CI>1: antagonism; CI=1: additivity. A more detailed CI range is presented in the table below_______________________________

Cl range Description Symbols <0.1 Very strong synergy ++++++ 0.1-0.3 Strong synergy + + + + 0.3-0.7 Synergy +++ 0.7-0.85 Moderate synergy ++ 0.85-0.90 Light synergy + 0.90-1.10 Practically additivity +/- 1.10-1.20 Light antagonism - 1.20-1.45 moderate antagonism -- 1.45-3.3 Antagonism — 3.3-10 Strong antagonism — >10 Very strong antagonism

Example 2 Combination studies - RSV with Renilla reporter gene.

RSV expressing Renilla luciferase (A2-RL-linel9F) was developed by Dr. Martin Moore of Emory University, Atlanta, Georgia, USA. In vitro viral kinetics of A2-RL-line19F is similar to that of wild-type RSV. The HEp-2 host cell line was purchased from ATCC (cat no. CCL-23) and cells were cultured in 1x 50/50 DMEM/Ham's F-12 medium containing L-glutamine and 15 mM HEPES (Mediatech, cat no. 10-092-SM). Additional 5% (v/v) FBS (Mediatech, cat. no. 35-010-CV) and 1% (v/v) penicillin/streptomycin (Mediatech, cat. no. 30-002) were added to the medium. -CI). HEp-2 cells were maintained at 37°C in a humidified atmosphere with 5% CO2.

Drug treatment and administration of virus doses.

The following procedure was followed to determine the effect of the compound combination. On the first day, 20,000 HEp-2 cells/well were seeded in a 96-well plate. The next day, the test samples were solubilized in 100% DMSO to the desired final test concentration of 200x. Compound (A) or a pharmaceutically acceptable salt thereof was then serially diluted (1:3) to give 9 different concentrations horizontally in a 96-well plate, and compound (B) or a pharmaceutically acceptable salt thereof was serially diluted (1:3) to give 7 different concentrations in the vertical direction in a 96-well plate. Serially diluted 200x test samples were then diluted 1:10 in cell culture medium to obtain 20x test samples. A 5 µl aliquot of 20x test samples was added staggered to cells with 90 µl of existing medium. Space was also allocated to titrate each of the compounds separately for use as reference controls. After a 12 hour pre-incubation of the test samples, A2-RL-line19F was added to the plate at a MOl of 0.5 and further incubated for 2 days at 37° C. in 5% CO ₂ .

Determination of activity against RSV.

The Renilla Luciferase Assay System (Promega, Cat # E2820) was used to measure anti-RSV activity. Analyzed tablets were prepared as described above. Luminescence was recorded using a Perkin Elmer Victor3V multi-mark counter.

Cell viability analysis.

CellTiter-Glo fluorescent cell viability assay (Promega, Cat # G7572) was used to measure cell viability. The CellTiter-Glo® Fluorescent Cell Viability Assay is a single-phase method for determining the number of viable cells in culture based on the amount of adenosine triphosphate (ATP) present, which signals the presence of metabolically active cells. The assay plates were prepared under the same conditions as for the RSV assay, except that no virus was added in the cell viability assay. A 100 µl aliquot of CellTiter-Glo reagent was added to each well and incubated at room temperature for 8 minutes. Luminescence was recorded using a Perkin Elmer Victor3V multi-mark counter.

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Data analysis.

Each experiment was performed at N=5 in terms of both anti-RSV activity and cell viability. The average percent inhibition of replicon values from 5 experiments was obtained and anti-RSV activity was analyzed using two drug interaction analysis models, isobologram analysis and/or Pritchard's model.

Analysis of the isobologram.

The effects of drug-drug combinations were assessed using a Levé additivity model in which experimental data were analyzed using CalcuSyn (Biosoft, Ferguson, MO), a computer program based on the Zhou-Talalay method. For each experimental combination, the value of the additivity index (CI) and the isobologram were calculated. CI values of <1, 1, and >1, respectively, indicate synergy, additive effect, and antagonism. Within the synergy category: CI<0.1 implies very strong synergy; CI 0.1-0.3 - strong synergy; CI 0.3-0.7 - synergy and CI 0.7-0.85 - moderate synergy. Isobologram analysis, which graphically represents the additive, synergistic and antagonistic effects of drugs, has also been used to model the interaction of antiviral activities. In this representation, the effective concentration (EC) value of one drug is plotted on one axis, and the corresponding EC value of the second drug is plotted on the second axis; the line connecting these two points is the amount of each drug in the combination that may be required to achieve an equivalent EU value, provided that their effects are additive.

Pritchard Model (Mac Synergy II).

MacSynergy II software was kindly provided by Dr. M. Pritchard (University of Michigan). This program allows a three-dimensional study of drug interactions at all data points obtained from a checkerboard combination of two inhibitors using the Bliss model of independent analysis of drug interactions. Confidence limits were determined from duplicated data. If the 95% confidence limits (CL) do not overlap the theoretical additive surface, then the interaction between the two drugs is substantially non-additive. The dimensions of synergy or antagonism can be defined and plotted in three dimensions and represent the relative amount of synergy or antagonism per change in the concentrations of the two drugs. The dimensions of synergy and antagonism are based on the Bliss model of independent drug interaction analysis, which assumes that both compounds act independently of each other on different targets. The set of calculated fractional responses faAB according to the Bliss model of independent analysis of drug interactions is calculated as faAB=faA+faB-faAxfaB, while faA and faB represent the fraction of possible responses, for example, % inhibition of compounds A and B, respectively, at amounts of dA and dB , and it describes the % inhibition of the combination of compounds A and B at the amount of (dA+dB). If faAB>faA+faB-faAxfaB, then Bliss synergy is obtained; if faAB<faA+faB-faAxfaB, then Bliss antagonism is obtained. The 95% synergy/antagonism measures are the sum of the differences between the observed inhibition and the 95% confidence limit in predicting faAB according to the Bliss independent drug interaction analysis model. In table. 1 shows dimensions and corresponding dimension descriptions for the results of an independent Bliss drug-drug interaction analysis. MacSynergy II was used for data analysis.

Table 1. Dimensional descriptions using MacSynergy II

Size (µM ² %) Size Description < 25 additivity 25-50 minor synergy 50-100 significant synergy > 100 strong synergy

Example 3 Test Combination Experiments for RSV.

In vitro combination studies with JNJ-53718678 (compound A) and various compounds with different mechanisms of action (compound B) were performed to evaluate their combined antiviral effect against RSV. Briefly, 9 concentrations of Compound A were combined with 6 or 7 concentrations of a specific Compound B. HeLa cells were infected with rgRSV224, a developed strain of RSV that encodes eGFP (Hallak L.K., Spillmann D., Collins P.L., Peeples M.E. Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection; Journal of Virology (2000), 74(22), 10508-13), in the presence of various concentrations of Compound A and Compound B alone or in combination.

Stock solutions of compounds were 5 mM in 100% DMSO with the following exceptions: ribavirin, 20 mM; A 60444, 10 mM; CR9501, 141 μM in PBS; palivizumab, 156 μM in PBS; MDT

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637, 100 μM in methanol. First, the compounds were serially diluted in 3-fold dilution series in DMSO; CR9501 and palivizumab were serially diluted in cell culture medium and MDT 637 in methanol. The concentration series was diluted 100-fold in cell culture medium to achieve a combined matrix. 10 μl of these compound solutions were transferred to black 384-well tissue culture-treated clear bottom plates. Then, 10 μl of HeLa cell suspension was added at a concentration of 150,000 cells per ml to achieve a concentration of 3,000 cells per well. Finally, 10 µl of the diluted virus stock was added to achieve an MOI of 1. The final DMSO concentration in all wells was 0.5% DMSO except for the MDT 637 experiment where it was 0.25% DMSO+0. 25% methanol. Each combination of compound concentrations was tested in 4 replicates on 3 separate plates for a total of 12 replicates. The plates were incubated for 3 days at 37°C in a humidified atmosphere at 5% CO ₂ . The GFP signal was measured with a PerkinElmer EnVision device using a 405 nm filter.

For statistical analysis, the observed GFP signal was normalized using viral controls (VC) and cell controls (CC) on the appropriate plate: virus) and VC was calculated as the median GFP for all control wells (no compound).

Synergy was assessed using the Harbron method (Statistics in Medicine, 2010, DOI:10.1002/sim3916). Using Leve's definition, two compounds are defined as being additive, synergistic, or antagonistic using the expression = 1, additivity di d'2 —--1- —— < 1, synergy > 1. antagonism, where d1 and d ₂ are doses of two compounds that in combination provide the effect y, and D _y ,1 and D _y , ₂ are doses of two compounds that provide the same effect y when administered as monotherapy. A 3PL (3-parameter logistic-logistic model) was applied to monotherapy dose-response data, where the baseline response rate was set at one.

Following Harbron's approach, the following models were fitted to the data: additive: assuming no interaction between the two compounds;

homogeneous: based on a constant interaction index τ in all combinations of doses;

linear A: based on the fact that the interaction index is linearly dependent on the log ₁₀ dose of compound A: τ=τ1+τ2 log ₁₀ (d _A );

separate A: based on the fact that the interaction index τ takes a separate value for each dose of compound A.

The last two models were also considered for compound B:

linear B: based on the fact that the interaction index τ linearly depends on the log ₁₀ dose of compound B: τ=τ1+τ2 log ₁₀ (d _B );

separate B: based on the fact that the interaction index τ takes a separate value for each dose of compound B.

A single model A was selected from all tested combinations based on the Akaike information criterion (AIC). Test results for combinations of Compound A with various Compounds B are shown below.

Combination: compound A and pulmicidal compound (N-(2-fluoro-6-methylphenyl)-6-(4-(5-methyl-2(7-oxa-2-azaspiro [3.5] nonan-2-yl)nicotinamido) benzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine2-carboxamide) as compound B.

Table 2 Normalized Data for Compound A in Combination with a Pulmonary Compound (Mean of 12 Replicates)

A=0 A=0.19 A=0.57 A=1.7 A=5.1 A=15 A=4 6 A=13 9 A=417 A=1250 v=o 0.94 0.96 0.92 0.78 0.56 0.43 0.36 0.20 0.11 0.06 B=0.058 0.99 0.99 0.93 0.81 0.56 0.39 0.28 0.17 0.08 0.04 B=0.17 0.99 0.99 0.91 0.74 0.51 0.37 0.23 0.16 0.08 0.04 B=0.52 1.04 0.96 0.87 0.70 0.45 0.31 0.23 0.14 0.07 0.04 B=1.6 0.91 0.87 0.75 0.46 0.27 0.18 0.11 0.06 0.04 0.02 B=4.7 0.20 0.14 0.12 0.06 0.03 0.01 0.01 -0.00 -0.00 -0.00 B=14 0.02 0.01 -0.00 -0.01 -0.01 -0.01 -0.02 -0.01 -0.01 -0.01 B=42 0.02 0.01 0.01 0.00 -0.00 -0.01 -0.01 -0.00 -0.00 -0.00

In table. 3 shows that the interaction index (τ) is estimated with 95% confidence intervals (CI) for a single A model. Provided that the degree of synergy varies depending on the doses of compound A, a significant degree of synergy is found at doses of 0.57 nM and above. In general, the degree of synergy increases with increasing dose levels, with a plateau from about 5.1 to 139 nM.

Table 3. Estimated Interaction Scores at 95% CI for Individual Model A. The interaction score at EC ₅₀ was approximated by linear interpolation of the estimates of τ

Dose (nM) Grade 95% CI τι 0.19 1.013 (0.955, 1.075) _τ2 0.57 0.93 (0.875, 0.989) τ ₃ 1.7 0.636 (0.601, 0.674) _τ4 5, 1 0.479 (0.449, 0.51) ₅ 15 0.484 (0.443, 0.528) _τ6 46 0.544 (0.479, 0.618) ₇ 139 0.51 (0.412, 0.632) _τ8 417 0.234 (0.198, 0.277) ₉ 1250 0.073 (0.06, 0.088) τ EU 50 9.79 0.481 (n.d., n. d . )

CLAIM

Claims (2)

1. Combination for the treatment or attenuation of respiratory syncytial virus (RSV) infection containing 3-({5-chloro-1-[3-(methylsulfonyl)propyl]-1H-indol-2yl}methyl)-1-( 2,2,2-trifluoroethyl)-1,3-dihydro-2H-imidazo[4,5-c]pyridin-2-one or a pharmaceutically acceptable salt thereof as compound A and M-(2-fluoro-6-methylphenyl )-6-(4-(5-methyl-2-(7-oxa-2azaspiro[3.5]nonan-2-yl)nicotinamido)benzoyl)-5,6-dihydro-4H-benzo[b]thieno[2, 3-b]azepine-2-carboxamide or a pharmaceutically acceptable salt thereof as compound B, where the amount of compound A corresponds to a concentration value of 0.19 to 1250 nM, and the amount of compound B corresponds to a concentration value of 0.058 to 42 nM. 2. Pharmaceutical composition with activity against RSV, containing a therapeutically effective amount of the combination according to claim 1 and a pharmaceutically acceptable carrier.