WO1986003412A1

WO1986003412A1 - Improvements relating to the treatment control and prevention of rhinovirus infections

Info

Publication number: WO1986003412A1
Application number: PCT/GB1985/000555
Authority: WO
Inventors: David Arthur John Tyrrell
Original assignee: Individual
Current assignee: Individual
Priority date: 1984-12-06
Filing date: 1985-12-06
Publication date: 1986-06-19
Anticipated expiration: 1987-06-06
Also published as: GB8530152D0; NO863165L; EP0207116A1; NO863165D0; ES549671A0; AU5234586A; GB2168608A; ES8700940A1

Abstract

The synergistic combination of a non-interferon antiviral substance active against rhinoviruses and an interferon for simultaneous, sequential or separate use in the treatment or prevention of rhinovirus infection.

Description

IMPROVEMENTS RELATING TO THE TREATMENT CONTROL AND PREVENTION OF RHINOVIRUS INFECTIONS

This invention relates to the treatment, control and prevention of viral respiratory infections and especially the common cold infections caused by rhinoviruses.

Attempts to produce antiviral substances useful in combatting respiratory infections have been pursued for many years with relatively limited success. Substances having powerful antiviral activity have indeed been discovered but the problem of achieving effective distribution of these drugs in the respiratory tract so that they reach the target cells has not yet been solved. This is equally true of the interferons which from the earliest time after their discovery in 1957 offered a prospect of controlling common cold and other respiratory infections and were subjected to various clinical trials. The inherent ability of the interferons to suppress colds and to be effective in prophylaxis when applied in sufficient amounts has undoubtedly been established and with increasing knowledge of the various species of interferon which exist, alpha, beta and gamma, antiviral activity against respiratory viruses has been confirmed with all known members of the alpha species and is likely to be similarly proven for beta and gamma species of interferon.

In relation to other viruses the possibility of achieving greater success in the control of virus infections by means of synergistic combinations of various agents has been considered and various experiments have been reported. In general the results have been less than clearcut and in some cases disappointing and even a contra-indication for therapy or prophylaxis.

It has now been found possible to obtain unexpected and remarkably high synergistic effects when interferons are combined with non-interferon antiviral agents.

Accordingly the present invention comprises the synergistic combination of a non-interferon antiviral substance active against rhinoviruses and an interferon for simultaneous, separate or sequential use in the treatment or prevention of- rhinovirus infection. Also included within the scope of the present invention is the use of a non-interferon antiviral substance active against rhinoviruses and an interferon for the manufacture of a synergistic medicament combination of the antiviral substance and the interferon for application in the treatment or prevention of a rhinovirus infection.

Additionally, there is included within the scope of the present invention a method of treating or preventing rhinovirus infection in which a non-interferon antiviral substance active against rhinoviruses and an interferon are administered to a patient whereby rhinovirus infection in the patient is treated or prevented by synergistic activity against the rhinovirus of the non-interferon activiral substance and the interferon.

Significant and in some cases exceptionally high levels of synergism have been obtained with human type alpha, beta and gamma interferons when combined with important non-interferon antiviral drugs which are at present still under evaluation as antiviral agents in their own right. It will be a relatively simple matter in the light of experimental data presented here to determine the circumstances in which synergism may result from the combination of any interferon and any antiviral agent active against rhino¬ viruses. This will appear clearly from the specific examples which follow. Example 1

This example shows the inhibition of replication of a rhino¬ virus type 2 by gamma interferon in combination with the drug enviroxime in WISH cells. The experimental results are presented in Table 1 which shows the antiviral activities of human type IFNγ and enviroxime when applied separately and the levels of these substances required to produce the same antiviral effect when applied in combination. The effects on virus yield are shown of progressive dilution of the active substances when applied separately or in combination. Part A of Table 1 shows the effect of progressively reducing IFNγ concentrations in the absence of - 3 - enviroxime whereas part B reproduces this for enviroxime in the absence of IFNγ. In Part C of Table 1 progressive dilution of each of the components of a mixture of the two substances is shown. Part A of Table 1 shows a hundred-fold reduction in virus yield (from 6.5 to 4.5) in the presence of two units/ml of IFNγ. Part B shows a similar reduction in the presence of between 0.03 and 0.15 mg/ml of enviroxime (2-amino-1-(isopropylsulphonyl)-6- (α-hydroxyimiobenzyl) benzimidazole; equal proportion of syn and anti isomers) . Part C of Table 1 shows that the same reduction is produced by a mixture containing 0.03 units of IFNγ and about 0.0003 (deduced) mg of enviroxime. This dramatic effect is also shown by the two right-hand columns which give the dilution reductions from stock solutions of the two components.

Table 1 also shows the Combination Index (CI) which is calculated as described by Spector et al (1982), Am. J. Med. J__ suppl 1A, 36-39, from the equation (Drug 1) (Drug 2)/[Drug 1 + Drug 2) (VC)] . (Drug 1), (Drug 2), (Drug 1 + Drug 2) and (VC) are yield of treated virus with (Drug 1), (Drug 2) combination of Drug 1 and Drug 2 and untreated virus control respectively. Taking the natural log of the above equation yield the CI. If CI - In 1 = 0 the interaction between the drugs is additive, if CI > 0 the interaction is syner¬ gistic and, if CI < 0 the interaction between drugs is antagonistic. In this case Drug 1 is HuIFNγ and Drug 2 enviroxime.

TABLE 1

Inhibition of replication of RV„ by IFNγ in combination with enviroxime in WISH cells

Ratio of virus yield

IFNγ Enviroxime Virus yield Fold reduction of untreated/ Combination Index

IU/ml μg/ml Log 10 IFNγ Enviroxime treated (NA *•*• Not applicable)

0 0 6.5 1 NA

8 0 1.5 100,000 NA

4 0 4 2 320 NA

2 0 4.5 4 100 NA

1 0 5 8 32 NA I

0.5 0 6 16 3 NA

0.25 0 6.5 32 1 NA

0 0.1 25 1 .5 100, 000 NA 0 0.06 2 2 32, 000 NA 0 0.03 4 4 320 NA 0 0.015 5 8 32 NA 0 0.0075 6.5 16 1 NA

TABLE 1 continued

Inhibition of replication of RV₂ by IFNγ in combination with enviroxime in WISH cells

Rates of virus yield

IFNγ Enviroxime Virus yield Fold reduction of untreated/

IU/ml μg/ml Log 10 IFNγ Enviroxime treated Combination Index

0.25 0.0015 1.5 32 32 100,000 11.5

0.125 0.0008 1.5 64 64 100,000 11.5 ^•

0.06 0.0004 1.5 128 128 100,000 11.5

0.06 0.0002 3 128 256 32,000 8.0

0.06 0.0001 5 128 512 32 3.4

0.03 0.0004 3.5 256 128 1,000 6.9 e

0.03 0.0002 5.5 256 256 10 2.3

0.01 0.0001 6 512 512 3 1.1

0.007 0.0001 6.5 1,024 512 1 0

0.01 0.00008 6.5 512 1,024 1 0

IFN was added 24 hours before virus. Enviroxime was added with virus. The cultures were inoculated with moi 10 TCID__ of^'RVg, harvested after 24 hours and then titrated. Recombinant human IFNγ was E.coli-derived (D0002) with a titre of 6.8 x 10⁷ IU/ml. WISH cells were grown at 37°C in MEM(Gibco) supplemented with 10% foetal bovine serum (FBS), 1% glutamine, penicillin 100 unit/ml and streptomycin 0.1 mg/ml. A laboratory passaged strain of RV_ was grown in Ohio HeLa cell monolayers maintained in BME supplemented with 2% FCS. Cultures were harvested at full cytopathic effect (CPE), frozen and thawed, clarified by centrifugation and the supernatant was stored at -70C.

Example 2

This example demonstrates the inhibition in WISH cells (grown as described in Example 1) of a rhinovirus type 9 (grown and harvested as for RV in Example 1) (100 TCID_₀/ml) by human type gamma interferon in combination with each of the three antiviral drugs enviroxime, ,6-dichloroflavan (DCF) and a chalcone (4'-ethoxy- 2^,-hydroxy-4,6^,-dimethoxy-chalcone. The results are presented in Table 2. In these experiments serial dilutions of both drugs were made, each in the presence of the other, in the form of a chequer board on a microtitre tray. The numbers in the first two columns indicate the end points of one drug in the presence of the indicated concentration of the other, and again it is seen that over a wide range of concentrations the amount of both drugs which is required is greatly reduced compared with that when they are acting alone. The size of these reductions is shown directly in columns 3 and 4 and the combined inhibitory effect is also expressed as the FIC index, as shown in the last column. The FIC index = (MIC of drug A in combination)/(MIC of drug A alone) + (MIC of drug B in combination)/(MIC of drug B alone). The interpretation of the index is as follows: FIC index <0.5: significant synergism; FIC index 0.5-0.9: suggestive of synergism; FIC index _*A : Effects are additive; FIC index 1.1-1.9: Indifference or^' partial antagonism; FIC index >2: Antagonism.

The results obtained in this example are also shown graphi¬ cally by means of Figure 1, sections (a) , (b) and (c) of which relate to the chalcone, dichloroflavan and enviroxime, respectively.

The broken line shows the merely additive effect on the inhibition of virus replication to be expected from the interferon and non-interferon antiviral and the unbroken line the actual synergistic effect.

A similar graphical presentation is given in Figure 2 which shows the inhibition of a rhinovirus type 9 (100 TCID _n/ml) by enviroxime in combination in turn with human type alpha, beta and a hybrid beta-like interferon, (hybrid IFNβ x 410: normal HuIFNβ in which some of the N-terminal sequence has been removed and replaced with HuIFNα- sequence) 90% pure and containing 5 x 10 IU/ml) the results being presented, respectively, in sections (a), (b) and (c) of the Figure.

Figure 3 shows the counterpart experiments to those of Figure 1 but using a rhinovirus type 2 (100 TCID-₀/ml), sections (a), (b) and (c) of the Figure relating, respectively, to the chalcone, dichloroflavan and enviroxime.

TABLϊ5 2

Inhibition of (100 TCID_5Q/inl) RV₉ IFNγ in combination with

Enviroxime. , DCF or Chalcone

IFNγ Drug/yg/ml 2-fold reduction of

IU/ml Enviroxime IFNγ Enviroxime FIC index

4 0 - 0 1

0 0.125 0 - 1

0.1 0.001 32 64 0.033

0.06 0.001 64 64 0.023

0.03 0.003 128 32 0.031

0.01 0.0075 256 16 0.062

0.01 0.01 256 8 0.082

IFNγ DCF IFNγ DCF

0 0.06 0 - 1

0.01 0.001 32 32 0.019

0.06 0.001 64 32 0.032

0.03 0.001 128 32 0.024

0.03 0.003 128 16 0.057

0.01 0.007 256 8 0.013

IFNγ Chalcone IFNγ Chalcone

0 0.06 0 - 1

■0.1 0.001 32 32 0.0416

0.06 0.001 64 32 0.0316

0.03 0.003 128 16 0.057

0.01 0.007 256 8 0.127

0.01 0.01 256 4 0.169 In the above Examples enviroxime, DCF, chalcone Ro-09-0410 HuIFN-γ, HuIFNα-2, HuIFNβ and hybrid HuIFNβ x 401 as appropriate, were first tested individually to determine their MIC and studied in binary combinations by chequerboard titration. Two-fold serial dilutions of one drug starting at 2 MIC were made and added in unit volume to rows of wells in microtitre plates containing confluent monolayers of WISH cells. Similar dilutions of a second drug were added to the columns of wells in order to produce all possible combinations within the chosen range of concentrations.

When IFNs were used the plates were incubated overnight, medium was removed and the second drug and virus were added.

To each well was added 100 TCID_₀ of RV. or RV_ and the plates were incubated at 33 C. The end point was that at which CPE was completely prevented. All experiments were run in triplicate and usually repeated two or three times. The results were usually identical or differed at most by two-fold (i.e. one dilution) . All tests included controls to detect activity of the virus inoculum and any cytotoxity resulting from the antiviral.

Certain drug combinations were tested in organ cultures of human embryonic nasal epithelium and human embryonic trachea and the results were assessed by virus yield. The method for such cultures was based on that of Tyrrell and Blamire, 1967 Br. J. Exp. Path_jK2), 217-227; specimens were obtained from The Tissue

Bank, Institute of Cancer Research, The Royal Marsden Hospital,

2 London, UK. One piece (about 1 cm ) of nasal epithelium with the underlying cartilage of septum or turbinate or one or two rings of human embryonic trachea were maintained in 1 ml maintenance media MEM, supplemented with 0.2% BPA and 10 U/ml penicillin and 0.1 mg/ml streptomycin.

All cultures were incubated in roller tubes at 33 C and the organs were examined for ciliary activity and only those showing good ciliary activity 24 hours after preparation of the culture were used. HuIFN was added 24 hours before virus. Enviroxime was added at the same time as virus. The medium was then harvested daily for up to five days and replaced by fresh medium containing the same concentration of enviroxime but no IFN. The organs were examined daily for ciliary activity and the harvested fluids titrated for virus in Ohio HeLa cells.

The results are summarised in Table 3.

TABLE 3

Synergistic effect of HuIFNγ and enviroxime on yield of RV„ in organ cultures of human embryonic nasal or tracheal epithelium

Log_*ιn virus yield from indicated culture on day

Concentration of drug in medium

Nasal Tracheal HuIFNγ Enviroxime

U/ml μg/ml

1 2 3 4 5 1 2 3 4 5

0 0 4.8 6.5 5.1 5.25 5 0 4.5 5.5 4.7 4.6

0.3 0 4.0 6.0 6.1 6.5 - 0 4.5 4.5 5.0 -

2.5 0 3.5 3.75 3.2 4.0 4 0 3 3.2 4.2 4.5

10 0 0 2.5 1.0 3.5 4 0 0.5 1.8 4.2 4.8

40 0 0 0.5 0.5 1.0 1.5 0 0 0.6 2.5 3

160 0 0 0 0 0 0 0 0 0 0 0

0 0.004 0 5.5 4.5 5.7 -

0 0.125 0 0 0.5 ^' 5.7 -

0.3 0.004 0 0 0 0 - 0 0 0 0 -

0.15 0.002 0 0 0 0 - 0 0.25 0 0.3 -

0.03 0.002 2 3.7 4.5 5.0 - 0 3.4 4.5 5.0 -

Non-interferon antiviral substances which are particularly suitable for carrying the invention into effect are organic compounds such as the chalcones and enviroximes which contain no heavy metals and which have a low degree of mammalian toxicity relative to the dose synergistically effective with the interferon to prevent or treat rhinovirus infection. The cytotoxic concen¬ tration of such substances in vitro is generally at least one and preferably at least three orders of magnitude greater than the effective concentration of the substance alone.

It will be appreciated that in the practical application of the invention the interferon and the other antiviral substance will be administered at about the same time either separately or in compounded form and in proportions calculated to achieve a pronounced synergistic effect. They will be administered to the patient by any of the known methods for achieving protection against the common cold e.g. by nasal drops or sprays or powders although orally administrable forms are also envisaged.

In particular, the combination of the present invention may be presented in the form of a pack which comprises two phases for simultaneous, separate or sequential oral or intranasal application,^" the said phase being isolated from one another, one phase comprising a non-interferon antiviral substance active against rhinoviruses together with a vehicle and the other phase comprising an interferon together with a vehicle, the construction of a container housing the phases or the arrangement of 'the phases relative to one another being such as to facilitate and encourage either simultaneous application of the phases or the application of first one of said phases and then the other, whereby in normal use of the contents of the pack a dose of the non-interferon antiviral substance and the interferon will be applied which is synergistically effective to treat or prevent rhinovirus infection. The expression "normal use" is intended to imply natural and reasonable use of the pack and contents according to the invention, having regard to any accompany¬ ing directions or indications and being used with the object of achieving simultaneous separate or sequential application of the two phases. It is envisaged that in practice an inteferon such as INFα or INFB will be applied, suitably intranasally, in a dose ranging from 0.02-50 MU per day and the non-interferon antiviral, e.g. enviroxime, in a dose ranging from 0.008-5 mg per day. Typically the doses of interferon and non-interferon antiviral are divided between nostrils.

For prevention of infection, somewhat lower doses of interferon (e.g. INFα or 6) and antiviral (e.g. enviroxime) can be used than for treatment and a daily dose of interferon in the range 0.02-3 MU in combination with a daily dose of antiviral in the range 0.008-1.40 mg is envisaged. The daily dosage of inter¬ feron is usually divided into 2 or 3 applications and of the antiviral into 3 to 5 applications.

When an infection is already present, a daily dose of inter¬ feron in the range 0.15-50 MU spread over 3 to 10 applications and of antiviral in the range 0.20 mg-5 mg spread over 4 to 10 appli¬ cations is envisaged. Treatment is normally discontinued after 2 or 3 days.

In clinical applications of particular interest for prophy¬ laxis, interferon (e.g. INFα with a potency of 2.5 MU/ml) is administered at a daily dosage 1.50 MU generally spread over 3 applications and typically to a total dosage 6.5 MU, the non- interferon antiviral (e.g. enviroxime) being administered at a daily dosage 1.12 mg which is itself spread over 4 applications and typical to a total dosage 6.72 mg.

Claims

1. The synergistic combination of a non-interferon antiviral substance active against rhinoviruses and an interferon for simultaneous, sequential or separate use in the treatment or prevention of rhinovirus infection.

2. The combination according to Claim^'1, in the form of a pack which comprises two phases for simultaneous, separate or sequential oral or intranasal application, the said phases being isolated from one another, one phase comprising a non-interferon antiviral substance active against rhinovisus together with a vehicle and the other phase comprising an interferon together with a vehicle, the construction of a container housing the phases or the arrange¬ ment of the phases relative to one another being such as to facilitate and encourage either simultaneous application of the phases or the application of first one of said phases and then the other, whereby in normal use of the contents of the pack a dose of the non-interferon antiviral substance and the interferon will be applied which is synergistically effective to treat or prevent rhinovirus infection.

3. The combination according to either preceding claim, in which the interferon is α-interferon.

4. The combination according to any preceding claim, in which the non-interferon antiviral substance is an organic compound.

5. The combination according to any preceding claim, in which the non-interferon antiviral substance contains no heavy metals.

6. The combination according to any preceding claim, in which the non-interferon antiviral substance is such that the cytotoxic concentration thereof _in vitro is at least one order of magnitude greater than the effective concentration of the substance when administered alone.

7. The combination according to any preceding claim, in which the non-interferon antiviral substance is an enviroxime.

8. The combination according to Claim 7, in which the enviroxime is 2-amino-1-(isopropylsulphonyl)-6-(α-hydroxyiminobenzyl) benzimidazole.

9. The combination according to Claim 8, in which the enviroxime comprises both syn and anti isomers.

10. The combination according to any preceding claim, in which the non-interferon antiviral substance is a chalcone or 4,6-dichloroflavan.

11. The combination according to Claim 10, in which the ^lethoxy-2^l- hydroxy-4,6'-dimethoxy chalcone.

12. The use of a non-interferon antiviral substance active against rhinoviruses and of interferon for the manufacture of a synergistic medicament combination of the antiviral substance and the interferon according to any preceding claim, for application in the treatment or prevention of a rhinovirus infection.

13. A method of treating or preventing rhinovirus infection in which a non-interferon antiviral substance active against rhino¬ viruses and an interferon are administered to a patient whereby rhinovirus infection in the patient is treated or prevented by synergistic activity against the rhinovirus of the non-interferon antiviral substance and the interferon.