WO1986000901A1

WO1986000901A1 - Immunoregulatory and anti-viral compound

Info

Publication number: WO1986000901A1
Application number: PCT/US1984/001168
Authority: WO
Inventors: Charles O. Gauntt; Kelvin K. Ogilvie
Original assignee: Gauntt Charles O; Ogilvie Kelvin K
Priority date: 1984-07-20
Filing date: 1984-07-20
Publication date: 1986-02-13
Also published as: EP0190123A1

Abstract

The compound 6-chloro-9- AD AD2-benzyloxy-1-(benzyloxy-methyl) ethoxy BDmethyl BD guanine of formula (I) exerts an immunoregulatory effect on mammals, by apparently reducing the cytotoxic activity of splenic lymphocytes and altering the T suppressor/T cytotoxic lymphocyte and NK cell sub-populations.

Description

IMMUNOREGULATORY AND ANTI-VIRAL COMPOUND

This invention relates to pharmaceuticals, and more specifically to a compound having anti-viral and immuno regulatory activities.

The lymphocyte cells of the mammalian body are of two general groups, namely, T-cells, which are thyπvus derived and mediate hypersensitivity, and B-cells, which derive from bone marrow and are responsible for antibody production. B-cells invoke an antibody response. They can be further sub-divided into cytotoxic T-lymphocytes, which kill foreign cells, and suppressor T-lymphocytes which modu¬ late or suppress the activity of the cytotoxic T-lympho¬ cytes. The natural immune system may also involve NK (natu¬ ral killer) cells, which are also cytotoxic but are distinct from T-lymphocytes.

There are situations where it would be desirable to be able to alter or regulate the natural and/or specific immune system of the body. For example, there are instances where the cytotoxic T-lymphocytes recognize cells as for¬ eign, and hence combat them, to an undesirable extent. For example, childhood infections of coxsackievirus can cause heart muscle damage, leading to myocarditis and lesions in the myocardium. The cells lining the blood vessels on the heart muscle cells can become altered, as a consequence of the viral infection. These altered cells are recognized as foreign, by the natural immune system, so that they are combatted by cytotoxic T-lymphocyte and perhaps NK cells, resulting in Dossible destruction of the heart muscle. The rejection of transplated organs by the living body is, of course, commonly caused by the action of the immune system.

O PI Previous attempts to modify the body's immune system have involved administration to the body of proteins (e.g. interferons) , bacterial excretions, T-cell growth factor (TCGF) etc. Levamisole, a phenyl-substituted imidazothiazole, is administered as an immunopotentiator, but markedly exacerbates coxsac ie irus-induced myocarditis.

The present invention provides a substituted guanine compound which exhibits im unoregulatory activity. The compound of the invention has the formula:

6-chloro-9-[ [2-benzyloxy-l- (benzyloxymethyl)-ethoxy]methyl]- guanine.

Studies conducted on this compound iτ vivo on laboratory mice have indicated that the compound exerts antimyocarditic activity by immunomodulatory mechanisms which appear to involve T suppressor/T cytotoxic lymphocyte and NK cell subpopulations. The compound has a degree of antiviral activity against coxsackievirus 3, which may or may not be distinct from its immunoregulatory activity.

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T e compoun may e prepare rom -c oroguan ne, a commercially available compound, by reaction with l,3-dibenzyloxy-2-chloromethoxy propane (preparable by reaction of l,3-dibenzyl-2-hydroxy propane and paraformaldehyde with hydrochloric acid) , 6-chloroguanine being suitably protected e.g. with silyl groups. The reagent l,3₇dibenzyl-2-hydroxy propane can be made by reaction of l,3-dichloro-2-propanol with sodium benzylate.

For use as an anti-viral drug or as an immunoregulant, the compound is suitably administered interthecally, from a suitable sterile solution. Effective unit doses for administration are suitably from about 0.1-100 mg of compound per kg mammal body weight, preferably from about 0.1 to about 20 mg per kg, on the basis of a dosage administered 2-4 times per day. Injectable solutions may be made up in distilled water or saline, e.g. isotonic solutions, and optionally buffered with phosphate, of concentration 1000-5000 mg per ml. Alternatively, the drug may be dissolved in dimethyl sulfoxide, diluted in MEM and calf serum, ready for injection.

Oral administration, as tablets or capsules along with a suitable carrier at the aforementioned approximate dosages, is also within the scope of the present invention.

The immunoregulatory activity of the compound was demonstrated by in vivo testing in laboratory mice. Groups of mice infected with coxsackievirus B3 and injected with the com¬ pound showed fewer lesions, as compared with similar groups of mice infected with coxsackievirus B3 but not injected with the compound. In_ vitro testing of the compound has shown that it has some antiviral activity, but not sufficient to account for its effects against coasackievirus-induced myocarditic lesions in vivo. Tests conducted on the groups of mice, after sacrifice, have indicated that the compound does not noticeably increase the production of interferons from the cells of mammals-, to account for its activity. In fact, it is found that the administration of the compound has the effect of reducing the number of cytotoxic T-cells whilst stimulating the activity of

NK cells and possibly T-suppressor cells.

The compound is accordingly useful in treatment or alleviation of conditions in living mammals which are adversely affected by the cytotoxic action of cytotoxic T-cells of the mammalian immune system. The acceptance and toleration of coxsackievirus variated cells is an example of such a condition, but the use of the compound of the present invention is not restricted thereto.

The invention is further illustrated in the following specific examples.

Example 1 - Synthesis

6-chloroguanine (200 g, 0.11 moles) 1,1,1,3,3,3- hexa ethyldisilazane (HMDS) (200 ml) and ammonium sulfate (300 mg) were combined and brought to reflux with stirring. After 1.75 h more ammonium sulfate was added. After a further hour the mixture was clear. The excess HMDS was distilled under reduced pressure to yield a solid. The solid was dissolved in

. ι -~--- Λ ^'ζ O PI dry benzene (200ml) , mercuric cyanide (34 g,0.135 moles) was added and 1,3-dibenzyloxy-2-chloromethoxy propane (0.110 moles) was added. The stirred mixture was brought to reflux. Reflux was continued for 5 hours. The mixture was cooled and filtered and the residue was washed with benzene. The filtrate and washings were combined and evaporated under reduced pressure to yield a syrup. The syrup was dissolved in methylene chloride

(400 ml) and washed successively with water (400 ml) , 30% aqueous potassium iodide (4 x 200 ml) , water (200 ml) and aqueous bicarbonate (2 x 200 ml) . The methylene chloride phase was dried with sodium sulfate and evaporated to yield 54 g of syrup. The rest of the syrup was dissolved in chloroform (50 ml) and applied to a prep, silica column (19.5 x 6.3 cm) . The column was eluted with 1% methanol in chloroform and fractions were collected. The desired material, namely 9-[ [2-benzyloxy-l-

(benzyloxoymethyl)ethox ]meth ll -6-chloroquanine, of formula:

O ^cμ-- °^~CH~

was isolated fom the appropriate fractions issuing from the column.

OMH Example 2 - In vitro tests

A my ¹carditic strain of coxsackievirus B3 (CVB3m) was propagated and plaque-assayed in HeLa cells by the method describe by Trousdale et al, "Biochem. Biophys. Res. Comm.",

76:368-375 (1977) . An assay to establish the effective dose of the compound of Example 1 that reduces plaque number by 50%

(ED--_n) was performed as follows. Duplicate confluent monolayers of cells were challenged with 50-100 pfu of a myocarditic variant of the virus CVB3m (see Gauntt et al, "J.

Med. Virol." 3_:207-220, 1979) . After 45 minutes at 37°C for adsorption, a nutrient-agar overlay ⁽MEM plus 1% Bactoagar, from Difco Labs, Detroit) with or without various concentrations of the compound of Example 1 was added. After 24-36 hours of incubation, the plaques were counted. Plaque number at the highest non-toxic level tested (20 ug/ml) in HeLa cells were reduced by 70-84%.

OMPI ^■me results are given in Table I below:

TABLE I

Concentration No. of plaques Experiment No. (ug/ml) (mean/2 dishes) % Reduction

10 21 79

5 59 41

1 1 82 19

0.1 111 0

0 101 -

20 21 79

15 43 57

2 10 49 50

5 88 11

2 95 4

1 64 36

0 99 -

20 14 84

15 34 61

3 10 34 62

5 59 32

2 67 23

1 82 6

0 87- -

The ED,.,, values calculated for experiments 1-3 respectively were 6, 11 and 7 ug/ml. Based on results from 4 experiments, the ED,, was 8.5 — 1.1 ug/ml.

The compound was cytotoxic to 25-75% of the cells at 25 ug/ml but was not cytotoxic at a level of 20 ug/ml after 48 hours of incubation. Cells exhibiting cytotoxicity-were rounded up and/or detached. In neonatal mouse skin fibroblasts, toxicity was evidenced by slow growth and elongation of the cells. The drug was toxic to 5-25% of the fibr-oblasts at 15 ug/ml after 48 hours.

OMP The fibroblasts used in this and other examples reported herein were prepared and cultured from CD-I or BALB/c neonatal mice born to breeder mice maintained in controlled animal laboratory facilities.'

The effect of the compound of Example 1 on virus yields in HeLa and neonatal skin fibroblast cultures was assessed in confluent monolayer cultures inoculated with 50 pfu/cell of

CVB3m. Virus was allowed to absorb for 45-60 minutes and after extensive washing, virus growth medium (MEM containing 1% fetal bovine serum) containing 10,5 or 1 ug/ml of the compound was added to duplicate cultures. After 40 hours incubation at 37°C the cultures were harvested, frozen and thawed three times and the lysates assayed for virus by the plaque πw.thod. The results are shown in Table II below.

TABLE II

% Control Yield

Cell Concentration Virus Yield Virus Yield (Drug) _o Type ug/ml PFU/ml Virus Yield (No Drug)

HeLa 10 1.04 X lo -i 57

1.46 X 10? 80

1 1.78 X 10⁷ 98

None 1.82 X 10⁷

CD-I neonatal skin fibroblas ;ttss 10 8.8 X 10² 2

5 7.2 X 10³ 18

1 1.78 X 10⁴ 45

None 4.0 X 10⁴

BALB/c neonataill 10 6.3 X 103 8

5 1.3 X 10⁴ 16

1 9.7 X 10⁴ 116

None 8.35 X 10⁴

OMPI These results indicate that the drug of Example 1 exhibits greater antiviral effect on virus yields in mouse neonatal skin fibroblasts than in HeLa cells.

Example 3 - In vivo tests

The effect of the compound on CVB3-induced myocarditis was examined using male mice. The compound for administration was prepared by dissolving the powder in dimethyl sulfoxide, diluting in MEM with 5% calf serum to 2000 ug/ml and storing at -20°C. Male mice in groups of 3-5 were injected intraperitoneally with 0.16 or 4.0 g/kg body weight of the compound, 2 1/2 - 4 hours prior to virus inoculation. The mice were then inoculated with 10 _. or 10 plaque-forming units of

CVB3m. Group^rs of mice inoculated with MEM only or compound only were included in these experiments as controls. Mice were sacrificed at 8 days post-inoculation.

Heart tissue from the sacrificed animals was examined for lesions, and assayed for virus. Lesions were counted as described in the aforementioned Gauntt et al paper. Focal area consisting of mononuclear leukocytes and myocytes undergoing necrosis were counted in 2-4 coronal sections per heart, taken one-third the distance from the apex of the heart. Portions of hearts were fixed in phosphate-buffered 10% formalin solution, pH 7.4, the sections were stained with hematoxylin and eosin and examined for lesions.

The heart tissues were also assayed for virus. The procedure for disruption of the heart tissues, and other cells,

to effect release of virus was as described in Gauntt et al,

"Infect. Immun." 3^:851-864, 1983 and elsewhere. Virus titers in heart tissues were measured on the apical l/3rd tip of each heart. Virus was assayed by the plaque method.

Serum from retroorbital sinus bleedings was assayed for antibody by a standard plaque reduction assay, described by Gauntt et al, "J. Med. Virol." 3-207-220., 1979. The reciprocal of the dilution at which 90% or greater reduction in plaque number of 1000 plaque-forming units (PFU) of CVB3 occurred was considered the antibody titer.

In thirteen separate experiments performed, the drug of

Examp^rle 1 ameliorated CVB3m-induced mycocarditis in 6 different species of mice. An experiment involving a group of adult Z mice qave a somewhat anomolous result, in that the lower dose of drug significantly reduced the extent of myocarditis induced by CVB3m, whereas the. higher dose increased the severity of myocarditis in these mice.

The results of the virus titers on the heart tissues ' showed that the drug did not suppress virus replication _i_n vivo in heart tissues, as titers of virus were either higher than, or not significantly different from, titers in the control group of mice. Again, the group of adult Z mice gave an anomolous result, showing a high virus titer at high drug dose.

Sera pooled from mice in four experiments or individually assayed in two experiments for anti-CVB3 neutralizing antibody titers were not significantly different in drug-treated virus-inoculated mice versus virus-inoculated mice

indicating that the humoral immune response was not depressed by the drug.

Interferon assays were also conducted, on sera from the mice, using vesicular stomatitis virus (VSV) as described in the aforementioned Gaunt et al paper from "J. Med. Virol.", 1979.

One unit of interferon reduces plaque formation of 90-120 pfu of

VSV by 50%, and the titer of interferon in a sample is a reciprocal of the dilution of a sample at end point. Interferon titers in sera pooled from 3 mice per sample, determined at 3, 5 and 7 days post injection, were not_ι significantly different from control samples, indicating that the antimyocarditic activity of the druq is not attributable to induction of interferon.

Example 4 - Effect on T-lymphocytes in splenic populations

Flow icrofluorometry was used to determine the proportion of T-cells in splenic lymphocyte populations pooled from BALB/c strain mice, 3-4 animals per group, in the presence and in the absence of the drug. Leukocytes were harvested from spleen by the method described by Gudvanqen et al, "Infect. Immun.", 41; 1157-1165. (1983) . The splenic lymphocytes were incubated for two one-hour periods at 37°C in complete RPMI 1640 (HEPES buffer at 20mM fetal bovine serum at 10%, gentamycin at 50 ug/ml and 2-mercaptoethanol at 5 x 10 M) to remove adherent cells. The cells were then harvested by centrifugation at 350 x g, washed twice in and taken up in HBSS containing 3% fetal bovine serum and 0.1% sodium nitride to 20 x 10 cell in 1 ml. Fifty icroliters of hybrido a supernatant fluid

containing anti-B or anti-T eel monoclonal antibody was added and incubation at 0°C on ice carried out for 30 minutes. After two washes with the latter buffer, the cells were incubated with a fluorescein-conjugated SLJ anti-rat kappa chain monoclonal antibody for 30 minutes on ice, washed twice in the previous buffer and resuspended in 0.5 ml. These cell preparations were then analyzed by flow microfluorometry (FMF) on a fluorescence-activated cell sorter (FACS IV, Becton Dickinson, Mountain View, CA) as modified by Kung e_t a_l Immunol. Rev. , 69 , 51-68 (1982) . Conditions of analysis were as reported by Sharrow e_t al, J. Immunol. 125: 2263-2268 (1968) . Histograms of fluorescence distribution were obtained by analyzing 20,000 cells per sample. Percentage of cells positive for a given surface marker were obtained after subtraction of control- values. Cells stained with fluoresceinated (SLJ) anti-rat IgG served as controls for cells indirectly stained with a monoclonal antibody followed by fluoresceinated SLJ anti-rat IgG.

Using monoclonal antibodies and analyzing 20,000 cells per group, there was found an increase in the proportion of cells staining positive for Thy 1 antigen in drug-treated, virus-inoculated mice, compared to normal mice. After corrections for proportion of positive cells staining with only SLJ anti-rat IgG, the proportions of positive cells staining with onoclonals directed against mouse IgM, Thy 1, Lyt i or Lyt 2 surface antigens and fluorescein-tagged SLJ anti-rat IgG were calculated in two experiments. An increase in proportion of Thy 1-bearing splenic lymphocytes was observed from the

O PI druq-treated, CVB3m-inoculated mice, compared with similar cell populations from the other three groups of mice.

An increase in proportion of Lyt2 but not Lytl antigen-positive cells was also found in splenic lymphocytes from drug-treated, CVB3m-inoculated mice. Decreases in proportions of IgM-positive B cells were observed in both virus-inoculated and drug-treated virus-inoculated groups compared to mice in normal or drug-treated groups.

Cytotoxic reactivities of splenic lymphocytes from the four groups of animals were assessed on normal and

CVB3m-inoculated murine fibroblast targ³et cells. The results showed that splenic lymphocytes from drug-treated

CVB3 -inoculated animals with reduced myocarditis, have reduced reactivity against infected target cells, when compared with lymphocytes from virus-inoculated controls. An increase in reactivity of T-lymphocyte-depleted splenic cells from drug-treated, CVB3 -inoculated mice suggested the presence of NK cells, as well as data from all three experiments which showed that splenic cells from drug-treated mice had reactivity against infected target cells.

Cells from the four groups were assayed for NK cells reactivity -against YAC-1 cells (obtained from the American type culture collection, Rockville,- Maryland, and cultured in complete RPMI 1640 medium supplemented w^'ith 10% fetal bovine serum, 50 ug/ml gentamycin sulphate, 100 mM sodium piruvate, 2-mercaptoethanol and .-glutamine) . -Cells from mice given the drug and inoculated with CVB3 were slightly higher in

reactivity against YAC-1 target cells than cells than the other three groups which exhibited lower similar reactivities. These data suggest that the drug enhances NK cell activity in infected mice.

Thus, administration of the compound of the present in_¬ vention to adolescent and adult mice of several strains effects a reduction in the number of lesions induced by CVB3 . Cytotoxic T-lymphocyte reactivities ±n vitro were reduced in splenic pop¬ ulations taken from compound-treated, CVB3 inoculated mice, compared to splenic lymphocytes from CVB3 inoculated mice, indicating that the compound according to the invention may ameliorate the coxsackievirus-induced myocarditis by altering . the in vivo activity of a subset of lymphocytes that are con-- sidered part of the mechanism of pathology. The compound also shows a stimulating effect on NK cell activity in CVB3 inoculated mice. An increase in the proportion of splenic T-lymphocytes bearing Thyl and Lyt2 surface markers was effected by the compound of the invention in CVB3 -inoculated animals.

Accordingly, the present invention provides a process for alleviating coxsackievirus-induced myocarditis in mammals, which comprises administering to the mammal an effective amount of 6-chloro-9-[[2-benzyloxy-l-(benzyloxymethyl) ethoxy] methyl] guanine.

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Claims

CLAIMS :

1. 6-chloro-9-[ [2-benzyloxy-l- (benzyloxymethyl) ethoxy] - methyl]guanine.

2. A pharmaceutical composition useful in treatment of coxsackievirus infections in mammals and myocarditic after-effects thereof, comprising the compound of claim 1 in admixture with a pharmaceutically acceptable carrier.

3. The composition of claim 2 in injectable, solution form.

4. The use of the compound of claim 1 in modifying the immune response system of a mammal.

5. A process of modifying the immune response system of a mammal, which comprises administering to the mammal an effective amount of the compound of claim 1.

6. A process of alleviating coxsackievirus-induced myocarditis in a mammal, which comprises administering to the mammal an effective amount of the compound of claim 1.

OMH