Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D473/00—Heterocyclic compounds containing purine ring systems

Definitions

• 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 thy ⁇ vus 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.
• cytotoxic T-lymphocytes recognize cells as for ⁇ eign, and hence combat them, to an undesirable extent.
• 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.
• the present invention provides a substituted guanine compound which exhibits im unoregulatory activity.
• the compound of the invention has the formula:
• 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.
• 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,3 7 dibenzyl-2-hydroxy propane can be made by reaction of l,3-dichloro-2-propanol with sodium benzylate.
• 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.
• 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 are derived from 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.
• 6-chloroguanine 200 g, 0.11 moles
• 1,1,1,3,3,3- hexa ethyldisilazane (HMDS) 200 ml
• ammonium sulfate 300 mg
• 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.
• 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.
• 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.'
• Example 1 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 ⁇ w.thod. The results are shown in Table II below.
• 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
• 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, were also assayed for virus.
• 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.
• 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
• VSV vesicular stomatitis virus
• One unit of interferon reduces plaque formation of 90-120 pfu of
• VSV by 50%
• 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 ⁇ significantly different from control samples, indicating that the antimyocarditic activity of the druq is not attributable to induction of interferon.
• 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
• 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.
• 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.
• NK 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
• 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.
• 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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• 1984
  • 1984-07-20 EP EP19840903011 patent/EP0190123A1/de not_active Withdrawn
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