Patent Application: US-29458702-A

Abstract:
the present invention relates to agent for suppressing infection and proliferation of human immunodeficiency syndrome virus , which infects immunocompetent cells such as macrophage or dendritic cell and causes destruction of immune system , and use of known compounds , macrolide derivatives for suppression of infection and proliferation of human immunodeficiency syndrome virus in m type macrophage derived from human monocytes . the present invention is useful for treatment of patients with hiv - 1 infection by low cost chemotherapeutic agents , and in addition , is clinically used as supplement agent in haart .

Description:
the present invention will be explained concretely with the following examples , but is not limited within these examples . in order to show growth suppression effect of the present invention , among macrolide derivatives , em , em201 , em202 , em703 , cam , em722 , em730 , em732 , em736 , em734 , em735 , em747 , em748 , em743 , em746 , em750 or em751 was dissolved in dmso with each 100 mm concentration , and used in dilution with the medium thereafter . a control group was m - mφ treated only with dmso used for dissolving macrolide derivatives . m - mφ and gm - m φ were subjected to contact infection with hiv - 1 bal strain for 2 hours , added 30 μm of em , em201 , em202 , em703 , cam , em722 , em730 , em732 , em736 , em734 , em735 , em747 , em748 , em743 , em746 , em750 or em751 , incubated for 14 days and amount of p24 protein in the cultured supernatant was assayed in the time - dependent manner . effect of em , em201 , em202 , em703 or cam on proliferation of hiv - 1 in m - mφ and gm - mφ . as compared with the control group ( dmso ), in m - mφ added with em201 or em703 , almost no p24 was detected and viral generation was strongly suppressed on 14 days of the culture ( refer to fig1 ). in m - mφ added with cam , approximately { fraction ( 1 / 2 )} suppression was recognized , but in the group added with em or em 202 , suppression of viral generation was recognized , though not so strong as compared with em201 and em703 ( refer to fig1 ). in gm - mφ , in the group added with em , em201 , em202 , em703 or cam , almost no viral generation was recognized at all assay points similar to the control group ( dmso ) ( refer to fig1 ). in the experiment using m - mφ derived from human monocytes collected from three adult human volunteers , the same results as shown in fig1 were obtained ( refer to fig1 ). the suppressive action of em201 and em703 on hiv - 1 bal in m - mφ is concentration dependent manner and completely inhibited viral generation at 3 μm or more ( refer to fig1 ). although viral proliferation at 300 μm was observed not only in em201 and em703 but also in em , em202 and cam , since it was observed in the control group ( dmso ), there might be due to cytotoxicity of dmso used for dissolving agents , the future experiments were conducted at 30 μm . effect of em , em201 , em202 , em703 or cam on cytopathy of hiv - 1 infected m - mφ . m - mφ was subjected to contact infection with hiv - 1 bal strain for 2 hours , added 30 μm of em , em201 , em202 , em703 or cam , incubated and observed the cell morphology . in the control group ( dmso ) and the group added with em , em202 or cam , formation of mgc was slightly recognized in accord with increase in amount of p24 protein as shown in example 1 , but in the group added with em201 and em703 , no cytopathic effect such as mcg formation was observed ( refer to fig1 ). effect of em , em201 , em202 , em703 or cam on tyrosine kinase hck protein of hiv - 1 infected m - mφ . as previously described , m - mφ expressed strongly tyrosine kinase hck protein , and regulating the expression of tyrosine kinase hck protein using antisense oligonucleotide could suppress proliferation of hiv - 1 in m - mφ . this result indicated that the expression of tyrosine kinase hck protein was essential for proliferation of hiv - 1 in m - mφ . since em201 and em703 suppressed proliferation of hiv - 1 in m - mφ , it was suggested that the expression of tyrosine kinase hck protein was suppressed in m - mφ treated with these agents . consequently , m - mφ was subjected to contact infection with hiv - 1 bal strain for 2 hours , added 30 μm of em , em201 , em202 , em703 or cam , incubated and expression of tyrosine kinase hck protein was observed on day 2 after the infection by western blotting . in the group of em , em202 and cam , which exhibited not so strong suppressive action on viral proliferation , the suppression of tyrosine kinase hck protein was observed , though not so strong , as compared with the control group ( treated only with dmso ) ( refer to fig1 ). in em201 and em703 , which showed strong suppressive action on viral proliferation , the expression of tyrosine kinase hck protein was strongly suppressed ( refer to fig1 ). effect of em , em201 , em202 , em703 or cam on activation of p38mapk and erk1 / 2 of hiv - 1 infected m - mφ . the p38mapk and erk1 / 2 ( p42 / 44mapk ) involve in various responses on intracellular signal transduction mechanism . tyrosine phosphorylation of p38mapk and erk1 / 2 in m - mφ infected with hiv - 1 bal strain was examined by western blotting . as a result , the tyrosine phosphorylation of p38mapk was strongly induced by viral infection , but the tyrosine phosphorylation of erk1 / 2 was weak . these results indicated that the activation of p38mapk was important for viral proliferation in m - mφ . consequently , effect of em , em201 , em202 , em703 and cam , which have suppressive action for viral proliferation in m - mφ , on the activation of p38mapk and erk1 / 2 was examined . thirty μm of em , em201 , em202 , em703 or cam were added to m - mφ infected with hiv - 1 bal strain , tyrosine phosphorylation of p38mapk was detected on day 2 of the culture by western blotting . in the group added with em , em202 and cam , in which the suppressive action on viral proliferation was not so strong , the tyrosine phosphorylation of p38mapk was recognized to decrease , though not so strong as compared with the control group ( dmso ). in the group added with em201 and em703 , which strongly suppressed viral proliferation , the tyrosine phosphorylation of p38mapk was significantly decreased [ refer to fig1 ( a )]. in any groups , total amount of p38mapk ( total sum of phosphorylated and non - phosphorylated p38mark ) was equal . these results suggested that the suppression of viral proliferation of em , em201 , em202 , em703 and cam in m - mφ was involved in the suppression of p38mapk . on the other hand , in the group added with em , em202 and cam , which were not so strong suppressive action of viral proliferation , tyrosine phosphorylation of erk1 / 2 was weakly recognized as same in the control group ( dmso ), but in the group added with em201 and em703 , which showed strong suppressive action of viral proliferation , promotion of tyrosine phosphorylation of erk1 / 2 was recognized . in any groups , total amount of erk1 / 2 ( total sum of phosphorylated and non - phosphorylated p38mark ) was equal [ refer to fig1 ( b )]. effect of p38mapk inhibitor on proliferation of virus in hiv - 1 infected m - mφ suppressed by addition of em , em201 , em202 , em703 and cam used in the present invention . from the results of example 4 , it was suggested that p38mapk activation is important for proliferation of hiv - 1 in m - mφ , and the suppressive action on hiv - 1 proliferation by em , em201 , em202 , em703 and cam is caused by suppressive action of these substances on activation of p38mapk . consequently , p38mapk inhibitor , sb203580 , ( 4 -[ 4 - fluoro - phenyl ]- 2 -[ 4 - methylsulfinylphenyl ]- 5 -[ 4 - pyridyl ]- 1h - imidazole ) and erk1 / 2 inhibitor , pd98059 , ( 2 -[ 2 - amino - 3 - methoxyphenyl ]- 4h - 1 - benzopyran - 4 - one ), 10 μm in concentration , were added in m - mφ infected with hiv - 1 bal strain and examined effect on virus . when sb203580 , 10 μm , was added in m - mφ infected with hiv - 1 bal strain and incubated , no p24 protein was almost detected even on day 14 and proliferation of virus was inhibited ( refer to fig2 ). on the other hand , when erk1 / 2 inhibitor , pd98059 , 10 μm , was added , amount of p24 protein was not different in the control group and viral proliferation was recognized ( refer to fig2 ). from these experimental results , the proliferation of macrophage directed hiv - 1 strain in m - mφ essentially required activation of p38mapk , and involvement of erk1 / 2 was thought to be low . effect of em722 , em730 , em732 and em736 used in the present invention on proliferation of hiv - 1 in m - mφ . m - mφ was subjected to contact infection with hiv - 1 bal strain for 2 hours , added various concentrations of em722 , em730 , em732 or em736 , incubated for 10 days and amount of p24 protein in the cultured supernatant was assayed in the time - dependent manner . a group treated only with dmso used for dissolving macrolide derivatives is set as the control group . increase in the amount of p24 protein was observed in the cultured supernatant of the control group added only with dmso depending on passing the culture days and proliferation of hiv - 1 was recognized . however , as compared with the control group , in the group added with em722 , em730 , em732 or em736 , production of p24 protein was suppressed depending on the concentration of agent and the suppression of viral production was recognized . especially , in the concentration of 30 μm , any agents of em722 , em730 , em732 and em736 were recognized to inhibit almost completely viral production ( refer to fig2 ). in the agents , em703 , em727 , em744 , em745 , em742 , em740 , em721 , em723 , em724 , em725 , em728 , em729 , em731 , em738 , em739 , em733 , em749 and em726 , similar results as in fig2 were obtained . further , in the experiment using m - mφ derived from human monocytes collected from three adult healthy volunteers , the same results as in fig2 were obtained . effect of em734 , em735 , em747 and em748 used in the present invention on proliferation of hiv - 1 in m - mφ . m - mφ was subjected to contact infection with hiv - 1 bal strain for 2 hours , added various concentrations of em734 , em735 , em747 and em748 , incubated for 10 days and amount of p24 protein in the cultured supernatant was assayed in the time - dependent manner . a group treated only with dmso used for dissolving macrolide derivatives is set as the control group . increase in the amount of p24 protein was observed in the cultured supernatant of the control group added only with dmso depending on passing the culture days and proliferation of hiv - 1 was recognized . however , as compared with the control group , in the group added with em734 , em735 , em747 and em748 , production of p24 protein was suppressed depending on the concentration of agent and the suppression of viral production was recognized . especially , in the concentration of 30 μm , any agents of em734 , em735 , em747 and em748 were recognized to inhibit almost completely viral production ( refer to fig2 ). further , in the experiment using m - mφ derived from human monocytes collected from three adult healthy volunteers , the same results as in fig2 were obtained . effect of em743 used in the present invention on proliferation of hiv - 1 in m - mφ . m - mφ was subjected to contact infection with hiv - 1 bal strain for 2 hours , added various concentrations of em743 , incubated for 10 days and amount of p24 protein in the cultured supernatant was assayed in the time - dependent manner . a group treated only with dmso used for dissolving macrolide derivatives is set as the control group . increase in the amount of p24 protein was observed in the cultured supernatant of the control group added only with dmso depending on passing the culture days and proliferation of hiv - 1 was recognized . however , as compared with the control group , in the group added with em743 , production of p24 protein was suppressed depending on the concentration of agent and the suppression of viral production was recognized . especially , in the concentration of 30 μm , em743 was recognized to inhibit almost completely viral production ( refer to fig2 ). further , in the experiment using m - mφ derived from human monocytes collected from three adult healthy volunteers , the same result as in fig2 was obtained . effect of em746 , em750 and em751 used in the present invention on proliferation of hiv - 1 in m - mφ . m - mφ was subjected to contact infection with hiv - 1 bal strain for 2 hours , added various concentrations of em746 , em750 and em751 , incubated for 10 days and amount of p24 protein in the cultured supernatant was assayed in the time - dependent manner . a group treated only with dmso used for dissolving macrolide derivatives is set as the control group . increase in the amount of p24 protein was observed in the cultured supernatant of the control group added only with dmso depending on passing the culture days and proliferation of hiv - 1 was recognized . however , as compared with the control group , in the group added with em746 , em750 and em751 , production of p24 protein was suppressed depending on the concentration of agent and the suppression of viral production was recognized . especially , in the concentration of 30 μm , any agents of em746 , em750 and em751 were recognized to inhibit almost completely viral production ( refer to fig2 ). further , in the experiment using m - mφ derived from human monocytes collected from three adult healthy volunteers , the same results as in fig2 were obtained . effect of em754 used in the present invention on proliferation of hiv - 1 in m - mφ . m - mφ was subjected to contact infection with hiv - 1 bal strain for 2 hours , added various concentrations of em754 , incubated for 10 days and amount of p24 protein in the cultured supernatant was assayed in the time - dependent manner . a group treated only with dmso used for dissolving macrolide derivatives is set as the control group . increase in the amount of p24 protein was observed in the cultured supernatant of the control group added only with dmso depending on passing the culture days and proliferation of hiv - 1 was recognized . however , as compared with the control group , in the group added with em7454 , production of p24 protein was suppressed depending on the concentration of agent and the suppression of viral production was recognized . especially , in the concentration of 30 μm , em754 was recognized to inhibit almost completely viral production ( refer to fig2 ). further , in the experiment using m - mφ derived from human monocytes collected from three adult healthy volunteers , the same result as in fig2 was obtained . as described hereinabove , the macrolide derivatives used in the present invention were recognized to have suppressive action on macrophage directed hiv - 1 proliferation in m - mφ derived from human monocytes . especially , em201 and em703 , which were recognized to have action for strongly inhibiting proliferation of macrophage directed hiv - 1 in m - mφ , inhibited almost completely the proliferation of virus even at concentration of 3 μm . the suppressive action of em201 and em703 on the proliferation of virus was recognized to exhibit sufficient suppressive effect with 95 % or more even on day 14 after the incubation by only adding em201 or em703 in the primary culture medium after contact infection of virus . further , em , em202 and cam also exhibited suppression of viral production depending upon drug concentration , and the same suppressive action was recognized in em722 , em730 , em732 , em736 , em734 , em735 , em747 , em748 , em743 , em746 , em750 em751 and em754 , at concentration of 30 , μm for almost complete inhibition . from this fact , the suppressive action was recognized to be involved in the pharmacological properties of macrolides showing accumulation in the tissue macrophage with long term acting . as described previously , the expression of tyrosine kinase hck protein was essential for proliferation of hiv - 1 in m - mφ , and since various macrolide derivatives of the present invention suppressed expression of tyrosine kinase hck protein in m - mφ , the suppressive action was suggested to be one of the mechanism of action of the suppressive action of these substances on hiv - 1 proliferation . further , since various macrolide derivatives of the present invention , which exhibit suppressive action on the proliferation of virus , inhibited tyrosine phosphorylation of p38mapk , the suppression of hiv - 1 proliferation was suggested to be based on the suppression of p38mapk activation by these substances . as described hereinabove , the present invention relates to use of macrolides derivatives for suppression of infection and proliferation of human immunodeficiency syndrome virus in the macrophage derived from the human monocytes . the present invention is not only useful as agents for suppression of infection and proliferation of human immunodeficiency syndrome virus but also expectative for clinical use as supplement agent in haart . 1 ): tae - 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