Patent Application: US-81757206-A

Abstract:
the use of oligodeoxyribonucleotides having a molecular weight of 4000 - 10000 dalton as an anti - tumour agent , alone or in combination with other active ingredients with anti - tumour action , is described . the oligotide may be produced by extraction from animal and / or vegetable tissues , in particular , from mammalian organs , or may be produced synthetically . the tumors which can be treated are preferably angiogenesis - dependent tumors , such as multiple myeloma or breast carcinoma .

Description:
we have recently developed a model for an alternative pathway of tumor angiogenesis . in addition to the endothelial cell sprouting from pre - existing vessels , we suggest that blood borne endothelial cells might also give rise to the tumor vasculature . these endothelial - like cells ( elc ) can transdifferentiate from tumor - associated dendritic cells under specific culture conditions ( 11 ). briefly , monocytes are elutriated from leukapheresis products of healthy human blood donors and cultured in the presence of granulocyte - macrophage - colony stimulating factor ( gm - csf ) and interleukin 4 ( il - 4 ) to stimulate the differentiation of dendritic cells ( dc ). in addition , cells are treated with a cocktail specifically released by tumor cells ( m - csf , il . 6 and lactate , gottfried et al ., manuscript submitted ) to promote the outgrowth of tumor - associated dendritic cells ( tudc ). these tudc - elc acquire the phenotype of endothelial cells ( factorviii related ag , vwf ) while they lose monocytic ( cd14 ) and dendritic cell markers ( cd1a ). importantly , they do not express cd34 , nor cd133 or cd146 which proves that they are real transdifferentiation products and no contaminants of either circulating endothelial progenitors ( cd34 , cd133 ) or mature circulating endothelial cells ( cd146 ). in addition , they are able to form tube - like structures in matrigel ™, an in vitro assay of angiogenesis . the matrigel ™ assay is one of the most popular and widely used in vitro angiogenesis assays ( 22 ). matrigel ™ is a semisolid synthetic mixture of extracellular matrix proteins which simulate the matrix that physiologically exist beneath the endothelial cell wall of a blood vessel . when the cells of question are seeded onto this matrix in microscopic chamber slides , they are activated to form tubular structures in 3 - 7 days , but only in the case that they have an endothelial phenotype . therefore , this assay is suitable to show the potential capacity of cells to give rise to a tumor vasculature . our data data demonstrate that oligotide and / or defibrotide in clinical and subclinical concentrations can inhibit tube formation of transdifferentiating elc ( tudc - elc ) in matrigel ™. tudc - elc and mature , differentiated endothelial cells , [ human umbilical vene ( huvec ) or microvascular endothelial cells ( hmec ) as “ stable ” controls ] were incubated in the presence or absence of oligotide or defibrotide ( 10 μg / ml each ) for 7 days . importantly , after a single addition of defibrotide , huvec and hmec are not affected in their tube formation potential , suggesting that defibrotide and / or oligotide only target transdifferentiating endothelial cells ( fig1 a ). however , when defibrotide was added repeatedly , it could also block angiogenesis of mature , fully differentiated endothelial cells ( see below ). by the help of a complimentary software from the nih ( image j , http :// rsb . info . nih . gov / ij /), we are able to quantify these effects , the total length of tubes and the area of the photograph are assessed , the microvascular density ( mvd ) is then given in total length / area [ pix - 1 ]. df significantly ( p = 0 . 02 , ttest ) downregulates mvd of tudc - elc ( fig1 b ). to support these data with an alternative angiogenesis assay the sprouting of rat aorta endothelial cells in matrigel ™ was prevented by nearly 100 %, when df was applied on a daily basis ( fig2 ), suggesting that df not only acts on transdifferentiating , but also on mature , fully differentiated endothelial cells . the aortic ring assay investigates macrovascular endothelial cells . but often , the tumor vasculature consists of microvascular endothelial cells . therefore , a third in vitro angiogenesis assay was performed on the basis of microvascular endothelial cells vascularizing through a layer of dermal fibroblasts after 9 - 11 days of culture . these vessel - like structures can subsequently be visualized by staining for cd31 and vwf . as demonstrated in fig3 ( a and b ), df can also block angiogenesis of human microvascular endothelial cells with a superiority for the daily application . interestingly , concentrations around 10 μg / ml appear to be the most effective . a single application of df could not significantly block angiogenesis . taken together , our data strongly suggest that defibrotide and / or oligotide can block angiogenesis of tumor - associated transdifferentiating endothelial cells and those that arise from already existing vascular cells . it is subject to ongoing studies whether oligotide and defibrotide also inhibit angiogenesis in vivo . we are currently performing a dorsal skin chamber assay ( 14 ) that investigates the effect of defibrotide in a highly vascularized human gastric carcinoma mouse model ( xenograft system ). first data clearly show that the microvascular density ( mvd ) of df - treated tumors is lower than that of control tumors . this set of experiments will be reproduced in due time . the mechanism of action by which df can block angiogenesis remains to be elucidated , but preliminary evidence from western blot analyses suggest a downregulating effect of df on activated p70s6 kinase ( p - p70s6 ), a mitogen - activated protein kinase . additional evidence for the impact of p70s6 kinase was obtained from another tube formation assay with hmec incubated in the presence or absence of the p70s6 kinase inhibtor drb . there are also first clinical data available for patients ( pts .) having received allogeneic stem cell transplantation ( sct ): in a cohort of 17 defibrotide - treated pts a striking decline in serum vegf levels has been seen , also suggesting that defibrotide might act through growth factor withdrawal for sprouting tumor endothelial cells . defibrotide and oligotide are strong candidates for a therapy of angiogenesis - dependent tumors and might be used alone or in combination with other anti - angiogeneic agents , such as rapamycin ( 14 ). interestingly , rapamycin has the negative side effect of pro - thrombotic activity ( 15 ) that could be attenuated by the simultaneous application of the anti - thrombotic and fibrionolytic defibrotide . 6 . &# 39 ; t veer , l . j ., et al . 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