according to the present invention , achieving effective inhibition of pfc uptake by macrophages will make it possible to achieve higher therapeutic concentrations of circulating pfc while administering smaller doses of pfc . blockade of the macrophages of the res is a well - known experimental phenomenon , but has not been employed as a therapeutic modality , largely because the res functions as a primary defense against circulating pathogens . nevertheless , materials , mainly lipids , exist which are preferentially taken up and stored within the res without ill - effect . to be a therapeutically effective component of a pfc dosage regimen blockage of the res uptake of pfc must not impede the ability of the res to remove circulating pathogenic organisms from the blood stream . blocking materials that are toxic to macrophages or significantly inhibit their ability to phagocytose pathogens are therefore not suitable for use in the present invention ( van rooijenet al , 1990 ). the empty , small vesicles ( esv ) for use in the present invention are preferably liposomes that are synthetic , multilamellar and / or unilamellar lipid microspheres that are preferentially engulfed by macrophages . for example , the synthesis of liposomes that vary in the extent to which they are taken up the res by varying size , dosage , and lipid composition have been described ( wessef et al , 1984 ; liu et al , 1992 ; brand et al , 1194 , which are hereby incorporated by reference in their entirety ). the present invention utilizes the initial enhancement , rather than suppression of phagocytosis of liposomes by the res , thereby blocking the uptake by macrophages of pfc that is subsequently administered . it is the object of this invention to increase the half - life of circulating pfc incorporated within a lipid emulsion by inhibiting the uptake of such pfc by macrophages without significant injury to the macrophages . this can be accomplished by the administration of empty , negatively charged liposomes or liposomes with modified lipid bilayers that are preferentially taken up by macrophages ( proffitt et al , 1983 , van etten , 1998 ) prior to the administration of pfc . macrophages engulf empty , negatively charged liposomes such as those made with the classic formulation of phosphatidylcholine , phosphatidylserine and cholesterol in a molar ratio of 40 : 10 : 50 more quickly and effectively than neutral lipid vesicles . for example , when a dose of such empty liposomes is administered to mice at a concentration of 80 micromole of lipid / kg , 90 % of the liposomes are taken up by the res in 30 min , a level of clearance that required 40 to 48 hours for other liposome formulations . moreover , clearance of bacteria from the blood stream , a key function of the res , is not impaired , even with a dose of negatively charged liposomes as high as 400 micromols / kg . k . pneumonaie and s . aureus were cleared from the blood stream as effectively as in control animals that did not receive any liposomes ( van etten et al 1998 ). effective , rapid blockade of the res is also achieved by the use of empty , small , 0 . 1 micrometer , unilamellar liposomes that have 6 - aminomannose incorporated in their lipid envelope . these vesicles are composed of distearoylphosphatidylcholine ( dspc ), cholestorol ( ch ) and 6 - aminomannose ( 8 : 3 : 1 ). when neutral lipid vesicles ( dspc : ch : diacetyl phosphate , 4 : 1 : 1 ) are subsequently injected they escape phagocytosis by the res . ( proffitt et al , 1983 ). the cited studies demonstrate that it is possible to block the res with the administration of empty liposomes ( sev ) without diminishing the res capacity to clear circulating pathogens and permit subsequently injected pfc to largely escape capture by the res , thereby enhancing the circulating concentrations of the oxygen carrier in the bloodstream . this invention , which utilizes the prior administration of empty liposomes as a first step of a two - step process in the therapeutic administration of pfc in order to inhibit the uptake of pfc by the res contrasts sharply with the disclosure of u . s . pat . nos . 5 , 679 , 394 and 5 , 624 , 220 , both entitled , “ method of extending the vascular dwell time of particulate therapeutic and particulate diagnostic agents ” ( long jr ., d . m . & amp ; long , r . a . 1993 , 1997 ). in these patents , liposomes infused with metabolically active particulates are administered simultaneously with the pfc emulsion in a one step procedure . by design their contents that ( 1 ) are deleterious to macrophages , ( 2 ) render the recipients susceptible to systemic infections , and ( 3 ) are co - administered together with the pfc . by contrast , in the methods of the present invention , ( 1 ) the blocking liposomes ( esv ) are given prior to the infusion of pfc to inhibit the subsequent uptake of pfc and ( 2 ) the esv are empty and devoid of materials that interfere with the metabolic activities of macrophages , to permit the res to carry out its primary function as scavenger of circulating pathogens . as discussed above , the present invention relates to a method of increasing the half - life and / or concentration in the circulation of perfluorocarbon ( pfc ) oxygen - carrying emulsions . the perfluorocarbon ( pfc ) oxygen - carrying emulsions that can be used in the present invention include , for example , the perfluorocarbon ( pfc ) oxygen - carrying emulsions disclosed in u . s . pat . nos . 4 , 859 , 363 , 5 , 536 , 753 , 5 , 621 , 144 , 5 , 635 , 539 , 5 , 628 , 930 and 7 , 383 , 395 , which are hereby incorporated by reference in their entirety . therapeutic applications of such pfcs include , but are not limited to : ( a ) red cell replacement following trauma occurring on the battlefield or in accidents or in the course of surgery , ( b ) delivery of oxygen to ischemic tissues damaged by stroke and infarction ( c ) scavenging of oxygen bubbles in the circulation such as those generated during cardiac by - pass surgery that are responsible for mini - strokes with deleterious physical and psychiatric consequences ( newman et al 2001 ) and ( d ) delivery of high concentrations of oxygen to enhance the sensitivity of tumors to be subjected to radiation therapy . these therapeutic applications that require freely circulating pfc are limited by its short dwell time in the vascular tree . metabolically inert pfc is readily engulfed and stored by res macrophages necessitating the administration of excessive amounts of the oxygen carrier . the prolonged storage causes hepato - and splenomegaly and stimulates the release of tumor necrosis factor and other cytokines that are responsible for the deleterous side effects of pfc treatment . liposomes useful in the present invention are preferably small ( generally under ten microns in diameter ) multilamellar or unilamellar phospholipid vesicles and are preferentially engulfed by macrophages as described above . empty , small vesicles ( esv ) for use in the present invention can be prepared by standard techniques including , for example , as described in u . s . pat . nos . 4 , 814 , 270 , 6 , 066 , 331 and 6 , 156 , 337 , which are hereby incorporated by reference in their entirety . the affinity of esv for macrophages is the basis for the methods of the present invention . the first step is the administration of empty , small vesicles ( esv ) that are devoid of particulates , antibiotics , metabolites or other materials that are toxic to the res , but are selectively taken up by macrophages of the res before treatment with pfc . the presence of phagocytosed esv within macrophages inhibits their capacity to engulf subsequently administered pfc emulsions ( profitt , r . t . 1983 ; lutz , j . 1994 ; nanrooijen , n . 1990 ). pfc will then remain within the circulation enabled to perform its intended function , the systemic transport and delivery of oxygen . the res retains its ability to degrade the esv and to clear the circulation of pathogenic organisms such as klepsiella pneumococcus or staphyococcus aureus ( van etten , 1998 ). in another embodiment , the present invention is directed to a method to enhance the oxygen transport capability of intravascularly injected perfluorocarbon ( pfc ) artificial oxygen - carrying emulsions by increasing the half - life ( dwell - time ) of pfc in the vascular system . this method is accomplished by the prior intravascular administration of empty , small vesicles ( esv ) that are sequestered by the macrophages of the reticular endothelial system ( res ), thereby inhibiting the capacity of the res to phagocytose subsequently injected emulsions of pfc and increasing the freely circulating concentrations of pfc and its therapeutic efficacy . in yet another embodiment , the present invention is directed to a method to diminish the fever , flu - like symptoms and other adverse phenomena that occur following the administration of pfc emulsions and their phagocytosis by the res . these symptoms occur as a consequence of the enhanced release by res macrophages of interleukin - 1 , tumor necrosis factor and other cytokines . because pfc is inert and cannot be metabolized both its storage within macrophages and their consequent release of cytokines is unduly prolonged . this method is accomplished by the intravascular administration of pfc only after the prior injection of esv . the esv are readily engulfed by the res and the phagocytosis of pfc is blocked . as the esv are readily broken down the stimulus for cytokine release is minimized . in yet another embodiment , the present invention is directed to a method to diminish the hepatomegaly and splenomegaly that may occur following the administration of pfc . this method is accomplished by the intravascular administration of esv prior to the injection of pfc , thereby inhibiting the uptake and retention of pfc by macrophages of the res . esv are non - toxic and unlike pfcs will be readily degraded by macrophage lysosomes . the esv for use in the present invention can be of varying sizes up to fifty or more microns in diameter and bounded by a unilamellar or a multilamellar membrane . the membrane may incorporate an aminomannose derivative of cholesterol , cholesterol , phosphtidylcholine or related lipids . the esv are also devoid of any metabolite , antibiotic , enzyme or any other biologically active material . again , the preparation of the esvs can be prepared by conventional techniques . the quantities of esv for use in the present invention will vary depending upon size and nature of the site targeted for pfc and the condition to be treated . a small , defined site such as the heart , brain , tumor or ischemic focus will require less pfc and esv than what will be required to treat massive hemorrhage and battlefield trauma . in summary , the life - saving potential for an artificial , oxygen - carrying substitute for red blood cells has resulted in the development of numerous perfluorocarbon ( pfc ) emulsions with an extraordinary capacity to bind and transport oxygen . unfortunately , the clinical usefulness of pfc has been limited to a great extent by its short half - life in the circulation , mainly a consequence of the rapid clearance and prolonged storage of pfc by macrophages of the reticulo - endothelial system ( res ). this invention relates to an improved method of delivering pfc to human subjects , thereby increasing its therapeutic efficacy , reducing the required amount of pfc and diminishing the adverse effects induced by pfc . pfc is inert and not metabolized , consequently storage within macrophages is greatly prolonged and causes hepatomegaly , splenomegaly and the sustained release of tumor necrosis factor and other cytokines . brandt , m . et al . 1994 ; depression of the phagocytic activity of the res by liposomes . pharm pharmacol letter . 4 : 1 - 4 . lane , t . a . 1995 ; perfluorochemical - based artificial oxygen carrying red cell substitutes . transfus . sci . 16 : 19 - 31 . liu , d . et al . 1992 ; role of liposome size and res blockade in controlling biodistribution and tumor uptake of gm1 - containing liposomes . bioch biophys acta . 1104 : 95 - 101 . lutz , j . et al . 1995 ; acute toxicity and depression of phagocytosis in vivo by liposomes : influence of lysophosphatidylcholine . life sciences . 56 : 99 - 106 . newman , n . f ., et al . 2002 ; longitudinal assessment of neurocognitive function after coronary - artery bypass surgery . new eng j of med . 344 : 395 - 402 . proffitt , r . t . et al . 1983 : liposomal blockade of the reticuloendothelial system : improved tumor imaging with small unilamellar vesicles science . 220 : 502 - 505 . spahn , d . r . 1999 ; blood substitutes . artificial oxygen carriers : perfluorocarbon emulsions . crit care 3 : r93 - r97 . van etten , e . m . w . et al . 1998 ; administration of liposomal agents and blood clearing capacity of the mononuclear phagocytic system . antimicrob agents chemother 42 : 1677 - 81 . van rooijen , n . et al . 1990 ; depletion and repopulation of macrophages in spleen and liver of rat after intravenous treatment with liposome - encapsulated dichloromethylene diphosphate . cell tissue res . 260 : 215 - 22 . wassef , n . m . et al . 1984 ; suppression of phagocytic function and phospholipids metabolism in macrophages by phosphatidylinositol liposomes . pnas usa . 9 : 2665 - 2669 . winslow , r . w . 2006 ; current status of oxygen carriers (‘ blood substitutes ’) 2006 . vox sanguinis 91 : 102 - 110 .
this invention describes a novel , two - step method for administering pfc . the first step is designed to block the res by administration of empty , small , liposomal vesicles that are rapidly and preferentially engulfed by macrophages , thereby inhibiting their phagocytosis of subsequently infused pfc emulsions . the second step is the subsequent injection of pfc . esv are devoid of materials that interfere with the macrophage &# 39 ; s metabolic processes and do not impair their ability to clear the circulation of pathogenic organisms . inhibition of the removal of pfc from the blood stream by the res will achieve increased circulating pfc , enhanced binding and transport of oxygen throughout the blood stream and consequential reduction of undesirable consequences such as organomegaly and cytokine toxicity .