the present invention involves a method for treating a variety of brain and central nervous system diseases , including cns tumors , cerebrovascular lesions , cns infections , and degenerative processes of the cns . all of these diseases are characterized by the formation of brain lesions . as previously described , the endothelial cells of the bbb capillary tissues normally permit a low degree of transendotheial passage of foreign materials . however , research has discovered that brain lesions cause an increase in bbb permeability . this increase allows the passage of larger molecular weight materials across the bbb . for example , the bbb will normally prevent molecules larger than 180 daltons from entry into the brain . during and after the onset of brain lesions , the bbb will permit the increased passage of molecules having a molecular weight of about 500 to 1 , 000 daltons . examples of drugs which may pass through the modified bbb i . e ., blood - brain lesion barrier include the antimetabolite methotrexate , the antibiotic gentamicin , and fluorescein . because of the modified bbb structure during and after lesion formation , it is now possible to administer therapeutic agents which would not pass through a normal bbb . following administration , it is important that therapeutic agents circulating outside the bbb do not reach toxic levels . this is often a problem since high doses of drugs used to treat brain and cns lesions are often required , especially if multiple lesions are involved . to control drug toxicity , the present invention involves administration of an &# 34 ; inactivating &# 34 ; or &# 34 ; neutralizing &# 34 ; material ( e . g ., drug antibody ) specific to the drug initially administered . the neutralizing material will have characteristics designed to prevent its passage through the modified bbb , thus ensuring that concentrations of the drug inside the bbb remain active . such characteristics can include lipid solubility , molecular weight , electrical charge , and others . in terms of molecular weight , neutralizing materials having a molecular weight in excess of about 1 , 000 daltons typically will not effectively pass through the modified bbb . after administration , such neutralizing material will typically act by binding with the drug circulating outside the bbb , thereby forming a complex conjugate which is either inactive and / or cleared from the circulatory system . similarly , certain neutralizing agents of lower molecular weight will be prevented from crossing the bbb due to some other characteristics such as lipid solubility or electrical charge . antibodies or antibody fragments ( i . e ., fab or ( fab &# 39 ;) 2 ) usable in the invention are prepared conventionally in either a polyclonal or monoclonal form . thus , suitable antibodies for numerous therapeutic agents , including methotrexate and gentamicin are readily available . in summary , the method of the present invention utilizes the modified character of the bbb caused by lesion formation to deliver therapeutic agents into tissues of the brain , while insuring that subsequently administered drug neutralizing substances remain outside of the bbb . as described above , this enables circulating amounts of the drug to be precisely controlled . administration of a therapeutic agent , followed by administration of a neutralizing material to reduce toxicity , across the bbb in a subject animal having a brain lesion is described below in the following example : adult , female , sprague - dawley rats at six days post inoculation intracerebrally with 2 . 0 × 10 6 cfu e . coli bacteria were anesthetized with sodium pentobarbital ( 50 mg / kg , intraperitoneal ). a catheter filled with heparinized sodium chloride was tied into the right external carotid artery . evans blue and fluorescein were administered intravenously ( 2 . 0 %, 2 ml / kg and 10 %, 0 . 12 ml , respectively ) to evaluate the integrity of the abscess barrier . subsequently , dosages of 125 i - gentamicin were administered intravenously . one hour thereafter , a pre - antibody serum sample was collected to evaluate the non - specific binding of gentamicin to serum proteins . antigentamicin antibody ( 0 . 06 ml antisera + 0 . 2 ml pbs ) or nonimmune sera , was administered intravenously . fifteen minutes later , a post antibody serum sample was collected to evaluate the extent of binding for circulating gentamicin . the rat was then sacrificed by perfusion with 0 . 9 % nacl to clear the vascular bed of radioactivity . tissue samples consisting of abscess ( ab ), brain around abscess ( baa ), brain distant to the abscess ( bfa ), and contralateral hemisphere ( lh ) were weighed and then homogenized in 0 . 5 ml of saline . to precipitate immune complexes , 2 ml of isopropyl alcohol was added to aliquots of tissue homogenates and 1 ml of alcohol to serum , centrifuged for 15 minutes at 1200 × g , and the resulting fractions counted for activity . summaries of the data obtained in the above experiment are described as follows in tables i and ii . table i______________________________________immune serumab baa bfa lh pre post p - per c1______________________________________rat g - 70 ; dose : 6 . 0 × 10 . sup . 6 cpmcpm / gm 7298 3292 2159 1501 99 % bound 52 42 54 457 22 97 43 % del . . 12 . 06 . 04 . 03doserat g - 73 ; dose : 13 . 0 × 10 . sup . 6 cpmcpm / gm 17 , 074 5703 1436 1000 97 % bound 44 31 49 49 22 96 75 % del . . 13 . 04 . 01 . 01doserat g - 80 ; dose : 13 . 0 × 10 . sup . 6 cpmcpm / gm 43 , 305 12 , 413 2895 2684 92 % bound 49 45 55 55 36 94 70 % del . . 14 . 04 . 01 . 01doserat g - 85 ; dose : 11 . 8 × 10 . sup . 6 cpmcpm / gm 11 , 728 6823 2488 1245 95 % bound 63 59 60 52 26 96 79 % del . . 10 . 06 . 02 . 01doserat g - 87 ; dose : 14 . 0 × 10 . sup . 6 cpmcpm / gm 12 , 694 5694 4428 814 94 % bound 73 61 68 41 24 96 75 % del . . 09 . 04 . 03 . 01doserat g - 88 ; dose : 14 . 1 × 10 . sup . 6 cpmcpm / gm 15 , 254 8457 4073 1012 96 % bound 56 53 65 50 23 96 77 % del . . 11 . 06 . 03 . 01dose______________________________________ legend : ab : abscess baa : brain around abscess bfa : brain distant to the abscess lh : left or non abscess bearing hemisphere pre : precipitable radioactivity from serum prior to antibody administration post : precipitable radioactivity from serum after antibody administration pper : precipitable radioactivity from serum after perfusion of rat c1 : percent clearance of activity from serum postperfusion × 100 preperfusion cpm / gm : counts per minute of radioactivity per gram tissue % bound : percent precipitable radioactivity from tissue sample % del . dose : percent delivered dose of radioactivity per gram of tissue table ii______________________________________ p - ab baa bfa lh pre post per c1______________________________________non immune serumrat g - 89 ; dose : 18 . 4 × 10 . sup . 6 cpmcpm / gm 12 , 304 11 , 236 5920 4991 84 % bound 44 45 48 47 26 27 21 % del . . 07 . 06 . 03 . 03doserat g - 90 ; dose : 18 . 4 × 10 . sup . 6 cpmcpm / gm 22 , 460 9417 3038 1389 86 % bound 30 44 43 50 22 21 3 % del . . 12 . 05 . 02 . 01doserat g - 91 ; dose : 20 × 10 . sup . 6 cpmcpm / gm 17 , 789 10 , 247 2036 1180 85 % bound 31 36 48 36 24 22 2 % del . . 09 . 05 . 01 . 01dose______________________________________mean valuesimmune serum % bound 56 49 59 51 26 96 70 96standard 4 5 3 2 2 . 4 6 1error % del . 0 . 12 0 . 05 0 . 02 0 . 01dosestandard 0 . 1 . 004 . 01 . 003errornon immune serum % bound 35 42 46 44 24 23 9 85standard 5 3 2 4 1 2 6 . 6error % del . 0 . 09 0 . 05 0 . 02 0 . 01dosestandard . 02 . 003 . 01 . 01error______________________________________ in rats given non - immune serum , the per cent of delivered dose per gram of tissue in abscess was 0 . 09 % indicating the presence of a cerebral abscess . in brain around abscess ( baa ), brain distant to abscess ( bfa ), and contralateral hemisphere ( lh ), the values were 0 . 05 %, 0 . 02 %, and 0 . 01 %. these values indicate that in areas of increased vascular permeability ( i . e ., bbb incompetence ), a marked elevation in drug delivery can be seen ( cerebritic brain 0 . 09 % versus normal brain 0 . 01 %). pre and post serum precipitation values of 24 % and 23 % indicate that gentamicin nonspecifically precipitates with serum proteins , and that the addition of non - immune serum does not alter precipitation characteristics . the precipitation of activity from brain tissues were 35 % ( ab ), 42 % ( baa ), 46 % ( bfa ), and 44 % ( lh ). these values suggest that there is more nonspecific precipitation activity from the brain since serum nonspecific binding was appreciably lower . in rats given anti - gentamicin antibody , the pre and post serum precipitation values of 26 % and 96 % indicate that sufficient antibody was administered to adequately bind a significant fraction of circulating drug . again , the per cent delivered dose per gram of tissue data indicates the presence of a cerebral abscess ( 0 . 12 % ( ab ) versus 0 . 01 % ( lh )). the precipitation of activity (% bound ) in the antibody - administered rats was high , as in the control group , but no difference could be seen between the abscess ( 56 %) and normal brain ( 51 %). this suggests that if the antibody had been able to penetrate the abscess , more precipitation should have been seen due to the addition of immunoprecipitation . having herein described a preferred embodiment of the invention , it will be anticipated that suitable modifications may be made by those skilled in the art that fall within the scope of the invention . thus , the scope of the invention shall be only construed in accordance with the following claims .
a method for the delivery of therapeutic agents across the blood brain barrier is disclosed which utilizes drug neutralization technology , and the selective permeability o this invention was made with support by the preuss foundation and with government support by the veterans administration and under grant no . 31770 awarded by the national institutes of health . the government has certain rights in this invention .