the present invention relates to new molecules that can act as vectors or carriers for transporting an agent , medicine or other molecule to the brain and / or central nervous system ( cns ). agents , medicines or other molecules which are unable or ineffective at crossing the blood - brain barrier by themselves , will be transported across the blood - brain barrier when attached or coupled ( conjugated ) to the vector or carrier . alternatively , an agent that is able to cross the blood - brain barrier by itself may also see its transport increase when conjugate to the carrier of the present invention . such conjugates can be in the form of a composition , such as a pharmaceutical composition , for treatment of a condition or disease . in international publication no . wo2004 / 060403 , the inventors have disclosed that angiopep - 1 ( seq id no . : 67 ) and aprotinin ( seq id no . : 98 ) are effective vectors for transporting desirable molecules across the blood brain barrier . the inventors herein demonstrate that other molecules could also be used as carriers for transporting an agent across the blood brain barrier . accordingly , peptides having similar domains as aprotinine and angiopep - 1 and a modified form of angiopep - 1 ( amidated , peptide no . 67 ) were therefore conceived as potential carrier vectors . these derived peptides resemble aprotinine and angiopep - 1 but comprise different amino acid insertions and bear different charges . thus far , 96 peptides presented in table 2 as well as additional peptides listed in the sequence listing were tested for their potential as carrier . it is to be understood herein that in the following experiments , peptides have been selected based on their higher activity compared to others . those which have not been selected for further experimentations are by no means being disclaimed and are not intended to be regarded as non - functional . these peptides show substantial activity and have utility has ( biologically active ) carriers and are also encompassed by the present invention . an in vitro model was used for screening assay and for mechanistic studies of drug transport to the brain . this efficient in vitro model of the blood - brain barrier was developed by the company cellial ™ technologies . yielding reproducible results , the in vitro model was used for evaluating the capacity of different carriers to reach the brain . the model consists of a co - culture of bovine brain capillary endothelial cells and rat glial cells . it presents ultrastructural features characteristic of brain endothelium including tight junctions , lack of fenestration , lack of transendothelial channels , low permeability for hydrophilic molecules and a high electrical resistance . moreover , this model has shown a good correlation coefficient between in vitro and in vivo analysis of wide range of molecules tested . to date , all the data obtained show that this bbb model closely mimics the in vivo situation by reproducing some of the complexities of the cellular environment that exist in vivo , while retaining the experimental advantages associated with tissue culture . many studies have validated this cell co - culture as one of the most reproducible in vitro model of the bbb . the in vitro model of bbb was established by using a co - culture of bbcecs and astrocytes . prior to cell culture , plate inserts ( millicell - pc 3 . 0 μm ; 30 - mm diameter ) were coated on the upper side with rat tail collagen . they were then set in six - well microplates containing the astrocytes and bbcecs were plated on the upper side of the filters in 2 ml of co - culture medium . this bbcec medium was changed three times a week . under these conditions , differentiated bbcecs formed a confluent monolayer 7 days later . experiments were performed between 5 and 7 days after confluence was reached . the permeability coefficient for sucrose was measured to verify the endothelial permeability . primary cultures of mixed astrocytes were prepared from newborn rat cerebral cortex ( dehouck m . p ., meresse s ., delorme p ., fruchart j . c ., cecchelli , r . an easier , reproductible , and mass - production method to study the blood - brain barrier in vitro . j . neurochem , 54 , 1798 - 1801 , 1990 ). briefly , after removing the meninges , the brain tissue was forced gently through an 82 μm nylon sieve . astrocytes were plated on six - well microplates at a concentration of 1 . 2 × 10 5 cells / ml in 2 ml of optimal culture medium ( dmem ) supplemented with 10 % heat inactivated fetal bovine serum . the medium was changed twice a week . bovine brain capillary endothelial cells ( bbcecs ) were obtained from cellial technologies . the cells were cultured in the presence of dmem medium supplemented with 10 % ( v / v ) horse serum and 10 % heat - inactivated calf serum , 2 mm of glutamine , 50 μg / ml of gentamycin , and 1 ng / ml of basic fibroblast growth factor , added every other day . originally , at a first level of selection , 96 peptides as described in table 2 were tested as carrier with the in vitro model of the bbb . each peptide was added to the upper side of the inserts covered or non - covered with endothelial cells for 90 minutes at 37 ° c . after the incubation , the peptides in the lower side of the chambers were resolved by electrophoresis . electrophoresis gels were stained with coomassie blue to visualize the peptides as illustrated with some peptides ( without limitation ) in fig1 . angiopep - 1 ( either seq id no . : 67 or peptide no . 67 ( amidated form )) is often used herein as a reference or for comparison purpose . in fig1 , each initial peptide applied to the upper side of the filters was loaded on electrophoresis gel ( ini ) as control . after 90 minutes of transcytosis , a volume of 50 μl from the basolateral side of the filters covered with endothelial cells (+) or non - covered (−) was also loaded on tricine gels . to visualize the peptides gels were stained with coomassie blue . following the first level of screening , peptides detected in the lower side of the chambers by coomassie blue staining ( 5 , 8 , 45 , 67 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 81 , 82 , 90 and 91 ) were selected for further study with the iodinated peptides . briefly , the selected peptides were iodinated with standard procedures using iodo - beads from sigma . two iodo - beads were used for each protein . these beads were washed twice with 3 ml of phosphate buffer ( pb ) on a whatman ™ filter and resuspended in 60 μl of pb . 125 i ( 1 mci ) from amersham - pharmacia biotech was added to the bead suspension for 5 min at room temperature . the iodination for each peptide was initiated by adding 100 μg ( 80 - 100 μl ) of the bead suspension . after an incubation of 10 min at room temperature , the supernatants were applied on a desalting column prepacked with 5 ml of cross - linked dextran ™ from pierce and 125 i - proteins were eluted with 10 ml of pbs . fractions of 0 . 5 ml were collected and the radioactivity in 5 μl of each fraction was measured . fractions corresponding to 125 i - proteins were pooled and dialyzed against ringer / hepes buffer , ph 7 . 4 . the efficiency of radiolabeling was between 0 . 6 - 1 . 0 × 10 8 cpm / 100 μg of protein . the iodinated peptides were also investigated with the in vitro model of the bbb . each peptide was added to upper side of the inserts covered or non - covered with endothelial cells for 90 minutes at 37 ° c . after the incubation , peptides in the lower side of the chambers were tca precipitated . results were expressed as cpm ratios . for each [ 125 i ]- peptide the number of cpm in the bottom chamber was divided by the total number of cpm added to filter covered with endothelial cells (+ cells / initial ) or uncovered (− cells / initial ). the ratio between the number of [ 125 i ]- peptide found in the bottom chamber of filters covered with or without endothelial cells was also calculated (+ cells /− cells ). a very low − cells / initial ratio indicates that filters may interfere with the peptides ( peptides 5 and 8 ). a high + cells / initial and + cells /− cells ratio indicate a better passage of the peptides across the brain endothelial cells . the results for the previously selected 18 peptides are shown in table 3 . from these results , 12 peptides with + cells /− cells ratios generally higher than 0 . 35 were selected namely ; 5 , 8 , 67 , 75 , 76 , 77 , 78 , 79 , 81 , 82 , 90 and 91 . peptides # 91 and # 77 were also selected for further investigation because of their + cells /− cells ratios (& gt ; 0 . 2 ). the 12 selected peptides were then investigated by assessing their permeability coefficients using the in vitro bbb model . the effect of each selected peptide at 250 nm on the bbb integrity was determined by measuring [ 14 c ] sucrose permeability in the bbb model on bbcec monolayers grown on filters in the presence of astrocytes . to achieve this test , brain endothelial cell monolayers grown on inserts were transferred to 6 - well plates containing 2 ml of ringer - hepes per well ( basolateral compartment ) for two hours at 37 ° c . ringer - hepes was composed of 150 mm nacl , 5 . 2 mm kcl , 2 . 2 mm cacl 2 , 0 . 2 mm mgcl 2 , 6 mm nahco 3 , 5 mm hepes , 2 . 8 mm hepes , ph 7 . 4 . in each apical chamber , the culture medium was replaced by 1 ml ringer - hepes containing the labeled [ 14 c ]- sucrose . at different times , inserts were placed into another well . [ 14 c ] sucrose passage was measured at 37 ° c ., on filters without cells or with filters coated with bbcec cells . the peptides are added at the start of the experiment at time zero . the results were plotted as the sucrose clearance ( μl ) as a function of time ( min ). clearance ⁢ ⁢ ( μl ) = [ c ] ⁢ a × va [ c ] ⁢ l the slope of the linear variation ( μl / min ) is the sucrose permeability coefficient for the filter without cells ( psf ) and one with coated with bbcec cells ( pst ) in the presence of the peptide . 1 / pe =( 1 / pst − 1 / psf )/ filter area ( 4 . 2 cm 2 ) the peptides with highest pe were selected : 67 , 76 , 90 , 91 , 5 , 79 , 8 , and 78 . the in situ cerebral perfusion ( in mice ) was used as the fourth level of selection to select the best peptides . this procedure also distinguishes between compounds remaining in the brain vascular compartment from those having crossed the abluminal endothelial membrane to enter the brain parenchyma . indeed , the technique of post - perfusion capillary depletion allows to measure whether the molecule really crosses the endothelium to enter the brain parenchyma . using this technique it is demonstrated herein that specific peptides tend to accumulate in the brain parenchyma fraction ( see table 4 ). four peptides , namely 5 , 67 , 76 and 91 , showed the highest levels of distribution in the parenchyma with a volume higher than 20 ml / 100 g and which represents at least 25 % of the volume found for the total brain ( homogenate ), thus showing the highest potential as carrier for use as transport vectors . peptide 79 was eliminated because of its lower volume of distribution in the brain parenchyma ( 18 ml / 100 g ). peptide 67 represents the amidated form of angiopep - 1 described in the previous application that the inventors filed . amidation of a peptide affect the overall charge of the peptide . as is apparent in tables 2 and 3 , two peptides having a different charge do not have necessary the same activity . the vector or carrier of the present invention may thus be used in a method for transporting an agent across the blood - brain barrier comprises administering to an individual an agent that comprises an active ingredient or a pharmaceutical agent attached to a carrier , such as aprotinin or a functional derivative thereof ( i . e ., an aprotinin analog , an aprotinin fragment , an aprotin derivative , an analogue of an aprotinin fragment ). the carrier and conjugate may be administered intra - arterially , intra - nasally , intra - peritoneally , intravenously , intramuscularly , sub - cutaneously , transdermally or per os to the patient . the agent may be , for example , an anti - angiogenic compound . the agent may have a maximum weight of 160 , 000 daltons . as discussed herein , the agent may be a marker or a drug such as a small molecule drug , a protein , a peptide or an enzyme . the drug may be adapted to treat , for example , a neurological disease or a central nervous system disorder of a patient . the drug may be a cytotoxic drug and the marker may be a detectable label such as a radioactive label , a green fluorescent protein , a histag protein or β - galactosidase . the agent may be delivered , for example , into the central nervous system of a patient . according to another embodiment , the uses , methods , compounds , agents , drugs or medicaments therein mentioned may not alter the integrity of the blood - brain barrier of the patient . according to a further embodiment of the present invention the peptide may be selected from the group consisting of aprotinin , an aprotinin fragment ( seq id no . : 1 ) and any one of the peptides defined in seq id no . : 1 to 97 , 99 , 100 or 101 . for example , peptides 5 , 76 , 91 , 97 and 97 as well as peptide 67 may be used in the present invention by linking them to an agent or a compound for transporting the agent or compound across the blood - brain barrier of a patient . the agent or compound may be adapted to treat a neurological disease or to treat a central nervous system disorder . the carrier of the present invention , such as for example , peptides 5 , 76 , 91 and 97 as well as peptide 67 may be linked to or labelled with a detectable label such as a radioimaging agent , such as those emitting radiation , for detection of a disease or condition , for example by the use of a radioimaging agent - antibody - carrier conjugate , wherein the antibody binds to a disease or condition - specific antigen . other binding molecules besides antibodies and which are known and used in the art are also contemplated by the present invention . alternatively , the carrier or functional derivative thereof of the present invention or mixtures thereof may be linked to a therapeutic agent , to treat a disease or condition , or may be linked to or labelled with mixtures thereof . treatment may be effected by administering a carrier - agent conjugate of the present invention to an individual under conditions which allow transport of the agent across the blood - brain barrier . a therapeutic agent as used herein may be a drug , a medicine , an agent emitting radiation , a cellular toxin ( for example , a chemotherapeutic agent ) and / or biologically active fragment thereof , and / or mixtures thereof to allow cell killing or it may be an agent to treat , cure , alleviate , improve , diminish or inhibit a disease or condition in an individual treated . a therapeutic agent may be a synthetic product or a product of fungal , bacterial or other microorganism , such as mycoplasma , viral etc ., animal , such as reptile , or plant origin . a therapeutic agent and / or biologically active fragment thereof may be an enzymatically active agent and / or fragment thereof , or may act by inhibiting or blocking an important and / or essential cellular pathway or by competing with an important and / or essential naturally occurring cellular component . examples of radioimaging agents emitting radiation ( detectable radio - labels ) that may be suitable are exemplified by indium - 111 , technitium - 99 , or low dose iodine - 131 . detectable labels , or markers , for use in the present invention may be a radiolabel , a fluorescent label , a nuclear magnetic resonance active label , a luminescent label , a chromophore label , a positron emitting isotope for pet scanner , chemiluminescence label , or an enzymatic label . fluorescent labels include but are not limited to , green fluorescent protein ( gfp ), fluorescein , and rhodamine . chemiluminescence labels include but are not limited to , luciferase and β - galactosidase . enzymatic labels include but are not limited to peroxidase and phosphatase . a histag may also be a detectable label . it is contemplated that an agent may be releasable from the carrier after transport across the blood - brain barrier , for example by enzymatic cleavage or breakage of a chemical bond between the carrier and the agent . the release agent may then function in its intended capacity in the absence of the carrier . the present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope . the following examples have been given with aprotinin . however , it has been demonstrated herein the molecules of the present invention share common properties with aprotinin with respect to their potential as carrier for transporting an agent across the blood brain barrier . these examples thus apply to the molecules of the present invention . for conjugation , paclitaxel ( taxol ™) has 2 strategic positions ( position c2 ′ and c7 ). fig2 illustrates the method of attachment of the vector or carrier of the present invention to paclitaxel . briefly , paclitaxel is reacted with anhydride succinic pyridine for 3 hours at room temperature to attach a succinyl group in position 2 ′. such 2 ′- succinyl paclitaxel has a cleavable ester bond in position 2 ′ which upon cleavage can simply release succinic acid . this cleavable ester bond can be further used for various modifications with linkers , if desired . the resulting 2 ′- o - succinyl - paclitaxel is then reacted with edc / nhs in dmso for 9 hours at room temperature , followed by the addition of the carrier or vector in ringer / dmso for an additional reaction time of 4 hours at room temperature . the reaction of conjugation depicted in fig2 is monitored by hplc . each intermediate , such as paclitaxel , 2 ′- o - succinyl - paclitaxel and 2 ′- o — nhs - succinyl - paclitaxel , is purified and validated using different approaches such as hplc , thin liquid chromatography , nmr ( 13 c or 1 h exchange ), melting point , mass spectrometry . the final conjugate is analyzed by mass spectrometry and sds - polyacrylamide gel electrophoresis . this allows determining the number of paclitaxel molecules conjugated on each vector . transcytosis capacity of aprotinin - paclitaxel conjugate was determined and is reported below in table 5 . as seen in table 5 , conjugation of paclitaxel to aprotinin still was able to cross the in vitro model of the blood brain barrier without affecting the sucrose integrity , thus proving that the molecules ( also referred herein as vectors or carriers ) of the present invention still retain their activity when conjugated to a large chemical entity such as paclitaxel . survival study in the rat brain tumor model was then conducted to verify whether the paclitaxel that was conjugated is still active in vivo . for the rat brain tumor model , rats received an intra - cerebral implantation of 50 000 cns - 1 glioma cells . three ( 3 ) days after , animals received treatment with vehicle ( aprotinin ), paclitaxel ( 5 mg / kg ) or paclitaxel - aprotinin ( 5 mg / kg ) by intravenous injection . treatment was then administered every week until animal was sacrificed ( see fig3 ). rats were monitored every day for clinical symptoms and weight loss . according to the protocol of good animal practice , animals were sacrificed when a weight loss was observed for 3 consecutive days or before if the weight loss was more than 20 % of the animal initial weight . using the same experimental protocol , paclitaxel when injected alone at the maximal tolerated dose ( 54 mg / kg ) was unable to increase mouse survival ( laccabue et al ., 2001 cancer , 92 ( 12 ): 3085 - 92 ). survival study was also conducted in mice implanted with a human brain tumor xenograft . for the mice brain tumor model , mice received an intra - cerebral implantation of 500 000 human u87 glioma cells . 3 days after implantation animals received treatment with paclitaxel - angiopep1 ( 5 mg / kg ) or vehicle by intravenous injection . treatment was then administered every week until animal was sacrificed . mice were monitored every day for clinical symptoms and weight loss . according to the protocol of good animal practice , animals were sacrificed when a weight loss was observed for 3 consecutive days or before if the weight loss was more than 20 % of the animal initial weight . it was now observed that the medium survival for the control group was 19 ± 2 days . for the statistical analysis a 20 % increase in survival was considered significant . as can be seen in fig4 , the conjugate paclitaxel - angiopep - 1 retained its activity , having a statistically significant effect . the survival time of the paclitaxel - angiopep1 treated animals is significantly extended when compared to control group ( p & lt ; 0 . 05 , n = 8 ). results obtained in the two survival studies indicate that the conjugation of paclitaxel with the vector of the present invention increases the animal survival . since proteins generally have several amino groups available for conjugation , amine coupling using sulfo - nhs / edc activation is be used to cross - link therapeutic antibodies with the vectors ( carriers ) of the present invention . this approach was chosen because it is a fast , simple and reproducible coupling technique , because the resulting conjugate is stable while still retaining the biological activity of the antibody and it has a high conjugation capacity that can be reliably controlled and a low non - specific interaction during the coupling procedures . antibodies or antibody fragments ( fab and fab ′ 2 ) have been conjugated with the vector of the present invention to increase their delivery to the brain . various conjugation approaches have been used to first conjugate iggs with aprotinin , having proven that the carriers of the present invention behave exactly as aprotinin . different cross - linkers , such as bs 3 [ bis ( sulfosuccinimidyl ) suberate ], nhs / edc ( n - hydroxysuccinimide and n - ethyl - n ′( dimethylaminopropyl ) carbodimide or sulfo - emcs ([ n - e - maleimidocaproic acid ] hydrazide ) have been tested for the conjugation of igg . bs 3 is a homobifunctional n - hydroxysuccinimide ester that targets accessible primary amines . nhs / edc creates a conjugation of primary amine groups with carboxyl groups . sulfo - emcs are heterobifunctional reactive groups ( maleimide and nhs - ester ) that are reactive toward : sulfhydryl and amino groups . conjugation of igg with aprotinin using the cross - linker bs 3 ( fig5 ) or sulfo - emcs ( fig6 ) was first assessed . transport of igg or igg - conjugates across the bbb was then tested . the uptake of [ 125 i ]- igg to the luminal side of mouse brain capillaries was measured using the in situ brain perfusion method adapted in the inventor &# 39 ; s laboratory for the study of drug uptake in the mouse brain ( dagenais et al ., 2000 , j . cereb . blood flow metab . 20 ( 2 ): 381 - 386 ). the bbb transport constants were determined as previously described by smith ( 1996 , pharm . biotechnol . 8 : 285 - 307 ). igg uptake was expressed as the volume of distribution ( vd ) from the following equation : where q * br is the calculated quantity of [ 125 i ]- igg or [ 125 i ]- igg - aprotinin conjugate per gram of right brain hemisphere and c * pf is the labelled tracer concentration measured in the perfusate . the results of this experiment indicate that there is higher brain uptake for [ 125 i ]- igg - aprotinin conjugate than that of unconjugated [ 125 i ]- igg ( see fig7 ). the conjugation of iggs with aprotinin increases their accumulation in the brain parenchyma in vivo . this study with taxol - angiopep - 2 ( herein referred to peptide no . 97 ( angiopep2 is not amidated ) was conducted to determine whether conjugation of taxol to angiopep - 2 could increase mice survival . the structure of angiopep - 2 is illustrated in seq id no . : 97 . for this experiment , mice received an intra - cerebral implantation of 500 000 human u87 glioma cells . after 3 days following implantation , animals were treated with the vehicle ( dmso / ringer - hepes 80 : 20 v / v ( i . e ., control )) or taxol - angiopep - 2 conjugate ( 3 : 1 , i . e ., ratio of 3 taxol molecules for each peptide ; txlan2 ( 5 mg / kg )) by tail vein injections ( fig8 ). mice were monitored every day for clinical symptoms and weight loss . treatments were administered until animals were sacrificed . as shown in table 6 , we observed that the median survival was 18 days for the control group whereas the median survival for mice receiving the taxol - angiopep - 2 conjugate was 21 days ( fig8 ). survival curve obtained for mice treated with taxol - angiopep - 2 conjugate ( in red ) indicates that the median survival was significantly increased by 17 % ( fig8 ). the statistical analysis presented also in table 6 indicates that administration of taxol - angiopep - 2 conjugate significantly increased survival by 17 % ( p values = 0 . 048 ). although the present invention has been described in details herein and illustrated in the accompanying drawings , it is to be understood that the invention is not limited to the embodiments described herein and that various changes and modifications may be effected without departing from the scope or spirit of the present invention . while the invention has been described in connection with specific embodiments thereof , it will be understood that it is capable of further modifications and this application is intended to cover any variations , uses , or adaptations of the invention following , in general , the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth , and as follows in the scope of the appended claims .
the invention relates to improvements in the field of drug delivery . more particularly , the invention relates to polypeptide derived from aprotinin and from aprotinin analogs as well as conjugates and pharmaceutical compositions comprising these polypeptides . the present invention also relates to the use of these polypeptide for transporting a compound or drug across the blood - brain barrier of an individual and in the treatment and diagnosis of neurological diseases .