Patent Publication Number: US-8535656-B2

Title: Amphiphilic polymer-protein conjugates and methods of use thereof

Description:
This application is a divisional application of U.S. patent application Ser. No. 12/488,846, filed on Jun. 22, 2009, now U.S. Pat. No. 8,168,222, which is a continuation-in-part of U.S. patent application Ser. No. 10/935,293, filed on Sep. 7, 2004, now U.S. Pat. No. 8,017,151. U.S. patent application Ser. No. 12/488,846 also claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/132,614, filed on Jun. 20, 2008; U.S. Provisional Patent Application No. 61/208,489, filed on Feb. 25, 2009; and U.S. Provisional Patent Application No. 61/208,537, filed on Feb. 25, 2009. The foregoing applications are incorporated by reference herein. 
    
    
     This invention was made with government support under Grant No. R01 NS051334 awarded by the National Institutes of Health. The government has certain rights in the invention. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the transport of biologically active proteins across biological membranes, particularly across the blood-brain barrier. 
     BACKGROUND OF THE INVENTION 
     The blood-brain barrier (BBB) is one of the most restrictive barriers in biology. Numerous factors work together to create this restrictive barrier. Electron microscopy studies have demonstrated that tight junctions between brain vascular endothelial cells and other endothelial cell modifications (e.g., decreased pinocytosis, lack of intracellular fenestrae) prevented the formation of a plasma ultrafiltrate. Enzymatic activity at the BBB further limits entry of some substances, especially of monoamines and some small peptides (Baranczyk-Kuzma and Audus (1987) J. Cereb. Blood Flow Metab., 7:801-805; Hardebo and Owman (1990) Pathophysiology of the Blood-Brain Barrier, pp. 41-55 (Johansson et al., Eds.) Elsevier, Amsterdam; Miller et al. (1994) J. Cell. Physiol., 161:333-341; Brownson et al. (1994) J. Pharmacol. Exp. Ther., 270:675-680; Brownlees and Williams (1993) J. Neurochem., 60:793-803). Saturable, brain-to-blood efflux systems, such as p-glycoprotein (Pgp), also prevent the accumulation of small molecules and lipid soluble substances (Taylor, E. M. (2002) Clin. Pharmacokinet., 41:81-92; Schinkel et al. (1996) J. Clin. Invest., 97:2517-2524). Peripheral factors such as protein binding/soluble receptors, enzymatic degradation, clearance, and sequestration by tissues also affect the ability of a substance to cross the BBB by limiting presentation; these factors are especially important for exogenously administered substances (Banks and Kastin (1993) Proceedings of the International Symposium on Blood Binding and Drug Transfer, pp. 223-242 (Tillement et al., Eds.) Fort and Clair, Paris). 
     Substances are able to cross the BBB by way of a few pathways. Such pathways include: 1) saturable transporter systems, 2) adsorptive transcytosis wherein the compound to be transported is internalized by a cell in the BBB and routed to the abluminal surface for deposition into the brain intracellular fluid compartment, 3) transmembrane diffusion wherein the substance dissolves into the lipid bilayer which forms the membranes of the cells comprising the BBB, and 4) extracellular pathways wherein compounds exploit the residual leakiness of the BBB. 
     The hydrophilicity, the lack of stability due to enzymatic or chemical degradation, and the lack of transport carriers capable of shuttling polypeptides across cell membranes all play a part in precluding most polypeptides from transport into the brain. Several approaches to modify polypeptides to alter their BBB permeability have been attempted including: 1) conjugation with proteins that naturally cross the BBB (Raub and Audus (1990) J. Cell. Sci., 97:127-138; Banks and Broadwell (1994) J. Neurochem., 62:2404-2419; Bickel et al. (2001) Adv. Drug Deliv. Rev., 46:247-279); 2) modifying the polypeptide with cationic groups, i.e. “cationization” (Kumagai et al. (1987) J. Biol. Chem., 262:15214-15219; Triguero et al. (1989) Proc. Natl. Acad. Sci., 86:4761-4765; Triguero et al. (1991) J. Pharmacol. Exp. Ther., 258:186-192; Bickel et al. (2001) Adv. Drug Deliv. Rev., 46:247-279); 3) modifying the polypeptides with polyethylene glycol (PEG; Delgado et al. (1992) Crit. Rev. Ther. Drug. Carrier. Syst., 9:249-304; 4) linking the polypeptides to antibodies targeting certain cellular receptors (Zhang and Pardridge (2001) Brain Res., 889:49-56; Zhang and Pardridge (2001) Stroke, 32:1378-1384; Song et al. (2002) J. Pharmacol. Exp. Ther., 301:605-610) and 5) stearoylation of the polypeptide (Chekhonin et al. (1991) FEBS Lett., 287:149-152; Chopineau et al. (1998) J. Contr. Release, 56:231-237; Chekhonin et al. (1995) Neuroreport., 7:129-132). 
     Another method employed to increase BBB transport is by the use of Pluronic® block copolymers (BASF Corporation, Mount Olive, N.J.). Pluronic® block copolymers (listed in the US and British Pharmacopoeia under the name “poloxamers”) consist of ethylene oxide (EO) and propylene oxide (PO) segments arranged in a basic A-B-A structure: EO a -PO b -EO a . This arrangement results in an amphiphilic copolymer, in which altering the number of EO units (a) and the number of PO units (b) can vary its size, hydrophilicity, and lipophilicity. A characteristic of Pluronic® copolymers is the ability to self-assemble into micelles in aqueous solutions. The noncovalent incorporation of drugs into the hydrophobic PO core of the Pluronic® micelle imparts to the drug increased solubility, increased metabolic stability, and increased circulation time (Kabanov and Alakhov (2002) Crit. Rev. Ther. Drug Carrier Syst., 19:1-72; Allen et al. (1999) Coll. Surfaces, B: Biointerfaces, 16:3-27). 
     Pluronic® micelles conjugated with antibody to alpha 2GP have been shown to deliver neuroleptic drugs and fluorescent dyes to the brain in mice (Kabanov et al. (1989) FEBS Lett., 258:343-345; Kabanov et al. (1992) J. Contr. Release, 22:141-157). Additionally, selected Pluronic® block copolymers, such as P85, are potent inhibitors of p-glycoprotein (Pgp) and increase entry of the Pgp-substrates to the brain across BBB (Batrakova et al. (1998) Pharm. Res., 15:1525-1532; Batrakova et al. (1999) Pharm. Res., 16:1366-1372; Batrakova et al. (2001) J. Pharmacol. Exp. Ther., 296:551-557). The mechanism of the Pluronic® effect in the latter case involves decreases in membrane microviscosity by Pluronic® and depletion of intracellular ATP, which together blocked the Pgp efflux function in brain endothelial cells (Batrakova et al. (2001) J. Pharmacol. Exp. Ther., 299:483-493; Batrakova et al. (2003) J. Pharmacol. Exp. Ther., 304:845-854). Pluronic® did not induce toxic effects in the BBB as revealed by the lack of alteration in paracellular permeability of the barrier (Batrakova et al. (1998) Pharm. Res., 15:1525-1532; Batrakova et al. (2001) J. Pharmacol. Exp. Ther., 296:551-557). It has been noted that Pluronic® fluorescently labeled with FITC can accumulate in the brain following systemic administration in mice (Kabanov et al. (1992) J. Contr. Release, 22:141-157). Furthermore, selected Pluronic® copolymers can cross the membranes of the brain endothelial cells (Batrakova et al. (2003) J. Pharmacol. Exp. Ther., 304:845-854). 
     Notably, Pluronic® copolymers have also been used in combination with anticancer drugs in the treatment of multidrug resistant (MDR) cancers (Alakhov et al. (1996) Bioconjug. Chem., 7:209-216; Alakhov et al. (1999) Colloids Surf., B: Biointerfaces, 16:113-134; Venne et al. (1996) Cancer Res., 56:3626-3629). Indeed, Phase I and II clinical trial are being performed on doxorubicin formulated with Pluronic® (“SP1049C”) for the treatment of adenocarinoma of esophagus and soft tissue sarcoma, both cancers with high incidence of MDR (Ranson et al. (2002) 5th international symposium on polymer therapeutics: from laboratory to clinical practice, pp. 15, The Welsh School of Pharmacy, Cardiff University, Cardiff, UK). 
     SUMMARY OF THE INVENTION 
     The present invention broadly relates to compositions and methods for the delivery of biologically active proteins across biological membranes, specifically, the blood brain barrier, wherein the protein is conjugated with an amphiphilic polymer. 
     According to one aspect of the invention, a method for delivering a protein across the blood-brain barrier into the central nervous system of an animal is provided. The method comprises administering to the animal a conjugate comprising the protein conjugated to an amphiphilic block copolymer by a cleavable linker moiety. In a preferred embodiment, the linker is cleaved or substantially cleaved either while traversing the BBB or once having crossed the BBB. 
     In a particular embodiment of the invention, the amphiphilic block copolymer is a copolymer comprising at least one poly(oxyethylene) segment and at least one poly(oxypropylene) segment. In yet another embodiment, the amphiphilic block copolymer is of the formula selected from the group consisting of Formulas I-VI, as shown below. In still another embodiment, the amphiphilic block copolymer is a Pluronic®, such as Pluronic® L121. 
     According to another aspect of the invention, the amphiphilic block copolymer of the instant invention has a hydrophilic-lipophilic balance (HLB) of less than or equal to 20, preferably less than or equal to 16, more preferably less than or equal to 12, and most preferably less than or equal to 8. 
     According to yet another aspect of the invention, the amphiphilic block copolymer of the instant invention has a hydrophobe molecular weight greater than or equal to 1500 Da, preferably greater than or equal to 2000 Da, more preferably greater than or equal to 2500 Da, and most preferably greater than or equal to 3000 Da. 
     In accordance with another aspect of the present invention, the cleavable linker moiety linker of the conjugate of the instant invention comprises a disulfide bond. Alternatively, the cleavable linker moiety linker comprises a recognition site for a protease. Exemplary proteases include, without limitation, endosomal cathepsins, cathepsin B, lysosomal proteases, and colagenase. 
     According to another aspect of the invention, the protein of the conjugates of the instant invention is a therapeutic protein. Exemplary therapeutic proteins include, without limitation, cytokines; growth factors such as, without limitation, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF); amyloid beta binders; enkephalin; modulators of α-, β-, and/or γ-secretases; Glial-derived neutrotrophic factor (GDNF); vasoactive intestinal peptide; acid alpha-glucosidase (GAA); acid sphingomyelinase; iduronate-2-sultatase (I2S); α-L-iduronidase (IDU); β-Hexosaminidase A (HexA); Acid β-glucocerebrosidase; N-acetylgalactosamine-4-sulfatase; and α-galactosidase A. In a particular embodiment, the therapeutic protein is a superoxide dismutase (SOD), (including, but not limited to, Cu/Zn SOD or SOD1 as well as other isoforms of SOD that can be used intra- and extracellularly for elimination of reactive oxygen species (ROS) under inflammatory and other disease conditions) and leptin, a protein that can be used for the treatment of obesity. 
    
    
     
       BRIEF DESCRIPTIONS OF THE DRAWING 
         FIG. 1  is a scheme for the synthesis of Pluronic®-HRP conjugates. Pluronic®-Amine is activated by bifunctional reagents (DSP or DSS) to obtain hydroxysuccinimide derivatives of Pluronic® (A and B, respectively) capable of reacting with the protein aminogroups (Step 1). The excess of the reagents was removed by gel filtration, and the activated polymers were immediately reacted with HRP (Step 2). 
         FIGS. 2A and 2B  are graphs of the chromatographic isolation of HRP-Pluronic® conjugates. The peaks correspond to (1) bis-substituted HRP; (2) mono-substituted HRP; and (3) unmodified HRP. The substitution degree in each fraction was confirmed by the TNBS assay. 
         FIG. 3  is a scheme showing alternative methods for synthesis of Pluronic®-HRP conjugates with degradable links. In Method 1, N-hydroxybenzotriazolyde derivative of Pluronic® is prepared by reacting with bis-(N-hydroxybenzotriazolyl)phenylphosphate and then used for modification of HRP. In Method 2, Pluronic®-succinate is obtained by reacting the polymer with succinic anhydride and then activated by water-soluble ethyl dimethylaminopropyl carbodiimide (EDC) to modify HRP. 
         FIG. 4  is a bar graph of the level of binding of unmodified HRP and Pluronic® HRP conjugates, described in Table 6 below, with BBMEC monolayers in 60 minutes. Data are mean±SEM (n=4). (*) indicates significant difference compared to HRP. 
         FIGS. 5A and 5B  are graph demonstrating the apical to basolateral permeability of HRP and Pluronic®-HRP in BBMEC monolayers as a function of time. Data are mean±SEM (n=4). 
         FIG. 6  is a bar graph showing the effects of various Pluronic® copolymers on the viscosity of BBMEC monlayers. 
         FIG. 7A  is a graph depicting the binding of unmodified HRP and stearoylated (St-HRP) with BBMEC monolayers as a function of time. Data are mean±SEM (n=4). (*) shows significant difference between HRP and St-HRP groups.  FIG. 7B  is a graph depicting the apical to basolateral permeability of HRP and St-HRP in BBMEC monolayers as a function of time. Data are mean±SEM (n=4). (*) shows significant difference between HRP and St-HRP groups. 
         FIG. 8A  provides a schematic for the conjugation of SOD1 and Pluronics® via a non-degradable linker.  FIG. 8B  provides a schematic for the generation of mono-amine Pluronics®. 
         FIG. 9A  provides an image of an SDS-PAGE of SOD1 and SOD1/P85 conjugates.  FIG. 9B  provides an image of a native, in-gel SOD1 activity assay of SOD1 and SOD1/P85 conjugates.  FIG. 9C  is a graph of SOD1 enzymatic activity based on pyrogallol autoxidation. 
         FIGS. 10A and 10B  provide the native, in-gel SOD1 activity assay of SOD1/P85 conjugates uptake within CATH.a neuronal cells ( FIG. 10A ) and SOD1/L81 conjugates uptake within BBMEC ( FIG. 10B ) at various incubation time courses. Bar graphs are provided of the activity. 
         FIGS. 11A and 11B  provide the cellular localization of SOD1 and SOD1/P85 labeled by Alex Flour 680 within CATH.a neuronal cells ( FIG. 11A ) and MDCK cells ( FIG. 11B ) at various time intervals. 
         FIG. 12A  provides multiple-time regression analysis of  125 I-leptin(ss)-P85 transport across the BBB. Ki (slope) was measured to be 0.272±0.037 μl/g-minute. Vi (intercept)=19.21±1.69 μl/g, r=0.92, p&lt;0.0001 and n=2 mice/time point. The brain/serum ratio of  125 I-leptin(ss)-P85, and +nonradioactive leptin(ss)-P85 10 minutes after i.v. injection is shown in the inset (data are means±SE).  FIG. 12B  demonstrates that nonradioactive rleptin (1 μg/mouse) did not inhibit the brain/serum ratio of  125 I-leptin(ss)-P85 at 10 minutes after i.v. injections significantly.  FIG. 12C  is a graph showing that recombinant leptin (1 μg/mouse) did inhibit the influx of simultaneously injected  131 I-rleptin (*p&lt;0.0003, n=10 mice/group). 
         FIG. 13A  demonstrates the clearance of  125 I-leptin(ss)-P85 from blood after i.v. injection. The inset shows the initial distribution phase was linear and the half-time disappearance from blood was 32.35 minutes.  FIG. 13B  is a graph of the percent of intravenously injected dose of  125 I-leptin(ss)-P85 taken up by each gram of brain tissue from 0 to 60 minutes after injection. The maximal value was estimated by a one-site binding model to approach 0.263% Inj/g. 
         FIG. 14A  shows the acid precipitation of  125 I-leptin(ss)-P85 from brain and serum. *p&lt;0.05 when compared with 6, 18, and 24 hour groups (n=3 mice/group).  FIG. 14B  is the SDS-PAGE gel electrophoresis of  125 I-leptin(ss)-P85 extracted from blood and brain 10 minutes after i.v. bolus injection compared with  125 I-leptin(ss)-P85 ( FIG. 14C ) in buffer as a standard. Molecular weight markers (in kDa) are shown on the left. S=Serum and B=Brain. 
         FIG. 15A  is a graph of capillary depletion with vascular washout 10 minutes after i.v. injection. The brain parenchyma/serum ratio (μl/g) was measured to be 3.78±0.26 μl/g (n=8 mice) and was significantly higher than the capillary/serum ratio of 3.04±0.18 μl/g (n=8 mice, p&lt;0.05).  FIG. 15B  provides the time courses of the brain/perfusate ratio of  125 I-leptin-P85 (Ki=0.892±0.236 μl/g-min, r=0.88, p&lt;0.05, n=3/time point).  FIG. 15C  demonstrates the inclusion of nonradioactive leptin(ss)-P85 or rleptin (100 ng/ml) in perfusion buffer did not alter the brain/perfusion ratio of  125 I-leptin(ss)-P85 at 5 minutes after i.v. injection. The brain/perfusion ratio as measured by brain perfusion was 21.82±4.04 μl/g with  125 I-leptin(ss)-P85 only, was 19.32±3.34 μl/g with +nonradioactive rleptin, and was 21.88±5.35 μl/g with +nonradioactive leptin(ss)-P85 (n=5/group). 
         FIG. 16A  provides a schematic for the modification of leptin with Pluronic®.  FIG. 16B  provides a schematic for the production of Pluronic® mono-sulfone reagent.  FIG. 16C  provides a schematic for the conjugation of Pluronic® to leptin via disulfide bridging.  FIG. 16D  provides a scheme for the conjugation of Pluronic® to the N-terminus of leptin. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In accordance with the present invention, compositions and methods are provided for the transport of biologically active proteins across biological membranes, particularly across the blood-brain barrier. Specifically, the proteins are conjugated to amphiphilic polymers for transport across the BBB. The conjugation of amphiphilic polymers to the proteins preferably results in i) increased stability of the protein in circulation by means of the formation of a protective hydrophilic polymer layer around the protein and ii) increasing the interaction of the protein with the BBB due to the influence of the lipophilic portions of the amphiphilic polymer. 
     In a preferred embodiment of the instant invention, the protein is conjugated to the amphiphilic polymer via a degradable linker. Preferably, the linker is cleaved or substantially cleaved during transport through the BBB and/or after crossing the BBB. Such cleavable linkers allow for the release of free protein in the central nervous system (CNS). 
     In a particular embodiment of the invention, the amphiphilic polymers are conjugated to the protein via modification of a free amino, thiol, or carboxyl group on the protein. The amphiphilic polymer may be mono-activated through a linker. 
     In yet another embodiment of the invention, methods are provided for the administration of amphiphilic polymer-protein conjugates comprising a therapeutic protein to a patient in order to treat conditions in which the therapeutic protein is known to be effective. In a particular embodiment, the conjugates are administered systemically. According to another aspect, the conjugates are administered in combination with free amphiphilic polymer. 
     In accordance with another embodiment of the invention, fatty acids are conjugated to the protein of interest and the resultant conjugates are employed in the methods described hereinabove for the amphiphilic polymer-conjugates. In a preferred embodiment, the fatty acids are essential fatty acids such as, without limitation, linoleic acid and alpha-linoleic acid. 
     I. DEFINITIONS 
     The following definitions are provided to facilitate an understanding of the present invention: 
     As used herein, the term “polymer” denotes molecules formed from the chemical union of two or more repeating units or monomers. The term “block copolymer” most simply refers to conjugates of at least two different polymer segments, wherein each polymer segment comprises two or more adjacent units of the same kind. 
     As used herein, the term “lipophilic” refers to the ability to dissolve in lipids. 
     As used herein, the term “hydrophilic” means the ability to dissolve in water. 
     As used herein, the term “amphiphilic” means the ability to dissolve in both water and lipids. Typically, an amphiphilic compound comprises a hydrophilic portion and a lipophilic portion. 
     The term “substantially cleaved” refers to the cleavage of the amphiphilic polymer from the protein of the conjugates of the instant invention, preferably at the linker moiety. “Substantial cleavage” occurs when at least 50% of the conjugates are cleaved, preferably at least 75% of the conjugates are cleaved, more preferably at least 90% of the conjugates are cleaved, and most preferably at least 95% of the conjugates are cleaved. 
     “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. 
     A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., Tween 80, Polysorbate 80), emulsifier, buffer (e.g., Tris HCl, acetate, phosphate), bulking substance (e.g., lactose, mannitol), excipient, auxilliary agent or vehicle with which an active agent of the present invention is administered. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. The compositions can be incorporated into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes or micelles. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of components of a pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention can be prepared, for example, in liquid form, or can be in dried powder form (e.g., lyophilized). Suitable pharmaceutical carriers are described in “Remington&#39;s Pharmaceutical Sciences” by E. W. Martin (Mack Publishing Co., Easton, Pa.); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, 20th Edition, (Lippincott, Williams and Wilkins), 2000; Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999. 
     II. AMPHIPHILIC POLYMERS 
     Amphiphilic polymers according to the instant invention are preferably amphiphilic block copolymers. Generally, amphiphilic block copolymers can be described in terms of having hydrophilic “A” and hydrophobic “B” block segments. Thus, for example, a copolymer of the formula A-B-A is a triblock copolymer consisting of a hydrophilic block connected to a hydrophobic block connected to another hydrophilic block. 
     Amphiphilic block copolymers which may be used in the practice of this invention are exemplified by the block copolymers having the formulas: 
     A-B-A type: 
                         
A-B type:
 
                         
B-A-B type:
 
                         
in which x, y, z, i, and j independently of each other have values from about 2 to about 800, preferably from about 5 to about 200, more preferably from about 5 to about 80, and wherein for each R 1 , R 2  pair, as shown in formula (IV) and (V), one is hydrogen and the other is a methyl group. Formulas (I) through (III) are oversimplified in that, in practice, the orientation of the isopropylene radicals within the B block will be random. This random orientation is indicated in formulas (IV) and (V), which are more complete. Such poly(oxyethylene)-poly(oxypropylene) compounds have been described by Santon (Am. Perfumer Cosmet. (1958) 72(4):54-58); Schmolka (Loc. cit. (1967) 82(7):25-30), Schick, ed. (Non-ionic Suifactants, Dekker, N.Y. 1967 pp. 300-371). A number of such compounds are commercially available under such generic trade names as “lipoloxamers”, “Pluronics®,” “poloxamers,” and “synperonics.” Pluronic® copolymers within the B-A-B formula, as opposed to the A-B-A formula typical of Pluronics®, are often referred to as “reversed” Pluronics®, “Pluronic® R” or “meroxapol.”
 
     The “polyoxamine” polymer of formula (IV) is available from BASF under the tradename Tetronic®. The order of the polyoxyethylene and polyoxypropylene blocks represented in formula (IV) can be reversed, creating Tetronic R®, also available from BASF (see, Schmolka, J. Am. Oil. Soc. (1979) 59:110). 
     Polyoxypropylene-polyoxyethylene block copolymers can also be designed with hydrophilic blocks comprising a random mix of ethylene oxide and propylene oxide repeating units. To maintain the hydrophilic character of the block, ethylene oxide would predominate. Similarly, the hydrophobic block can be a mixture of ethylene oxide and propylene oxide repeating units. Such block copolymers are available from BASF under the tradename Pluradot™. 
     The hydrophobic/hydrophilic properties of a given block copolymer depend upon the ratio of the number of oxypropylene groups to the number of oxyethylene groups. For a composition comprising an A-B-A type block copolymer of poly(oxyethylene)-poly(oxypropylene), for example, this relationship, taking into account the molecular masses of the central hydrophobic block and the terminal hydrophilic blocks, can be expressed as follows:
 
 n =( H/L )(1.32)
 
in which H is the number of oxypropylene units and L is the number of oxyethylene units. In the general case of a block copolymer containing hydrophobic B-type segments and hydrophilic A-type segments, the hydrophobic hydrophilic properties and micelle-forming properties are related to the value n (which is related to the hydrophilic-lipophilic balance (HLB) described hereinbelow) as defined as:
 
 n =(| B|/|A |)×( b/a )
 
where |B| and |A| are the number of repeating units in the hydrophobic and hydrophilic blocks of the copolymer, respectively, and b and a are the molecular weights for the respective repeating units.
 
     Selecting a block copolymer with the appropriate n value depends upon the hydrophobic/hydrophilic properties of the specific agent, or the composite hydrophilic/hydrophilic properties of a mixture of agents to be formulated. One aspect of the present invention involves utilizing a mixture of different block-copolymers of poly(oxyethylene)-poly(oxypropylene) to achieve a prescribed hydrophobic-hydrophilic balance. For example, a first block copolymer may have an n of 1.0 whereas a second may have a value of 1.5. If material having an n of 1.3 is desired, a mixture of one weight portion of the first block copolymer and 1.5 weight portion of the second block-copolymer can be employed. 
     Thus, a more generalized relationship for such mixtures can be expressed as follows:
 
 N =(1.32)[( H   1   m   1 )/(( L   1 )( m   1   +m   2 ))+( H   2   m   2 )/(( L   2 )( m   1   +m   2 ))]
 
in which H 1  and H 2  are the number of oxypropylene units in the first and second block copolymers, respectively; L 1  is the number of oxyethylene units in the first block copolymer; L 2  is the number of oxyethylene units in the second block copolymer; m 1  is the weight proportion of the first block-copolymer; and m 2  is the weight proportion of the second block copolymer.
 
     An even more general case of a mixture of K block copolymers containing hydrophobic B-type block copolymers and hydrophilic A-type block copolymers, the N value can be expressed as follows: 
             N   =       (     b   /   a     )     ⁢       ∑     i   =   1     k     ⁢     [       (            B        i     /          A        i       )     ,     (       m   i     /   M     )       ]               
where |A| i  and |B| i  are the numbers of repeating units in the hydrophilic (A-type) and hydrophobic (B-type) blocks, respectively, of the i-th block copolymer, m is the weight proportion of this block copolymers, M is the sum of weight proportions of all block copolymers in the mixture
 
             M   =       ∑     i   =   1     k     ⁢     m   i             
and a and b are the molecular weights for the repeating units of the hydrophilic and hydrophobic blocks of these block copolymers, respectively.
 
     If only one block copolymer of poly(oxyethylene)-poly(oxypropylene) is utilized, N will equal n. An analogous relationship will apply to compositions employing more than two block copolymers of poly(oxyethylene)-poly(oxypropylene) (EO-PO). Where mixtures of block copolymers are used, a value N will be used, which value will be the weighted average of n for each contributing copolymer, with the averaging based on the weight portions of the component copolymers. The value N can be used to estimate the micelle-forming properties of a mixture of copolymers. The use of the mixtures of block copolymers enhances solubility and prevents aggregation of more hydrophobic block copolymers in the presence of serum proteins. 
     A number of Pluronic® copolymers are designed to meet the following formula: 
                         
The ordinarily skilled artisan will recognize that the values of x, y, and z will usually represent a statistical average and that the values of x and z are often, though not necessarily, the same. The characteristics of a number of Pluronic® copolymers corresponding to formula (I) are as follows:
 
     
       
         
           
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                 Hydrophobe 
                 CMC 
                 Hydrophobe 
               
               
                 Copolymer 
                 Weight 
                 (% w/v) 
                 percentage 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 Pluronic ® L61 
                 1750 
                 0.0003 
                 90 
               
               
                 Pluronic ® L64 
                 1750 
                 0.002 
                 60 
               
               
                 Pluronic ® F68 
                 1750 
                 4-5 
                 20 
               
               
                 Pluronic ® P85 
                 2250 
                  0.005-0.007 
                 50 
               
               
                 Pluronic ® F127 
                 4000 
                  0.003-0.005 
                 30 
               
               
                 Pluronic ® F108 
                 3250 
                 0.0035-0.007 
                 20 
               
               
                   
               
            
           
         
       
     
     These critical micelle concentrations (CMC) values were determined by the surface tension method described in Kabanov et al. (Macromolecules (1995) 28: 2303-2314). 
     These block copolymers can be prepared by the methods set out, for example, in U.S. Pat. No. 2,674,619 and are commercially available from BASF under the trademark Pluronic®. Pluronic® block copolymers are designated by a letter prefix followed by a two or a three digit number. The letter prefixes (L, P, or F) refer to the physical form of each polymer, (liquid, paste, or flakeable solid). The numeric code defines the structural parameters of the block copolymer. The last digit of this code approximates the weight content of EO block in tens of weight percent (for example, 80% weight if the digit is 8, or 10% weight if the digit is 1). The remaining first one or two digits designate the molecular mass of the central PO block. To decipher the code, one should multiply the corresponding number by 300 to obtain the approximate molecular mass in daltons (Da). Therefore Pluronic nomenclature provides a convenient approach to estimate the characteristics of the block copolymer in the absence of reference literature. For example, the code ‘F127’ defines the block copolymer, which is in solid flake form, has a PO block of 3600 Da (12×300) and 70% weight of EO. The precise molecular characteristics of each Pluronic® block copolymer can be obtained from the manufacturer. 
     Additional specific poly(oxyethylene)-poly(oxypropylene) block copolymers which can be used in practicing this invention include Pluronic® and Pluronic®-R block copolymers, such as those listed in Table 2. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                 Hydro- 
                   
                   
                 Hydro- 
               
               
                   
                 Hydrophobe 
                 phobe 
                 Pluronic- 
                 Hydrophobe 
                 phobe 
               
               
                 Pluronic ® 
                 Weight 
                 % 
                 R ® 
                 Weight 
                 % 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 L31 
                 950 
                 90 
                 10R5 
                 1000 
                 50 
               
               
                 L35 
                 950 
                 50 
                 10R8 
                 1000 
                 20 
               
               
                 F38 
                 900 
                 20 
                 12R3 
                 1200 
                 70 
               
               
                 L42 
                 1200 
                 80 
                 17R1 
                 1700 
                 90 
               
               
                 L43 
                 1200 
                 70 
                 17R2 
                 1700 
                 80 
               
               
                 L44 
                 1200 
                 60 
                 17R4 
                 1700 
                 60 
               
               
                 L61 
                 1750 
                 90 
                 17R8 
                 1700 
                 20 
               
               
                 L62 
                 1750 
                 80 
                 22R4 
                 2200 
                 60 
               
               
                 L63 
                 1750 
                 70 
                 25R1 
                 2500 
                 90 
               
               
                 L64 
                 1750 
                 60 
                 25R2 
                 2500 
                 80 
               
               
                 P65 
                 1750 
                 50 
                 25R4 
                 2500 
                 60 
               
               
                 F68 
                 1750 
                 20 
                 25R5 
                 2500 
                 50 
               
               
                 L72 
                 2050 
                 80 
                 25R8 
                 2500 
                 50 
               
               
                 P75 
                 2050 
                 50 
                 31R1 
                 3100 
                 90 
               
               
                 F77 
                 2050 
                 30 
                 31R2 
                 3100 
                 80 
               
               
                 L81 
                 2250 
                 90 
                 31R4 
                 3100 
                 60 
               
               
                 P84 
                 2250 
                 60 
               
               
                 P85 
                 2250 
                 50 
               
               
                 F87 
                 2250 
                 30 
               
               
                 F88 
                 2250 
                 20 
               
               
                 L92 
                 2750 
                 80 
               
               
                 F98 
                 2750 
                 20 
               
               
                 L101 
                 3250 
                 90 
               
               
                 P103 
                 3250 
                 70 
               
               
                 P104 
                 3250 
                 60 
               
               
                 P105 
                 3250 
                 50 
               
               
                 F108 
                 3250 
                 20 
               
               
                 L121 
                 4000 
                 90 
               
               
                 L122 
                 4000 
                 80 
               
               
                 L123 
                 4000 
                 70 
               
               
                 F127 
                 4000 
                 30 
               
               
                   
               
            
           
         
       
     
     Other specific poly(oxyethylene)-poly(oxypropylene) block copolymers which can be included in compositions described herein are the Tetronic® and Tetronic® R nonionic surfactants of formula (IV) and (V), above, which are tetrafunctional block copolymers derived from the addition of ethylene oxide and propylene oxide to ethylenediamine. Tetronic® and Tetronic® R copolymers include, without limitation, those set forth in Table 3. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Tetronic ® 
                 Form 
                 HLB 
                 Average MW 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 304 
                 Liquid 
                 16 
                 1650 
               
               
                   
                 701 
                 Liquid 
                 3 
                 3600 
               
               
                   
                 704 
                 Liquid 
                 15 
                 5500 
               
               
                   
                 901 
                 Liquid 
                 3 
                 4700 
               
               
                   
                 904 
                 Liquid 
                 15 
                 6700 
               
               
                   
                 908 
                 Solid 
                 31 
                 25000 
               
               
                   
                 1107  
                 Solid 
                 24 
                 15000 
               
               
                   
                 1301  
                 Liquid 
                 2 
                 6800 
               
               
                   
                 1307  
                 Solid 
                 24 
                 18000 
               
               
                   
                  90R4 
                 Liquid 
                 7 
                 7240 
               
               
                   
                 150R1  
                 Liquid 
                 1 
                 8000 
               
               
                   
                   
               
            
           
         
       
     
     In selecting PEO-PPO copolymers for use in the instant invention, the poly(oxyethylene) units making up the first segment need not consist solely of ethylene oxide as previously mentioned. Nor is it necessary that all of the poly(oxypropylene) segment consist solely of propylene oxide units. Instead, in the simplest cases, for example, at least one of the monomers in segment A may be substituted with a side chain group. 
     In addition, the present invention can also be practiced using diamine-linked polyoxyethylene-polyoxypropylene polymers of formula: 
                         
wherein the same number and sequence of polyether moieties extend symetrically from each nitrogen; i and j have values from about 2 to about 800, preferably from about 5 to about 200, more preferably from about 5 to about 80; R* is an alkylene of about 2 to about 6 carbons, a cycloalkylene of about 5 to about 8 carbons or phenylene; R 1  and R 2 , either (a) both represent hydrogen or (b) one represents hydrogen and the other represents methyl; R 3  and R 4  either (a) both are hydrogen or (b) one is hydrogen and the other is methyl; if both of R 3  and R 4  represent hydrogen, then one of R 5  and R 6  represents hydrogen and the other is methyl; and both of R 5  and R 6  represent hydrogen when one of R 3  and R 4  represents methyl.
 
     Over 30 Pluronic® copolymers with different lengths of hydrophilic ethylene oxide (N EO ) and hydrophobic propylene oxide (N PO ) blocks are available from BASF Corp. (see, for example, Tables 2 and 4). These molecules are characterized by different hydrophilic-lipophilic balance (HLB) and CMC (Kozlov et al. (2000) Macromolecules, 33:3305-3313; see, for example, Tables 3 and 4). The HLB value, which typically falls in the range of 1 to 31 for Pluronic® block copolymers, reflects the balance of the size and strength of the hydrophilic groups and lipophilic groups of the polymer (see, for example, Attwood and Florence (1983) “Surfactant Systems: Their Chemistry, Pharmacy and Biology,” Chapman and Hall, New York) and can be determined experimentally by, for example, the phenol titration method of Marszall (see, for example, “Parfumerie, Kosmetik”, Vol. 60, 1979, pp. 444-448; Rompp, Chemistry Lexicon, 8th Edition 1983, p. 1750; U.S. Pat. No. 4,795,643). HLB values for Pluronic® polymers are available from BASF Corp. HLB values can be approximated by the formula: 
               HLB   =         -   36     ⁢           ⁢     y       2   ⁢   x     +   y         +   33       ,         
wherein y is the number of hydrophobic propylene oxide units and x is the number of hydrophilic ethylene oxide units, though HLB values provided by BASF are preferred. Notably, as hydrophobicity increases, HLB decreases.
 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Pluronic ® 
                 MW (a)   
                 N PO   (b)   
                 N EO   (b)   
                 HLB (a)   
                 CMC, μM (c)   
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 L31 
                 1100 
                 17.1 
                 2.5 
                 5 
                 1180 
               
               
                 L35 
                 1900 
                 16.4 
                 21.6 
                 19 
                 5260 
               
               
                 F38 
                 4700 
                   
                   
                 31 
                   
               
               
                 L42 
                 1630 
                   
                   
                 8 
                   
               
               
                 L43 
                 1850 
                 22.3 
                 12.6 
                 12 
                 2160 
               
               
                 L44 
                 2200 
                 22.8 
                 20.0 
                 16 
                 3590 
               
               
                 L61 
                 2000 
                 31 
                 4.5 
                 3 
                 110 
               
               
                 L62 
                 2500 
                 34.5 
                 11.4 
                 7 
                 400 
               
               
                 L63 
                 2650 
                   
                   
                 11 
                   
               
               
                 L64 
                 2900 
                 30 
                 26.4 
                 15 
                 480 
               
               
                 P65 
                 3400 
                   
                   
                 17 
                   
               
               
                 F68 
                 8400 
                 29 
                 152.7 
                 29 
                 480 
               
               
                 L72 
                 2750 
                   
                   
                 7 
                   
               
               
                 P75 
                 4150 
                   
                   
                 17 
                   
               
               
                 F77 
                 6600 
                   
                   
                 25 
                   
               
               
                 L81 
                 2750 
                 42.7 
                 6.2 
                 2 
                 23 
               
               
                 P84 
                 4200 
                 43.4 
                 38.2 
                 14 
                 71 
               
               
                 P85 
                 4600 
                 39.7 
                 52.3 
                 16 
                 65 
               
               
                 F87 
                 7700 
                 39.8 
                 122.5 
                 24 
                 91 
               
               
                 F88 
                 11400 
                 39.3 
                 207.8 
                 28 
                 250 
               
               
                 L92 
                 3650 
                 50.3 
                 16.6 
                 6 
                 88 
               
               
                 F98 
                 13000 
                 44.8 
                 236.4 
                 28 
                 77 
               
               
                 L101 
                 3800 
                 58.9 
                 8.6 
                 1 
                 2.1 
               
               
                 P103 
                 4950 
                 59.7 
                 33.8 
                 9 
                 6.1 
               
               
                 P104 
                 5900 
                 61.0 
                 53.6 
                 13 
                 3.4 
               
               
                 P105 
                 6500 
                 56.0 
                 73.9 
                 15 
                 6.2 
               
               
                 F108 
                 14600 
                 50.3 
                 265.4 
                 27 
                 22 
               
               
                 L121 
                 4400 
                 68.3 
                 10.0 
                 1 
                 1 
               
               
                 L122 
                 5000 
                   
                   
                 4 
                   
               
               
                 P123 
                 5750 
                 69.4 
                 39.2 
                 8 
                 44 
               
               
                 F127 
                 12600 
                 65.2 
                 200.4 
                 22 
                 2.8 
               
               
                 10R5 
                 1950 
                   
                   
                 15 
                   
               
               
                 10R8 
                 4550 
                   
                   
                 19 
                   
               
               
                 12R3 
                 1800 
                   
                   
                 7 
                   
               
               
                 17R1 
                 1900 
                   
                   
                 3 
                   
               
               
                 17R2 
                 2150 
                   
                   
                 6 
                   
               
               
                 17R4 
                 2650 
                   
                   
                 12 
                   
               
               
                 17R8 
                 7000 
                   
                   
                 16 
                   
               
               
                 22R4 
                 3350 
                   
                   
                 10 
                   
               
               
                 25R1 
                 2700 
                   
                   
                 2 
                   
               
               
                 25R2 
                 3100 
                   
                   
                 4 
                   
               
               
                 25R4 
                 3600 
                   
                   
                 8 
                   
               
               
                 25R5 
                 4250 
                   
                   
                 10 
                   
               
               
                 25R8 
                 8550 
                   
                   
                 13 
                   
               
               
                 31R1 
                 3250 
                   
                   
                 1 
                   
               
               
                 31R2 
                 3300 
                   
                   
                 2 
                   
               
               
                 31R4 
                 4150 
                   
                   
                 7 
               
               
                   
               
               
                   (a) The average molecular weights and HLB provided by the manufacturer (BASF Co.); 
               
               
                   (b) The average numbers of EO and PO units were calculated using the average molecular weights of the blocks; 
               
               
                   (c) Critical micelle concentration (CMC) values at 37° C. were determined using pyrene probe (Kozlov et al. (2000) Macromolecules, 33:3305-3313). 
               
            
           
         
       
     
     The transport of non-conjugated Pluronic® copolymers in brain microvessel endothelial cells depends on the hydrophobicity of the block copolymers (Batrakova et al. (2003) J. Pharmacol. Exp. Ther., 304:845-854). Copolymers, with intermediate hydrophobicity, such as P85 (HLB=16), are easily transported inside the cells. The most hydrophobic copolymers, such as L121 (HLB=1), may remain bound with the membrane structures. Additionally, the relatively hydrophilic copolymers (HLB&gt;20) may not readily penetrate cells. Notably, following conjugation with polypeptides, the transport characteristics of the conjugates may not match exactly those of the unconjugated block copolymer molecules alone. Thus, the hydrophobicity/lipophilicity of the protein prior to conjugation may be taken into account when selecting a polymer to conjugate to the protein. 
     Preferably, the amphiphilic polymer of the instant invention is a copolymer of poly(oxyethylene) and poly(oxypropylene), more preferably the amphiphilic polymer is a Pluronic® copolymer. The preferred amphiphilic polymer possesses an HLB of less than or equal to 20, with an HLB of less than or equal to 16 being more preferred, with an HLB of less than or equal to 12 being more preferred and an HLB of less than or equal to 8 being most preferred. In a particular embodiment, the amphiphilic polymers contain long hydrophobic blocks, wherein the molecular weight of the hydrophobic block is, for example, greater than 1500, greater than 2000, greater than 2500, or greater than 3000. In still another embodiment, the hydrophobe molecular weight is from about 500 to about 5000, particularly about 1600 to 4000, and more particularly about 2200 to about 3000. In another embodiment, the hydrophobic block is a poly(oxypropylene) block. 
     Commercial Pluronics® have a symmetric poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) triblock structure. Diblock and multiblock copolymers can also be used in this invention. For example, an alternative asymmetric poly(oxyethylene)-poly(oxypropylene) polymer or an asymmetric poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) copolymer may also be used to enhance protein transport and maintain protein activity. Mono amino, carboxyl or other functional group derivatives of asymmetric poly(oxyethylene)-poly(oxypropylene) block copolymers may be used for protein modification. For example, the poly(oxyethylene)-poly(oxypropylene) di-block copolymer is synthesized by sequential polymerization of poly(oxyethylene) and poly(oxypropylene). The preferred copolymers contain a functional group at the poly(oxyethylene) or poly(oxypropylene). The lengths of the poly(oxypropylene) block (NPO) are preferably from about 10 to about 100, more preferably from about 30 to about 70, still more preferably from about 40 to about 60. The preferred length of the poly(oxyethylene) block (NEO) are from about 3 to about 300, more preferred from about 3 to about 100, still more preferred from about 6 to about 60. The copolymer is used for attachment to a protein via poly(oxypropylene) or poly(oxyethylene) segment. If the polymer is attached via poly(oxyethylene) block, a short poly(oxyethylene) sequence of about 1 to about 20 ethylene oxide units can be introduced to “cap” the distant poly(oxypropylene) end of the copolymer, which can increase the solubility of block copolymer-protein conjugates. Many methods available in art can be use to introduce such a terminal poly(oxyethylene) segment. 
     In a preferred embodiment, the conjugated proteins contain from about 1 to about 10 block copolymer moieties linked to one protein, more preferably from about 1 to about 7 block copolymer moieties linked to one protein, still more preferably from about 1 to about 3 block copolymer moieties. In the preferred embodiments, the conjugated proteins can contain mixture of conjugates with different number of block copolymers conjugated to the protein or the mixtures of non-conjugated proteins with one or several conjugated proteins containing with the preferred degree of conjugation. As a result the average number of conjugated block copolymer moieties in such protein mixtures may be a fraction number or even less than one, for example, in a mixture of unconjugated protein and protein conjugated with one or several block copolymer moieties. In addition to block copolymers conjugated to the proteins the mixtures can contain unconjugated block copolymers of the same or different structure. 
     III. PROTEINS 
     While the preferred embodiment of the instant invention involves conjugating proteins to the amphiphilic polymers in order to mediate crossing of a biological membrane, it is also within the scope of the instant invention to conjugate other therapeutic agents or compounds of interest to the amphiphilic polymer. Such agents or compounds include, without limitation, polypeptides, peptides, glycoproteins, nucleic acids, synthetic and natural drugs, lipids, and the like. 
     In a preferred embodiment of the instant invention, the proteins conjugated to the amphiphilic polymers are therapeutic proteins, i.e., they effect amelioration and/or cure of a disease, disorder, pathology, and/or the symptoms associated therewith. The proteins may have therapeutic value against, without limitation, neurological degenerative disorders, stroke, Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, trauma, infections, meningitis, encephalitis, gliomas, cancers (including brain metastasis), HIV, HIV associated dementia, HIV associated neurocognitive disorders, paralysis, amyotrophic lateral sclerosis, CNS-associated cardiovascular disease, prion disease, obesity, metabolic disorders, inflammatory disease, and lysosomal diseases (such as, without limitation Pompe disease, Niemann-Pick, Hunter syndrome (MPS II), Mucopolysaccharidosis I (MPS I), GM2-gangliosidoses, Gaucher disease, Sanfilippo syndrome (MPS IIIA), and Fabry disease). Examples of specific proteins include, without limitation, cytokines, enkephalin, growth factors (e.g., epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), nerve growth factor (NGF)), amyloid beta binders (e.g. antibodies), modulators of α-, β-, and/or γ-secretases, Glial-derived neutrotrophic factor (GDNF), vasoactive intestinal peptide, acid alpha-glucosidase (GAA), acid sphingomyelinase, iduronate-2-sultatase (I2S), α-L-iduronidase (IDU), β-Hexosaminidase A (HexA), Acid β-glucocerebrosidase, N-acetylgalactosamine-4-sulfatase, α-galactosidase A. Certain of the proteins are exemplified in Table 5. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Protein 
                 Ref. 
                 Disease 
                 Function 
               
               
                   
               
             
            
               
                 Glial-derived 
                 Schapira, A. H. 
                 Parkinson&#39;s and 
                 Neuroprotection and 
               
               
                 neutrotrophic factor 
                 (2003) Neurology 
                 Alzheimer&#39;s 
                 neurorestoration 
               
               
                 (GDNF) 
                 61:S56-63 
                 diseases 
                   
               
               
                   
                   
                 Stroke 
                   
               
               
                 Epidermal growth 
                 Ferrari, G., et al. 
                 Parkinson&#39;s and 
                 Stimulates dopaminergic 
               
               
                 factor (EGF) 
                 (1990) Adv Exp Med 
                 Alzheimer&#39;s 
                 development 
               
               
                   
                 Biol. 265:93-99 
                 diseases 
                   
               
               
                   
                   
                 Stroke 
                   
               
               
                 Basic fibroblast 
                 Ferrari, G., et al. 
                 Parkinson&#39;s and 
                 Stimulates proliferation 
               
               
                 growth factor 
                 (1991) J Neurosci 
                 Alzheimer&#39;s 
                 and migration of neutral 
               
               
                 (bFGF) 
                 Res. 30:493-497 
                 diseases 
                 stem cells 
               
               
                   
                   
                 Stroke 
                   
               
               
                 Nerve growth factor 
                 Koliatsos, V. E., et al. 
                 Parkinson&#39;s and 
                 Protects cholinergic cells 
               
               
                 (NGF) 
                 (1991) Ann Neurol. 
                 Alzheimer&#39;s 
                 from injury-induced death 
               
               
                   
                 30:831-840 
                 diseases 
                   
               
               
                   
                   
                 Stroke 
                   
               
               
                 Vasoactive intestinal 
                 Dogrukol-Ak, D., et 
                 Alzheimer&#39;s 
                 Promote neuronal survival, 
               
               
                 peptide 
                 al. (2003) Peptides 
                 diseases 
                 prevent exitotoxic cell 
               
               
                   
                 24:437-444 
                 Stroke 
                 death 
               
               
                 Acid alpha- 
                 Amalfitano, A., et al. 
                 Pompe (lysosomal 
                 Enzyme replacement 
               
               
                 glucosidase (GAA) 
                 (2001) Genet Med. 
                 disease) 
                 therapy 
               
               
                   
                 3:132-138 
                   
                   
               
               
                 Acid 
                 Simonaro, C. M., et 
                 Niemann-Pick 
                 Enzyme replacement 
               
               
                 sphingomyelinase 
                 al. (2002) Am J Hum 
                 (lysosomal disease) 
                 therapy 
               
               
                   
                 Genet. 71:1413-1419 
                   
                   
               
               
                 Iduronate-2-sultatase 
                 Muenzer, J., et al. 
                 Hunter syndrome 
                 Enzyme replacement 
               
               
                 (I2S) 
                 (2002) Acta Paediatr 
                 (MPS II) (lysosomal 
                 therapy 
               
               
                   
                 Suppl. 91:98-99 
                 disease) 
                   
               
               
                 α-L-iduronidase 
                 Wraith, J. E., et al. 
                 Mucopolysaccharidosis 
                 Enzyme replacement 
               
               
                 (IDU) 
                 (2004) J Pediatr. 
                 I (MPS I) 
                 therapy 
               
               
                   
                 144:581-588 
                 (lysosomal disease) 
                   
               
               
                 β-Hexosaminidase 
                 Wicklow, B. A., et al. 
                 GM2-gangliosidoses 
                 Enzyme replacement 
               
               
                 A (HexA) 
                 (2004) Am J Med 
                 (lysosomal disease) 
                 therapy 
               
               
                   
                 Genet. 127A:158-166 
                   
                   
               
               
                 Acid β- 
                 Grabowski, G. A., 
                 Gaucher disease 
                 Enzyme replacement 
               
               
                 glucocerebrosidase 
                 (2004) J Pediatr. 
                 (lysosomal disease) 
                 therapy 
               
               
                   
                 144:S15-19. 
                   
                   
               
               
                 N- 
                 Auclair, D., et al. 
                 Sanfilippo syndrome 
                 Enzyme replacement 
               
               
                 acetylgalactosamine- 
                 (2003) Mol Genet 
                 (MPS IIIA) 
                 therapy 
               
               
                 4-sulfatase 
                 Metab. 78:163-174 
                 (lysosomal disease) 
                   
               
               
                 α-galactosidase A 
                 Przybylska, M., et al. 
                 Fabry (lysosomal 
                 Enzyme replacement 
               
               
                   
                 (2004) J Gene Med. 
                 disease) 
                 therapy 
               
               
                   
                 6:85-92 
               
               
                   
               
            
           
         
       
     
     Additionally, the therapeutic protein optionally does not exhibit any appreciable therapeutic activity prior to cleavage/removal of the linker and/or amphiphilic polymer. In other words, prior to cleavage the therapeutic protein does not produce its intended therapeutic effect. 
     A. Superoxide Dismutase (SOD) 
     In a particular embodiment, the protein is superoxide dismutase (SOD). In a preferred embodiment, SOD is linked to the polymer via a non-degradable linker (e.g., the linker remainder from conjugation with DSS or EDC). The polymer conjugated SOD may be administered to a subject (e.g., in a composition comprising at least one pharmaceutically acceptable carrier) in order to treat inflammation, neurodegeneration, neurological disorders and other disorders of the central nervous system (including, but not limited to, Alzheimer&#39;s disease, Parkinson&#39;s disease, neurocardiovascular disease/dysregualtion) as well as for immune enhancement and as an anti-aging agent. In a particular embodiment, the polymer conjugated SOD is administered to a subject in need thereof to treat a neurodegenerative disease (e.g., Alzheimer&#39;s disease, Parkinson&#39;s disease, Lewy Body disease, amyotrophic lateral sclerosis, and prion disease). In a particular embodiment, the disease is stroke, traumatic brain injury, hypertension (including in chronic heart failure), or obesity. 
     The need for delivery of therapeutic polypeptides to affected brain tissues in Alzheimer&#39;s and Parkinson&#39;s diseases (AD and PD) (Brinton, R. D. (1999) Int. J. Fertil. Womens Med., 44:174-85; Gozes, I. (2001) Trends Neurosci., 24:700-5; Kroll et al. (1998) Neurosurgery 42:1083-100), infections (meningitis, encephalitis, prion disease, and HIV-related dementia) (Bachis et al. (2005) Ann. N.Y. Acad. Sci., 1053:247-57; Wang et al. (2003) Virology 305:66-76), stroke (Koliatsos et al. (1991) Ann. Neurol., 30:831-40; Dogrukol-Ak et al. (2003) Peptides 24:437-44), lysosomal storage (Desnick et al. (2002) Nat. Rev. Genet., 3:954-66; Urayama et al. (2004) Proc. Natl. Acad. Sci., 101:12658-63), obesity (Banks, W. (2003) Curr. Pharm. Des., 9:801-809; Banks et al. (2002) J. Drug Target., 10:297-308), and other metabolic and inflammatory diseases of the CNS is immediate and cannot be overstated. 
     An important component of metabolic and degenerative diseases of the nervous system involves inflammation (Perry et al. (1995) Curr. Opin. Neurobiol., 5:636-41). Such inflammatory activities are profound, as they lead to excessive production of pro-inflammatory products and ROS that lead in part, to cell death and neurodegeneration. By affecting neuroinflammatory activities during disease, such as through the use of targeted antioxidants or drugs that inhibit the production or formation of proinflammatory cytokines and eicosanoids, the levels of ROS as well as other neurotoxins can be reduced, resulting in improved disease outcomes (Prasad, et al. (1999) Curr. Opin. Neurol., 12:761-70). However, such approaches have been limited, as drugs must not only penetrate the BBB but also find themselves in sufficient concentrations to affect ongoing disease mechanisms. Moreover, as inflammatory mechanisms are a likely early event for disease, therapeutic modalities must be used early and frequently. The limitation of drug delivery is one major obstacle confronting the development of new treatment paradigms for nervous system disorders. 
     One such disease is PD, the second most prevalent neurodegenerative disorder in people over 65. This disease is characterized by lack of the neurotransmitter dopamine due to a loss of dopaminergic neurons within the SNpc and their innervations to the striatum. PD neuropathology involves brain inflammation, microglia activation, and subsequent secretory neurotoxic activities, including ROS production, that play crucial roles in cell damage and death (McGeer et al. (1988) Neurology 38:1285-91; Busciglio et al. (1995) Nature 378:776-9; Ebadi et al. (1996) Prog. Neurobiol., 48:1-19; Wu et al. (2003) Proc. Natl. Acad. Sci., 100:6145-50). PD brains show reduced levels of antioxidant enzymes and antioxidants (Ambani et al. (1975) Arch. Neurol., 32:114-8; Riederer et al. (1989) J. Neurochem., 52:515-20; Abraham et al. (2005) Indian J. Med. Res., 121:111-5) resulting in a reduced capacity to manage oxidative stress and associated neurodegeneration. Mounting evidence supports the notion that antioxidants can inhibit inflammatory responses and protect dopaminergic neurons in laboratory and animal models of PD (Wu et al. (2002) J. Neurosci., 22:1763-71; Du et al. (2001) Proc. Natl. Acad. Sci., 98:14669-74; Kurkowska-Jastrzebska et al. (2002) Int. Immunopharmacol., 2:1213-8; Teismann et al. (2001) Synapse 39:167-74; Ferger et al. (1999) Naunyn Schmiedebergs Arch. Pharmacol., 360:256-61; Ferger et al. (1998) Naunyn Schmiedebergs Arch. Pharmacol., 358:351-9; Peng et al. (2005) J. Biol. Chem., 280:29194-8). Catalase catalyzes the conversion of hydrogen peroxide, a known ROS, to water and molecular oxygen with one of the highest turnover rates for all known enzymes. Mounting evidence suggests that antioxidants can inhibit the inflammatory response and protect up to 90% of dopaminergic neurons in vitro and in vivo (Wu et al. (2002) J. Neurosci., 22:1763-71; Du et al. (2001) Proc. Natl. Acad. Sci., 98:14669-74; Kurkowska-Jastrzebska et al. (2002) Int. Immunopharmacol., 2:1213-8; Teismann et al. (2001) Synapse 39:167-74; Ferger et al. (1999) Naunyn. Schmiedebergs Arch. Pharmacol., 360:256-61; Ferger et al. (1998) Naunyn. Schmiedebergs Arch. Pharmacol., 358:351-9; Peng et al. (2005) J. Biol. Chem., 280:29194-8). In an in vitro model of PD, catalase was shown to rescue primary cultured cerebellar granule cells from ROS toxic effects (Prasad et al. (1999) Curr. Opin. Neurol., 12:761-70; Gonzalez-Polo et al. (2004) Cell Biol. Int., 28:373-80). Furthermore, low molecular mass catalase activator, rasagiline, induced neuroprotection in a mouse model of PD (Maruyama et al. (2002) Neurotoxicol. Teratol., 24:675-82). Few clinical trials have been performed using low molecular mass antioxidants, of which the most extensive used is R-tocopherol and deprenyl to inhibit the rate of PD progression (Group, T. P. S. (1993) N. Engl. J., 328:176-183). However, and as described above, most of the trials failed to show significant improvements because of restricted transport of R-tocopherol across the BBB and the time following the disease the drugs were used (Pappert et al. (1996) Neurology, 47:1037-42). 
     The antioxidant enzyme superoxide dismutase (SOD), particularly, SOD1 (also called Cu/Zn SOD) are known to catalyze the dismutation of superoxide (O 2   − ). Thus, SOD, particularly SOD1, can be used in antioxidant therapy, particularly for neurocardiovascular-related diseases, if peripherally administered it penetrates the blood brain barrier (BBB) and the plasma membrane of neurons. It is demonstrated herein that conjugating SOD1 with poly(ethylene oxide)-poly(propylene oxide) block copolymer (Pluronics®) improves SOD1 delivery to the brain and provide therapeutic effects. 
     An alternative modification route is to target the unique N-terminal amino group of SOD1. This can be achieved by using an aldehyde functionalized Pluronic® analogue and SOD (e.g., SOD1), containing a free N-terminal amino group (commercial sample deprotected enzymatically). 
     Notably, the commercial P85 and L81 contain ca. 10% wt. of lower molecular mass polymer admixtures. The polymers can be purified by gel-permeation chromatography and characterized to determine a) the number- and weight averaged molecular masses (Mn, Mw) and polydispersity (D=Mw/Mn) by MALDI-TOF mass spectroscopy, and b) the PEG/PPG composition by nuclear magnetic resonance (NMR). 
     Additionally, the reaction scheme of the instant invention excludes the possibility of SOD cross-linking. However, aggregation of SOD-Pluronic® is possible upon storage. To address this, protein molecular mass may be measured using sedimentation equilibrium analysis and, if needed, agents to prevent aggregation (glycerol, ethanol, etc.) may be utilized. 
     B. Leptin 
     In yet another embodiment of the instant invention, the protein is leptin. Leptin conjugates to block copolymers can be synthesized, for example, by primary amine modification using disulfide bond, disulfide bride insertion, or protein N-terminal attachment. In a particular embodiment, SOD is linked to the polymer via a non-degradable linker (e.g., the remainder from conjugating with DSS) or a degradable linker (e.g., disulfide containing linkers such as the remainder from conjugating with DSP). The conjugation of the block copolymers to leptin enhance bioavailability and efficient transport across the blood brain barrier. The polymer conjugated leptin may be administered to a subject (e.g., in a composition comprising at least one pharmaceutically acceptable carrier) in order to treat obesity (e.g., to reduce the subject&#39;s weight). 
     Leptin is a 16 kDa regulatory protein, secreted by fat cells, which acts within the brain to control appetite and thermogenesis (Zhang et al. (1994) Nature, 372:425-432; Ahima et al. (1996) Nature, 382:250-252; Friedman and Halaas (1998) Nature, 395:763-770). Leptin crosses the BBB by way of a specific, saturable transport system (Banks et al. (1996) Peptides, 17:305-311). In normal body weight animals, transport across the BBB allows leptin to access its CNS receptors. With obesity, the leptin transporter becomes increasing impaired, resulting in a resistance to circulating leptin (Banks et al. (1999) Peptides, 20:1341-1345; Banks and Farrell (2003) Am. J. Physiol. Endocrinol. Metab., 285:E10-15; Kastin et al. (1999) Peptides, 20:1449-1453; Hileman et al. (2002) Endocrinology, 143:775-783). 
     Several lines of evidence in humans and rodents show that impaired BBB transport is important in the maintenance and in the progression of obesity (Van Heek et al. (1997) J. Clin. Invest. 99:385-390; Banks and Farrell (2003) Am. J. Physiol. Endocrinol. Metab. 285:E10-15). In normal body weight rats and mice, in which obesity is induced with diet (that is, strains without inherent defects in leptin protein or receptor expression or downstream circuitries), leptin transporter defects predominate over brain receptor defects early on. Calculations based on CSF and serum levels of leptin indicate that in advanced obesity in humans (leptin levels of about 40 ng/ml), transporter defects account for about ⅔ of the resistance to peripheral leptin (Banks, W. A. (2003) Curr. Pharm. Des. 9:801-809). Transporter defects are acquired, reversible, and mediated only partly by excess of endogenous leptin (Banks et al. (1999) Peptides, 20:1341-1345; Banks and Farrell (2003) Am. J. Physiol. Endocrinol. Metab., 285:E10-15). Because the leptin transporter is impaired in obesity, high doses of peripherally administered leptin have to little or no effect (Heymsfield et al. (1999) JAMA, 282:1568-1575; Farooqi et al. (1999) N. Engl. J. Med., 341:879-884; Fujioka et al. (2000) NAASO Annual Meeting; Halaas et al. (1997) Proc. Natl. Acad. Sci., 94:8878-8883; Van Heek et al. (1997) J. Clin. Invest., 99:385-390; Heymsfield et al. (1999) JAMA, 282:1568-1575; Pelleymounter et al. (1998) Am. J. Physiol., 275:R950-959). Thus, the delivery of leptin into the CNS would be effective in the treatment of obesity. 
     As stated above, leptin is transported across the BBB by a saturable mechanism. Leptin is transported into all regions of the brain and by both the vascular and epithelial barrier, but the rate of transport, the saturation kinetics, and maximal transport vary among brain regions. The hypothalamus, which contains the arcuate nucleus, takes up the most leptin in thin and normal body weight animals, but the pons medulla and hippocampus take up the most in obese animals (Heymsfield et al. (1999) JAMA 282:1568-1575). With increasing obesity, transport of exogenously administered leptin (and the efficiency with which endogenous circulating leptin is transported) progressively decreases. Even in thin animals, the amount of leptin circulating in blood partially saturates the leptin BBB transporter (Banks et al. (2000) Am. J. Physiol. Endocrinol. Metab., 278:E1158-1165). This is consistent with ideas that leptin evolved to communicate with the CNS especially during low leptin states. The BBB transporter in outbred mice is at about half of maximal saturation in most brain regions at blood levels of 10-15 ng/ml; a level of 5-12 ng/ml is the range for ideal body weight. Therefore, even in the absence of accepted norms of ideal body weight in Western societies, the leptin transporter is already substantially saturated, which means it is beyond its most efficient level of activity. Studies with wild baboons in East Africa support the idea that ancestral levels of leptin were much lower than those considered normal (Banks et al. (2001) J. Clin. Endocrinol. Metab., 86:4315-4320; Banks et al. (2003) J. Clin. Endocrinol. Metab., 88:1234-1240). As a result, blood levels of leptin must be increased to produce an ever diminishing increase in CNS leptin levels. For example, it has been calculated that increasing serum leptin levels 200% from 10 ng/ml (reported as the value seen in “normal” individuals (Caro et al. (1996) Lancet 348:159-161)) to 30 ng/ml (moderate obesity) results in only a 68% increase in CNS levels, from 555 to 934 μg/ml of CSF (Banks, W. A. (2003) Curr. Pharm. Des., 9:801-809). 
     Impaired leptin transport may be acquired. In rodents made obese with a high fat diet, the defect in transport precedes the defect in brain receptor function (Halaas et al. (1997) Proc. Natl. Acad. Sci., 94:8878-8883; Van Heek et al. (1997) J. Clin. Invest., 99:385-390). In rodents with an inborn defect in brain receptor function, the leptin transporter defect is acquired in tandem with diet-induced obesity (Levin et al. (2004) Am. J. Physiol. Regul. Integr. Comp. Physiol., 286:R143-150). In outbred obese mouse, the BBB defect is to some degree reversible with loss of body weight (Banks et al. (2003) Am. J. Physiol. Endocrinol. Metab., 285:E10-15). The defect in leptin transporter capacity is not simply caused by increased levels of circulating leptin (Banks et al. (1999) Peptides 20:1341-1345). Both obesity and starvation impair leptin transporter activity by release of triglycerides (Banks et al. (2004) Diabetes 53:1253-1260). Both endogenous and exogenous triglycerides impair leptin transport. Lowering triglycerides with pharmacologic agents enhances leptin transport. As a result of these factors, obese mice and humans are much less responsive, or even unresponsive, to peripherally administered leptin (Halaas et al. (1997) Proc. Natl. Acad. Sci., 94:8878-8883; Van Heek et al. (1997) J. Clin. Invest., 99:385-390; Heymsfield et al. (1999) JAMA 282:1568-1575; Pelleymounter et al. (1998) Am. J. Physiol., 275:R950-959). For example, 10 mg/kg of leptin injected i.p. resulted in a 12% loss of body weight in thin mice but only a non-significant 3.1% in diet-induced CD-1 obese mice when compared to controls (Pelleymounter et al. (1998) Am. J. Physiol., 275:R950-959). The thin mice lost 5.3 g of fat (46% of total body fat) whereas the obese mice lost 1 g of fat (a non-significant 5% of total body fat). 
     IV. LINKERS 
     Examples of certain linkers and methods of conjugating the linker with the protein of interest and the amphiphilic polymer are provided in Examples 4, 9 and 11. The linker moiety joining the amphiphilic polymer and the protein of the conjugate may be non-biodegradable or biodegradable. In a particular embodiment, the linker is cleaved in vivo as the conjugate either passes through the BBB, or upon completion of the transfer across the BBB. In a certain embodiment of the instant invention, the linker moiety comprises amino acids that constitute a protease recognition site or other such specifically recognized enzymatic cleavage site. Exemplary protease recognition sites include, without limitation, amino acid sequences cleavable by endosomal cathepsin, such as cathepsin B (e.g., Gly-(Phe)-Leu-Gly (SEQ ID NO: 1); see, e.g., DeNardo et al. (2003) Clinical Cancer Res. 9:3865s-72s); sequences cleavable by lysosomal proteases (e.g., Gly-Leu-Gly and Gly-Phe-Leu-Gly (SEQ ID NO: 1); see, e.g., Guu et al. (2002) J. Biomater. Sci. Polym. Ed. 13:1135-51; Rejmanova et al. (1985) Biomaterials 6:45-48); and sequences cleavable by collagenase (e.g., GGGLGPAGGK (SEQ ID NO: 2) and KALGQPQ (SEQ ID NO: 3); see, e.g., Gobin and West (2003) Biotechnol. Prog. 19:1781-5; Kim and Healy (2003) Biomacromolecules 4:1214-23). 
     In another embodiment the linker region comprises a disulfide bond. Preferably, the disulfide bond is stable in the blood, but hydrolyzable by reductases present in the BBB. Representative examples of linker moieties comprising a disulfide bond include, without limitation: 
     —OC(O)NH(CH 2 ) 2 NHC(O) (CH 2 ) 2 SS(CH 2 ) 2 C(O)NH—; 
     —OC(O)NH(CH 2 ) 2 SS(CH 2 ) 2 N═CH—; and 
     —OC(O)NH(CH 2 ) 2 SS(CH 2 ) 2 NH—. 
     In another embodiment the linker region comprises a hydrolyzable ester. Preferably, the hydrolyzable ester is stable in the blood, but hydrolyzable by hydrolases present in the BBB. 
     In a preferred embodiment, the linker moiety is completely cleaved or substantially cleaved, effecting the removal of the amphiphilic polymer from the protein. In yet another embodiment, the linker moiety is completely cleaved or substantially cleaved resulting in the removal of the amphiphilic polymer from the protein and most, if not all, of the linker region. 
     Additionally, the linkage between the protein and the amphiphilic polymer can be a direct linkage between a functional group at a termini of the polymer and a functional group on the protein. 
     Generally, the linker is a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches the therapeutic protein to the amphiphilic copolymer. The linker can be linked to any synthetically feasible position of the therapeutic protein and the polymer. In a preferred embodiment the linker is attached at a position which avoids blocking the activity of the therapeutic protein. Exemplary linkers may comprise at least one optionally substituted; saturated or unsaturated; linear, branched or cyclic alkyl group or an optionally substituted aryl group. The linker may also be a polypeptide (e.g., from about 1 to about 20 amino acids, particularly about 1 to about 10). The linker may be biodegradable under physiological environments or conditions. The linker may also be non-degradable and may be a covalent bond or any other chemical structure which cannot be substantially cleaved or cleaved at all under physiological environments or conditions. 
     As used herein, the term “biodegradable” or “biodegradation” is defined as the conversion of materials into less complex intermediates or end products by solubilization hydrolysis under physiological conditions, or by the action of biologically formed entities which can be enzymes or other products of the organism. The term “non-degradable” refers to a chemical structure that cannot be cleaved under physiological conditions. 
     The term “alkyl,” as employed herein, includes both straight and branched chain hydrocarbons containing about 1 to about 20 carbons, particularly about 1 to about 15, particularly about 5 to about 15 carbons in the main chain. The hydrocarbon chain of the alkyl groups may be interrupted with heteroatoms such as oxygen, nitrogen, or sulfur atoms. Each alkyl group may optionally be substituted with substituents which include, for example, alkyl, halo (such as F, Cl, Br, I), haloalkyl (e.g., CCl 3  or CF 3 ), alkoxyl, alkylthio, hydroxy, methoxy, carboxyl, oxo, epoxy, alkyloxycarbonyl, alkylcarbonyloxy, amino, carbamoyl (e.g., NH 2 C(═O)— or NHRC(═O)—, wherein R is an alkyl), urea (—NHCONH 2 ), alkylurea, aryl, ether, ester, thioester, nitrile, nitro, amide, carbonyl, carboxylate and thiol. 
     The term “aryl,” as employed herein, refers monocyclic and bicyclic aromatic groups containing 6 to 10 carbons in the ring portion. Aryl groups may be optionally substituted through available carbon atoms. The aromatic ring system may include heteroatoms such as sulfur, oxygen, or nitrogen. 
     V. ADMINISTRATION OF CONJUGATES 
     The amphiphilic polymer-protein conjugates described herein will generally be administered to a patient as a pharmaceutical preparation. The term “patient” as used herein refers to human or animal subjects. These amphiphilic polymer-protein conjugates may be employed therapeutically, under the guidance of a physician. 
     The pharmaceutical preparation comprising the amphiphilic polymer-protein conjugates of the invention may be conveniently formulated for administration with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. The concentration of amphiphilic polymer-protein conjugates in the chosen medium will depend on the hydrophobic or hydrophilic nature of the medium, as well as the size and other properties of the amphiphilic polymer-protein conjugates. Solubility limits may be easily determined by one skilled in the art. 
     As used herein, “biologically acceptable medium” or “carrier” includes any and all solvents, dispersion media and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation, as exemplified in the preceding discussion. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the amphiphilic polymer-protein conjugate to be administered, its use in the pharmaceutical preparation is contemplated. 
     The dose and dosage regimen of an amphiphilic polymer-protein conjugate according to the invention that is suitable for administration to a particular patient may be determined by a physician considering the patient&#39;s age, sex, weight, general medical condition, and the specific condition for which the amphiphilic polymer-protein conjugate is being administered and the severity thereof. The physician may also take into account the route of administration of the amphiphilic polymer-protein conjugate, the pharmaceutical carrier with which the amphiphilic polymer-protein conjugate is to combined, and the amphiphilic polymer-protein conjugate&#39;s biological activity. 
     Selection of a suitable pharmaceutical preparation will also depend upon the mode of administration chosen. For example, the amphiphilic polymer-protein conjugates of the invention may be administered by direct injection into an area proximal to the BBB. In this instance, a pharmaceutical preparation comprises the amphiphilic polymer-protein conjugates dispersed in a medium that is compatible with the site of injection. 
     Amphiphilic polymer-protein conjugates may be administered by any method such as intravenous injection into the blood stream, oral administration, or by subcutaneous, intramuscular or intraperitoneal injection. Pharmaceutical preparations for injection are known in the art. If injection is selected as a method for administering the amphiphilic polymer-protein conjugates, steps must be taken to ensure that sufficient amounts of the molecules reach their target cells to exert a biological effect. The lipophilicity of the amphiphilic polymer-protein conjugates, or the pharmaceutical preparation in which they are delivered, may have to be increased so that the molecules can arrive at their target location. Furthermore, the amphiphilic polymer-protein conjugates may have to be delivered in a cell-targeting carrier so that sufficient numbers of molecules will reach the target cells. Methods for increasing the lipophilicity of a molecule are known in the art. 
     Pharmaceutical compositions containing a conjugate of the present invention as the active ingredient in intimate admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, direct injection, intracranial, and intravitreal. In preparing the amphiphilic polymer-protein conjugate in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form in which solid pharmaceutical carriers are employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. Additionally, the conjugate of the instant invention may be administered in a slow-release matrix. For example, the conjugate may be administered in a gel comprising unconjugated poloxamers. 
     A pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. 
     Dosage units may be proportionately increased or decreased based on the weight of the patient. Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art. 
     In accordance with the present invention, the appropriate dosage unit for the administration of amphiphilic polymer-protein conjugates may be determined by evaluating the toxicity of the molecules in animal models. Various concentrations of amphiphilic polymer-protein conjugate pharmaceutical preparations may be administered to mice, and the minimal and maximal dosages may be determined based on the beneficial results and side effects observed as a result of the treatment. Appropriate dosage unit may also be determined by assessing the efficacy of the amphiphilic polymer-protein conjugate treatment in combination with other standard drugs. The dosage units of amphiphilic polymer-protein conjugate may be determined individually or in combination with each treatment according to the effect detected. 
     The pharmaceutical preparation comprising the amphiphilic polymer-protein conjugates may be administered at appropriate intervals, for example, at least twice a day or more until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level. The appropriate interval in a particular case would normally depend on the condition of the patient. 
     The following examples provide illustrative methods of practicing the instant invention, and are not intended to limit the scope of the invention in any way. While certain of the following examples specifically identify a certain type of Pluronic® block copolymer (e.g., Pluronic® P85), the use of any amphiphilic polymer is within the scope of the instant invention. 
     Example 1 
     Generation of Pluronic®-Amines 
     The conjugates of Pluronic® copolymers can be synthesized by first generating mono-amino derivatives of Pluronic® copolymers, which are referred to herein as “Pluronic®-amines.” The “Pluronic®-amines” were generated as described in Vinogradov et al. (Bioconjug. Chem. (1998) 9:805-812). Essentially, the method of Vinogradov et al. is a two step procedure. 
     First, 2 to 3 g of Pluronic® (L81, P85, L121 and P123) were dried overnight in vacuo at 50° C. and then dissolved in 15 ml of anhydrous acetonitrile. One molar equivalent of 4′-methoxytrityl chloride (MTr-Cl) were dissolved in 10 ml of anhydrous pyridine and added to the Pluronic® solution. Acetonitrile was then removed in vacuo, and the mixture was allowed to stand for 3 hours at 25° C. The reaction was then stopped by adding 1 ml of methanol, and pyridine was removed in vacuo by co-evaporation with 2×15 ml of toluene. The monosubstituted MTr-Pluronic® was isolated from non-reacted polymer and bis-MTr-Pluronic® by adsorption chromatography on Silicagel column (4.5×10 cm) using dichloromethane-methanol stepwise gradient elution. The products were then analyzed by thin-layer chromatography (TLC) in chloroform-MeOH, 9:1, and detected in trifluoroacetic acid vapors (test to the presence of MTr-group). Product yields were typically 70-85% of theoretical. 
     In the second step, 1-1.5 g of mono-MTr-Pluronic® was dried by co-evaporation with 2×15 ml of anhydrous acetonitrile in vacuo and then reacted with 5-fold molar excess of 1,1′-carbonyldiimidazole (CDI) in 10 ml of anhydrous acetonitrile for 1 hour at 40° C. The reaction mixture was then treated with 0.2 ml of water for 20 minutes to neutralize the unreacted CDI, and then added to a solution of ethylenediamine (20-fold molar excess) in 40 ml of ethanol upon stirring. The mixture was kept overnight at 25° C., diluted by 50 ml of water and dialyzed for 18 hours using a membrane with 2,000 kDa cutoff against 10% aqueous ethanol (2×2 L). After the dialysis, the polymer was concentrated in vacuo, co-evaporated twice with methanol (10 ml), and re-dissolved in 50 ml of 2% trifluoroacetic acid (TFA) in dichloromethane. After incubation for 1 hour at 25° C., the bright yellow solution was concentrated in vacuo and neutralized by 5 ml of 10% triethylamine in methanol. The resulting Pluronic®-amine was isolated by gel filtration on Sephadex LH-20 column (2.5×30 cm) and analyzed by TLC in chloroform-MeOH, 9:1, mixture. The Pluronic®-amines develop blue color after spraying with 1% ninhydrine solution in ethanol (as a test for the presence of an aminogroup). The product yields were about 70-80% of theoretical. About 85 to 95% of Pluronic® molecules contain primary amino groups as determined by 2,4,6-trinitrobenzolsulfonic acid (TNBS) titration. 
     The TNBS titration can be performed as follows. A 5% solution of TNBS (Sigma-Aldrich, St. Louis, Mo.) was diluted 100 times in 1M Na-borate buffer, pH 9.5. The native and modified compounds were dissolved in the same buffer at 1 mg/ml. The reaction was then initiated by adding the compound and TNBS solutions (20 μl each) to 180 μl of 0.1M Na-borate buffer, pH 9.5 in 96-well plates. After a 1 hour incubation at 20° C., the optical density (405 nm) was recoded on a Microplate reader, in triplicate. The number of modified groups per protein is determined as S=N×(1−A m C n /A n C m ), where N is the number of primary amino groups in the native compound, A n  and A m  are the optical densities for the native and modified compounds, and C n  and C m  are the concentrations of native and modified compounds, as determined, for example, by the Pierce BCA assay (Rockford, Ill.) using dilutions a standard BSA solution for calibration. 
     Example 2 
     Conjugation of Pluronic®-Amines with Proteins 
     “Pluronic®-Amine” derivatives, as obtained in Example 1, were then conjugated to horseradish peroxidase (HRP) using the two-step procedure shown in  FIG. 1 . HRP was chosen as a lead model for these experiments because its size of 40 kDa is similar to molecules of therapeutic interest and because its BBB characteristics are well known. HRP does not cross the BBB by saturable or transmembrane mechanisms. Only a small amount enters brain from the circulation by way of the extracellular pathways, which are the same pathway used by albumin. In other words, it essentially does not penetrate the BBB. 
     In the first step of the conjugation procedure, Pluronic®-amines were activated with Loman&#39;s reagent, dithiobis(succinimidyl propionate) (DSP), or disuccinimidylsuberate (DSS). Specifically, Pluronic®-amine (50-70 μmol) in 2.5 ml of methanol was added dropwise to a stirred solution of 3-fold molar excess of dithiobis(succinimidyl propionate) (DSP) to generate a cleavable linker or disuccinimidyl suberate (DSS) for a nondegradable, stable linker, in 2.5 ml of dimethylformamide and kept for 30 minutes at 25° C. The resulting hydroxysuccinimide derivatives of Pluronic® were isolated by gel filtration on Sephadex LH-20 column (2.5×30 cm) in methanol and analyzed by TLC developed in iodine vapors. 
     Second, the products of the above reactions were linked to the aminogroups of HRP. The DSP reagent was used to introduce a cleavable or degradable disulfide linkage between the HRP and Pluronic® molecules, while the DSS linkage was not degradable. Specifically, pooled fractions were collected, concentrated in vacuo, and dissolved in 1.5 ml of 20% ethanol. HRP (1 μmol) was dissolved in 0.5 ml of 0.1 M borate buffer, pH 9.5, and mixed with a previously prepared solution (1.5 ml) of activated Pluronic® to a obtain total volume of 2 ml. The homogeneous reaction mixture was kept overnight at 4° C. Modified HRP was then precipitated in 50 ml of cold ethanol (−10° C., 0.5 hour), and separated from the excess of polymer by centrifugation at 3000 rpm for 20 minutes at 4° C. Yellow precipitate was washed by cold ethanol (2×10 ml) and dried in vacuo. Pluronic®-HRP conjugate was purified by cation exchange chromatography using glass column (1×15 cm) with TSK CM-650M resin. Pluronic®-HRP conjugate was eluted in 50 mM sodium acetate, pH 4.4, 0.03M sodium chloride and 2.5% ethanol with detection at 280 nm ( FIGS. 2A and 2B ). Residual unmodified HRP was eluted in a gradient of the sodium chloride (0.03 to 0.15M). Collected fractions were desalted in dialysis tubes with 20 kDa cutoff against water at 4° C. and freeze-dried. 
     To vary the number of copolymer chains per protein molecule, different excesses of activated Pluronic® were used. Overall, four conjugates with stable or degradable linkages and different modification degrees were synthesized as shown in Table 6. TNBS analysis demonstrated that the products contained mainly mono- and bis-substituted protein. The fractions of unmodified HRP did not exceed 25%. The unmodified HRP was discarded, while the fractions of modified protein (peaks 1 and 2) were combined for each conjugate and used in subsequent studies. The characteristics of the Pluronic®-HRP conjugates obtained are summarized in Table 6. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                 Pluronic ® 
                 Type of  
                 Modification 
                 Residual 
               
               
                 Conjugate 
                 excess 
                 link 
                 degree (a)   
                 activity, % (b)   
               
               
                   
               
             
            
               
                 HRP-P85 (1) 
                 50 
                 degradable 
                 1.4 
                 97 
               
               
                 HRP-P85 (2) 
                 50 
                 stable 
                 1.6 
                 100  
               
               
                 HRP-P85 (3) 
                 70 
                 degradable 
                 2.0 
                 89 
               
               
                 HRP-L121 
                 70 
                 degradable 
                 2.1 
                 95 
               
               
                   
               
               
                   (a) Average number of Pluronic ® chains per HRP molecule determined using TNBS titration assay. 
               
               
                   (b) Compared to the native HRP, o-phenylenediamine reaction. 
               
            
           
         
       
     
     Example 3 
     Alternative Methods for Generating Pluronic®-Polypeptide Conjugates 
     Two alternative methods for introduction of degradable linkages in Pluronic®-polypeptide conjugates are presented in  FIG. 3 . In both methods Pluronic® can be taken in excess to avoid activation of both ends. 
     One method involves the introduction of a phosphoramidate linkage, which is susceptible to a mild acidic hydrolysis in endosomes following the cleavage of phosphomonoesteri group within the cells (Wagner et al. (2000) Med. Res. Rev. 20:417-451). Bis-(N-hydroxybenzotriazolyde)-phenylphosphate can be generated in situ by reacting dichlorophenylphosphate (30 μmol), N-hydroxybenzotriazole (60 μmol) and diisopropylethylamine (60 μmol) in 5 ml of anhydrous THF for 1 hour at 4° C. This reagent can be added dropwise to a stirred solution of excess of Pluronic® (90 μmol, dried overnight in vacuo at 50° C.) in 5 ml of THF and the mixture can be kept for 4 hours at 25° C. The activated Pluronic® can be isolated from unreacted block copolymer by gel filtration on Sephadex LH-20 column (2.5×30 cm) in methanol and analyzed by TLC developed by exposure to iodine vapors. Pooled fractions can be concentrated in vacuo, and dissolved in 1.5 ml of 20% ethanol before reaction with polypeptides. 
     The second method involves preparation of succinate-Pluronic® derivative, which is then conjugated to HRP using water soluble carbodiimide. Notably, the ester bond is hydrolytically degradable (Wagner et al. (2000) Med. Res. Rev. 20:417-451). A solution of succinic anhydride/dimethylaminopyridine (30 μmol:3 μmol) in 1 ml of anhydrous pyridine can be added dropwise to a stirred solution of excess of Pluronic® (90 μmol) in 5 ml of THF and the mixture can be kept overnight at 25° C. The solvent can be removed in vacuo. The activated Pluronic® can be purified by gel filtration on Sephadex LH-20 column (2.5×30 cm) in methanol and analyzed by TLC. Pooled fractions can be concentrated in vacuo, and dissolved in 1.5 ml of 20% ethanol and used for reaction with HRP (4-6 h, 25° C.) in the presence of a 3 molar equivalents of ethyl dimethylaminopropyl carbodiimide. 
     Example 4 
     Examples of Pluronic®-Protein Conjugate Preparations 
     The following are examples of synthetic procedures for the preparation of Pluronic®-protein conjugates with degradable linkers. The last example provides methods used to characterize the resultant conjugates. Method 1 gives outlines of monosubstituted methoxytrityl Pluronic® and the synthesis of five types of monofunctional polymers (Pluronic®) including linkers with the following structures: 
     (1) Polymer-OC(O)NH(C 1-12 ) 2 NH 2    
     (2) Polymer-OC(O)NH(CH 2 ) 2 SS(CH 2 ) 2 NH 2    
     (3) Polymer-OC(O) (CH 2 ) 2 COOH 
     (4) Polymer-OP(O)(OPh)OH 
     (5) Polymer-OC(O)NH(CH 2 ) 2 NHC(O)CH 2 C(OH)(COOH)CH 2 COOH 
     Methods 2-6 enable the generation of Pluronic®-protein conjugates having the following structures: 
     
         
         
           
             (1) Polymer-OC(O)NH(CH 2 ) 2 NHC(O)(CH 2 ) 2 SS(CH 2 ) 2 C(O)NH-Protein 
             (2) Polymer-OC(O)NH(CH 2 ) 2 SS(CH 2 ) 2 N═CH-Protein 
             (3) Polymer-OC(O)(CH 2 ) 2 C(O)NH-Protein 
             (4) Polymer-OP(O)(OPh)NH-Protein 
             (5) Polymer-OC(O)NH(CH 2 ) 2 NHC(O)CH 2 C(OH) (COOH)CH 2 C (O)NH-Protein
 
Method 1. Synthesis of Monofunctional Pluronic® Block Copolymers
 
           
         
       
    
     1. Monosubstituted methoxytrityl Pluronic®. 3 g of Pluronic® was dried in vacuo at 50° C. and then dissolved in 15 mL of anhydrous acetonitrile. One molar equivalent of methoxytrityl chloride (Aldrich) was dissolved in 10 mL of anhydrous pyridine and added to the Pluronic® solution. Acetonitrile was removed by evaporation in vacuo and the mixture was allowed to stand for 3 hours at 25° C. The reaction was stopped by adding 1 mL of methanol and pyridine was removed in vacuo by coevaporating twice with 15 mL of toluene. The monosubstituted product was isolated from nonreacted polymer and by products using adsorption chromatography on a Silicagel column (4.5×10 cm) in dichloromethane. Stepwise gradient elution with dichloromethane and 2%, 5%, and 10% methanol in dichloromethane (150 mL each) was used, and fractions of 8 mL were collected. Yields: 2.5-3 g (70-85% of theoretical). Thin layer chromatography (TLC): R f  0.6 (Silicagel plates (Merck, Whitehouse Station, N.J.), dichloromethane-methanol, 9:1). The product developed yellow coloring on the TLC plate in acid vapors (test on the presence of methoxytrityl group). 
     2. Monosubstituted Pluronic® ethylamine. The monosubstituted methoxytrityl Pluronic® was dried in vacuo over phosphorus oxide and treated by 5 molar equivalents of 1,1′-carbonyldiimidazole (CDI) (Aldrich, St. Louis, Mo.) in 10 mL of anhydrous acetonitrile for 2 hours at 40° C. Water (0.2 mL) was added to quench the excess of CDI, and 20 minutes later, this solution was added to a 40 mL of stirring 10% solution of ethylenediamine in ethanol. The reaction mixture was stirred overnight at 25° C. and then was diluted by the same volume of water (50 mL). Low molecular weight compounds have been removed by dialysis in the membrane tube with cutoff 2,000 in 2 L of 10% aqueous ethanol for 18 hours at 4° C. with two changes of the solution. The dialysate was concentrated in vacuo and redissolved in 50 mL of 2% trifluoroacetic acid in dichloromethane. Following the incubation for 1 hour at 25° C., bright yellow solution was concentrated in vacuo and neutralized by 5 mL of 10% triethylamine in methanol. The monosubstituted Pluronic® ethylamine was isolated by gel filtration on Sephadex LH20 column (2.5×30 cm) in methanol collecting fractions: 8 mL/4 minute. Yields: 2.0-2.2 g of white solids (70-80% of theoretical). TLC: R f  0.4 (Silicagel plates, dichloromethane-methanol, 9:1). The product developed blue color after spraying the TLC plates with 1% ninhydrine solution in ethanol, to test for the presence of amino group. 
     3. Monosubstituted Pluronic®-cystamine. The monosubstituted methoxytrityl Pluronic® was dried in vacuo over phosphorus oxide and treated by 5 molar equivalents of 1,1′-carbonyldiimidazole (CDI) in 10 mL of anhydrous acetonitrile for 2 hours at 40° C. Water (0.2 mL) was added to quench the excess of CDI, and 20 minutes later, this solution was added to a 40 mL of stirring 20% solution of cystamine, in the form of free amine, in ethanol. The reaction mixture was stirred overnight at 25° C. and then was diluted by the same volume of water (50 mL). Low molecular weight compounds were been removed by dialysis in the membrane tube with cutoff 2,000 in 2 L of 10% aqueous ethanol for 18 hours at 4° C. with two changes of the solution. The dialysate was concentrated in vacuo and redissolved in 50 mL of 2% trifluoroacetic acid in dichloromethane. Following the incubation for 1 hour at 25° C., bright yellow solution was concentrated in vacuo and neutralized by 5 mL of 10% triethylamine in methanol. The monosubstituted Pluronic® cystamine was isolated by gel filtration on Sephadex LH 20 column (2.5×30 cm) in methanol collecting fractions: 8 mL/4 minute. Yields: 2.0-2.2 g of white solids (70-80% of theoretical). TLC: R f  0.4 (Silicagel plates, dichloromethane-methanol, 9:1). The product developed blue color after spraying the TLC plates with 1% ninhydrine solution in ethanol. 
     4. Monosubstituted Pluronic® succinate. The monosubstituted methoxytrityl Pluronic® was dried in vacuo over phosphorus oxide and treated by 5 molar equivalents of succinic anhydride (Aldrich) in 10 mL of anhydrous pyridine in the presence of catalytic quantity of 4-dimethylaminopyridine for 4 hours at 40° C. Methanol (0.5 mL) was added to quench the excess of reagent for 30 minutes at 25° C. Low molecular weight compounds have been removed by dialysis in the membrane tube with cutoff 2,000 in 2 L of 10% aqueous ethanol for 18 hours at 4° C. with two changes of the solution. The dialysate was concentrated in vacuo and redissolved in 50 mL of 2% trifluoroacetic acid in dichloromethane. Following the incubation for 1 hour at 25° C., bright yellow solution was concentrated in vacuo and neutralized by 5 mL of 10% triethylamine in methanol. The monosubstituted Pluronic®-succinate was isolated by gel filtration on Sephadex LH-20 column (2.5×30 cm) in methanol collecting fractions: 8 mL/4 minute. Yields: 2.4-2.5 g of white solids (&gt;85% of theoretical). TLC: R f  0.3 (Silicagel plates, dichloromethane-methanol, 9:1). 
     5. Monosubstituted Pluronic® phenylphosphate. The monosubstituted methoxytrityl Pluronic® was dried in vacuo over phosphorus oxide and treated by 2 molar equivalents of phenyl phosphorodichloridate in the presence of 2.2 equivalents of diisopropylethylamine in 20 mL of anhydrous dioxane for 1 hour in an ice bath. Then an excess of 1% aqueous hydrochloric acid (100 mL) was added and the reaction mixture was stirred for 1 hour at 25° C. After adjusting pH to 7, low molecular weight compounds have been removed by dialysis in the membrane tube with cutoff of 2,000 in 2 L of 10% aqueous ethanol for 18 hours at 4° C. with two changes of the solution. The monosubstituted Pluronic® phenylphosphate was freeze-dried. Yields: 2.0 g of white solids (70% of theoretical). TLC: R f  0.25 (Silicagel plates dichloromethane-methanol, 9:1). 
     6. Monosubstituted Pluronic® citrate. For modification of Pluronic® block copolymer with citric acid, 330 mg of Pluronic®-amine was first dissolved in 2.5 mL of methanol and added dropwise to a stirring solution of 110 mg of citric acid incubated with 55 mg of N,N′ dicyclohexylcarbodiimide in 5 mL of DMF for 1 hour at 25° C. Following the 4 hour reaction, the modified Pluronic® was isolated by gel filtration on Sephadex LH 20 column (2.5×30 cm) in methanol (fractions 8 mL/4 min). Yields: 0.3 g of white solids (90% of theoretical). TLC: R f  0.35 (Merck Silicagel plates, dichloromethane methanol, 9:1). 
     Method 2. Synthesis of Protein Conjugate with Pluronic® Ethylamine (DSP Method) 
     For activation of Pluronic® block copolymer, 330 mg of Pluronic® amine was first dissolved in 2.5 mL of methanol and added dropwise to a stirring solution of 80 mg of dithiobis(succinimidyl) propionate (DSP) in 2.5 mL of DMF at 25° C. Following the 30 minute reaction, the DSP modified Pluronic® was isolated by gel filtration on Sephadex LH 20 column (2.5×30 cm) in methanol (fractions 8 mL/4 min). TLC: R f  0.5-0.6 (Merck Silicagel plates, dichloromethane-methanol, 9:1). The product developed yellow color after exposure of TLC plates to iodine vapors (test on the presence of Pluronic® polymers). Polymer-containing fractions were collected and concentrated in vacuo. The N-hydroxysuccinimidocarboxyl (NHS) product (Pluronic®-NHS) was dissolved in 1.5 mL of 20% ethanol immediately before reaction with protein. 
     44 mg of horseradish peroxidase (HRP type VI, MW 44 KDa; Sigma, St. Louis, Mo.) was dissolved in 0.5 mL of 0.1 M borate, pH 9.5, and mixed with a previously prepared solution (1.5 mL) of Pluronic®-NHS to obtain total volume of 2 mL. Homogeneous reaction mixture was kept overnight at 4° C. Modified HRP was precipitated in 40 mL of cold (−10° C.) ethanol, incubated in a refrigerator for 30 min, and separated from excess of Pluronic®-NHS by centrifugation at 3000 rpm for 20 minutes at 4° C. Brown precipitate was washed by cold ethanol (2×10 ml) and dried in vacuo. Yield: 75-90 mg. 
     Pluronic®-HRP conjugate was purified by cation exchange chromatography using glass column (2×8 cm) with TSK CM-650M resin in 10 mM sodium acetate, pH 4.4 10 mM sodium chloride and 5% ethanol at elution rate 1.5 mL/minute. First, the column was washed by this eluent, and then elution with sodium chloride (gradient from 0.01 to 0.15M over 60 minutes) was used for isolation of the Pluronic®-HRP and separation of residual (unreacted) HRP. Collected fractions were desalted in dialysis tubes with a cutoff of 20 KDa against water overnight at 4° C. and freeze-dried. The obtained conjugate was analyzed by protein and salt contents using the analytical procedures described below. 
     Method 3. Synthesis of Protein Conjugate with Pluronic® Cystamine (Periodate Method) 
     To a solution of 44 mg of horseradish peroxidase (Sigma, HRP type VI) in 1 mL of water, 50 μl, of freshly prepared 0.1 M aqueous solution of sodium metaperiodate was added to oxidize the vicinal hydroxyl groups of carbohydrate moieties of HRP and generate HRP-aldehyde. The reaction mixture was vortex mixed and kept for 30 minutes at 25° C. in the dark. Activated HRP (HRP-aldehyde) was passed through a small NAP-20 column previously equilibrated with 10 mM ammonium carbonate, pH 9.3, to remove excess of metaperiodate. The brown colored fraction of activated HRP was directly collected in a vial containing 300 mg of Pluronic®-cystamine dissolved in the above buffer (2 ml); the reaction mixture was vortex mixed and kept overnight at 4° C. to form a Schiff base. After overnight incubation, 50 μL of 5 M sodium cyanoborohydride was added, and reaction mixture was vortex mixed and kept for 3 h at 4° C. The solution was degassed and passed through a Sephadex G-25 column (2.5×30 cm) previously equilibrated with 10 mM PBS. The brown colored fractions containing Pluronic®-HRP conjugate were collected, desalted and purified by cation exchange chromatography as described above. Isolated Pluronic®-HRP conjugate was desalted and freeze dried. Yield: 85-90 mg. The conjugate was analyzed by protein and salt contents using the analytical procedures described below. 
     Method 4. Synthesis of Protein Conjugate with Pluronic® Succinate 
     For activation of Pluronic® block copolymer, 330 mg of Pluronic®-succinate and 50 mg of N-hydroxysuccinimide were first dissolved in 5 mL of dry dioxane, then 45 mg of N,N′-dicyclohexylcarbodiimide (DCC) added to the solution at 25° C. Following the overnight reaction, an N-hydroxysuccinimidocarboxyl Pluronic® derivative (Pluronic®-NHS) was isolated by gel filtration on Sephadex LH-20 column (2.5×30 cm) in methanol (fractions 8 mL/4 min). TLC: R f  0.7 (Merck Silicagel plates, dichloromethane-methanol, 9:1). The product developed yellow color after exposure of TLC plates to iodine vapors (test on the presence of Pluronic® polymers). Polymer containing fractions were collected and concentrated in vacuo. The product was dissolved in 1.5 mL of 20% ethanol immediately before reaction with protein. 
     44 mg of horseradish peroxidase (Sigma, HRP type VI) was dissolved in 0.5 mL of 0.1 M borate, pH 9.5, and mixed with a previously prepared solution (1.5 mL) of Pluronic®-NHS to obtain total volume of 2 mL. Homogeneous reaction mixture was kept overnight at 4° C. Modified HRP was precipitated in 40 mL of cold (−10° C.) ethanol, incubated in a refrigerator for 30 minutes and separated from excess of Pluronic®-NHS by centrifugation at 3000 rpm for 20 minutes at 4° C. Brown precipitate was washed by cold ethanol (2×10 ml) and dried in vacuo. Yield: 80-100 mg. 
     Pluronic®-HRP conjugate was purified by cation exchange chromatography using glass column (2×8 cm) with TSK CM-650M resin in 10 mM sodium acetate, pH 4.4, 10 mM sodium chloride and 5% ethanol at elution rate 1.5 ml/minutes. First, the column was washed by this eluent, and then elution with sodium chloride (gradient from 0.01 to 0.15M over 60 min) was used for isolation of the Pluronic®-HRP and separation of residual (unreacted) HRP. Collected fractions were desalted in dialysis tubes with a cutoff 20 KDa against water overnight at 4° C. and freeze-dried. The obtained conjugate was analyzed by protein and salt contents, using the analytical procedures described below. 
     Method 5. Synthesis of Protein Conjugate with Pluronic® Phenylphosphate 
     330 mg of Pluronic®-phenylphosphate and 60 mg of 1 hydroxybenzotriazole were dissolved in 5 ml of aqueous dioxane, and 40 mg of ethyl diisopropylaminocarbodiimide (EDC) was added to the solution at 25° C. The reaction was performed for 4 hours at 25° C. and the hydroxybenzotriazole-modified Pluronic® (Pluronic®-HBT) was isolated by gel filtration on Sephadex LH-20 column (2.5×30 cm) in methanol (fractions 8 ml/4 min). The product developed yellow color after exposure of TLC plates to iodine vapors (test on the presence of Pluronic® polymers). Polymer containing fractions were collected and concentrated in vacuo. The product was dissolved in 1.5 mL of 20% ethanol immediately before reaction with protein. 
     44 mg of horseradish peroxidase (Sigma, HRP type VI) was dissolved in 0.5 mL of 0.1 M borate, pH 9.5, and mixed with a previously prepared solution (1.5 mL) of Pluronic® HBT to obtain total volume of 2 mL. Homogeneous reaction mixture was kept overnight at 4° C. Modified HRP was precipitated in 40 mL of cold (−10° C.) ethanol, incubated in refrigerator for 30 minutes and separated from excess of Pluronic®-HBT by centrifugation at 3000 rpm for 20 minutes at 4° C. Brown precipitate was washed by cold ethanol (2×10 ml) and dried in vacuo. Yield: 80-100 mg. 
     Pluronic® HBT conjugate was purified by cation exchange chromatography using a glass column (2×8 cm) with TSK CM-650M resin in 10 mM sodium acetate, pH 4.4, 10 mM sodium chloride and 5% ethanol at elution rate 1.5 mL/minute. First, the column was washed by this eluent, and then elution with sodium chloride (gradient from 0.01 to 0.15 M over 60 min) was used for isolation of the Pluronic®-HBT and separation of residual (unreacted) HBT. Collected fractions were desalted in dialysis tubes with a cutoff of 20 KDa against water overnight at 4° C. and freeze-dried. The obtained conjugate was analyzed by protein and salt contents using the analytical procedures described below. 
     Method 6. Synthesis of Protein Conjugate with Pluronic® Citrate 
     300 mg of Pluronic®-citrate and 50 mg of N-hydroxysuccinimide were first dissolved in 5 mL of dry dioxane, then 45 mg of N,N′-dicyclohexylcarbodiimide added to the solution at 25° C. Following the overnight reaction, an N-hydroxysuccinimidocarboxyl Pluronic® derivative (Pluronic®-citrate-NHS) was isolated by gel filtration on Sephadex LH-20 column (2.5×30 cm) in methanol (fractions 8 mL/4 min). TLC: R f  0.7 (Merck Silicagel plates, dichloromethane-methanol, 9:1). The product developed yellow color after exposure of TLC plates to iodine vapors. Polymer containing fractions were collected and concentrated in vacuo. The product was dissolved in 1.5 mL of 20% ethanol immediately before reaction with protein. 
     44 mg of horseradish peroxidase (Sigma, HRP type VI) was dissolved in 0.5 mL of 0.1 M borate, pH 9.5, and mixed with a previously prepared solution (1.5 mL) of Pluronic®-citrate-NHS to obtain total volume of 2 mL. Homogeneous reaction mixture was kept overnight at 4° C. Modified HRP was precipitated in 40 mL of cold (−10° C.) ethanol, incubated in refrigerator for 30 minutes and separated from excess of Pluronic®-NHS by centrifugation at 3000 rpm for 20 minutes at 4° C. Brown precipitate was washed by cold ethanol (2×10 ml) and dried in vacuo. Yield: 70-85 mg. 
     Pluronic®-citrate-HRP conjugate was purified by cation exchange chromatography using glass column (2×8 cm) with TSK CM-650M resin in 10 mM sodium acetate, pH 4.4, 10 mM sodium chloride and 5% ethanol at elution rate 1.5 mL/minute. First, the column was washed by this eluent, and then elution with sodium chloride (gradient from 0.01 to 0.15M over 60 min) was used for isolation of the Pluronic®-citrate-HRP and separation of residual (unreacted) HRP. Collected fractions were desalted in dialysis tubes with a cutoff of 20 KDa against water overnight at 4° C. and freeze-dried. The obtained conjugate was analyzed by protein and salt contents using the analytical procedures described below. 
     Method 7. Analysis of Pluronic® Protein Conjugates 
     1. Protein Assay. Pierce BCA Kit was used to measure protein content in the Pluronic® conjugates. Protein sample was dissolved in water (1.0 mg/mL). Standard solutions of bovine serum albumin (BSA) have been used to obtain calibration curve. Three parallel aliquots were taken per sample and each standard solution. The kit&#39;s Solution A (10 mL) was mixed with the kit&#39;s Solution B (0.2 mL, 1150 dilution), then 0.02 mL of sample solution and 0.2 mL of summary mixture AB were added into 96 well plate by multi channel pipette (8 tips). Mixtures were incubated for 30 minutes at 37° C., and an absorbance of purple solutions was measured at 550 nm. Results were compared with calibration curve to calculate the protein content. 
     2. Pluronic®/protein molar ratio. The Pluronic®/protein molar ratio was determined by substitution rate of amino groups in protein. Solutions of initial and modified protein (1 mg/ml) were first prepared. Commercial 5% solution of 2,4,6 trinitrobenzosulfonic acid (TNBS) (Sigma) was dissolved in 100 times by 0.1M Na-borate buffer, pH 9.5, before analysis. Reaction was performed with 20 μL of sample and 20 μL of TNBS solution in 160 μL of 0.1 M Na borate buffer, pH 9.5, in 96 well plate. Three parallel aliquots were taken per each sample. Reaction mixtures were incubated for 1 h at 25 C, and an absorbance of brown solutions was determined at 405 nm in Microplate reader. HRP contains 6 primary amino groups. To calculate the substitution rate, the amino group content (A) was divided by protein content (N). The following equation calculates the number of polymer molecules per protein S i =4N 0 (A 0 N 0 −A i /N i )/A 0 . 
     3. HRP enzymatic activity. Samples (20 μL) with protein concentration 0.1 mg/mL obtained by dilution of initial solutions (1 mg/mL) in 10,000 times by water were taken in 96 well plate. 200 μL of the solution of o-phenylenediamine (5 mg/mL) in 0.1 M citrate buffer, pH 5, containing 1 mg/mL BSA, 0.1% Triton and 0.02% of hydrogen peroxide was used as color substrate in reaction for 5 minutes at 37° C. 30 μL of a Stop solution (0.5% sodium sulphite dissolved in a 2 N sulfuric acid) was immediately added, and absorbance measured at 450 550 nm to determine the HRP activity. Calibration curve for HRP activity per mg of protein was plotted and used in these calculations. 
     Example 5 
     Binding of Pluronic®-HRP Conjugates to Brain Endothelial Cells 
       FIG. 4  summarizes the results of the study of binding of various Pluronic®-HRP conjugates with bovine brain microvessel endothelial cell (BBMEC) monolayers. The primary cultured BBMEC retain many of the morphological and biochemical characteristics of the BBB and display transport systems present in vivo (Miller et al. (1992) J. Tissue Cult. Methods, 14:217-224). These cells form tight junctions and possess low pinocytic activity and therefore possess low permeability for macromolecules, including unmodified HRP (Karyekar et al. (2003) J. Pharm. Sci., 92:414-423). At the same time, these cells express receptor systems that enable transcellular transport (transcytosis) of selected macromolecules (Miller et al. (1994) J. Cell. Physiol., 161:333-341; Maresh et al. (2001) Life Sci., 69:67-73). Thus, primary cultured BBMEC are well suited for identifying and characterizing the transport processes in the BBB at the cellular level. 
     BBMECs were isolated from cow brains, obtained at a local meat processing plant, using mechanical and enzymatic disruption of brain matter coupled with gradient separation as described in Miller et al. (J. Tissue Cult. Methods (1992) 14:217-224). Cells were seeded onto either 1) collagen coated, fibronectin-treated 24-well culture plates at a density of 50,000 cells/cm 2  or 2) polycarbonate membrane inserts, 24-mm, 0.4 μm pore size (Fisher Scientific, Pittsburgh, Pa.) at a density of 250,000 cells/insert. The cells were grown using media consisting of 45% minimum essential medium (MEM), 45% Ham&#39;s F-12 (F12), and 10% horse serum supplemented with antibiotics and heparin sulfate and used after reaching confluency (typically 10-12 days). 
     The BBMECs were incubated with unmodified HRP or the conjugates for 60 minutes. The cell monolayers were washed with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) and then washed in PBS. Notably, the experiments can be conducted with and without 10% fetal calf serum to assess possible effects of serum binding on the transport of the modified HRP. The cells were then lysed in 1% Triton X100 and the net amount of the cell-bound enzyme was determined by colorimetric reaction with o-phenylenediamine in 0.1M citrate buffer containing 0.1% Triton X100, 1 mg/ml BSA, and 0.02% hydrogen peroxide. The amount of HRP was normalized by the amount of cell protein as determined by the Pierce BCA assay. 
     There was little difference in binding of unmodified HRP and HRP-P85(2) conjugate, containing a stable linkage. However, the binding of conjugates containing a cleavable disulfide linkage (HRP-P85(3) and HRP-L121) was increased significantly. The most pronounced effect (over 6-fold increase of binding) was observed in the case of a hydrophobic Pluronic® L121 conjugate. This result was consistent with the earlier report suggesting strong binding of this hydrophobic block copolymer with the BBMEC membranes (Batrakova et al. (2003) J. Pharmacol. Exp. Ther., 304:845-854). HRP-P85(3) displayed about a 2-fold increase in binding with the cells. Therefore, modification of HRP with Pluronic® can increase the overall binding of this protein with brain microvessel endothelial cells. 
     Example 6 
     Permeability of Pluronic®-HRP in BBMEC Monolayers 
     The following study evaluated permeability of various Pluronic®-HRP conjugates in BBMEC monolayers. The method entailed radioactively labeling the native or modified polypeptides with  125 I using Iodobeads (Pierce, Rockford, Ill.) and purifying by Sephadex G10 column chromatography. Confluent BBMEC monolayers were grown on polycarbonate membrane inserts placed in Side-Bi-Side diffusion cells from Crown BioScientific, Inc. (Somervile, N.J.) and maintained at 37° C.±0.1° C. and preincubated with an assay buffer (122 mM NaCl, 25 mM NaHCO 3 , 10 mM glucose, 3 mM KCl, 1.2 mM MgSO 4 , 0.4 mM K 2 HPO 4 , 1.4 mM CaCl 2  and 10 mM HEPES) for 30 minutes. The confluent monolayers mean resistance was no less than 120.0 Ω·cm 2 . For luminal to abluminal transport studies, the assay buffer at the luminal (apical) side was removed and replaced with the transport buffer containing solutions of the modified or native polypeptides radioactively labeled with  125 I (10 6  cpm). Notably, the experiments can be conducted with and without 10% fetal calf serum to assess possible effects of serum binding on the transport of the modified HRP. The aliquots at the abluminal (basolateral) side of the monolayers was removed at 15, 30, 45, 60, 90 and 120 minutes and immediately replaced with the same volume of the assay buffer. The radioactivity was measured in a gamma counter. In the case of HRP, the enzyme activity was also measured using o-phenylenediamine as a substrate to determine activity of the protein. All transport experiments were conducted in triplicate. The trans-epithelial electrical resistance (TEER) of the monolayers was recorded as an index of cell viability and monolayer integrity. At the end of the experiment, the monolayers were solubilized in 0.5 ml of 1% Triton X100 and the amount of cell associated radioactivity was measured along with the radioactivity from solutions in donor and receiver chambers. The liquid samples were precipitated by 30% trifluoroacetic acid (TFA) and the radioactivity in the pellet and supernatant was determined. The percent of the intact material was defined as [cpm pellet/(cpm pellet+cpm supernatant)]×100%. 
     Notably, neither the conjugated nor unmodified HRP caused alteration in TEER suggesting that the integrity of the monolayer&#39;s tight junctions was not affected. At the same time there were substantial differences in permeability displayed by different conjugates. The transport of unmodified HRP was quite slow. Modification of HRP with P85 via non-degradable linkage (HRP-P85 (2)) resulted in approximately two-fold increase in the permeability of the protein ( FIG. 5 ). The conjugate containing the cleavable linkage, HRP-P85 (3), was transported almost six times fasted than the unmodified HRP ( FIG. 5 ). Finally, the conjugate with the hydrophobic Pluronic® L121, also containing a cleavable linkage, displayed the highest rates of transport across the BBMEC monolayers ( FIG. 5 ). The rate of its transport was over 10-times higher than that of the unmodified HRP. Overall, the conjugation of the enzyme with highly hydrophobic Pluronic® L121 via the biodegradable linkage resulted in the greatest increase in permeability across the brain endothelial cells. 
     Example 7 
     Microviscosity Studies in BBMEC 
     To examine effects of Pluronic®s on the fluidity properties of the cell plasma membranes in BBMEC, a fluorescent compound, 1[4(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA DPH) was used (Prendergast et al. (1967) Biochemistry, 20:7333-7338). For these studies, the BBMEC suspension was incubated with various Pluronic® conjugates for 2 hours at 37° C., washed twice and incubated with 2 μM TMA-DPH (Molecular Probes, Eugene, Oreg.) for 10 minutes. Then, the cells were washed twice to remove extracellular probe, and re-suspended in an appropriate volume of PBS and changes in fluorescent polarization were recorded. 
     Fluorescence intensities were measured with a Hitachi F5000 spectrophotometer equipped with a polarizer set. This instrument detects fluorescence intensity (I) with the relative position of the polarizer and analyzer (parallel, I ∥ , or perpendicular, I ⊥ ) and fluorescence anisotropy r, was calculated according to Eq. 1:
 
 r =( I   ∥   −I   ⊥ )/=( I   ∥ +2 I   ⊥ )
 
An excitation wavelength of 365 nm and an emission wavelength of 425 nm were used for both probes. Cell suspensions were gently mixed before each reading. In all cases, corrections for stray light and intrinsic fluorescence were made by subtracting the values for I ∥  and I ⊥ , of unlabeled samples from those of identical but labeled samples. Microviscosities (η) were derived as described previously TMA-DPH (Chazotte, B. (1994) Biochim. Biophys. Acta., 1194:315-328) by the method based on the Perrin equation (Eq. 2) for rotational depolarization of a nonspherical fluorophore:
 
 r   0   /r= 1+ C ( r )τ/η
 
where r 0  and r are limiting and measured fluorescence anisotropies, T is the absolute temperature, and τ is the exited state lifetime. The value of r 0  and τ were 0.362 and 7 ns, respectively. C(r) is a molecular shape parameter equal to 15.3×10 5  poise·deg −1 s −1 .
 
     The obtained results suggest that more lipophilic PBC have stronger fluidization effect on BBMEC membranes ( FIG. 6 ). 
     Example 8 
     Fatty Acid Conjugates 
     Artificial hydrophobization of polypeptides with a small number of fatty acid residues (e.g. stearate or palmitate) has been shown to enhance cellular uptake (Kabanov et al. (1989) Protein Eng. 3:39-42). Specifically, this approach involves point modification of lysine or N-terminal amino groups with one or two fatty acid residues per protein molecule. As a result of such modification the protein molecule remains water-soluble but also acquires hydrophobic anchors that can target even very hydrophilic proteins to cell surfaces (Slepnev et al. (1995) Bioconjug. Chem. 6:608-615). Fatty acid modification of water-soluble polypeptides, such as HRP, results in enhanced polypeptide binding with the cell membranes and internalization in many cell types (Slepnev et al. (1995) Bioconjug. Chem. 6:608-615). 
     Previous studies have shown that modification of the Fab fragments of antibodies against gliofibrillar acid protein (GFAP) and brain specific alpha 2-glycoprotein (alpha 2GP) with stearate led to an increased accumulation of the modified Fab fragments in the brain in a rat (Chekhonin et al. (1991) FEBS Lett. 287:149-152; Chekhonin et al. (1995) Neuroreport. 7:129-132). Furthermore, a neuroleptic drug conjugated with the stearoylated antibody Fab fragments was much more potent compared to the free drug. In comparison, fatty acylated Fab fragments of non-specific antibodies did not accumulate in the brain but instead accumulated in the liver, while stearoylated Fab fragments of brain-specific antibodies displayed preferential accumulation in the brain. Subsequent studies using bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of BBB demonstrated that stearoyaltion of ribonuclease A (approx. 13.6 kDa) increases the passage of this enzyme across the BBB by almost 8.8-fold (Chopineau et al. (1998) J. Control Release 56:231-237). Of the three fatty acid derivatives analyzed-myristic, palmitic and stearic, the latter was the most active. A possible mechanism for the entry of the fatty acylated polypeptides to the brain is adsorptive endocytosis. Given the characteristics of the BBB transport for nonessential free fatty acids as reviewed elsewhere, it is unlikely that the modified polypeptides are able to use those transporters (Banks et al. (1997) Permeability of the blood-brain barrier to circulating free fatty acids. in: Handbook of Essential Fatty Acid Biology: Biochemistry, Physiology, and Behavioral Neurobiology, pp. 3-14 (Yehuda, S, and Mostofsky, D. I., Eds.) Humana Press, Totowa, N.J.). Thus, use of free fatty acid receptor mediated transport by the modified polypeptides is not likely. However, essential fatty acids, such as linoleic, are more readily transported (Edmond, J. (2001) J. Mol. Neurosci. 16:181-193; discussion 215-221; Edmond et al. (1998) J. Neurochem. 70:1227-1234). 
     While stearic acid is exemplified herein, the instant invention contemplates the conjugation of essential fatty acids directly or through a cleavable linker to the protein to be transported across the BBB. 
     The enzyme horseradish peroxidase (HRP) contains six amino groups available for chemical modification. To modify these groups with stearic acid residues, the enzyme was acylated with stearoyl chloride in the reverse micelle system of Aerosol OT in octane (Slepnev et al. (1995) Bioconjug. Chem. 6:608-615). Based on the titration of the remaining free amino groups of the protein with 2,4,6-trinitrobenzenesulfonic acid, an average of 1.1 stearic acid residue per protein molecule was introduced onto HRP under these conditions. The solubility of the stearoylated HRP (St-HRP) was about 1 mg/ml, its RZ was 2.7 and the catalytic activity −50% of the native HRP activity (RZ of native HRP −3.0). 
     The effect of stearic acid acylation on the HRP overall binding with brain endothelial cells was studied using primary bovine brain microvessel edothelial cells (BBMEC) as an in vitro model of the BBB. Briefly, the BBMEC monolayers were grown to confluency (10-13 days), and then exposed for various times to the solutions of unmodified or stearoylated HRP (St-HRP) (40 μg/ml). After that the cell monolayers were washed (with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS), and then in PBS) and lysed in 1% Triton X100. The HRP activity in the lysates was determined by colorimetric reaction with o-phenylenediamine in 0.1 M-citrate buffer, containing 0.1% Triton X-100, 1 mg/ml BSA, and 0.02% hydrogen peroxide. The amount of the HRP was normalized by the cell protein determined using the Pierce-BCA assay. The results are presented in  FIG. 7A . Modification of HRP resulted in about a 3 fold increase of its overall binding with the BBMEC. 
     BBMEC monolayers grown to confluency on membrane inserts were placed in Side-Bi-Side diffusion cells. The unmodified or St-HRP (40 μg/ml) was placed in the donor chamber at the apical side of the monolayers. The appearance of the enzyme in the receiver chamber at the basolateral side of the monolayers was registered by measuring the activity of HRP. The trans-epithelial electrical resistance (TEER) of the monolayers was recorded as an index of cell viability and monolayer integrity. As shown in  FIG. 7B , modification increased the apparent permeability coefficient (P app ) about 2-fold (0.4×10 8  cm/s) when compared with the non-modified HRP (0.24×10 8  cm/s). There were no changes in TEER or permeability of a paracellular marker,  3 H-mannitol, demonstrating that the integrity of the monolayers was not altered. 
     Example 9 
     Modification of Superoxide Dismutase (SOD) by Pluronics® 
     Introduction 
     The antioxidant enzyme superoxide dismutase (SOD) exerts a crucial protective role in pathophysiological conditions induced by elevated reactive oxygen species (ROS), such as inflammation, neurodegeneration and neurocardiovascular dysregulation. For example, SOD1 (also called Cu/ZnSOD), the SOD isoform that exists primarily in cytoplasm, is known to exert anti-hypertensive effects by scavenging superoxide (O 2   − ) in the brain. Thus, SOD is an antioxidant therapy for neuroncardiovascular-related diseases, if peripherally administered SOD1 can penetrate the blood brain barrier (BBB) and the plasma membrane of neurons. It is demonstrated herein that conjugating SOD with poly(ethylene oxide)-poly(propylene oxide) block copolymer (Pluronics®) improves SOD delivery to the brain and provide therapeutic effects. 
     Experimental Procedures 
     
         
         
           
             Conjugation of SOD1 and Pluronics® 
           
         
       
    
     The conjugation was initiated by the generation of mono-amine Pluronics®, followed by activation with N-hydroxysuccinimide (NHS)-containing crosslinker disuccinimidyl suberate (DSS). The activated Pluronics® was gel filtrated on a NAP™-25 column (GE Healthcare) in 20% ethanol aqueous solution to remove excess of DSS. The conjugates were precipitated in cold acetone to remove excess non-reacted copolymers. 
     More specifically, mono-amine P85 (50 mg) in 0.5 ml of methanol was mixed with DSS (20 mg, 6-fold molar excess) solution in 0.5 ml of DMF stored over molecular sieves (4 Å). The mixture was supplemented with 0.1 ml sodium borate buffer (0.1 M, pH 8) and incubated for 30 minutes at 25° C. After excess DSS was removed by gel filtration, activated copolymer was mixed with SOD1 (4 mg) in 0.5 ml of 0.1 M sodium borate (pH 8). The homogeneous reaction mixture was incubated overnight at 4° C. Cold acetone was then used to precipitate the conjugates.
         Generation of Mono-Amine Pluronics®       

     The hydroxyl group at one end of the Pluronics® chain was protected by methoxyltrityl chloride (MTr-Cl) and followed by activating by carbonyldiimidazole (CDI) and reacting with ethylenediamine (EDA). The product was dissolved in trifluoroacetic acid (TFA) to deprotect the hydroxy group. The final product was purified in gel filtration column to remove all small molecules including EDA. The reaction and purity of the products were assayed by thin liquid chromatography (TLC). The amine modification degree was assayed by ninhydrin titration.
         Enzymatic Activity Assay Based on Pyrogallol Autoxidation       

     Pyrogallol autoxidation occurs rapidly at pH&lt;9.5 and involves O 2   −  as a chain-propagating species. Superoxide dismutase (SOD) catalyzes the dismutation of the superoxide radical (O 2   − ) into hydrogen peroxide and elemental oxygen (O 2 ). Thus SOD inhibits pyrogallol autoxidation and, as such, provides a method to determine SOD activity. Briefly, 0.0002 to 200 μg/ml (based on Micro BCA™ protein assay) SOD1 or SOD1/P85 conjugates were mixed with 20 μl of a fresh pyrogallol (0.05 mg/ml) in Tris/HCl buffer (0.1 M, pH 8.0) containing 1 mM DPTA. The reaction mixture was added to 96-well plates to a final volume of 200 μl in Tris/HCl buffer (0.1M, pH 8.0). The rate of autoxidation was measured immediately by recording the increase in absorbance of 420 nm up to 10 minutes in SpectraMax® M5 (Molecular Devices). The data was interpreted as the inhibition rate (%) vs concentration using the equation: {[(S1−S3)−(SS−S2)]/(S1−S3))×100, wherein S1=slope of water; S2=slope of solution dissolving sample and without pyrogallol; S3=slope of water without pyrogallol; and SS=slope of sample.
         Cellular Uptake       

     CATH.a cells, a catecholaminergic neuronal cell culture model, were cultured in RPMI 1640 media supplemented with 8% horse serum, 4% FBS, and 1% penicillin-streptomycin. CATH.a neurons were differentiated for a total of 6 days by adding fresh N-6,2′-O-dibutyryladenosine 3′,5′-cycle-monophosphate (1 mM) to the culture media every 2 days. Bovine brain microvessel endothelial cells (BBMEC) were isolated from the gray matter of the bovine cerebral cortex by enzymatic digestion followed by subsequent centrifugations. Cells were seeded onto collagen-coated, fibronectin-treated 25 cm 2  plate and grown to confluence (typically 10-12 days) in a culture completed media. CATH.a cells were pretreated in serum free media for 24 hours and then exposed to SOD1 or SOD1/P85 (80 μg/ml) in serum free media for various time intervals at 37° C. CATH.a cells were collected after washing with cold phosphate-buffered saline and incubating with 0.25% trypsin for 5 minutes. BBMEC were pretreated in serum free media for 1 hour and incubated with SOD1 or SOD1/L81 conjugates (80 μg/ml) for various time course. BBMEC were collected by scraping the flask without trypsin digestion. The collected cell pellet was lysed by sonication and cell protein content was measured using Bio-Rad protein assay. Sample (50 μg or 100 μg) was loaded to 12% home-made native gel and electrophoresis was performed routinely. The SOD1 in-gel activity was determined by staining with solution containing 2.43 mM nitroblue tetrazolium, 28 mM TEMED and 28 uM of riboflavin.
         Confocal Microscopy       

     CATH.a neuronal cell in two-well chambered coverglasses (Fischer Scientific, Waltham, Mass.) were incubated with 80 μg/ml Alexa Fluor 680 labeled SOD1 or SOD1/P85 conjugates. CATH.a cells were cultured as described above. MDCK cells were plated in four-well chamber at a density of 40,000 cells/well and grown for 2 days to reach 70% confluence in a culture media DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were incubated with SOD1 or SOD1/P85 conjugates for various time course at 37° C. The medium with the protein was removed and the cells were rinsed twice with PBS. The cells were covered with PBS and the images were recorded using confocal microscope (Carl Zeiss LSM 510 Meta, Peabody, Mass.). 
     Results 
       FIG. 8A  provides a schematic for the conjugation of SOD1 and Pluronics® via a non-degradable linker.  FIG. 8B  provides a schematic for the generation of mono-amine Pluronics®. The Pluronics® used were L81 and P85. The average formula of L81 is EO 3 -PO 43 -EO 3  with an average molecular weight of 2750, and HLB of 2, and a critical micelle concentration (CMC) value of 0.006% at 37° C. (as determined by pyrene probe). The average formula of P85 is EO 26 -PO 40 -EO 26  with an average molecular weight of 4600, and HLB of 16, and a critical micelle concentration (CMC) value of 0.03% at 37° C. (as determined by pyrene probe). 
       FIG. 9A  provides an image of an SDS-PAGE of SOD1 and SOD1/P85 conjugates.  FIG. 9B  provides an image of a native, in-gel SOD1 activity assay of SOD1 and SOD1/P85 conjugates.  FIG. 9C  is a graph of SOD1 enzymatic activity based on pyrogallol autoxidation. The IC 50  for SOD1 is 2.14; IC 50  for SOD1-DSS-P85 is 7.12; and IC 50  for SOD1-EDC-P85 is 6.30. 
       FIGS. 10A and 10B  provide the native, in-gel SOD1 activity assay of SOD1/P85 conjugates uptake within CATH.a neuronal cells ( FIG. 10A ) and SOD1/L81 conjugates uptake within BBMEC ( FIG. 10B ) at various incubation time courses. The gels were analyzed using ImageJ software to convert the lane profile into the bar graphs. The cellular bound/internalized SOD1 conjugates smeared in lanes 3 and 5 in  FIG. 10A  and lanes 5 and 7 in  FIG. 10B , similar to the profile of SOD1 conjugates alone in  FIG. 9B . Native SOD1 did not show enhanced binding and activity. These data indicate that Pluronics® modification increases SOD1 binding and internalization in both neurons and brain endothelial cells. 
       FIGS. 11A and 11B  provide the cellular localization of SOD1 and SOD1/P85 labeled by Alex Flour 680 within CATH.a neuronal cells ( FIG. 11A ) and MDCK cells ( FIG. 11B ) at various time intervals. 
     Theses data demonstrate that SOD1 and Pluronic® polymers can be conjugated such that the activity of SOD1 is maintained (see, e.g., DSS and EDC links). Pluronic® modification allowed SOD1 to penetrate neuron cell membranes and resulted in an increase in SOD1 activity. Pluronic® L81 modification also enhanced SOD1 uptake in primary brain endothelial cells, thereby indicating that such modifications increase SOD1 brain delivery and can be used for therapeutic treatment of neurocardiovascular diseases. 
     Example 10 
     Transport Across the Blood-Brain Barrier of Pluronic Leptin 
     Introduction 
     The incidence and prevalence of obesity has increased substantially in the past decade and is now regarded as a global epidemic associated with other morbid conditions. Leptin is a product of the ob (obese) gene and is a 16 kDa protein, secreted into circulation and crosses the blood-brain barrier (BBB) to interact with its receptors to affect appetite and energy expenditure. Leptin is transported to the central nervous system by a saturable, unidirectional system that involves binding of leptin to the leptin receptor expressed by endothelial cells of the BBB that function as leptin transporters. Impaired transport of leptin across the BBB can lead to leptin resistance, which is a cause of obesity. Therapy to mitigate obesity conditions includes the use of leptin. However, native leptin has had limited clinical use because of such problems as short half-life in circulation and the block in the BBB transport that arises in obesity. Pluronic® block copolymer conjugations (structural/chemical modifications of peptides with macro-molecular polymers) improve the efficiency of peptide-based therapeutics and overcome many of the problems associated with them. Such protein modifications have been shown herein to enhance peptide stability and solubility, membrane transport, circulation, while reducing systemic clearance and degradation. It has been demonstrated herein that modified horseradish peroxidase with one of the Pluronic® copolymer P85 enhance the delivery of this protein across the BBB in vivo and brain microvessel endothelial cells in vitro. 
     The effect of chemical modifications of leptin conjugated with Pluronic® P85 [leptin(ss)-P85] on BBB uptake and stability while reducing systemic clearance is studied below. 
     Experimental Procedures 
     Iodination 
     Pluronic® P85-leptin conjugate and recombinant murine leptin (rleptin) were synthesized. Pluronic® P85-modified leptin was radioactively labeled with  125 I (Perkin Elmer, Mass.) by the chloramine T method and purified on a column of G-10 sephadex. Recombinant leptin was radio iodinated with  131 I by the lactoperoxidase method. 
     Blood-to-Brain Influx Rate 
     Multiple-time regression analysis was applied to calculate the blood-to-brain unidirectional influx rate (Ki). Male CD-1 mice were given an injection into the jugular vein of 0.2 ml of lactated Ringer&#39;s solution with 1% BSA (LR-BSA) containing of  125 I-leptin(ss)-P85. Blood and brain were collected between 2 to 60 minutes after i.v. injection. The levels of radioactivity in serum (50 μl) and brain samples were counted in a gamma counter for 3 minutes. The brain/serum ratios (μl/g) for  125 I-leptin(ss)-P85 were plotted against their respective exposure times (Expt). Exposure time was calculated from the equation:
 
 Am/Cpt=Ki[∫   t   0   CP ( t ) dt]/Cp ( t )+ Vi  
 
where Am is cpm/g of brain, Cpt is cpm/μL of arterial serum at time t, and exposure time (Expt, in minutes) is measured by the term [∫ t   0 CP(t)dt]/Cp(t). The linear portion of the relation between the brain/serum ratios versus Expt was used to calculate Ki (μl/g-min) and Vi (μl/g). The slope of the linearity measures Ki and is reported with its error term. The y-intercept of the linearity measures Vi, the initial volume of distribution in brain at t=0.
 
     To determine whether brain uptake of  125 I-leptin(ss)-P85 was saturable, either 1 μg/mouse nonradioactive Pluronic®-modified, leptin(ss)-P85 or nonradioactive rleptin (1 μg/mouse) was included in the i.v. injection of some mice. Blood and brain were harvested at 10 minutes after i.v. injection. 
     To determine the rate of clearance of  125 I-leptin(ss)-P85 from the serum, results were expressed as the percent of the injected dose in each milliliter of serum (% Inj/ml) and these values were plotted against time (minutes). The % Inj/ml was determined by the equation:
 
%  Inj /ml=100(CPM/ml serum/(mean CPM/injection)
 
     The percent of the injected dose entering each gram of brain (% Inj/g) corrected for the vascular space and Vi was calculated at each time point from the equation:
 
%  Inj /g=100( Am/Cpt−Vi ) Cpt/Inj  
 
where Inj is the dose of  125 I-leptin(ss)-P85 injected i.v.
 
Stability and Presence of  125 I-leptin-P85 in Blood and Brain
 
     Mice were given an injection into the jugular vein of 0.2 ml of LR-BSA containing of  125 I-leptin-P85. For acid precipitation, arterial blood and brain were collected at 10 minutes, 6, 18, or 24 hours after i.v. injection. The brain was homogenized in 1% BSA in 0.25 M PBS and the blood were centrifuged at 5400×g for 10 minutes at 4° C. The supernatant and the serum were precipitated with 30% trichloroacetic acid. The precipitation rate was the percentage of radioactivity in the pellet divided by the total radioactivity in the pellet and supernatant. 
     For the gel electrophoresis, blood and brain were collected at 10 minutes after i.v. injection. The supernatant of the brain homogenate was obtained by centrifugation at 9000×g for 15 minutes at 4° C. The supernatant was concentrated by a speed vacuum system. The supernatant and serum samples were heated at 60° C. to denature proteins before 12% Bis-Tris NuPAGE® electrophoresis (Invitrogen, Carlsbad, Calif.) was performed. The gel was dried and exposed to BioMax® film (Kodak, Rochester, N.Y.) overnight. 
     Octanol/Buffer Partition Coefficient 
     Lipid solubility of leptin(ss)-P85 was measured by adding  125 I-leptin(ss)-P85 to tubes containing 0.25 M chloride-free PBS (PH 7.5) and octanol. This solution was vigorously mixed and then centrifuged at 5400×g for 10 minutes to separate the two phases. The mean partition coefficient was expressed as the ratio of cpm in the octanol phase to the cpm in the buffer phase. 
     Capillary Depletion 
     Anesthetized mice received an i.v. injection  125 I-leptin(ss)-P85. At 10 minutes, arterial blood was collected from the abdominal aorta. The descending aorta was clamped, both jugular veins severed, and the mouse was perfused with 20 ml of lactated Ringer&#39;s solution through left ventricle of the heart before decapitation. The whole brain removed, weighed, and placed in an ice-cold glass homogenizer. The brain was homogenized (10 strokes) in 0.8 ml of physiological buffer (10 mM HEPES, 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl 2 , 1 mM MgSO 4 , 1 mM NaH 2 PO 4 , and 10 mM D-glucose, pH 7.4). Dextran solution (1.6 ml of a 26% solution) was added to the homogenate, mixed, and homogenized a second time (3 strokes). The homogenate was centrifuged at 5400×g for 15 minutes at 4° C. The resulting supernatant (brain parenchymal fraction) and pellet (capillary fraction) were separated. The parenchyma/serum and capillary/serum ratios (μl/g) were calculated by the equation:
 
Ratio=(cpm/g of tissue)/(cpm/μl of serum)
 
Brain Perfusion Studies
 
     Brain uptake of  125 I-leptin(ss)-P85 was determined by the brain perfusion method of Banks et al. Briefly,  125 I-leptin(ss)-P85 was diluted in Zlokovic&#39;s buffer (pH 7.4; 7.19 g/l NaCl, 0.3 g/l KCl, 0.28 g/l CaCl 2 , 2.1 g/l NaHCO 3 , 0.16 g/l KH 2 PO 4 , 0.17 g/l anhydrous MgCl 2 , 0.99 g/l D-glucose, and 10 g/l BSA added on the day of perfusion). The thorax was opened, the heart was exposed and the descending thoracic aorta was clamped. Both jugular veins were severed. A 26-gauge butterfly needle was inserted into the left ventricle of the heart, and the buffer containing  125 I-leptin(ss)-P85 was infused at a rate of 2 ml/minute. Perfusion was stopped after 1-10 minutes for each mouse, followed by decapitation (n=3/time point). The brain was collected, weighed and counted in a gamma counter for 3 minutes. The brain/perfusion ratio (μl/g) was calculated by the formula:
 
Brain/perfusion ratio=(cpm/g of brain)/(cpm/μl of perfusion)
 
     After the brain/perfusion ratio of  125 I-leptin(ss)-P85 was plotted against time (minutes), the slope of the regression line represented the influx rate (Ki). 
     To test inhibition, three groups of mice were studied by brain perfusion: 
     1) mice perfused with only  125 I-leptin(ss)-P85, 
     2) mice perfused with  125 I-leptin(ss)-P85+nonradioactive rleptin at 100 ng/ml, and 
     3) mice perfused with  125 I-leptin(ss)-P85+nonradioactive leptin(ss)-P85 at 100 ng/ml (n=5 mice/group). The brain/perfusion ratio (μl/g) of radioactivity was calculated. 
     Results 
       125 I-leptin(ss)-P85 rapidly entered the brain from the blood with an influx rate Ki=0.272±0.037 μl/g-minute. Unlabeled leptin(ss)-P85 or rleptin had no effect on the entry of  125 I-leptin(ss)-P85 into the brain but did inhibit the influx of simultaneously injected  131 I-rleptin after i.v. injection. As shown by SDS-PAGE and TCA acid precipitation, the radioactivity reaching the brain from the blood represents intact  125 I-leptin(ss)-P85 and it is stable in the brain and circulating blood after i.v. injection. Octanol/buffer partition coefficient calculated to be a log value of −1.92, showing that  125 I-leptin(ss)-P85 is hydrophilic. 
     Capillary depletion studies with vascular perfusion confirmed that, most of  125 I-leptin(ss)-P85 reached the brain parenchyma in 10 minutes. These studies show for the first time that the structure based Pluronic® modification of leptin increased metabolic stability and improved effectiveness. 
     More specifically,  FIG. 12A  provides multiple-time regression analysis of  125 I-leptin(ss)-P85 transport across the BBB. Ki (slope) was measured to be 0.272±0.037 μl/g-minute.  FIG. 12B  demonstrates that nonradioactive rleptin (1 μg/mouse) did not significantly inhibit the brain/serum ratio of  125 I-leptin(ss)-P85 at 10 minutes after i.v. injections.  FIG. 12C  is a graph showing that recombinant leptin (1 μg/mouse) did inhibit the influx of simultaneously injected  131 I-rleptin (*p&lt;0.0003, n=10 mice/group). 
       FIG. 13A  demonstrates the clearance of  125 I-leptin(ss)-P85 from blood after i.v. injection.  FIG. 13B  is a graph of the percent of intravenously injected dose of  125 I-leptin(ss)-P85 taken up by each gram of brain tissue from 0 to 60 minutes after injection. The maximal value was estimated by a one-site binding model to approach 0.263% Inj/g. 
       FIG. 14A  shows the acid precipitation of  125 I-leptin(ss)-P85 from brain and serum.  FIG. 14B  is the SDS-PAGE gel electrophoresis of  125 I-leptin(ss)-P85 extracted from blood and brain 10 minutes after i.v. bolus injection compared with  125 I-leptin(ss)-P85 ( FIG. 14C ) in buffer as a standard. 
       FIG. 15A  is a graph of capillary depletion with vascular washout 10 minutes after i.v. injection. The brain parenchyma/serum ratio (μl/g) was measured to be 3.78±0.26 μl/g (n=8 mice) and was significantly higher than the capillary/serum ratio of 3.04±0.18 μl/g (n=8 mice, p&lt;0.05).  FIG. 15B  provides the time courses of the brain/perfusate ratio of  125 I-leptin-P85 (Ki=0.892±0.236 μl/g-min, r=0.88, p&lt;0.05, n=3/time point).  FIG. 15C  demonstrates the inclusion of nonradioactive leptin(ss)-P85 or rleptin (100 ng/ml) in perfusion buffer did not alter the brain/perfusion ratio of  125 I-leptin(ss)-P85 at 5 minutes after i.v. injection. The brain/perfusion ratio as measured by brain perfusion was 21.82±4.04 μl/g with  125 I-leptin(ss)-P85 only, was 19.32±3.34 μl/g with +nonradioactive rleptin, and was 21.88±5.35 μl/g with +nonradioactive leptin(ss)-P85 (n=5/group). 
     Example 11 
     Preparation of Conjugated Leptin 
     Leptin conjugates are prepared using block copolymer Pluronics®. Pluronic® consists of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) and is arranged in a basic A-B-A structure: PEO-PPO-PEO. The Pluronic® chain may be attached to leptin using three different methods: primary amine modification using disulfide bond or non-degradable link, disulfide bridge insertion, and protein N-terminal attachment. The application of disulfide bond link between leptin and Pluronic® has the potential to maintain leptin activity in vivo due to the cleavage of disulfide bond. Whereas leptin modification using disulfide bridge insertion and protein N-terminal attachment decreases the heterogeneity of produced conjugates; as a result, these modified leptins provide better activity and less toxicity. The modifications via these three methods enhance leptin bioavailability and achieve efficient transport of leptin to the brain independently of leptin receptor or transporter. 
     1. Leptin primary amine modification by Pluronics® 
     Synthesis of PL-LepA starts with preparation of the monoamine Pluronic® derivative. This procedure is described herein as well as Batrakova et al. (Bioconjug. Chem. (2005) 16:793-802) and Yi et al. (Bioconjug. Chem. (2008) 19:1071-1077). Specifically, the Pluronic® molecule is protected at one end with 4′-methoxytrityl group (MTr). The remaining free hydroxyl group of the protected Pluronic® is activated with 1,1′-carbonyldiimidazole (CDI). Then, it is reacted with excess ethylenediamine. These mono amine Pluronic® derivatives will be further used to synthesize leptin-Pluronic conjugates using the synthetic strategies presented in  FIG. 16A . The molar ratio of the copolymer to leptin may be varied (e.g., 10:1, 20:1, 50:1, 100:1). DSP and DSS may be used as the linkers. The conjugates may be purified by ion exchange chromatography to separate the unmodified leptin and isolate leptin analogs with different modification degrees. The purified products may be analyzed by SOS-PAGE (with marker polypeptides of different molecular masses) and isoelectric focusing. The degree of modification may be determined by titration of the free amine groups on leptin and by MALDI-TOF spectroscopy. The points of attachment of Pluronic® to leptin may be determined via proteolytic digestion of the samples followed by peptide sequencing and mass spectroscopy. 
     2. Leptin Modification by Pluronics® Via “Disulfide Bridging” Insertion 
     Leptin can be modified by disulfide bridging via a unique S—S bond (Cys 96-Cys 146) that would maintain tertiary structure with little direct hindrance to its receptor binding (Brocchini et al. (2006) Nat. Protcol. 1:2241-2252). To adopt the disulfide bridging for modification of leptin with Pluronic®, the mono amino-Pluronic® may be activated using a bis-thiol alkylating reagent as shown in  FIG. 16B . The obtained Pluronic® mono-sulfone reagents (1 to 3 fold excess) may then be used for leptin modification in sodium phosphate buffer, pH 7.8 containing 2M L-arginine ( FIG. 16C ). The products may be purified and characterized as described above. 
     3. Leptin Modification by Pluronics® Via N-Terminal Attachment 
     To decrease the heterogeneity of produced conjugates, protein N-terminal modification by Pluronic® may be used ( FIG. 16D ). Specifically, the Pluronic® molecule may be protected at one end with 4′-methoxytrityl group (MTr). The remaining free hydroxyl group of the protected Pluronic® may be activated with 1,1′-carbonyldiimidazole (CDI), and react it with excess 3-amino-1,2-propanediol. After de-protection, the product is oxidized by sodium periodate and then reacted with leptin immediately in the solution (pH&lt;8) containing excess of sodium cyanoborohydride. The produced conjugates are further purified by gel filtration chromatograph and characterized as described above. 
     A number of publications and patent documents are cited throughout the foregoing specification in order to describe the state of the art to which this invention pertains. The entire disclosure of each of these citations is incorporated by reference herein. 
     While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.