Recent Developments in Peptide Drug Delivery to the Brain William M. Pardridge

Department of Medicine, Brain Research Institute, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A. (Received October I I , 1991; Accepted February 29, 1992) Peptide-based therapeutics are highly water-soluble compounds that do not readily enter brain from blood owing to poor transport through the brain capillary endothelial wall, i t . , the blood-brain barrier (BBB). Strategies available for peptide drug delivery to brain include: (a) neurosurgical-based (intraventriculardrug infusion, hyperosmotic opening of the BBB); (b) pharmacological-based(peptidelipidization, liposomes);and (c) physiological-based(biochemical opening of the BRB. chimeric peptides). Chimeric peptides are formed by the covalent coupling of a pharmaceutical peptide (that is normally not transported through the BBB) to a brain transport vector that undergoes absorptivemediated or receptor-mediated transcytosis through the BBB. The most efficient brain transport vector known to date is a monoclonal antibody to the transferrin receptor. and this vector achieves a brain volume of distribution approximately IX-fold greater than the plasma space by 5 hr after a single intravenous injection of antibody. The chimeric peptides are formed generally with chemical-based linkers. However, avidin/biotin-based linkers allow for high yield coupling of drug t o vector, and for the release of biologically-active peptide following cleavage of the chimeric peptide linker. These strategies may also be used for the delivery of antisense oligonucleotide-basedtherapeutics to brain. In conclusion, the development of efficacious neuropharmaceuticals in the future will require the development of both drug delivery and drug discovery strategies that operate i n parallel. Ahvrrmt;

,4hhrcrirr/ions: BBB, blood-brain barrier; BSA, bovine serum albumin; CSF, cerebrospinal fluid; D A B E , [D-Ala’] pendorphin; DTNB. 5.5’-dithio-bis (2)-nitrobenzoicacid; FPLC. fast protein liquid chromatography; HIV, human immuno-

deliciency virus; K, dissociation constant; NHS, N-hydroxysuccinimide;RSA, rat serum albumin; SPDP, N-succinimidyl3-[2-pyridyldithio(propionate)]; SATA, S-acetylthioaceticacid; V, volume of distribution.

Blood-borne peptide or monoclonal antibody pharmaceuticals, like other water-soluble drugs, have virtually no access to brain owing to the slow or negligible transport of most of these substances through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. The importance of developing new strategies for peptide drug delivery to the brain originates from parallell areas of neuroscience. First, there is a greater awareness of disorders that affect the nervous system, and of the importance of developing new neuropharmaceuticals for treatment of these diseases. Thesc illnesses include ethanol abuse, drug addiction, affective disorders, phobias. obscssivc-compulsive disorders. antisocial behaviour. schizophrenia. and impaired cognition (Regicr er ul. 1988). Other common brain disorders include migraine headache, Alzheimer’s disease, stroke. epilepsy, HIV infection and Parkinson’s disease. Thus, thc brain accounts for the principal morbidity in life, and the numbers of individuals affected dwarf that of heart disease and cancer combined, which are the principal causes of mortality. In parallel to the increasing recognition of common brain disorders is the discovery and elucidation o f nearly 100 different peptidergic neuromodulator systems in the central nervous system. Since the elucidation of the structure of angiotensin in 1945, the discovery and chemical elucidation of novel neuropeptides has grown exponentially, and new neuropeptide systems are discovered at the rate of several per year (Owman 1987). These peptides oftentimes could have important therapeutic effects in brain. However, most neuropeptides are barred from entry to brain by virtue of

a lack of transport of these water-soluble molecules through the BBB.

Methodologic considerations. The experimental evaluation of whether a given peptide or protein crosses the BBB requires that artifacts which support the apparent brain “uptake” of a blood-borne peptide be excluded. It must be shown that the peptide actually undergoes transport through the BBB and into brain interstitial fluid, rather than simple vascular sequestration. The latter may be caused by non-specific binding of the peptide to brain entothelium, o r may represent receptor-mediated or absorptive-mediated endocytosis into the brain capillary endothelium. In either case, a brain V, will be recorded for the peptide that is higher than the V, for the plasma volume marker, e.g., labeled mannitol, sucrose, inulin, or native albumin. However, this is not evidence that the peptide has been transported through the BBB. In order to differentiate transport through the BBB (transcytosis) from vascular bindingiendocytosis, a capillary depletion technique was developed (Triguero et ul. 1990). This technique for measuring transcytosis is faster and more easily quantified than is autoradiography (Duffy & Pardridge 1987). Initial studies with acetylated low density lipoprotein showed that this protein achieved a brain V, 3-fold higher than sucrose after 10 min. of carotid artery perfusion, but that all of this “uptake” represented vascular bindingiendocytosis (Fishman et ul. 1987; Triguero et ul. 1990). If experimental steps are taken, e.g., capillary depletion method, to differentiate transcytosis from binding/ endocy-

4

WILLIAM M. PARDRIDGE Table I .

Approaches to blood-brain barrier (BBB) drug delivery. Approach

Example

Neurosurgical

Intra-ventricular drug delivery Hyper-osmotic opening of BBB

Pharmacological

Lipidization Liposomes

Physiological

Chimeric peptides Biochemical opening of BBB

Adapted from Pardridge (1991).

tosis, additional experiments should be performed to show that the radiolabeled substance that gains access to the postvascular brain volume does not represent an artifact caused by vascular enzymatic degradation of the blood-borne peptide. For example, recent studies of the BBB transport of [‘251]P-endorphinreport a measurable brain V, of the labeled P-endorphin even following capillary depletion of the brain homogenate (Pardridge et al. 1990a & b). However, chromatographic examination of the post-vascular supernatant showed only evidence of degradation products and no evidence of unmetabolized P-endorphin in brain tyrosine is rapidly cleared (Pardridge et al. 1990a). The [1251] from P-endorphin by vascular aminopeptidase (Solhonne et 01. 1987). These recent studies showing the lack of transport of P-endorphin through the BBB confirm earlier reports (Bloom et al. 1978; Houghton et al. 1980). Another example of vascular metabolism of blood-borne peptide causing apparent “uptake” of the peptide is the case of thyrotropin releasing hormone (TRH). The inclusion of peptide inhibitors, e.g., bacitracin, in the carotid arterial perfusate essentially lowered the brain V, of labeled TRH to the blood volume V, (Zlokovic et al. 1988). In summary, endothelial binding, endocytosis, or metabolism of circulating peptide should be experimentally examined before claims are made regarding peptide transport through the BBB. No known paracellular pathways exist for peptide transport through the BBB. Therefore, circulating peptides gain access to brain interestitium via either receptor-mediated o r absorptivemediated transcytosis through the BBB. Since most neuropeptides do not engage specific transport systems within the BBB, the transport of neuropeptides from blood to brain interstitium requires the employment of a particular drug delivery system. Overview of strategies for peptide drug delivery to brain. The strategies for drug delivery to brain may be classified as neurosurgical-based, pharmacologic-based, and physiologic-based. Neurosurgical-based strategies include hyperosmolar BBB distruption (Neuwelt & Rapoport 1984) and intraventricular drug delivery. While intraventricular drug infusion is ideal for distributing drugs to the surface of the brain (Harbaugh 1989), the infusion of drugs into the ventricular space results in minimal distribution of drug down into brain parenchyma. There is approximately 140 ml of CSF in human brain, and this volume is turned over

MiniReview

completely every 4 5 hr, owing to continued CSF secretion by the choroid plexus and efflux via bulk flow of CSF from brain to the systemic venous circulation (Davson et al. 1987). Whereas CSF is exiting the brain via bulk flow, the only avenue for drug distribution to the brain parenchyma is diffusion, which occurs at a relatively slow rate. For example, drugs with the molecular weight of glucose (M, = 180) or myoglobin (M,= 17,500) have diffusion coefficients (cm2/sec.) of 6 x and 1. I x respectively. It may be calculated that drugs of this size will diffuse a distance of 5 mm in 12 hr and 2.7 days, respectively (Pardridge 1991). Thus, the rates of molecular diffusion are slow compared to the rate of CSF bulk flow exiting the brain. Consequently, the concentration of drug following intraventricular diffusion decreases logarithmically over the distance traversed from the surface to inner structures of the brain (Blasberg el al. 1975). Within the context of intraventricular drug delivery, it is also useful to discuss transnasal administration of peptide therapeutics. Lipid-soluble substances such as progesterone may gain access to CSF directly following transnasal administration (Kumar et al. 1982). Similarly, substances injected into the brain may exit via bulk flow (Bradbury et al. 1981) and gain access to nasal lymphatics. However, it is unlikely that peptides may gain access to CSF flow tracks following transnasal administration owing to the poor transport of peptides across the arachnoid membrane. Transnasal administration, however, is an effective bypass of the intestinal barrier, and recent studies have shown that the systemic bioavailability of peptides following transnasal administration is greatly enhanced by the inclusion of 5% cyclodextrins in the peptide vehicle (Merkus et al. 1991). Pharmacologic-based strategies for peptide delivery to brain include peptide lipidization (Tsuzuki et al. 1991) and liposomes (Gregoriadis et al. 1985). Peptide lipidization involves the blocking of polar functional groups on the peptide molecule with lipid-soluble constituents. While this approach is shown to be useful for small penta-peptides such as enkephalins (Tsuzuki et al. 1991), it is unlikely that lipidization of peptides much larger than enkephalins will lead to effective distribution to brain, owing to size exclusion phenomena related to BBB transport. For example, cyclosporin, a cyclic Il-amino acid peptide with a molecular weight of 1203, has an octanol Ringer’s partition coefficient of 991 f55, which is approximately equal to the lipid partition coefficient for highly lipid soluble steroid hormones such as testosterone (Cefalu & Pardridge 1985). However, the BBB transport of cyclosporin is very low, and in the presence of serum lipoproteins is essentially nil (Lemaire et al. 1988). Theories of molecular movement across lipid membranes include a pore model, wherein pores are caused by “kinks” in the lipid membrane owing to rotational movement about the carbon-carbon bonds of the free fatty acyl hydrocarbon chains in the membrane (Trauble 1971). Such a model would be consistent with size exclusion phenomena, and it appears that lipid-soluble molecules greater than molecular weight of 600-1,000 d o not cross the BBB at

PEPTIDE DRUG DELIVERY TO BRAIN

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rates commensurate with the lipid solubility of the compound. For these reasons, liposomes have generally not proven to be effective vehicles for drug transport to brain (Pardridge 1991). Exceptions include conditions such as brain injury (Chan et al. 1987), where there is pre-existing disruption of the BBB. Physiologic-based strategies for drug delivery to brain include biochemical opening of the BBB and the use of chimeric peptides. Biochemical opening of the BBB involves the administration of vasoactive substances such as leukotrienes (Baba et al. 1991). that create transient pores within the BBB membrane. Such compounds may be biochemical analogues of hyperosmotic opening of the BBB, wherein endothelial tight junctions are transiently opened by the intracarotid administration of water-soluble solute in high concentrations in the order of 2M (Neuwelt & Rapoport 1984). An important caveat related to biochemical opening of the BBB is that chronic neuropathologic changes take place in brain when the BBB is repetitively opened (Johansson et al. 1990). The chimeric peptide approach to drug delivery employs the use of carriers that transport drugs through the BBB by normal physiologic transport mechanisms related to receptor-mediated or absorptivemediated transcytosis through the BBB, and these transport processes operate while the BBB remains intact (Pardridge 1986, 1991). Overview of chimeric peptide approach to peptide drug delivery to brain. Chimeric peptides are formed when the non-transportable, pharmaceutical peptide, e.g., P-endorphin, is covalently coupled to a BBB transport vector, i.e., a peptide or modified plasma protein that undergoes receptor-mediated or absorptive-mediated transcytosis through the BBB. The four steps

0

0

1-1

endocytosis

exocytosis

1 1 BARRIER

BLOOD

of the overall chimeric peptide transport process are depicted in fig. 1. The first step is the receptor-mediated or absorptive-mediated transport of the chimeric peptide conjugate from the circulating compartment into brain capillary endothelial cytoplasm. The second step is the exocytosis of the conjugate from the endothelial cytoplasm to the brain interstitial fluid. The third step is the cleavage of the covalent bond, e.g., disulfide bond, that joins the two components of the chimeric peptide, and which results in the liberation of the free transport vector and of the unconjugated, pharmacologically active peptide. The fourth step is the binding of the pharmacologically active peptide with its respective receptor on brain cells. The two important development aspects of the chimeric peptide approach are (a) the development of brain-specific or semi-brain-specific transport vectors; and (b) the development of coupling strategies that result both in efficient, high yield coupling of the pharmaceutical peptide to its BBB transport vector, as well as the release of biologically active peptide following cleavage of the chimeric peptide. Brain transport vectors. Potential BBB drug transport vectors include compounds that are normally transporated into brain from blood via receptor-mediated or absorptive-mediated transcytosis. P e p tides that are transported through the BBB via receptormediated transcytosis include insulin (Duffy & Pardridge 1987), transferrin (Fishman et af. 1987), and possibly insulin-like growth factors (Duffy et al. 1988). The use of insulin as a transport vector is problematic, owing to the hypoglycaemia that would result following the administration of an insulin-based chimeric peptide. The use of insulin-like growth factor as a vector is problematic, owing to the very high degree of plasma protein binding of this peptide. The use of transferrin as brain transport vector is complicated by the very high concentration of circulating transferrin that is manyfold greater than the KD of the BBB transfemn receptor. However, these problems are circumvented by the use of a monoclonal antibody that reacts with an epitope of the transferrin receptor projecting into the extracellular space. Recent studies by Friden et af. (1991) show that a monoclonal antibody to the transferrin receptor is a highly efficient vector for transporting drugs to brain. This antibody targets brain to a greater extent than peripheral organs such as heart, lung, or kidney (fig. 2). Vectors that undergo absorptive-mediated transcytosis through the BBB include cationic proteins, such as cationized albumin (Kumagai et af. 1987),cationized antibodies (Triguero et al. 1989), or natural cationic proteins such as histone (Pardridge et af. 1989) or avidin (Pardridge & Boado 1991). The brain capillary endothelial cell is endowed with negative surface charges on both the lumenal and ablumenal membrane (Vorbrodt 1989). The interaction of cationic substances with these negative charges appears to trigger absorptive-mediated transcytosis through the BBB, as these compounds distribute into brain interstitial fluid following systemic administration (Triguero et af. 1990). Not all cat-

Hs*F

receptor @ bindlng

\\*

B-R

brain

BRAIN

Fig. 1. The delivery of chimeric peptide through the BBB is viewed

as a process composed of four steps: ( I ) receptor- or absorptivemediated endocytosis of the blood-borne chimeric peptide into brain endothelial cytoplasm;(2) exocytosis of the chimeric peptide into the brain interstitial fluid; (3) cleavage of the disulfide bond liberating unconjugated transport vector and pharmacologically active peptide; and (4) binding of the pharmacologically active peptide with

its receptor on brain cells. Abbreviations: A, transportable peptide, e.g., cationized albumin; B, non-transportable (pharmacologically active) peptide, e.g., P-endorphin; A-R, peptide A receptor; B-R, peptide B receptor. Reproduced from Pardridge (1991).

5

6

WILLIAM M. PARDRIDGE

ionic substances undergo absorptive-mediated transcytosis through the BBB. For example, the opioid peptide dynorphin A (1-8) is a basic peptide that does not undergo transport into brain (Terasaki et al. 1989). However, its analogue [N-methyl-Tyr', N-methyl-Arg', D-Leu'] dynorphin A (1-8) ethylamide, also called E2078, has recently been shown to undergo absorptive-mediated transcytosis through the BBB. The KD of E2078 binding to isolated brain capillaries is in the 1-10 pM range (Terasaki et al. 1989), similar to that found with other cationic substances (Pardridge 1991). Terasaki and co-workers have recently employed the combined use of intracarotid artery perfusion methods and intracerebral dialysis fiber methods to demon-

BLOOD-BRAIN BARRIER TRANSPORT VECTOR: MONOCLONAL ANTIBODY TO TRANSFERRIN RECEPTOR

Fig. 2. (A) Schematic of the transferrin receptor, which is a disulfidelinked homodimer of molecular weight of approximately 190,000. Six N-linked glycosylation sites are shown, C. Nearly 90% of the receptor projects into the extracellular space, and is released to plasma as a monomer following trypsin cleavage. Approximately 4%)of the receptor is transmembrane, and approximately 8% projects into the cytoplasmic compartment, where there are palmitoylation sites (F.A.). Reproduced from Seligman (1983). (B) Immunoperoxidase staining of cryostat sections of rat brain using the OX26 mouse monoclonal antibody to the transferrin receptor. The capillaries are intensely visualized owing to the enrichment of the transferrin receptor in the brain microvascular endothelium. (C) Selective uptake of a 'H-labeled mouse monoclonal antibody (OX26) to the rat transferrin receptor. The ratio of volume of distribution (V,) of the 'H-monoclonal antibody divided by the V, of '*'I-mouse IgGZa, the isotype control, in brain and four other organs is plotted versus the time after a single intravenous injection of both isotopes in ketamine-anaesthetized rats. Data are mean &S.E. (n= 3 rats per timepoint). The V, value of the mouse IgG2a control reached equilibrium within 15 sec. in all organs except the kidney (where the equilibrium V, was reached by 2 hr), and the average V, values for the mouse IgG2a are shown in the inset. By 5 hr after intravenous administration, the selective enrichment of the transport vector is greatest in brain of the live organs analyzed. Reproduced from Pardridge et al. (1991).

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strate that this cationic peptide distributes into brain interstitial fluid following carotid arterial administration (Terasaki et al. 1991). Moreover, this peptide has central analgesic properties in the tail-flick assay, and has an EDsosimilar to that of morphine (Nakazawa et al. 1990). In addition to cationic substances, lectins may also undergo absorptivemediated transcytosis through the BBB, and the subcellular organelles involved in this transcytosis pathway have been examined by employing a horseradish peroxidase wheat germ agglutinin conjugate (Broadwell et al. 1988). The vectors that have been studied in greatest detail include cationized rat albumin and the OX-26 monoclonal antibody to the transferrin receptor. Both vectors are semibrain-specific. Cationized rat albumin targets brain and kidney to a much greater extent that liver, heart, or lung (Pardridge et al. 1990b). The anti-transferrin receptor monoclonal antibody targets brain and liver to a greater extent than kidney, heart, or lung (Pardridge et al. 1991). Both vectors have favorable pharmacokinetic parameters. For example, the half-time of clearance of cationized rat albumin from the bloodstream in rats is 2.5 f0.4 hr. Conversely, the half-time of clearance of the OX-26 monoclonal antibody from the bloodstream in rats, following an initial rapid uptake in the liver, is 3.9 0.2 hours (Pardridge et al. 1990b & 1991). When rat albumin is mildly cationized to the extent that the protein is able to act as a brain drug transport vector, the cationic protein has minimal, if any, toxicity. Cationized rat albumin was administered daily to rats subcutaneously at a dose of 1 mg/kg for up to eight weeks, and no significant differences were observed in the body weight or organ weights of the animals receiving cationized RSA as compared to native RSA. In addition, there was no differences in organ histology, and there was no difference in the values of 20 different serum chemistries related to liver, renal, and metabolic function testing (Pardridge et al. 1990b). Finally, although heterologous proteins are known to be highly antigenic (Muckerheide et ul. 1987), a cationized homologous protein such as cationized RSA was found to have only weak immunogenicity in rats (Pardridge et al. 1990b). In order to minimize hepatic uptake of cationized RSA and potential renal toxicity of this cationic protein, only mildly cationized RSA was employed, and the use of mildly cationized RSA achieved a brain V, approximately fivefold greater than the plasma marker at 5 hours following a single intravenous injection (Pardridge et al. 1990b). Conversely, the brain VD of the mouse monoclonal antibody to the rat transferrin receptor reaches a value 18-fold greater than the plasma volume at 5 hr following systemic administration (fig. 2C). Since 90% of the transferrin receptor projects into the extracellular space (Seligman 1983), the circulating antireceptor antibody is readily accessible to the BBB transferrin receptor (fig. 2A). The transferrin receptor is highly enriched in brain microvessels as compared to brain parenchyma (Jeffries et al. 1984), and acts to shuttle endogenous transferrin from the blood to the brain compartments, (Fishman et al. 1987). Since the anti-transferrin receptor monoclonal

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PEF'TIDE DRUG DELIVERY TO BRAIN

antibody binds to an epitope removed from the transferrin binding site, the monoclonal antibody is able to "piggyback" the transferrin driven receptor, and to enter brain interstitial space from blood (Friden et al. 1991). Although a large amount of transferrin receptor is present in liver, there is minimal receptor in kidney, myocardium, or lung, and the anti-transferrin receptor antibody acts as a semibrain-specific transport vector (fig. 2). While vectors with a higher degree of brain specificity may be discovered in the future, cationized RSA or the monoclonal antibody to the transferrin receptor provide for the availability of relatively efficient brain transport vectors. Another important area for development in the chimeric peptide approach is the introduction of coupling strategies that allow for a high degree of conjugation of drug to vector, as well as for the release of biologically active peptide following cleavage of the chimeric peptide. Chimeric peptide coupling strategies. The chimeric peptide linker may be formed through a variety of covalent bonds that have seen reviewed recently and include ester linkages, thioether linkages, acid-labile linkages, Schiff-based linkers, and disulfide linkers (Pardridge 1991). In addition to these chemical linkers, two other fundamentally different linker strategies include the use of (a) avidin-biotin technology (Pardridge & Boado 1991), and (b) genetically engineered gene fusions constructs (Shin & Morrison 1990). In addition to classifying linkers as either chemical, avidin/ biotin-based, or genetic fusion constructs, linkers may also be classified as to whether the bonds are cleavable or noncleavable. Disulfide linkages are readily cleavable by cells following endocytosis (Feener et al. 1990). In addition, disulfide bonds have been shown to be relatively stable in brain capillaries (Pardridge 1991), which favours transport of intact chimeric peptide through the BBB. Since avidin-biotin linkers may either be disulfide-based or nondisulfide-based, avidin-biotin linkers may be classified as both cleavable or non-cleavable, depending on the biotin analogue that is used to biotinylate the peptide therapeutic (Pardridge 1991). The chimeric peptides produced from gene fusions are generally non-cleavable, although tryptic-sensitive regions between the two components of the fusion protein may be introduced through genetic engineering. Whether a cleavable or non-cleavable linker is employed depends on whether the receptor in brain will bind the peptide when it is still covalently coupled to the transport vector. It is likely that in many applications the interaction of the peptide with its receptor in brain will be optimal following cleavage of the peptide from its transport vector. Therefore disulfide-based linkers have been used to prepare chimeric peptides. In one approach, the cationized bovine serum albumin (BSA) vector was thiolated with N-succinimidyl-3[2-pyridyldithio(propionate)] (SPDP) and dithiothreitol (Kumagai et al. 1987; Pardridge et al. 1990a). Alternatively, amino groups on the vector compound may be thiolated with 2-iminothiolane, also called Traut's reagent, or N-

7

succimidyl S-acetylthioacetic acid (SATA) and hydroxylamine. The thiolated vector (or peptide) is then reacted with its complementary peptide (or vector) that contains an activated disulfide linkage that may react with the thiolated vector (or peptide). A surface cysteine sulfhydryl moiety may be activated with use of Ellman's reagent, which is 5,5'-dithio-bis(2)-nitrobenzoic acid (DTNB). If the peptide or vector does not contain a free surface cysteine sulfhydryl, then a primary amino group may be activated with either SPDP, 2-iminothiolane followed by Ellman's reagent, or SATA-hydroxylaminefollowed by Ellman's reagent (Pardridge 1991). The use of disulfide-based linkers presumes that disulfide bonds are relatively stable in blood and labile in brain. Previous studies have shown that disulfide linkers are in fact relatively stable in the circulation (Letvin et al. 1986), and a recent study has shown that the disulfide bond is rapidly cleaved in rat brain homogenate with a half-time of approximately 60 seconds at 37" (Pardridge et al. 1990a). In these studies, ['"I-Tyr, D-Ala'lbeta-endorphin (DABE) was covalently coupled to cationized BSA using SPDP. The chimeric peptide eluted at 10 ml on a Superose 12HR fast protein liquid chromatography (FPLC) gel filtration column (fig. 3). Within 60 sec. of incubation with rat brain homogenate, approximately 50% of the radioactivity co-migrated with the P-endorphin standard (fig. 3); by 15 min. of incubation, a principal peak of radioactivity co-migrated with iodotyrosine; and by 60 min. of incubation, this iodotyrosine peak was the principal radioactive moiety in the incubation (fig. 3). These studies indicate that the beta-endorphin cationized BSA chimeric peptide was cleaved at the disulfide bond at a faster rate than the P-endorphin was degraded by brain. In order for free P-endorphin to be liberated in brain with the chimeric peptide approach, the pharmacologically active peptide must be degraded slower than the chimeric peptide is cleaved. The studies described in fig. 3 indicate that the brain is endowed with relatively active disulfide reductases that are necessary for cleavage of the disulfide bond. Following disulfide cleavage, a mercaptoproprionate group remains covalently attached as a amide linkage to the amino group on the pharmacologically active peptide that participated in formation of the disulfide bond. Therefore, it is important to take the necessary steps to insure that the amino group participating in formation of the disulfide bond is not situated in a portion of the molecule that is critical for high affinity receptor binding. In the case of opioid peptides, a free N-terminal amino group is required for active opioid receptor binding (Bewley & Li 1983), and this amino group may participate in formation of the disulfide bond. Therefore, the mercapto-proprionate group will remain attached to the N-terminal amino group and prevent adequate opioid receptor binding. The strategies that are available for insuring retention of biologic activity of the peptide following disulfide cleavage have been reviewed recently (Pardridge 1991). Possible approaches include: (a) replacement of internal lysine moieties, when poss-

8

MiniReview

WILLIAM M. PARDRIDGE

ible, with arginine moieties to eliminate epsilon amino groups on the lysine residue from participating in disulfide formation; (b) protection of the N-terminal amino group with citraconic anhydride followed by acid cleavage at the appropriate step; (c) addition of a carboxyl terminal extended primary amino group that does not participate in receptor binding (Goldstein et al. 1988). Antisense oligonucleotide drug delivery to brain. Antisense oligonucleotides may exert pharmacologic effects by hybridizing with either genes to impair transcription or cytoplasmic mRNA species to impair translation (Weintraub 1990). These highly negative-charged compounds do not cross the BBB, which makes the problem of antisense oligonucleotide drug delivery to brain, similar to the problem of peptide drug delivery to brain. Since oligonucleotides

7

8

BUFFER

STREPTAWDIN

12 -

-~

U

z Q

I

0 m F'

i

1

-2

84-

% Fig. 4. Avidin-mediated uptake of biotinylated antisense oligonucleotide by isolated bovine brain capillaries in vitro. The percent uptake of "P-biotinylated oligonucleotide (bio-DNA) by the capillaries is plotted versus the incubation vehicle, which contains either buffer, 800 nM avidin, or 800 nM streptavidin. The incubation was performed at room temperature for 30 min. The antisense oligonucleotide used in these studies is a 21-mer complementary to the bovine GLUT-I glucose transporter mRNA (Boado & Pardridge 1990), and corresponds to nucleotides -9 to 12 ( f1 represents the first nucleotide at the methionine initiation codon), wherein the thymine base that pairs to 10 of the mRNA is replaced by 6-amino uracil, suitable for biotinylation near the 5'-end of the antisense oligonucleotide. The oligonucleotide was biotinylated with N-hydroxysuccinimidobiotin (NHS-biotin), and labeled at the ??-end with polynucleotide T4 kinase using [y-'*P]adenosine triphosphate. The labeled biotinylated antisense oligonucleotide was then mixed with either avidin or streptavidin prior to addition to the isolated bovine brain capillaries. Reproduced from Pardridge & Boado (1991).

+

6 5 4

+

3 2 I

5 4

.1

16 -

15 rnin

h

7

3

Q

2

X Y

I

E

b 4 3 2 I

50

0 min

olbumin endorphin

40 30 20 10

2 4 6 8 10 12 14 16 18 2 0 2 2 2 4 2 6

ml Fig. 3. Superose 12HR gel filtration fast protein liquid chromatography (FPLC) of ['251-Tyr,D-Ala*]beta-endorphin (DABE) covalently coupled to cationized bovine serum albumin (BSA) following incubation of the conjugate with rat brain homogenate for 0, 1, 15, or 60 min. at 37". The monoiodotyrosine is formed subsequent to the liberation of the unconjugated P-endorphin following cleavage of the cationized BSA chimeric peptide by rat brain homogenate enzymes. The migration of albumin, 0-endorphin, iodotyrosine, or iodide standards at 10, 16, 20, and 24 ml is shown. Reproduced from Pardridge el al. (1990a).

may be modified to introduce a single primary amino group, these agents may be covalently coupled to brain transport vectors using reactions similar to that employed for formation of chimeric peptides (Pardridge & Boado 1991). In addition, oligonucleotides, like peptides, may be biotinylated and thereby bind with very high affinity to avidin (Shimkus et al. 1985). Avidin is a cationic protein that has recently been found to undergo absorptive-mediated transport at brain capillaries similar to other cationic proteins (Pardridge & Boado 1991). Since avidin binds biotin with extremely high affinity, KD= lo-'' M (dissociation Tl,,=89 days) (Green 1990), avidin may serve as a stable brain drug transport vector for any biotinylated antisense oligonucleotide or peptide. Avidin binds to brain capillaries in a time-dependent, temperature-dependent, and saturable process that is inhibited by other polycationic proteins such as protamine (Pardridge & Boado 1991). Conversely, the bacterial homologue of avidin, streptavidin, which also binds biotin with very high affinity but is a slightly acidic protein (Green 1990), does not bind to brain capillaries. Fig. 4 demonstrates the enhanced brain capillary uptake of a 32P-labeledbiotinylated antisense oligonucleotide that is mediated by avidin but not by streptavadin. These preliminary studies suggest the avidin-biotin system may be used to facilitate the coupling of therapeutic substances to brain drug delivery vectors (Pardridge 1991).

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PEPTIDE DRUG DELIVERY TO BRAIN

Conclusions. Until relatively recently, there existed few, if any, practical strategies for delivering peptide-based therapeutics to the brain employing non-invasive, physiologic-based procedures. The development of the chimeric peptide strategy, the development of efficacious semi-brain-specific transport vectors such as a monoclonal antibody to the transferrin receptor, and the development of refined linker strategies for preserving biologic activity of the pharmacologically active peptide following its cleavage from the drug transport vector are all relatively recent events that auger for the successful delivery of peptide-based therapeutics t o brain in the future. The further development of successful transcellular drug delivery strategies is an important area that should proceed in parallel t o drug discovery. As ,,trial- and -error” design gives way t o “rational” drug design, it is likely that the drugs developed will be highly water-soluble substances that do not cross the BBB. Therefore, the development of neuropharmaceuticals in the future that are clinically efficacious may require the refinement of both drug delivery strategies and drug discovery strategies that operate in parallel. Acknowledgements Sherri J. Chien skillfully prepared the manuscript. Work in the author’s laboratory is supported by the National Institutes of Health of the U.S. Public Health Service.

References Baba, T., K . L. Black, K . lkezaki, K. Chen & D. P. Becker: Intracarotid infusion of leukotriene C, selectively increases blood-brain barrier permeability after focal ischemia in rats. J. Cereb. Blood Flow Meiabol. 1991, 11, 638-643. Bewley, T.A. & C . H. Li: Evidence for tertiary structure in aqueous solutions of human P-endorphin as shown by difference absorption spectroscopy. Biochemistry 1983, 22, 2671-2675. Blasberg, R. G., C . Patlak & J. D. Fenstermacher: Intrathecal chemotheraphy: brain tissue profiles after vetriculo-cisternal perfusion. J. Pharmacol. Exp. Therap. 1975, 195, 73-83. Bloom, F., D. Segal, N. Ling & R. Guillemin: Endorphins: profound behavioral effects in rats suggest new etiological factors in mental illness. Science 1978, 194, 630-632. Boado, R. J. & W. M. Pardridge: Molecular cloning of the bovine blood-brain barrier glucose transporter cDNA and demonstration of phylogenetic conservation of the 5’-untranslated region. Mol. Cell. Neurosci. 1990, 1, 224232. Bradbury, M. W. B., H. F. Cserr & R. J. Westrop: Drainage of cerebral interstitial fluid into deep cervical lymph of the rabbit. Amrr. J. Physiol. 1981, 240, F329-F3336. Broadwell, R. D., B. J. Balin & M. Salcman: Transcytotic pathway for blood-borne protein through the blood-brain barrier. Proc. Nail. Acad. Sci. USA 1988, 85, 632-636. Cefalu. W. T. & W. M. Pardridge: Restrictive transport of a lipid soluble peptide (cyclosporin) through the blood-brain barrier. J. Neurochem. 1985, 45, 19541956. Chan, P. H., S. Longar & R. A. Fishman: Protective effects of liposome-entrapped superoxide dismutase on posttraumatic brain edema. Ann. Neurol. 1987, 21, 540-547. Davson, H., K. Welch & M. B. Segal: Secretion of the cerebrospinal

9

fluid. In: The physiology and pathophysiology of the cerebrospinal fluid. Churchill Livingstone, London, 1987, p. 201. Duffy, K. R. & W. M. Pardridge: Blood-brain barrier transcytosis of insulin in developing rabbits. Brain Res. 1987, 420, 32-38. Duffy, K. R., W. M. Pardridge & R. G. Rosenfeld: Human bloodbrain barrier insulin-like growth factor receptor. Metabolism 1988,37, 136140. Feener, E. P., W.-C. Shen & H. J.-P. Ryser: Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes. J. Biol. Chem. 1990, 265, 18780-18785. Fishman, J. B., J. B. Rubin, J. V. Handrahan, F. R. Connor & R. E. Fine: Receptor-mediated transcytosis of transferrin across the blood-brain barrier. J. Neurosci. Res. 1987, 18, 299-304. Friden, P. M., L. Walus, G . F. Musso, M. A. Taylor, B. Malfroy & R. M. Starzyk: Antitransferrin receptor antibody and antibodydrug conjugates cross the blood-brain barrier. Proc. Natl. Acad. Sci. USA, 1991,88,47714775. Goldstein, A,, J. J. Nestor, Jr., A. Naidu & S . R. Newman: “DAKLI”: a multipurpose ligand with high affinity and selectivity for dynorphin ( x opioid) binding sites. Proc. Natl. Acad. Sci. USA 1988,85, 7375-7379. Green, N. M.: Avidin and streptavidin. Methods Enzymol. 1990, 184, 52-67. Gregoriadis, G., J. Senior, B. Wolff & C. Kirby: Targeting of liposomes to accessible cells in vivo. Ann. N. I:Acad. Sci. 1985, 446, 319-340. Harbaugh, R. E.: Nerve growth factor as a potential treatment in Alzheimer’s disease. Biomed. Pharmacotherap. 1989,43,483485. Houghton, R. A., R. W. Swann & C. H. Li: P-Endorphin: stability, clearance behavior, and entry into the central nervous system after intravenous injection of the tritiated peptide in rats and rabbits. Proc. Natl. Acad. Sci. USA 1980, 77, 4588-4591. Jeffries, W. A,, M. R. Brandon, S . V. Hunt, A. F. William, D. C. Gatter & D. Y. Mason: Transferrin receptor on endothelium of brain capillaries. Nature 1984, 312, 162-163. Johansson, B. B., C. Nordbor & I. Westergren: Neuronal injury after a transient opening of the blood-brain barrier: modifying factors. In: Pathophysiology of the blood-brain barrier. Eds.: B. B. Johansson, Ch. Owman & H. Widner. Elsevier Science Publishers, Amsterdam, 1990, pp. 145-157. Kumagai, A. K., J. Eisenberg & W. M. Pardridge: Absorptivemediated endocytosis of cationized albumin and a P-endorphincationized albumin chimeric peptide by isolated brain capillaries. Model system of blood-brain barrier transport. J. Biol. Chem. 1987, 262, 15214-15219. Kumar, T. C. A., G . F. X. David, A. Sankaranarayanan, V. Puri & K. R. Sundram: Pharmacokinetics of progesterone after its administration to ovariectomized rhesus monkeys by injection, infusion, or nasal spraying. Proc. Natl. Acad. Sci. USA 1982, 79, 4 1 8 5 4 189. Lemaire, M., W. M. Pardridge & G . Chaudhuri: Influence of blood components on the tissue uptake indices of cyclosporin in rats. J. Pharmacol. Exp. Therap. 1988, 244, 740-743. Letvin, N. L., V. S. Goldmacher, J. Ritz, J. M. Yetz, S. F. Schlossman & J. M. Lambert: In vivo administration of lymphocyte-specific monoclonal antibodies in nonhuman primates. J. Clin. Invest. 1986, 77, 977-984. Merkus, F. W. H. M., J. C. Verhoef, S . G. Romeijn & N. G . M. Schipper: Absorption enhancing effect of cyclodextrins on intranasally administered insulin in rats. Pharm. Res. 1991, 8, 588-592. Muckerheide, A., R. J. Apple, A. J. Pesce & J. G. Michael: Cationization of protein antigens. J. Zmmunol. 1987, 138, 833:837. Nakazawa, T., Y. Furuya, T. Kaneko, K. Yamatsu, H. Yoshino & S . Tachibana: Analgesia produced by E-2078, a systematically active dynorphin analog, in mice. J. Pharm. Exp. Therap. 1990, 252, 1247-1254. Neuwelt, E. A. & S . I. Rapoport: Modification of the blood-brain

10

WILLIAM M. PARDRIDGE

barrier in the chemotherapy of malignant brain tumors. Fed. Proc. 1984,43, 214219. Owman, C.: Regulatory peptides in cerebrovascular nerves and their effects upon the brain circulation-from a decade of research. In: Pepiidergic mechanisms in the cerebral circulation. Eds.: L. Edvinsson & J. McCulloch. Ellis Horwood Publishers, London, 1987, pp. 191-213. Pardridge, W. M.: Peptide drug delivery to the brain. Raven Press, New York, 1991, pp. 1-357. Pardridge, W. M.: Receptor-mediated peptide transport through the blood-brain barrier. Endocr. Rev. 1986, 7, 314330. Pardridge, W. M. & R. 3. Boado: Enhanced cellular uptake of biotinylated antisense oligonucleotide or peptide mediated by avidin, a cationic protein. FEBS Lett. 1991, 288, 30-32. Pardridge. W. M., J. L. Buciak & P. Friden: Selective transport of an antitransferrin receptor antibody through the blood-brain barrier in vivo. J. Pharmacol. Exp. Therap. 1991, 259, 66-70. Pardridge, W. M., D. Triguero & J. L. Buciak: Transport of histone through the blood-brain barrier. J. Pharmacol. Exp. Therap. 1989, 251. 821-826. Pardridge, W. M., D. Triguero & J. L. Buciak: P-Endorphin chimeric peptides: transport through the blood-brain barrier in vivo and cleavage of disulfide linkage by brain. Endocrinology 1990a, 126, 977 984. Pardridge, W. M., D. Triguero, J. L. Buciak & J. Yang: Evaluation of cationized rat albumin as a potential blood-brain barrier drug transport vector. J. Pharmacol. Exp. Therap. 1990b, 255, 893899. Regier. D. A,, J. H. Boyd, J. D. Burke Jr., D. S. Rae, J. K. Myers, M. Kramer, L. N. Robins, L. K. George, M. Karno & B. Z. Locke: One month prevalence of mental disorders in the United States. Arch. Gen. Psychiat. 1988, 45, 977-986. Seligman, P.: Structure and function of the transferrin receptor. Prog. Hematol. 1983, 13, 131-147. Shimkus, M., J. Levy & T. Herman: A chemically cleavable biotinylated nucleotide: usefulness in the recovery of protein-DNA complexes from avidin affinity columns, Proc. Nail. Acad. Sci. USA 1985, 82, 2593-2597.

MiniRevie w

Shin, S.-U. & S. L. Morrison: Expression and characterization of an antibody binding specificityjoined to insulin-like growth factor 1: potential applications for cellular targeting. Proc. Narl. Acad. Sci. USA 1990, 87, 5322-5326. Solhonne, B., C. Gros, H. Pollard & J. C. Schwartz: Major localization of aminopeptidase M in rat brain microvessels. Neuroscience 1987, 22, 225-232. Terasaki, T., Y. Deguchi, H. Sato, K. Hirai & A. Tsuji: In vivo transport of a dynorphin-like analgesic peptide, E-2078, through the blood-brain barrier: an application of brain microdialysis. Pharm. Rex 1991, 8, 815-820. Terasaki, T., K. Hirai, S. Hitoshi, Y. S. Kang & A. Tsuji: Absorptivemediated endocytosis of a dynorphin-like analgesic peptide, E2078, into the blood-brain barrier. J. Pharmacol. Exp. Therap. 1989, 251, 351-357. Trauble, H.: The movement of molecules across lipid membranes: a molecular theory. J. Membr. Biol. 1971, 4, 193-208. Triguero, D., J. B. Buciak & W. M. Pardridge: Capillary depletion method for quantifying blood-brain barrier transcytosis of circulating peptides and plasma proteins. J. Neurochem. 1990, 54, 1882-1 888. Triguero, D., J. B. Buciak, J. Yang & W.M. Pardridge: Blood-brain barrier transport of cationized immunoglobulin G. Enhanced delivery compared to native protein. Procl. Nail. Acad. Sci. USA 1989, 86, 47614765. Tsuzuki, N., T. Hama, T. Hibi, R. Konishi, S. Futaki & K. Kitagawa: Adamantane as a brain-directed drug carrier for poorly absorbed drug: anti-nociceptive effects of [D-Ala’] leu-enkephalin dervatives conjugated with 1 -adamantane moiety. Eiochem. Pharmacol. 1991, 41, R5-R8. Vorbrodt, A. W.: Ultracytochemical characterization of anionic sites in the wall of brain capillaries. J. Neurocyrol. 1989, 18, 359-368. Weintraub, H. M.: Antisense RNA and DNA. Sci. Am. 1990, 262, 4046. Zlockovic, B. V., M. N. Lipovac, D. J. Begley, H. Davson & L. Rakic: Slow penetration of thyrotropin-releasing hormone across the blood-brain barrier of an in situ perfused guinea pig brain. J. Neurochem. 1988, 51, 252-257.