J. Pharm. Pharmacol. 1996, 48:136-146
`Received April 21, 1995
`Accepted April 28, 1995
`The Blood-brain Barrier: Principles for Targeting Peptides
`and Drugs to the Central Nervous System
`
`0 1996 J. Pharm. Pharmacol.
`
`DAVID J . B E G L E Y
`Biomedical Sciences Division, King’s College London, Strand, London WC2R 2LS, UK
`
`Abstract
`The presence of the blood-brain barrier (BBB), reduces the brain uptake of many drugs, peptides and other
`solutes from blood. Strategies for increasing the uptake of drugs and peptide-based drugs include;
`structural modifications to increase plasma half-life; improving passive penetration of the BBB by
`increasing the lipophilicity of the molecule; designing drugs which react with transporters present in the
`BBB; and reducing turnover and efflux from the central nervous system (CNS).
`
`Introduction
`The blood-brain barrier (BBB) is a vital element in the
`regulation of the constancy of the internal environment of
`the brain. The composition of the extracellular fluid of the
`brain is controlled within very precise limits, largely inde-
`pendently of the composition of the circulating blood, to
`provide a stable environment in which the integrative neu-
`ronal functions of the brain can optimally take place. The
`blood-brain barrier is formed at the level of the endothelial
`cells of the cerebral capillaries. These cells are characterised
`by having
`tight continuous circumferential junctions
`between the cells of the capillaries thus abolishing any
`aqueous paracellular pathways between
`the
`cells
`(Brightman 1992). The endothelium is thus characterised
`by exhibiting a high transendothelial electrical resistance in
`the region of 1500-20000 cm2 (Butt et a1 1990). The
`presence of the tight junctions and the lack of aqueous
`pathways between cells greatly restricts the movement of
`polar solutes across the cerebral endothelium.
`Some regions within the central nervous system (CNS)
`lack a BBB and the capillaries are fenestrated allowing the
`free movement of solutes between the blood and the sur-
`rounding interstitial fluid. These areas are collectively
`termed the circumventricular organs (CVOs) and comprise
`the choroid plexus, the median eminence, the neurohypo-
`physis, the pineal gland, the organum vasculosum of the
`lamina terminalis, the subfornical organ, the subcommisural
`organ and the area postrema. Some of these structures, the
`median eminence, the neurohypophysis and the pineal are
`neurohaemal organs specialised for the release of neuroen-
`docrine secretion into the bloodstream. The other areas may
`be regarded as windows of the brain where a limited number
`of neurones within the immediate vicinity of the circumven-
`tricular organ have an unrestricted access to blood solutes.
`This access enables the brain to monitor closely the compo-
`sition of the blood and to react accordingly. The ependymal
`cells surrounding the circumventricular organs have what
`appear to be tight junctions between them presumably to
`enclose a volume of brain extracellular fluid (ECF) sur-
`rounding the CVO to prevent diffusion of interstitial solutes
`away from the region of the circumventricular organ
`
`(Brightman 1992). The relative surface area of the perme-
`able fenestrated capillaries of the circumventricular organs
`compared to the tight BBB capillaries is 1 : 5000. Given these
`considerations there is little possibility of these high perme-
`ability areas being able to influence the composition of the
`bulk of the brain extracellular fluid and the CVOs do not
`form a realistic route for drug entry into the brain.
`The presence of the BBB is easily demonstrated by study-
`ing the distribution of the inert hydrophilic marker inulin
`(Begley 1992a, 1994) (Table I). The percentage distribution
`spaces in brain regions with a BBB correspond to the plasma
`volume of the brain area. In the choroid plexus and the
`anterior and posterior pituitary where the capillaries are
`fenestrated the inulin space corresponds to the extracellular
`space of the tissue.
`Because of the presence of the BBB a number of specific
`transport mechanisms are required to be present in the
`cerebral endothelial cells to ensure that the brain receives
`an adequate supply of nutrients. These are illustrated and
`described in Fig. 1.
`Passive diffusion may occur across the endothelium,
`either through the cells themselves or through the tight
`junctions between cells. Movement across the BBB by
`passive mechanisms will be determined by considerations
`such as molecular weight and lipophilicity as discussed later.
`The entry of glucose into the brain is via a facilitated carrier
`GLUT-1 present in the endothelial cells. GLUT-1 is insulin
`insensitive and always shows a relatively high level of
`expression. It will however upregulate in chronic hypo-
`glycaemia and downregulate in chronic hyperglycaemia.
`Amino acids are transported across the BBB on a variety
`of transporters. Large neutral amino acids, such as tyrosine,
`phenylalanine, leucine, isoleucine, valine, histidine and
`methionine are transported into the brain by an energy-
`dependent transporter termed system-L which is present on
`the luminal and abluminal membranes and is principally
`directed from blood to endothelial cell and from endothelial
`cell into brain. System-ASC which transports as model
`substrates alanine, serine and cystine, plus a number of
`neutral amino acids such as threonine and asparagine is
`also expressed
`to a lesser extent and with the same
`directional properties as system-L. There is an obvious
`
`Petitioner Alembic Pharmaceuticals Limited - Exhibit 1031 - Page 1
`
`

`
`Table 1. Percent inulin spaces in various regions of the guinea-pig
`brain determined 10 min after intravenous bolus injection.
`
`Capillary lumen
`
`Brain ECF
`
`TARGETING PEPTIDES AND DRUGS TO THE CNS
`
`137
`
`Diffusion
`
`GLUT ,
`
`System L,
`
`System A X
`
`Receptors
`(h)
`
`Brain region
`
`Whole brain
`Olfactory bulb
`Hippocampus
`Caudate nucleus
`parietal cortex
`Hypothalamus
`Choroid plexus
`pituitary (anterior & posterior)
`
`Inulin space (YO)
`1.77 f 0.14
`2.30 f 0.37
`1.45 f 0.22
`1.26 f 0.18
`2.45 f 0.30
`20.80 f 1.10
`
`1.50 * 0.18
`26.70 * 2.90
`
`The differences between brain area possessing a blood-brain
`barrier and areas where the capillaries are fenestrated may be
`illustrated by injecting experimental animals with radiolabelled
`inulin (5000 Da). Where there is a blood-brain barrier the inulin
`occupies the plasma space of the tissue which is between 2.45 and
`1.26% depending on the region. Where the capillaries are fene-
`strated the inulin occupies the plasma and the extracellular space of
`the tissue giving spaces of approximately 25%. The inulin spaces
`were determined after an intravenous bolus injection of 5OmCi
`[3H]inulin in anaesthetised guinea-pigs. After 10 min, blood samples
`were taken by cardiac puncture and the animals were decapitated.
`Brain samples were then dissected out and the inulin space calcu-
`x 100. Mean f s.e.m.
`lated as: inulin space = Cplasma/Chrdln
`
`+ & - S y s t e m A
`
`overlap in the substrates acceptable to system-L and ASC.
`System-A, transporting principally glycine and proline is
`present on the abluminal membrane of the endothelial cells
`and is directed out of the brain (Betz & Goldstein 1978). A
`transporter for the dicarboxylic amino acids, glutamic and
`aspartic acid, is also present in the BBB and is directed out
`of the brain. Both system-A and system-ASC are sodium- as
`well as energy-dependent. A number of specific receptors for
`solutes also exists both on the luminal and abluminal
`surfaces of the endothelial cells. These receptors may be
`linked to second messengers such as CAMP or may mod-
`ulate the activity of channels or transporters in the BBB.
`There may also be transporters in the cell membrane for
`solutes such as small peptides.
`The level of endocytic activity in the BBB, compared with
`other endothelia, is minimal. However transcytosis for
`certain macromolecules may occur and form a low capacity
`transport mechanism for these solutes. Receptor-mediated
`endocytosis may be solute-specific and inducible by that
`solute.
`The BBB also has a physiological and biochemical dimen-
`sion in that the endothelial cells have a high density of
`mitochondria compared with other endothelia presumably
`reflecting a high level of oxidative ATP production. In
`addition the BBB is the site of a high level of enzyme activity
`directed towards the inactivation of centrally active blood-
`borne solutes and toxins (Audus et a1 1992; Grieg 1992). The
`high enzyme activities present in or on the cerebral endothe-
`ha1 cells include monoamine oxidase (MAO) types A and B,
`L-amino acid decarboxylase (AAD), catechol-0-methyl
`transferase (COMT), butyryl-cholinesterase (BChE) and 4-
`aminobutyrate aminotransferase. Levels of gamma-gluta-
`my1 transpeptidase activity are high in cerebral endothelial
`cells and are thought to be related to amino acid transport
`phenomena. Also membrane-bound epoxidehydrolase
`(mEH), UDP-glucuronosyl-transferase (UGT), benzoxyre-
`sorufin-0-de-ethylase, NADPH cytochrome P450 reductase
`
`FIG. I. Interactions of solutes with the blood-brain barrier. Solutes
`both in blood and in the brain extracellular fluid may interact with
`the BBB in a variety of ways. Solute which traverse the BBB by
`diffusion may either diffuse to a limited extent through the zona
`occludens (a) or directly across the endothelium (b). Polar solutes,
`for example glucose, employ a facilitated carrier Glut-I (c), in the
`cell membrane and neutral amino acids employ Systems-L and ASC
`(d and e). Glut-1, system-L and System ASC are represented in the
`same orientation in both the luminal and the abluminal membranes
`of the cerebral capillary endothelial cells. System-A (f) transports
`glycine and a system for the acidic amino acids (g) transports
`glutamic acid and aspartic acid from the brain extracellular fluid
`into the cerebral endothelial cells. There are also receptors for
`blood-borne solutes on the luminal surface of the endothelium
`(i and h). These receptors may be linked to intracellular messengers
`or control the activity of other channels in the luminal membrane
`and thus alter the activity of the endothelial cells. Specific peptide
`transporters have been demonstrated in the luminal endothelial
`membrane fj) transporting peptides into the endothelium. If these
`peptides are to reach the brain extracellular fluid by this route there
`must be equivalent transporters in the abluminal membrane. Larger
`peptides and proteins will be internalized by an endocytic mechan-
`ism (k). This may be receptor-mediated or non-specific in nature.
`The level of endocytic activity in the cerebral capillary endothelium
`is much lower than in other tissues endothelia and transcytosis
`remains a controversial topic. Multidrug resistance protein or
`P-glycoprotein is expressed constitutively in the luminal membrane
`of the cerebral endothelial cells (I) and transports a variety of
`structurally unrelated substrates out of the endothelium. Many of
`these are lipophilic and potentially neurotoxic and would otherwise
`enter the brain to a greater extent.
`
`and glutathione-S-transferase activities are high in brain
`microvessel preparations and in the choroid plexus. These
`enzymes are thought to play a part in the metabolism of
`drugs and xenobiotic compounds.
`The intrinsic membrane protein P-glycoprotein some-
`times referred to as multidrug resistance protein (mdr-
`protein) is also present in the luminal plasma membrane
`of the endothelial cells constituting blood-brain barrier.
`This is an efflux pump which appears to be a constituent
`part of the BBB and transports a wide range of structurally
`unrelated substances out of cells. Pgp is an ATP-dependent
`pump and is a member of a family of intrinsic membrane
`proteins which are normally expressed at the BBB, the
`intestine, the liver and the kidney (Cordon-Cardo et a1
`
`Petitioner Alembic Pharmaceuticals Limited - Exhibit 1031 - Page 2
`
`

`
`138
`
`DAVID .I. BEGLEY
`
`/
`/
`/
`,*r BCNU
`
`CCNU
`
`Vinbbstine
`
`Vincristine
`
`/' Epipodophyllotoxin
`,/. Sucrose
`8 1
`
`-4
`
`L
`-3
`
`I
`
`1
`
`I
`
`2
`
`I
`
`3
`
`I
`I
`0
`-2
`-1
`1
`Log octanollwater partition coefficient
`FIG. 2. The relationship of the octanol/water partition coefficient to blood-brain barrier permeability for some selected
`drugs. Substances falling on the dotted line would have a direct relationship between their brain uptake and their lipid
`solubility. Note that there is a wide scatter of points around the line. Vinblastine and vincristine are known substrates for
`the efflux pump P-glycoprotein expressed at the blood brain barrier and this limits their penetration into brain. Other
`substances which also lie well away from the line but are not shown on the plot are those which have carrier-mediated
`uptake mechanisms such as glucose and amino acids, which are fairly polar with partition coefficients between I x lo-'
`but with high BBB permeabilities.
`and I x
`BCNU = I .3-bis-chloro(2-chloroethyl)-l-nitrosourea; CCNU = I-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea; PCNU =
`1 -(2-chloroethyl)3-(2,6,-dioxo-3-piperidyl)-nitrosourea; 5FU = 5-fluorouracil. Adapted from Greig (1992).
`
`1989; Ruez & Gros 1994). The role of Pgp therefore appears
`to be a protective one transporting a range of potentially
`toxic substances out of the body. The expression of Pgp
`occurs in many tissues including malignant tissue after
`exposure to cytotoxic drugs where it confers resistance to
`a wide range of cytotoxic agents. Hence its alternative name
`multidrug resistance protein. In this situation its acts to
`maintain the intracellular levels of cytotoxic drugs below a
`toxic level and thus frustrates repeated cancer chemother-
`apy. Its constitutive role in the normal BBB is inferred to be
`a protective one reducing the entry of lipophilic and neuro-
`toxic substances into the CNS.
`
`Improving passive permeation of the BBB
`For many drugs that enter the brain by passive diffusion
`their brain-uptake and lipid solubility are well correlated
`(Fig. 2). Lipid solubility is most often determined as a
`partition coefficient between buffer and octanol and is
`quoted as the logarithm of the partition, logPo,,. Many
`small solutes entering the brain do so by dissolving in the
`lipid of the cell membrane. In-vitro studies using cultured
`cerebral endothelial cells have suggested that in this system
`the relationship between lipid solubility and brain uptake
`might be a sigmoidal one (Oldendorf 1974; Van Bree et a1
`1988). With substances having a logP between -2.00 and 0
`(partition coefficients of 0.01 and 1.00) lying on the more
`linear portion of the curve. Very hydrophobic substances
`show little further increase in brain uptake and hydrophilic
`substances which are polar and highly ionized at physio-
`logical p H also have a limited brain uptake.
`
`It has been suggested by Levin (1980) that large lipid-
`soluble substances with a molecular weight above 500Da
`become physically impeded from crossing the cell membrane
`and thus exhibit a molecular weight cut-off. This has been
`used to explain the low brain-uptake of substances such as
`vinblastine, vincristine and cyclosporin, which lie well off
`the regression line suggested in Fig. 2. However it is now
`well-established that these cytotoxic agents are substrates of
`P-glycoprotein (Tsuji et al 1992, 1993) which is actively
`extruding them from the cerebral endothelial cells. Surpris-
`ingly an increase in molecular volume may actually enhance
`brain uptake (Abraham et a1 1994).
`It has been suggested that delta logP, defined as the
`logPoctanol/bul€er minus the logPcyclohexane/huffer is a better pre-
`dictor of brain uptake (Young et al 1988). Delta logP reflects
`the hydrogen-bonding capacity of a substance and this prop-
`erty in turn influences brain uptake. In general in a series of
`related substances those with a lower-hydrogen bonding
`potential enter the brain the most readily. The hydrogen-
`bonding potential determines the activation energy necessary
`to pluck a molecule out of the aqueous phase and into the lipid
`phase of a membrane. Various physical factors influencing
`brain uptake have been incorporated into a predictive equa-
`tion for brain uptake (Abraham et al 1994).
`To maximize the brain uptake and the bioavailability of a
`substance to the brain a number of molecular manipulations
`may be carried out to alter the properties of a substance;
`these may be summarized as:
`1. Increasing the plasma stability and hence plasma half life.
`2. Improving lipid solubility.
`
`Petitioner Alembic Pharmaceuticals Limited - Exhibit 1031 - Page 3
`
`

`
`(a) pyroglutamyl-histidyl-prolinamide
`WH)
`
`R I = H , R 2 = H
`
`(b) pyroglutamyl-histidyl-3 methylprolinamide
`(Pyr-His-MepNH2. RX 74355)
`R l = H , R Z = C H ,
`
`(c) pyroglutamyl-histidy1-3,3 dimethylprolinamide
`(Pyr-His-Dmp-NH2. RX 77368)
`R1= CH,. R2 = CH,
`
`139
`TARGETING PEPTIDES AND DRUGS TO THE CNS
`(Table 2). The central nervous activity of the T R H
`analogues is also enhanced (Brewster et al 1981; Brewster
`1983), possibly as the result of increased brain penetration
`although an enhanced central potency should also be
`considered.
`Other simple modifications to peptides that may be made
`are to amidate the C-terminus and acetylate the N-terminus
`both of which confer an increased resistance to exopepti-
`dases. In addition acetylation of the N-terminus also
`increases lipid solubility significantly.
`An excellent example of increasing brain uptake by
`enhancing lipid solubility and reducing hydrogen bonding
`capacity is the chemical conversion of morphine to heroin
`(diacetyl-morphine). Substitution of the two hydroxyl
`groups of morphine by acetyl groups in heroin increases
`the brain uptake over twenty-fivefold (Oldendorf 1974). As
`a general rule for each pair of hydrogen bonds removed
`from a molecule there is a log order increase in BBB
`permeabilty. Once within the brain, heroin is rapidly con-
`verted to monoacetyl morphine and more slowly to mor-
`phine. Thus the rapid brain entry of heroin in comparison
`with morphine makes it a favoured drug of abuse and
`presumably enhances its addictive potential.
`In the case of many peptides the introduction of
`D-isomers of the naturally occurring amino acids in to the
`peptide sequence can greatly enhance the plasma half life of
`the peptide. Again the reactivity with plasma peptidases is
`greatly reduced. For example octreotide (D-Phe-Cys-Phe-
`D-Tyr-Lys-Thr-Cys-Thr-OH : sandostatin : SMS 20 1-995)
`is an octapeptide analogue of somatostatin Ala-Gly Cys-
`Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH).
`The
`attenuated peptide with the D substitutions, D-Phe at
`position two of somatostatin and D-Trp at position five,
`extends the first order half-life of somatostatin from 2-3 min
`in human plasma to 113 min (Lamberts 1987). The substitu-
`tion of D-Phe at position four also increases the growth
`hormone inhibiting potency of sandostatin compared with
`somatostatin by some 45 times. D-Amino acid substitutions
`also have marked effects on the plasma half-lives of
`encephalin analogues. Examples are two analogues of
`leucine encephalin (Tyr-Gly-Gly-Phe-Leu), namely DADLE
`
`FIG. 3. Structure of T R H and related analogues RX 74355 and
`RX 77368. The substitution of the methyl groups into the proline
`residue increases the plasma half-lives and the central effectiveness
`of the molecules.
`
`3. Enhancing or maintaining reactivity with existing BBB
`transport mechanisms.
`4. Retaining central nervous activity.
`5. Increasing the stability in brain extracellular fluid and
`reducing reactivity with efflux transport mechanisms in the
`CNS.
`
`A number of chemical modifications can be carried out to
`improve the CNS activity of drugs. For example the peptide
`thyrotropin
`releasing hormone
`(TRH-pyroglutamyl-
`histidyl-prolinamide) has a short plasma half life due to
`circulating peptidases. To extend the half-life, methyl
`groups may be added to the prolinamide residue to produce
`a 3-methylprolinamide (RX74355) and a 3,3-dimethylproli-
`namide (RX77368) (Brewster et a1 1981, 1983) (Fig. 3).
`These methyl substitutions increase the lipid solubility of
`the molecule and also the molecular volume. The substitu-
`tions also increase the first order half-lives in human plasma
`from 33 (TRH) to 210 (RX74355) and 1080min (RX77368)
`
`Table 2. First-order half-lives (min) of TRH, RX74355, and RX77368. There are
`significant species differences in the rates at which plasma and a brain homogenate
`will break down T R H and its analogues.
`
`T R H
`
`RX74355
`(Methylproline)
`
`RX77368
`(Dimethylproline)
`
`Rat
`Plasma
`Brain homogenate
`Dog
`Plasma
`Brain homogenate
`Man
`Plasma
`Brain homogenate
`Mouse
`Plasma
`Brain homogenate
`
`22
`9
`
`> 1500
`11
`
`33
`18
`
`215
`12
`
`From Brewster (1983).
`
`I I4
`54
`
`> 1500
`90
`
`210
`66
`
`> 1500
`28
`
`390
`190
`
`> 1500
`174
`
`1080
`168
`
`> 1500
`150
`
`Petitioner Alembic Pharmaceuticals Limited - Exhibit 1031 - Page 4
`
`

`
`140
`
`DAVID J. BEGLEY
`
`Ala-Gly-Cvs-Lvs-Asn-Phe-Phe-Tm-Lvs-Thr-Phe-Thr-Ser-Cvs-OH
`Somatostatin14
`
`D-Phe-Cvs-Phe-D-Tm-Lvs-Thr-Cvs-Thr-OH
`
`Ac-D-Phe-Cvs-Tvr-D-T~-Lvs-Val-Cvs-Thr-NH2
`
`0.23'
`
`D-Phe-CvsTvr-D-Tm-Lvs-Val-Cvs-TrpNHz
`
`RC160
`
`0 ,092'
`
`D-Phe-Cvs-Tvr-D-T~-Lvs-Val-Cvs-Thr-NHz
`
`RC121
`
`r
`0
`
`1
`0.05
`
`I
`0.1
`
`I
`0.15
`
`I
`0.2
`
`I
`0.25
`
`Unidirectional cerebrovascular
`influx constant
`(pL g-' min-1)
`FIG. 4. Unidirectional cerebrovascular permeability constants (K,") of some somatostatin analogues. The brain uptakes
`are determined by intravenous bolus injection techniques. The structure of the analogues is given with that of
`somatostatin for comparison. Unfortunately the brain uptake of somatostatin cannot be determined by a comparable
`method as it is very unstable in plasma. Data from Begley et al (1992b) and Banks et al (1990).
`
`(D-Ala2, D-Leu5-encephalin) and dalargin (Tyr-D-Ala-Gly-
`Phe-Leu-Arg) both of which have greatly extended half-lives
`in plasma compared with the parent peptide.
`A number of somatostatin analogues exist where the
`plasma half-life is extended and the lipid solubility is altered
`thus modifying two factors which influence brain uptake,
`plasma half life and biological potency of the molecules.
`Intravenous bolus injection studies have been carried out to
`measure the brain uptake of these peptides, (Banks et al
`1990; Begley 1992b, 1994). These somatostatin analogues
`are amphiphilic and their brain uptakes are non-saturable
`suggesting that their penetration into the brain will be
`passive. However changes in the molecular structure of the
`analogues produce significant changes in brain uptake. Fig. 4
`illustrates the brain uptakes of four octapeptide analogues
`of somatostatin, determined by an intravenous bolus injec-
`tion technique (Banks et al 1990; Begley 1992b, 1994) which
`have D-Phe and D-Trp substitutions in the molecule. Com-
`pared with RC 121 the substitution of a Trp residue at
`position 8 compared with RC 160, thus introducing an
`_ _
`additional aromatic side chain into the molecule, approxi-
`mately doubles
`the cerebrovascular permeability constant.
`Acetylating the
`N-terminus of RC 121 as in RC 161, and
`
`therefore increasing the lipid solubility, increases the perme-
`ability constant almost sixfold. The peptide RC 160 has
`marked and prolonged analgesic actions after intravenous
`administration (Eschalier et a1 1990).
`Using synthetic peptide sequences which have hydropho-
`bic properties may provide a mechanism for inserting a
`peptide into the cell membrane (Begley 1994). For example a
`number of naturally-occurring peptides have hydrophobic
`regions which demonstrate a natural affinity for cell mem-
`branes. Mellitin, a component of bee venom (Kaiser &
`Kezdy 1987), is a 26-amino acid peptide (Fig. 5). The N-
`terminal amino acids 1-20 contain two a-helical regions
`1-10 and 13-20 which are hydrophobic and insert the
`molecule into the cell membrane, the lysine residues 21 -26
`are highly cationic and are thought to open up pores thus
`permeabilizing the membrane (Kaiser & Kezdy 1987).
`Signal peptides are similar hydrophobic regions in a pre-
`pro-peptide or protein which enable the entire molecule to
`be inserted through the unit membrane of the endoplasmic
`reticulum. The signal amino acid sequence is not conserved
`between pre-pro-proteins and thus the common feature is
`the hydrophobicity (lipohilicity) of the region (Engelman &
`Steitz 1981; Emr & Silhavey 1983; Begley 1994). The
`
`5
`10
`N- ly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr Thr-Gly
`+
`
`15
`
`25
`IlleSerTrpllejLys- Arg-Lys- Arg-Gl~-Gln NH2
`+ + + +
`FIG. 5. Amino acid sequence of mellitin. The hydrophobic areas which insert into the cell membrane are boxed
`Positively-charged side chains which disrupt the cell membrane are indicated with + .
`
`Petitioner Alembic Pharmaceuticals Limited - Exhibit 1031 - Page 5
`
`

`
`TARGETING PEPTIDES AND DRUGS TO THE CNS
`
`141
`
`230 nm
`
`I
`
`I
`
`\
`Poly-butyl-cyanuacrylate nanoparticle
`
`FIG. 6. Schematic diagram of a nanoparticle. The dalargin is
`absorbed onto the surface of the nanoparticle which is then
`coated with polysorbate-SO.
`
`function of the signal sequence is thus to enable a large polar
`protein or peptide to traverse a unit membrane without
`damage to either the pre-pro-protein or the membrane.
`Indeed passive permeation of the BBB may not be limited
`to small molecules. Colloidal polymer particles (nanoparti-
`cles) may be formed from poly(butylcyanoacrylate) with an
`average diameter of 230 nm. Nanoparticles have been used
`to deliver the peptide dalargin to the CNS (Fig. 6) (Engel-
`man & Steitz 1981; Emr & Silhavey 1983; Kreuter et al
`1995). Dalargin is a hexapeptide analogue of encephalin
`which is stable in plasma but has little central analgesic
`action when injected intravenously. If dalargin is absorbed
`onto the surface of nanoparticles and these particles are then
`coated with the detergent polysorbate-80 and the complex
`injected into mice, a pronounced analgesic effect is obtained
`reaching a maximum in 45min (Fig. 7). Little effect is
`produced with dalargin, nanoparticles or polysorbate-80
`alone. The effect is also dose-dependent with a greater
`absorption of dalargin onto the particles and is reversible
`by naloxone (Engelman & Steitz 198 1; Emr & Silhavey 1983;
`Kreuter et al 1995). Presumably the detergent enables the
`particles to penetrate the BBB and the dalargin is released
`from the nanoparticles in effective amounts within the brain.
`In contrast, attempts to use liposomes to deliver drugs to the
`brain have been universally unsucessful (Pardridge 199 1).
`
`0
`
`15
`
`30
`
`45
`
`60
`
`75
`
`90
`
`Time (min)
`FIG. 7. Analgesia in mice produced by dalargin-loaded nanoparti-
`cles. Analgesia is expressed as the percent of the maximally possible
`effect after intravenous injection of the indicated dose. (n = 6,
`mean
`s.d.)
`
`Exploiting existing transporters
`As mentioned earlier the presence of a blood-brain barrier
`to polar molecules dictates that a number of transporters
`must be present in the cerebral endothelium in order to
`supply the brain with an adequate supply of nutrients. The
`kinetic properties of some of these transporters are shown in
`Table 3.
`The glucose carrier GLUT-I has a very large capacity to
`transport ,BD-glucose across the BBB. The intestinal Na ' 1
`D-glucose transporter will also transport /3-glycosides but
`not as effectively as the preferred substrate glucose. Given
`that GLUT-] and the intestinal transporter have a similar
`
`Table 3. Kinetic values for some transport systems present at the BBB.
`
`Representative substrate
`
`Transporter
`
`Glucose
`Lactic acid
`Phenylalanine (apparent)
`Phenylalanine (real)
`Arginine (apparent)
`Leucine encephalin
`Arginine vasopressin
`Adenosine
`Adenine
`
`Hexose: Glut- I
`Monocarboxylic acid
`System-LI
`System-L1
`Basic amino acid
`Peptide specific
`Peptide specific
`Nucleoside
`Purine base
`
`K, ( P M )
`11000 f 1400
`1800 f 600
`30.0 f 1.0
`1 1 .o f 2.0
`40.0 f 24
`39.0 f 3.2
`25.0 * 3.0
`2.08 f 0.32
`11.0f3
`
`V,,,
`
`(nmol min-1 g-1)
`1420 f 140
`91 f 3 5
`14.0 f 4.0
`25.0 f 6.0
`5.0 f 3.0
`160.0 f 22.0*
`5.49 f 0.74*
`0.75 f 0.08
`0.5 f 0.09
`
`K, (pL g-' min-I)
`
`44.0 f 14.0
`8.0 f 1.0
`0.062 f 0.086
`0.021 f 0.044
`
`*pmoI min-l g-l.
`The K, value IS the concentration of substrate at which the transporter is half-saturated and indicates the affinity of the substrate for the
`is the maximal transporting capacity of the system. The Glut-1 transporter has a high affinity and a high capacity for
`system. The V,,,
`glucose. As explained in the text many amino acids share a common transport system and will have different affinities (K,)
`for the systems;
`because of this they will compete with each other for transport. Thus an amino acid will have a real K, and V,,, where their kinetic values are
`determined in the absence of competing amino acids and apparent values when competing amino acids are present. The Kd is a measure of the
`Passive diffusional component to the movement of a solute and is significant in the case of the amino acids but is much smaller in the case of
`the more polar peptides. Means f s.e.m. Compiled from various sources.
`
`Petitioner Alembic Pharmaceuticals Limited - Exhibit 1031 - Page 6
`
`

`
`142
`
`DAVID J. BEGLEY
`
`1
`
`2
`
`3
`
`4
`
`5
`
`51
`rsH HSi
`ys-.?
`
`6
`
`7
`
`8
`
`9
`
`H,N-Tyr-Dben-Gly-Phe-Dben-Ser-Gly-&NH,
`D-Gk-p(l-0) _I
`
`0 II
`H,N-Tyr-DPen-Gly-PheDPen-Ser-Gly-C-NH,
`D-GIc-p(I
`
`0 II
`
`r S H H S i
`H2N-Tyr-DCys-Glyr-Phe-DCys-Ser-Gly-C-NH2
`b + P
`
`H d
`
`
`
`r S ---S
`
`1 II
`
`0
`
`H
`
`10
`
`H,N-Tyr-DPen-Gly-Phe-DPen-C-NH,
`
`DPDPE 6
`
`H,N-Tyr-DCys-Ser-Phe-DCys-Gly-C-NH,
`~ - ~ i c - p - ( i -q-J
`
`0
`
`rS-' 1 II
`H,N-Tyr-DCys-Ser-Phe-DCys-Gly-C-NH,
`~ - ~ i c - p - ( i -0) J
`rS-' 1 I1
`0
`H2N-Tyr-LCys-Gly-Phe-LCys-Ser-C-NH,
`D-Gic-p-(i-rO) J
`1 II
`
`0
`r S H HS
`H2N-Tyr-DCys-Gly-Phe-DCys-Ser-Gly-C-NH,
`6 + p
`
`D - G i c - p - ( i a d
`
`rs -' 1 FI
`
`D-Glc-&( 1 4
`
`FIG. 8. Some analogues of DPDPE (D-pen2.' encephalin). DPDPE is a &receptor agonist but is ineffective
`intraperitoneally. Analogues 4 and 5 and 8 and 9 are 6- and preceptor agonists. However only analogues 4 and 5
`produce analgesia determined by the tail flick test after intraperitoneal administration. From Polt et al (1994).
`
`structure it might be possible that peptide-P-D-glycoside
`conjugates may be acceptable to the brain glucose transpor-
`ter (Polt 1994). Some glycopeptides administered intra-
`peritoneally as L-serinyl-P-D-glycoside analogues of Met'
`encephalin (Fig. 8) have been shown to be transported
`across the BBB and bind to p - and 6-opioid receptors in
`the brain. The glycopeptide encephalin analogues 4 and 5
`produce a marked and long-lasting analgesia after intraper-
`itoneal administration as determined by tail-flick and hot-
`plate assays in mice (Fig. 9). This analgesia is reversible with
`naloxone (Polt 1994). It is suggested that GLUT-I is
`responsible for transporting the glycopeptide into the
`CNS. Interestingly, these glycopeptides have a reduced
`lipohilicity compared with the native encephalins but the
`conjugate has reactivity with both the glucose transporter
`
`1 O O r
`
`-
`
`0 6 . '
`w
`20
`0
`
`'
`40
`
`.
`
` ' *
`'
`80
`60
`Time (min)
`
`'
`100
`
`'
`
`120
`
`FIG. 9. Analgesia produced by DPDPE glycopeptide analogue 5.
`Intraperitoneal administration of this pegtide produces dose-related
`and long-lasting antinociception in a 55 C tail-flick test. From Polt
`et al (1994).
`
`and with opioid receptors within the brain (Polt 1994). The
`brain transport appears to be specific for P-0-linked glyco-
`sides as a-linked and N-linked and 0-acyl linked glucose
`conjugates (Fig. 8) d o not appear to cross the BBB and have
`no analgesic effects. Also only glycosides linked via the
`serine residue showed any CNS activity. In this connection
`it is interesting to note that morphine-6-glucuronide formed
`naturally in the liver is 10-50 times more potent in producing
`analgesia than morphine itself and that morphine may act as
`a prodrug with the 6-glucuronide producing a significant
`part of the analgesic effect. There is thus a good possibility
`that glycosylation might be applicable to the delivery of a
`range of drugs across the BBB.
`System-L transporting neutral amino acids into the CNS
`is another obvious target for drug analogues or complexes
`which might have a reactivity with this system. To date, the
`most successful exploitation of system-L is the delivery
`of L-dopa to the brain for