Petitioner Amerigen Pharmaceuticals Ltd.
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1031 - Page 1
`
`

`
`Petitioner Amerigen Pharmaceuticals Ltd.
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1031 - Page 2
`
`

`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1031 - Page 3
`Petitioner Amerigen Pharmaceuticals Ltd.
`
`

`
`TARGETING PEPTIDES AND DRUGS TO THE CNS
`
`139
`
`0
`
`O
`
`'
`
`C’
`
`0
`
`NH—CH—é/
`\N
`\
`CH2
`
`N
`H
`
`R1
`
`'92
`NH
`Q’C\NH2
`
`(a) pyrogluiamyl-histidyl-prolinamid2
`(TRH)
`R1 = H. R2 :H
`
`(b) pymglutamyl-histidyl-3 methylprolinamide
`(Pyr-His-MepNI-I2, RX 74355)
`R] : H, R2 = CH3
`
`(c) pyroglutamyl—hisIidyl-3.3 dimethylprolinamide
`(Pyr-His-Dmp-NH2. RX T7368)
`R1: CH3, R2 2 CH3
`
`FIG. 3. Structure of TRH 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 eflectiveness
`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 efllux 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-dimethy1proli-
`namide (RX77368) (Brewster et al 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 1080 min (RX77368)
`
`(Table 2). The central nervous activity of the TRH
`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 201-995)
`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 l 13 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 efieets on the plasma half-lives of
`encephalin analogues. Examples are two analogues of
`leucine encephalin (Tyr-Gly-Gly-Phe-Leu), namely DADLE
`
`Table 2. First-order half-lives (min) of TRH, RX74355, and RX77368. There are
`significant species diflerenees in the rates at which plasma and a brain homogenate
`will break down TRH and its analogues.
`
`TRH
`
`RX74355
`(Methylproline)
`
`RX77368
`(Dimethylproline)
`
`Rat
`Plasma
`Brain homogenate
`Dog
`Plasma
`Brain homogenate
`Man
`Plasma
`Brain homogenate
`Mouse
`Plasma
`Brain homogenate
`
`22
`9
`
`>l500
`11
`
`33
`18
`
`215
`12
`
`From Brewster (1983).
`
`I14
`54
`
`> 1500
`90
`
`210
`66
`
`> 1500
`28
`
`390
`190
`
`> 1 500
`174
`
`1080
`168
`
`> 1 500
`150
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1031 - Page 4
`Petitioner Mylan Pharmaceuticals Inc. — Exhibit 1031 — Page 4
`
`

`
`140
`
`DAVID J. BEGLEY
`
`Ala-GIy-Cys-Lys—Asn-Phe-PherTgLys-Thr—Phe—Thr~Ser-Cys—OH
`Somalostatin“
`
`D-Phe—Cys—Phe-D-TQLys-Thr-Cys-Thr—OH
`
`Ac—D-Phe—Cys—Tyr-D—T1;p-Lys-Val-Cys—Thr-NH;
`
`D»Phe»Cys—Tyr-D~T[p;Lys—Val-Cys-TrpNH2
`
`D-Phe—Cys-Tyr—D—Tgg-Lys-Val—Cys-Thr—Nl-lg
`
`RC12l
`
`0
`
`0.05
`
`0.1
`
`0.15
`
`0.2
`
`0.25
`
`Unidirectional cerebrovascular
`influx constant
`
`(/JL g" min“)
`
`FIG. 4. Unidirectional cerebrovascular permeability constants (Kin) 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 (l992b) and Banks et al (1990).
`
`(D-Alaz, D-Leus-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 12]
`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 al 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 aflfinity 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 :1-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
`
`10
`15
`N-E ily-lle—Gly-Ala—Val-Leu-LyS-Val—Leu-ThriThr—Gly-ll Eu-Pro-Ala-Leu-
`+
`
`25
`20
`lle—Ser—Trp-Ile Lys—Arg-Lys-Arg—Gln—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 Mylan Pharmaceuticals Inc. - Exhibit 1031 - Page 5
`Petitioner Mylan Pharmaceuticals Inc. — Exhibit 1031 — Page 5
`
`

`
`TARGETING PEPTIDES AND DRUGS TO THE CNS
`
`141
`
`(Dalargin) /'
`
`Tyr—DAla-Gly-Phe-Leu~Arg
`
`Poly—butyl—cyanoacrylate nanoparticle
`
`ba -80
`‘C
`
`Polywr
`
`Fig. 6. Schematic diagram of a nanoparticle. The dalargin is
`absorbed onto the surface of the nanoparticle which is then
`coated with polysorbate-80.
`
`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(buty1cyanoacrylate) with an
`average diameter of 230nm. 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 ofnanoparticles 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 45 min (Fig. 7). Little elfect
`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 1981; 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 1991).
`
`(%)
`
`
`Maximumpossibleeffect
`
`O
`
`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
`efl"ect after intravenous injection of the indicated dose. (11: 6.
`mean 3: 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-1 has a very large capacity to
`transport ,3-D-glucose across the BBB. The intestinal Na+/
`D-glucose transporter will also transport fi—glycosides but
`not as effectively as the preferred substrate glucose. Given
`that GLUT-1 and the intestinal transporter have a similar
`
`Table 3. Kinetic values for some transport systems present at the BBB.
`
`
`
` Representative substrate Transporter K,,, (MM) Vmx (nmol min“' gr‘) K) (ML g" min'')
`
`
`
`Glucose
`Hexose: Glut-l
`11000 :: 1400
`1420 :: 140
`Lactic acid
`Monocarboxylie acid
`1800 :: 600
`91 :: 35
`Phenylalanine (apparent)
`System-Ll
`30-0 :: 1-0
`14-0 :: 4-0
`Phenylalanine (real)
`System-Ll
`ll-0 :: 2-0
`25-0 :: 6-0
`Arginine (apparent)
`Basic amino acid
`40-0 i 24
`5-0 :: 3-O
`eucine encephalin
`Peptide specific
`39-0 :: 32
`160-0 :: 220*
`Arginine vasopressin
`Peptide specific
`2-08 :: 0-32
`5 -49 :: 0-74*
`Adenosine
`Nucleoside
`25-0 :: 3-0
`0-75 :: 0-08
`Adenine
`Purine base
`1 1-0 :: 3
`0-5 :: 0-O9
`
`44-0 :: 14-0
`8-0 3: 1-0
`
`0062 :: 0-086
`0-02] : 0044
`
`*pmol min" g".
`The Km value is the concentration of substrate at which the transporter is half-saturated and indicates the affinity of the substrate for the
`System. The Vmax is the maximal transporting capacity of the system. The Glut-l transporter has a high aflinity and a high capacity for
`glucose. As explained in the text many amino acids share a common transport system and will have ditferent affinities (Km) for the systems;
`bficause ofthis they will compete with each other for transport. Thus an amino acid will have a real Km and Vmax where their kinetic values are
`dfitermined in the absence ofeompeting amino acids and apparent values when competing amino acids are present. The Kd is a measure ofthe
`Passive diffusional component to the movement ofa solute and is significant in the case of the amino acids but is much smaller in the ease of
`the more polar peptides. Means i s.e.m. Compiled from various sources.
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1031 - Page 6
`Petitioner Mylan Pharmaceuticals Inc. — Exhibit 1031 — Page 6
`
`

`
`142
`
`DAVID J. BEGLEY
`
`1
`
`2
`
`3
`
`4
`
`5
`
`SH HS
`
`0
`ll
`7
`l_
`H2N-Tyr-DCys-Ser-Phe-DCys-Gly—C-NH2
`D-G|e—fl-(1->0)
`
`if
`rS—s7
`H2N-Tyr-DCys—Ser-Phe-DCys-Gly-C-NH2
`D-Glc-ll-(1—>O)
`
`s —— s
`
`0
`n
`_l
`F
`HZN-Tyr-LCys-Gly—Phe-LCys-Ser-C-NH2
`D-G|c—fi-(1-r0)
`
`E
`’—SH HST
`H2N-Tyr-DCys-Gly-Phe-DCys—Ser—GIy—C—NH2
`D-Glc-[5-(1—>O
`6 + p,
`
`if
`I"S“‘S‘l
`H,N-Tyr—DCys-Gly-Phe-DCys- er—GIy-C-NH2
`D-G|c—B~(1~>O
`5 + I‘
`
`6
`
`7
`
`8
`
`9
`
`10
`
`—
`
`2
`
`1?
`H81’
`H2N-Tyr-DPen-G|y-Phe-DPen—Ser-Gly-C-NH2
`D-Glc—fi-(1-O)——J
`r
`*rs—sw’
`H2N-Tyr-DPen-Gly-Phe-DPen-Ser—Gly-C-NH2
`D-G|c—p—(1~>O)
`
`H S
`
`S H
`
`O
`ll
`“l
`l_
`HZN-Tyr-Dcys-GlynPhe-DCys—Ser—Gly-C-NH2
`H
`6 + ll
`
`S —~ 3
`
`O
`I’
`‘l
`u
`H2N-Tyr-DCys-G|y-Phe-DCys-fer-Gly-C—NH2
`H
`5+1:
`
`HZN-Tyr-DPen—G|y-Phe—DPen—C—NH2
`DPDPE 6
`
`ineffective
`is
`FIG. 8. Some analogues of DPDPE (D-pen” encephalin). DPDPE is a 6-receptor agonist but
`intraperitoneally. Analogues 4 and 5 and 8 and 9 are 6- and H-receptor 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-fl-D-glycoside
`conjugates may be acceptable to the brain glucose transpor-
`ter (Polt 1994). Some glycopeptides administered intra-
`peritoneally as L-serinyl-,6-D-glyeoside analogues of Met5
`encephalin (Fig. 8) have been shown to be transported
`across the BBB and bind to u- 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-1 is
`responsible for
`transporting the glyeopeptide into the
`CNS.
`Interestingly,
`these glyeopeptides have a reduced
`lipohilicity compared with the native encephalins but the
`conjugate has reactivity with both the glucose transporter
`
`Antinociception
`
`(%)
`
`0
`
`20
`
`40
`
`60
`Time (min)
`
`80
`
`100
`
`120
`
`Fro. 9. Analgesia produced by DPDPE glycopeptide analogue 5.
`Intrapcritoneal administration of this pe tide produces dose-related
`and long-lasting antinociception in a 55 C tail-flick test. From Poll
`et al (1994).
`
`and with opioid receptors within the brain (Polt 1994). The
`brain transport appears to be specific for ,3-0-linked glyco-
`sides as oz-linked and N-linked and O-acyl linked glucose
`conjugates (Fig. 8) do 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 the treatment of Parkinsonism.
`Dopamine given orally is ineffective as it is not a substrate
`for system-L and thus does not enter the brain readily. It is
`also subject to rapid metabolism at the level of the BBB by
`MAO and COMT. Fortunately L-dopa is a substrate for
`system-L and the enzyme L-amino acid decarboxylase
`(AAD or dopa decarboyxlase) within the BBB will convert
`L-dopa to dopamine during passage through the barrier.
`The dopamine can then be taken up by cells in the substantia
`nigra to replace their missing neurotransmitter. Although
`some further dopamine must be lost as the result of MAO-B
`and COMT activity in the CNS suflicient appears to reach
`the nerve cells.
`
`The antineoplasic alkylating agent melphalan also has a
`reactivity with system-L (Grieg 1992). It
`is chemically a
`nitrogen mustard derivative of phenylalanine (Fig. 10).
`However its reactivity with system-L is only some 20% of
`that of phenylalanine and endogenous amino acids will
`compete for transport. The steric requirements for system-
`L are that the substrate must possess an O-8IT1lI1O group and
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1031 - Page 7
`Petitioner Mylan Pharmaceuticals Inc. — Exhibit 1031 — Page 7
`
`

`
`TARGETING PEPTIDES AND DRUGS TO THE CNS
`
`143
`
`Cfia
`
`>—:CH2{<
`
`Leucifle
`
`CH3
`
`H3N‘
`
`Coo‘
`
`H3N+
`
`H2
`
`Phenylalanine 000.
`
`C
`
`H
`
`H‘-,N*
`
`H2~—<
`
`L-DOPA
`
`COO
`
`\ H3N+
`C
`
`H
`
`,
`
`C
`9/ \C coo
`BCH : 2-amino-bicyclo[2,2,1]-heptnne-2-carboxylic acid
`Definitive specific substrate for the L-system of Christensen
`
`H3N*
`
`coo‘
`
`H3N*
`
`coo’
`
`Baclofen
`
`Gabapentin
`
`Cl
`
`CH2
`
`C|——CH2
`
`CH2
`
`CH2
`
`>~@-2+
`
`H3N"
`
`COO‘
`
`Melphalan
`
`CI-——CH,
`
`CH
`
`CI—C H2?CH2
`
`’>N
`
`H,N*
`
`COO‘
`
`D, L NAM : (D,L-Zaamino-7-bisl(2-ch|oroethyl)amino]-
`l,2,3,4-tetrahydro-2-napthoic acid)
`
`FIG. 10. Substrates known to be transported by system-L at the
`blood—brain barrier. All of the structures have in common an amino
`and a carboxylic acid group attached to a single carbon atom, with
`the exception of baclofen and gabapentin. In the case of these two
`drugs it is thought that the amino and carboxylic acid groups have
`suflicient flexibility to make the molecules sterically acceptable to
`the transport mechanism. BCH is the defining substrate for system-
`L and only reacts with that amino acid transporter.
`
`a carboxylic acid group attached to the same carbon atom
`plus a hydrophobic side group. However there are some
`known exceptions (Fig. 10). The size of the hydrophobic side
`group does not appear to be a hindrance to transport and
`the amino acids phenylalanine and tyrosine have a high
`aflinity for the transporter. The importance of the relative
`positions of the a-amino and carboxylic acids groups is
`emphasized by the fact that when the small peptide glycyl-
`ieucine is formed reactivity with system-L is lost. Other
`Substrates with significant afiinity for system-L have been
`designed (Grieg 1992). The nitrogen mustard DL-2-amino-
`7-bis((2—chloroethyl)amino)-1,2,3,4-tetrahydro-2-naphthoic
`acid (DL-NAM) is a good example. It possesses an addi-
`tional naphthoic side chain compared with melphalan
`(Fig. 10), which renders it more hydrophobic and confers
`0n the molecule 20 times the affinity for system-L than
`Phenylalanine and 100 times the affinity of Melphalan and
`L'd0pa for the transporter. Additional advantages of DL-
`NAM are that it has 1.5 times the alkylating activity of
`melphalan and it is only 20% bound to plasma proteins
`°°mpared with 80% for mephalan giving it
`a high
`
`therapeutic index (Grieg 1992). Its high aflinity for system-
`L means that it competes effectively with endogenous amino
`acids to achieve an eflicient brain uptake even though the
`Vmax is lower.
`The antiepileptic drug gabapentin (neurontin) also enters
`the CNS via system-L even though its does not fully satisfy
`the strict steric requirements suggested above. Its uptake
`into the CNS is inhibited by BCH the model substrate for
`system-L (Fig. 10). Gabapentin is not a substrate for the
`glutamate transporter and is thus unusual in that it is a 7-
`amino acid that is acceptable to system-L. It is suggested
`that the amino and carboxylic acid groups are sufliciently
`flexible to render it sterically acceptable to system-L. The
`binding site for gabapentin in nervous tissue is distinct from
`system-L as leucine is more potent in displacing it than is
`BCH. It acts as an inhibitor of branched-chain amino
`transferase and both it and leucine act to stimulate gluta-
`mate dehydrogenase in the CNS.
`A similar situation pertains for the muscle relaxant baclo-
`fen (4-amino-3-(p-chlorophenyl)-butyric acid (Fig. 10) (Van
`Bree et al 1988). Baclofen acts at the spinal and supraspinal
`levels and is transported into the CNS by system-L.
`A number of specific uptake mechanisms for peptides
`exist at the BBB (Begley 1994). Those for leucine encephalin
`and arginine vasopressin have been kinetically described as
`high aflinity low capacity systems (Zlokovic et al 1989, 1990)
`as shown in Table 3. The uptake mechanism for leucine
`encephalin is remarkably specific showing little afiinity for
`fragments of the molecule with almost the entire pentapep-
`tide being sterically required. Fig. 11 shows the results of
`inhibition studies carried out in an in—situ brain perfusion
`technique with the addition of a number of inhibitors and
`peptide fragments. Under the conditions of perfusion the
`
`Kml;IL9"min") UCLU
`
`3Q!
`._l
`
`+SmMTG
`
`+2mMBacitracin
`
`+0.5mMBestatin
`
`+SmMTyrosine
`
`+2mMLeuEnc
`
`+SmMTGG
`
`+SmMTGGP
`
`+SmMGGPL
`
`
`
`
`
`+0.044ICI174846
`
`
`
`+0.053mMDAGO
`
`1 1. Specificity of leucine encephalin uptake at the blood—brain
`1:16.
`barrier. Inhibition studies to determine the specificity of the uptake
`mechanism for leucine encephalin at the blood—brain barrier of the
`guinea-pig hippocampus determined by an in—situ vascular brain
`perfusion method. Vertical axis unidirectional cerebrovascular con-
`stant Kin pL g“ min'1:ks.e.m. Horizontal axis Kin for leucine
`encephalin tracer alone (control); plus 2mM bacitracin, 0.5mM
`bestatin. 5 mM L-tyrosine, 2mM leucine encephalin, 5 mM tyrosyl-
`glycine, 5mM tyrosyl-glycyl-glycine, 5mM_ t_yrosyl-glycyl-glycyl-
`phenylalanine, 0.044 mM ICI 174846 (a 6 Oplold ligand), 0.053 mM
`DAGO (a ,u opioid |igand.). Only the addition of 5mM leucine
`encephalin produces a significant
`inhibition of the Km value
`(P < 0-01) and 5 mM tyrosyl-glycyl-glycyl-phenylalanine (P < 005).
`Data from Zlokovic et al (1987, 1989). Means :: s.e.m.
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1031 - Page 8
`Petitioner Mylan Pharmaceuticals Inc. — Exhibit 1031 — Page 8
`
`

`
`144
`
`DAVID J. BEGLEY
`
`(3HTyr)-encephalin molecule is stable as the addition of
`2.0 mM bacitracin or 02 mM bestatin to the perfusate has no
`effect on the cerebrovascular permeability constant, Kin.
`Significant reduction of the Km value, indicating competi-
`tive inhibition, was only produced by intact leucine ence-
`phalin and des-leucine encephalin. DAGO and ICI 174846,
`ligands for the p- and 6-opioid receptors, respectively, had
`no effect in the K-,,, indicating that the carrier is distinct from
`the receptor producing the central nervous effects of these
`opioid peptides (Zlokovic et al 1989).
`Transporters for macromolecules also exist at the BBB
`which might be utilised as vectors to transport material into
`the CNS. Strategies utilizing the transferrin receptor in this
`way have been attempted (Friden et al 1991, 1993; Pardridge
`et al 1991). A monoclonal antibody,
`to the transferrin
`receptor OX26 has been utilised as this avoids competition
`between any administered transferrin and endogenous
`transferrin in the experimental animal. A variety of mole-
`cules can then be attached to the monoclonal antibody. In
`experimental animals this approach has been used to deliver
`horseradish peroxidase, nerve growth factor (NGF), meth-
`otrexate and vasoactive intestinal polypeptide (VIP), across
`the blood-brain barrier.
`In some respects these studies
`remain controversial as there is evidence to suggest that in
`the intra-endothelial transport of iron by transferrin at the
`BBB, the iron is decoupled from the transferrin within the
`cells and the transferrin molecule itself may not necessarily
`be transcytosed. (Taylor & Morgan 1990; Ueda et al 1993).
`However, the NGF-OX26-transferrin complex may act as a
`vector to allow large proteins such an NGF to enter a
`protected compartment within the endothelial cells and the
`whole complex may not necessarily have to be exocytosed at
`the abluminal membrane.
`
`Efflux mechanisms
`
`As mentioned earlier the efflux pump P-glycoprotein is
`expressed constitutively at the luminal surface of the cere-
`bral endothelial cells forming the BBB. A number of lipid
`soluble cytotoxic agents, such as Vincristine and vinblastine
`and other substrates for P-glycoprotein, do not exhibit the
`brain uptakes that would be predicted by their lipid solu-
`bility (Begley 1992) and are outliers on the plot shown in
`Fig. 2. One strategy for enhancing the uptake of substrates
`
`for P-glycoprotein is to employ non-competitive or compe_
`titive inhibitors of the eflfux pump. These agents will reverse
`multidrug resistance in peripheral tissues that have acquired
`it and thus have potentially a wide application in cancer
`chemotherapy. Table 4 shows the effect of a number of
`substrates and inhibitors of Pgp activity on the accumula-
`tion of [3H]colchicine in-vitro into a cultured monolayer of
`an immortalized clone of rat brain endothelial cells (Begley
`et al 1994). Similar results have also been obtained in-vivo
`by administering therapeutic doses of Vincristine to guinea-
`pigs (Begley & Evans 1992). In this study co-administration
`of Vincristine and [3H]colchicine increases BBB uptake of
`colchicine compared with control animals receiving tracer
`colchicine alone, suggesting an inhibition of Pgp activity in
`the cerebral endothelium.
`
`A spectrum of efflux pumps exists within the CNS and
`contributes to the homeostasis of the brain extracellular
`
`fluid. These pumps may be located in the cerebral endothe-
`lium and also at the choroid plexus. For example penicillin,
`p-aminohippuric acid and AZT (azidothymidine), are
`pumped out of cerebrospinal fluid (CSF) at the choroid
`plexus by an organic acid pump which is probenecid-sensi-
`tive (Spector & Lorenzo 1974). Probenecid can therefore be
`used to increase the CSF levels of these drugs. Amino acids
`are cleared rapidly from CSF by saturable transport
`mechanisms (Davson et al 1982) as are a number of peptides
`(Begley & Chain 1988, 1992; Banks & Kastin I992). Amino
`acids with neurotransmitter function have very low free
`concentrations in CSF. Thus the design of peptide- or
`amino acid-based drugs with a reduced affinity for these
`eflfux mechanisms or the inhibition of these efflux mechan-
`isms will lead to an increase in CNS levels. There are also free
`
`peptidases in CSF (Begley & Chain 1988, 1992; Begley 1994)
`which can hydrolyse peptides, for example leucine encepha-
`lin and angiotensin II. The substitution of D-isomers into the
`peptide confers resistance to the action of these enzymes in
`the same manner as with plasma peptidases.
`
`Opening and Permeabilizing the BBB
`
`A number of techniques exist which enable the permeability
`of the BBB to be modified. Most of these are non-selective
`
`and open the barrier to a range of solutes of varying
`molecular weight.
`
`Table 4. Inhibition of Pgp activity in RBE4 cells.
`
`Inhibitor
`Vincristine
`AZT
`Chlorpromazine
`Verapamil
`
`Concn (pm)
`50
`50
`50
`100
`
`[3H] Colchicine uptake (mL (pg protein)'*')
`
`Control
`136411-19
`141610-82
`12911093
`12-45] l~l9
`
`Experimental
`533918-62
`339313-24
`25751 1'88
`17461862
`
`% control
`39l**
`232**
`200**
`140*
`
`Cells were either pre-treated with inhibitor in HBSS for 30 min (experimental), or left for
`30min in HBSS alone (control). The incubation medium was then changed for one
`containing [3H]colchicine (18 nM, 1.38 mCi) plus inhibitor (experimental) or the same
`chemical and radioactive concentrations of [31-I]colchicine alone (control) and incubated for
`a further 30 min. An increase of [31-I]colchicine accumulation into the cells is interpreted as
`an inhibition of the efflux activity of Pgp. Mean ::s.e.m. Experimental values were
`1
`.
`sig9r(1)i4fi)cantly different from control values by unpaired Student’s t—teSL From Begley et al
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1031 - Page 9
`Petitioner Mylan Pharmaceuticals Inc. — Exhibit 1031 — Page 9
`
`

`
`TARGETING PEPTIDES AND DRUGS TO THE CNS
`
`145
`
`The most widely used technique is that of osmotic open-
`ing where a short
`infusion of a hyperosmotic solution,
`usually mannitol,
`is
`introduced into a carotid artery.
`Large molecules can be given access to the CNS by this
`technique and its therefore thought that the osmotic agent is
`affecting the integrity of the tight junctions between the
`cerebral endothelial cells. One explanation that has been
`offered is that the osmotic agent is shrinking the endothelial
`cells and physically opening paracellular pathways. Osmotic
`blood-brain barrier disruption coupled with the infusion of
`chemotherapeutic drugs has been used to treat a number of
`central nervous tumours with marked improvements in
`survival
`time and little apparent ill effect from the BBB
`disruption (Gumerlock & Neuwalt 1992). Osmotic BBB
`disruption can be used to deliver therapeutic substances
`that would otherwise be excluded from the CNS because of
`their size, including the possibility of introducing geneti-
`cally-engineered retroviruses in order to replace defective
`genes.
`The peptide bradykinin and some of its analogues
`increase BBB permeability by apparently permeabilizing
`the tight junctions via B2 receptors (Unterberg et al 1984);
`this general phenomenon is referred to as receptor—mediated
`permeabilization (RMP). Similar permeabilization can be
`achieved with leukotrienes, histamine and 5-hydroxytrypta-
`mine. In in-vitro cultures of monolayers of bovine cerebral
`endothelial cells leucine encephalin, a-adrenergics, arachi—
`donic acid, bradykinin, aluminium, phorbolmyristate esters,
`and or-thrombin all increase permeability of the monolayer,
`whilst angiotensin II, saralasin (an angiotensin analogue), B-
`adrenergics, lowered temperature, and 2-deoxyglucose all
`reduced the permeability of the monolayer (Grieg 1992).
`Some of these factors used to permeabilize the barrier are
`more selective than others. As previously mentioned, the
`BBB exists for the specific purpose of protecting the brain
`from potential toxins and providing a separate and stable
`environment within which the neurones can perform their
`integrative functions. Thus any non-selective opening of the
`barrier will transitorily disturb the brain fluid environment
`and cause possible long term damage.
`
`Conclusions
`
`Clearly then, there is considerable scope for optimizing drug
`delivery to the brain in order to treat a whole range of
`Central nervous diseases from neoplasm to degenerative
`disorders and psychiatric disturbance.
`There still remains a great need for a systematic study of
`molecular structure and properties of drugs in relation to
`their BBB penetration. For substances that enter passively,
`the aim is to extend the plasma half life,
`increase the
`Penetration of the blood brain barrier, retain and enhance
`central activity and reduce reactivity with central nervous
`mechanisms degrading or removing the drug from the CNS.
`In practice it is unlikely that all of these ideals will be
`Satisfied for a single drug, but optimizing some of them
`will probably produce gains in most cases.
`Specifically designing drugs which mimic the substrates
`f°T uptake mechanisms located in the BBB, appears to hold
`great promise, particularly if a range of chemical groups can
`be designed which can be attached to a variety of
`
`compounds rendering them substrates with a high aflinity
`for particular carriers. The two transporters GLUT-1 and
`system-L have the highest capacity and perhaps offer the
`best chance for delivering substances to the brain in quan-
`tity. However it may not be necessary to deliver large
`quantities of some substances to the brain which possess
`very potent central activities and which may influence, for
`example, behavioural activity. In these cases lower-capacity
`transporters or an enhanced passive diffusion might suflice.
`Opening and permeabilizing the barrier are generally non-
`selective and are useful
`in allowing an acute entry of
`substances into the brain where a once-only opening is
`required in order to introduce a genetic vector or potent
`drug. Non-selectively lifting the protective BBB repeatedly
`may carry some risks but these risks might be clinically