Patent Owner, UCB Pharma GmbH – Exhibit 2005 - 0001
`
`

`
`E. Callegari et al.
`
`Introduction
`
`Overactive bladder (OAB) is a syndrome defined by the
`International Continence Society as urgency, with or
`without urgency incontinence, usually with increased
`daytime frequency and nocturia [1]. OAB affects at least
`10% of the adult population [2] and the prevalence
`increases with age [3]. Postulated aetiologies for this con-
`dition include increased afferent activity, decreased inhi-
`bitory control and increased sensitivity of the detrusor
`muscle to efferent stimulations [4, 5]. Muscarinic receptors
`are thought to mediate the detrusor contractions of
`normal voiding, but in OAB the muscarinic receptors are
`associated with bladder contraction leading to urinary fre-
`quency, urgency and urgency incontinence. Antimuscar—
`inic agents, such as darifenacin, oxybutynin, solifenacin,
`trospium, tolterodine and fesoterodine have consistently
`demonstrated significant efflcacy for the treatment of OAB
`symptoms [6, 7]. However, treatment is associated with
`typical anticholinergic side effects such as dry mouth,con—
`stipation, somnolence and blurred vision. The adverse
`effects (AE5) of antimuscarinic drugs may occur because
`muscarinic receptors are located throughout the body [8]
`where inhibition of specific receptor subtypes is associated
`with side effects. For example, in the central nervous
`system (CNS), muscarinic receptors, particularly the M.
`subtype, are thought to play an important role in the con-
`solidation of long-tenn memory. Cognitive impairment is
`associated with anticholinergic therapy in the elderly [9]
`and the side-effect profiles seen with some antimuscarinic
`OAB drugs are consistent with inhibition of muscarinic
`receptors in the CNS [10]. Although the incidence of CNS
`AEs of antimuscarinic agents is generally much lower than
`that of dry mouth [6, 7], CNS AEs can be of great concern,
`particularly in the elderly [1 1]. The incidence of CNS AEs
`among the available antimuscarinic agents for OAB seems
`to differ. For instance, while darifenacin has been shown to
`
`have no significant effects on memory vs. placebo, oxybu-
`tynin ER caused significant memory deterioration which
`was deemed comparable with brain ageing of 10 years
`[12]. The ability of certain antimuscarinic OAB drugs to
`exert CNS-related pharmacological effects at therapeutic
`doses for OAB treatment depends on their ability to pen-
`etrate the CNS as well as relative affinity for relevant mus-
`carinic receptor subtypes in the CNS, particularly M.
`[13—1S]. CNS penetration of drugs depends on the perme-
`ability properties of the blood—brain barrier (BBB) [16, 17]
`and the influence of active efflux transporters present in
`brain tissue, such as P-glycoprotein (P-gp) [18, 19].There-
`fore, the relative permeability and affinity of OAB agents
`for P-gp is an important consideration in understanding
`their potential to exert AEs manifested in the CNS.
`The purpose of the present paper was to present a
`comprehensive set of non—clinica|
`in vitro and animal
`studies that investigated in parallel the CNS penetration
`potential of antimuscarinic OAB drugs. The following
`
`236 I 72:2 / Br] Clin Pharmacol
`
`studies were conducted: (i) physicochemical characteriza-
`tion, including lipophilicity; (ii) in vitro RRCK cell passive
`permeability assessment; (iii) in vitro P-gp mediated efflux
`in MDCK—MDR1 transcellular flux assay; and (iv) in vivo
`brain, plasma and CSF concentrations following a single
`subcutaneous dose in rats. Strategies for assessment of
`brain penetration of compounds have focused on deter-
`mination of unbound brain : unbound plasma concentra-
`tion ratios (Kp,free), and consideration of the involvement
`of transporter proteins at the BBB, in particular P-gp [19].
`Therefore, to understand further CNS disposition, the
`unbound brain : unbound plasma concentration ratios
`were estimated using brain and plasma binding experi-
`ments in vitro [19, 20].The overall aim of this package of
`data was to enable an understanding of the brain penetra-
`tion potential of antimuscarinic OAB drugs in relation to
`their physicochemical and permeability properties. The
`antimuscarinic agents included in these studies were
`5—hydroxymethyl tolterodine (5—HMT, the active metabo-
`lite of tolterodine as well as fesoterodine), darifenacin,
`
`oxybutynin, solifenacin, tolterodine and trospium. Fes-
`oterodine is a pro—drug that is rapidly and extensively con-
`verted to 5-HMT by esterases in vivo, and is not detectable
`after oral administration [21, 22]. Therefore, 5-HMT was
`
`evaluated as the relevant active moiety of fesoterodine in
`this study.
`
`Methods
`
`Materials
`
`Oxybutynin, N-methylscopolamine HBr and atropine
`(internal standard) were purchased from Sigma-Aldrich.
`5-hydroxymethyl tolterodine (5-HMT), solifenacin and tro-
`spium chloride were purchased from Toronto Research
`Chemicals Inc. (Ontario, Canada).
`
`Fesoterodine and scopolamine were obtained from
`Pfizer Global Research and Development central com-
`pound stores (Milwaukee, WI, USA).To|terodine was pur-
`chased from Sequoia Research Products (Pangbourne,UK).
`Darifenacin was purchased from Toronto Research Chemi-
`cals Inc. (Ontario, Canada) and Sequoia Research Products
`(Pangbourne, UK). Hanks's balanced salt solution (HBSS),
`cell culture media and supplements were purchased
`from lnvitrogen (Paisley, UK). All other reagents were
`obtained from Sigma-Aldrich (Poole, UK) or J.T. Baker
`(Phillipsburg, NJ. USA).
`
`Assessment of physical properties
`of compounds
`The definitions of physical properties of compounds were:
`log D, logarithm of the distribution coefficient between
`octanol and buffer at pH 7.4;c|og P, logarithm of the calcu-
`lated partition coefficient between octanol and water,
`polar surface area (PSA), the area of molecular surface
`belonging to polar atoms (units: A’) [17], rotatable bond
`
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`Patent Owner, UCB Pharma GmbH – Exhibit 2005 - 0002
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`count, count of all non—terrnina| single bonds and hydro-
`gen bond acceptor and donor counts, counts of all atoms
`in a molecule that are potentially involved in a hydrogen
`bond as acceptor or donor atoms, respectively. All calcu-
`lated parameters were determined within the proprietary
`Pfizer database (RGate), where clog P was determined
`using the BioByte program clog P, version 4.3 and PSA
`using a published method [17]. Log D values were taken
`from published sources [23].
`
`Monolayer efflux studies in MDCK
`(Madin-Darby canine kidney) and
`RRCK cell lines
`
`MDCK-MDR1 expressing P—gp were originally obtained
`from Netherlands Cancer Institute (Amsterdam, the Neth-
`
`erlands). RRCK cells were generated in house (Pfizer lnc.,
`Groton, MA, USA) as a subclone of MDCK wi|d—type (MDCK-
`WT) cells that displayed low expression of endogenous
`P—gp (approximately 1—2% of MDCK—WT cells, based on
`mRNA level). The rank order of permeability values for
`compounds whose transcellular flux was predominantly
`by passive diffusion were similar for RRCK and MDCK-WT
`(data not shown). Monolayer efflux studies were con-
`ducted as previously described in the literature for MDCK-
`MDR1 cells [24]. Cells were cultured in minimal essential
`
`medium on with supplements and passaged when 70-80%
`confluent. Cell monolayer flux studies were conducted 5
`days after seeding in 24-well transwell inserts [MDCK-
`MDR1 in 0.4-pm pore size (Corning Costar) at 1.8 x 105
`cells cm"; RRCK in 1-pm pore size (Becton Dickinson,
`Cowley, UK) at 4.2 x 10‘ cells cm"]. Donor and acceptor
`solutions were prepared from HBSS, containing HEPES at
`20 mM, pH 7.4. Stock solutions of test compounds were
`prepared at 10 mM in dimethyl sulphoxide (DMSO) and
`used to prepare donor solutions of 2 pM compound in
`0.05% (v/v) DMSO and also containing 2 pM nadolol used
`as monolayer integrity marker. Apparent permeability
`(Paw) of compounds was detennined in apical to basolat-
`eral (A—>B) and basolateral to apical (B—)A) directions in
`triplicate by incubation with compound for 2 h at 37°C.
`Samples of medium (20 pl) from both donor and acceptor
`chambers were analysed by tandem liquid chromatogra-
`phy and mass spectrometry (LC/MS-MS). The LC/MS-MS
`system consisted of a 2.1 x 15 mm C18 optilynx column
`(Optimize Technologies lnc., Oregon city, OR, USA) in line
`with Onyx monolithic C18 column 50 x 4.6 mm (Phenom-
`enex, Torrance, CA, USA) operating at a flow rate of
`3 ml min". The aqueous mobile phase consisted of 90%
`2 mM ammonium acetate, 10% methanol, 0.1 % formic acid.
`
`The organic mobile phase was 10% 2mM ammonium
`acetate, 90% methanol, 0.1 % formic acid. Mass spectrom-
`etry was performed on a SCIEX API 4000 triple quadrupole
`mass spectrometer (Applied Biosystems, Ontario, Canada),
`with turbo ion spray source. Data were acquired in positive
`ion mode with an ion spray probe voltage of 5.5 kV. The
`following selected reaction monitoring transitions, given
`
`CNS penetration potential of OAB agents
`
`as mass : charge ratio (m/z) were used to measure
`the compounds: tolterodine m/z 310—>201 and nadolol
`m/z 310—>254 at collision energy of 25 eV; darifenacin
`m/z 427—>147, oxybutynin m/z 358—>124, 5—HMT m/z
`342—>223, fesoterodine m/2 412—>223 and nadolol m/z
`
`310—>201 at a collision energy of 40 eV; solifenacin
`m/z 364—>110, trospium m/z 392—>182 and nadolol m/z
`310—)56 at a collision energy of 55 eV.
`P,,,,, values were calculated according to the equation
`P,,,,, = (Q/t) x 1/Co x 1/A, where Q is the sampled concen-
`tration in the acceptor compartment, t incubation time; Co
`is the initial concentration in the donor compartment and
`A is the area of the filter of the transwell plate. Monolayers
`with nadolol Paw values of less than 1 x 105cm s" were
`deemed intact.
`
`In vivo brain penetration study
`All procedures performed on animals were in accordance
`with US federal regulations and established NIH guidelines
`and were reviewed and approved by the Institutional
`Animal Care and Use Committee. Male Sprague-Dawley
`rats weighing approximately 250 g (n = 3) received a single
`dose of compound subcutaneously (0.3 or 1mg kg").
`Dosing solutions were prepared fresh in saline on the
`day of the study and were administered at 1 ml kg“. At
`1 h postdose, animals were euthanized using CO2 and
`approximately 3 ml of blood removed by cardiac puncture
`and plasma prepared by centrifugation. Cerebrospinal
`fluid (CSF) was drawn from the cistema magna using a 25
`gauge needle attached to polyethylene tubing (2 mm) and
`a syringe. Brains were removed, rinsed with saline and
`weighed. All samples were immediately frozen on dry ice
`after processing and stored at —20°C until analysis. Brain
`samples were homogenized in four volumes of phosphate
`buffered saline (1 in 5 tissue dilution). Plasma, CSF and
`
`brain samples were analysed using a LC/MS-MS method.
`Calibration standard and quality control
`(QC) samples
`were prepared in untreated rat plasma or brain homoge-
`nate. The lower limit of quantitation (LLOQ) for all com-
`pounds was
`0.1 ng ml“ while the upper
`limit of
`quantitation (ULOQ)
`ranged from 25 to 100 ng mf‘,
`depending on the dose received. Aliquots of plasma or
`brain (50 pl) were basified using 50 mM K2HPO4, pH 8.0
`(200 pl) and mixed with internal standard (atropine, 10 pl
`of 50 ng ml“) before loading onto a 96-well solid phase
`extraction plate (Waters Oasis HLB, 10 mg). Following
`several washes (400-pl water, followed by 400-pl metha-
`nol :water 10:90), compounds were eluted using methanol
`(400 pl) and eluant dried under nitrogen heated to 40°C.
`Samples were reconstituted in 50-pl 5mM ammonium
`acetate buffer containing 0.1% fonnic acid (v/v) before
`analysis by LC/MS-MS. Extracted samples (10 pl) were
`injected onto a Phenomenex Luna C18 analytical column
`(5 pM, 2.1 x 50 mm). The liquid chromatography system
`consisted of a gradient mixture of acetonitrile and 5 mM
`ammonium acetate pH adjusted with formic acid (0.1%,
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`Patent Owner, UCB Pharma GmbH — Exhibit 2005 - 0003
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`
`E. Callegari et al.
`
`v/v) maintained at a flow rate of 0.3 ml min"‘. Samples con-
`taining concentrations above the ULOQ were diluted with
`control matrix and re—ana|ysed. CSF samples (20 pl) were
`mixed with internal standard and ammonium acetate
`
`buffer [5 mM with 0.1% formic acid (v/v)] and were quanti-
`fied using a direct—inject method onto the LC/MS—MS
`system described above with standard curves prepared in
`untreated rat plasma ultrafiltrate. Data were acquired in
`positive ion mode with an ion spray probe voltage of
`5.5 kV using the same reaction monitoring transitions as in
`the transcellular flux experiments, with the internal stan-
`dard atropine monitored at m/z 290—>124.
`
`Plasma and brain binding
`Binding of compounds to rat plasma or rat brain was deter-
`mined by a previously described equilibrium dialysis
`method [20]. Briefly, untreated plasma or brain homoge-
`nate (5x diluted with PBS) wasfortified with test compound
`to yield a final concentration of 1 pM. Samples were placed
`in a 96-well equilibrium dialysis block (HTDialysis, Gales
`Ferry,CT, USA) fitted with Spectra—Por 2 membranes (Spec-
`trum Laboratories, Rancho Dominguez, CA, USA) and incu-
`bated at a temperature of 37°C, humidity of 95% and CO;
`concentration of 5%. After 6 h,a|iquots (10 pl) of buffer and
`matrix were removed and added directly to a 96-well
`polypropylene block containing internal standard in aceto-
`nitrile (atropine, 50 ng mF‘).An equal volume of the oppo-
`site matrix was added to each sample to yield unifonn
`sample composition. All samples were analysed using
`LC/MS—MS with conditions similar to those described
`
`above.The unbound fraction in plasma (f..,,) was calculated
`using the ratio of drug concentrations in buffer to matrix.
`Unbound fraction in brain (f...,) was calculated in a similar
`fashion but tissue dilution factor (D) was taken into account
`
`according to the correction described by Kalvass & Maurer
`[20], whereby f..., = 1/D/[(1/f..2) — 1] + I/D, where f..; =
`unbound fraction measured in diluted tissue homogenate.
`
`Analysis of in vivo brain penetration data
`The treatment of data obtained from in vivo brain penetra-
`tion studies has been discussed extensively in the litera-
`ture and has underlined the value of applying knowledge
`
`Table 1
`
`Physicochemical properties of various antimuscarinic agents
`
`of tissue binding in comparing tissue concentrations, and
`hence understanding brain penetration [19, 20, 25]. Fur-
`thermore, comparsion of results obtained using unbound
`brain tissue homogenate concentrations with in vivo
`microdialysis has supported the use of free concentrations
`in brain and CSF as surrogates for concentrations in brain
`interstitial fluid [26]. Therefore, the tissue concentration,
`
`plasma protein binding and brain binding data were used
`to calculate the following parameters:
`
`Brain : plasma ratio (8 : P):
`total concentration in brain (ng g"):
`total concentration in plasma (ng mF')
`
`Unbound concentration in plasma =
`total concentration in plasmaxfup
`
`Unbound concentration in brain:
`
`total concentration in brainxf,,.,
`
`Ratio of unbound fraction fractions
`
`in brain and plasma: f,,, :f.,,,
`
`Kp,free = unbound concentration in brain:
`unbound concentration in plasma
`
`CSF : free plasma ratio:
`concentration in CSF: unbound concentration in plasma
`
`Results
`
`Physicochemical property assessment of
`OAB agents
`The physicochemical properties that describe the lipophi-
`licity, hydrogen bonding potential, polarity and flexibility
`of the range of antimuscarinics studied are summarized in
`Table 1.The compounds used for this investigation possess
`relatively low molecular weight (range 325-426 Da),
`typical of small drug molecules and, with the exception of
`trospium, can be classed as lipophilic based on their log D
`and calculated log P values (ranges log D 0.74—>3.3 and
`
`Antimustarinlc agent
`
`Clog P
`
`Log D
`
`Hydrogen bond acceptors
`
`Hydrogen bond donors
`
`
`
`Rotatable bonds
`
`Molecular weight (Dal
`
`PSA (A2)
`
`For definition of parameters in the table, refer to Methods.
`
`238 / 72:2 / Br|C|in Pharrnacol
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`CNS penetration potential of OAB agents
`
`I
`
`mill:
`
`§6
`
`
`
`Pappx|0"cms" 33
`.
`
`Trosplum 5-I-IMT DarIfen- Solifen- ToItero- Oxybut-
`acln
`acin
`dine
`ynln
`
`I 3.7
`
`4.0
`
`I .3
`
`I .4
`
`0.6
`
`B-*A:A-B
`efflux ratio 4.3
`
`III9
`
`AO
`
`PappxI0'‘cms" —NWOO9
`
`0 T
`
`rvosplum 5-HMT Darlfe- SoIifen- ToIteno- Oxybut-
`nacin
`acin
`dine
`ynln
`
`Figure 1
`Passive transport of antimuscarinic agents in RRCK cells. Fluxes of corn-
`pounds aaoss RRCK cell monolayers in the apical to basolateral (A—)B)
`direction are shown. lncubations were performed in triplicate and error
`bars represent SDs
`
`clog P 3.6-4.9). The rank order of lipophilicity determined
`by log D [23] was oxybutynin > darifenacin > tolterodine =
`solifenacin z fesoterodine > SHMT > trospium.Trospium
`(log D -1 .22,c|og P -1.2) is a much more hydrophilic com-
`pound in comparison due to the presence of a quaternary
`amine group that is ionized at physiological pH. Hydrogen
`bonding potential evaluated as the sum of hydrogen
`bond donors and acceptors was broadly similar between
`compounds, with rank order darifenacin = 5-HMT (5) >
`oxybutynin = trospium (4) > tolterodine = solifenacin =
`fesoterodine (3). All the compounds possessed relatively
`low PSA values ranging from 23.5A’ for tolterodine to 55.6
`A’ for darifenacin.The number of rotatable bonds in a mol-
`
`ecule is a measure of the flexibility of the compound. The
`range in the number of rotatable bonds in this series of
`compounds is relatively large with solifenacin having the
`lowest value (4) and 5-HMT and darifenacin having the
`highest value (8).
`
`Transcellular flux across RRCK and MDR 1 -
`MDCK cell monolayers
`In vitro cell membrane permeability and the influence of
`P-gp were assessed in cell lines designed to measure tran-
`scellular flux in the presence and absence of P-gp. A stable
`population of MDCK cells was selected by flow cytometry
`to have little or no functional P-gp that would contribute
`to efflux of substrates of this protein. In this cell line, des-
`ignated RRCK, compounds displayed a range of flux
`values in the apical
`to basolateral direction (A—>B)
`(Figure 1), and compounds could be classed as possessing
`low (<5 x 10" cm 5''), moderate (5-1 5 x 10* cm s") or high
`(>15 x 10* cm s") transcellular flux Thus, trospium (P.,,,, =
`0.63 x 10" cm s") possessed low flux, 5-HMT moderate
`and solifenacin, tolterodine, darifenacin and oxybutynin
`high flux values.
`The ability of compounds to act as P-gp substrates was
`assessed in MDCK cells transfected with the human mdri
`
`gene that expresses P-gp (MDCK-MDR—1). The ratio of
`
`Figure 2
`Transcellular flux of antimuscarinic agents in MDCK-MDR1 cells. Fluxes of
`compounds across MDCK-MDR1 cell monolayers were measured in apical
`to basolateral (A—>B) and basolateral to apical (B—)A) directions.The ratio
`of A—)B/B~>A fluxes (efflux ratio) is shown under each compound. Incu-
`bations were perfonned in triplicate and error bars represent SDs.A—>B
`flux (Z ); B—>A (-)
`
`fluxes
`to apical
`to basolateral : basolateral
`apical
`(A—>B:B—>A efflux ratio) was used as an index of P—gp-
`mediated
`efflux
`across MDCK—MDR—1 monolayers
`(Figure 2). The efflux ratios shown by the range of com-
`pounds varied between 0.6 and 13.7. Using an efflux ratio
`of >2 to indicate significant P-gp—mediated efflux [27], it
`was found that trospium, 5-HMT and darifenacin were
`likely to be substrates of the P-gp transporter, whereas
`solifenacin, tolterodine and oxybutynin were unlikely to
`act as substrates.
`
`In vivo CNS penetration of OAB agents in rats
`Detennination of plasma, brain and CSF drug concentra-
`tions by specific LC/MS-MS afforded the required sensitiv-
`ity and selectivity for accurate detennination of drug
`concentration in tissues and ensured that metabolites did
`
`not contribute to apparent drug concentrations (the tissue
`concentration data used to determine brain penetration
`are shown in Table 2). CNS penetration was initially
`assessed by calculating the total brain : plasma concentra-
`tion ratios (B : P) and CSF :free plasma concentration ratios
`(CSF :free plasma). During collection of the brain in these
`experiments, blood remains in the tissue and following
`homogenization and LC/MS-MS analysis the residual
`blood can contribute an observed B:P of up to 0.04,
`reflecting presence of drug in the vasculature of the brain
`rather than true brain penetration [28]. Hence, values of
`B: P below 0.04 and CSF :free plasma approaching 0 are
`consistent with no significant brain penetration and no
`equilibrium between CSF and free plasma. The results of
`CNS penetration in vivo are summarized in Tab|e3 and
`displayed graphically in increasing order of their CNS pen-
`etration in Figure 3.
`In parallel experiments, scopolamine (0.3 mg kg") and
`N-methylscopolamine (0.3 mg kg") were administered as
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`Patent Owner, UCB Pharma GmbH — Exhibit 2005 - 0005
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`Patent Owner, UCB Pharma GmbH – Exhibit 2005 - 0005
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`

`
`E. Callegari et al.
`
`Table 2
`
`Mean concentrations and unbound fractions of antimuscarinic agents in rat tissues following subcutaneous dosing
`
`.
`.
`
`Compound
`
`Tiospiuln
`S-HMT
`Darifenacin
`solifenacin
`Tolterodine
`Oxybutynin
`N-Inethylsoopolamine
`Scopolamine
`
`Plasma
`concentration
`(ng ml-‘)
`
`106 1 85
`27.3 1 1.3
`39.0 1 5.8
`59.3 1 4.6
`20.8 1 2.7
`22.5 1 5.2
`45.9 1 6.3
`29.8 1 2.0
`
`Brain
`concentration
`(ng g")
`2.68 1 1.31
`4.38 1 0.58
`3.04 1 0.10
`180 1 55
`61.3 1 6.7
`141 1 27
`2.33 1 0.92
`102 1 6
`
`0.019 1 0
`
`CSF
`concentration
`(ng ml")
`0.33 1 0.34
`0.74 1 0.07
`0.17 1 0.04
`10.9 1 1.7
`1.07 1 0.17
`0.71 1 0.26
`0.38 1 0.25
`27.9 1 NA
`
`0.72 1 0.02
`0.72 1 0.03
`0.074 1 0.006
`0.13 1 0.02
`0.32 1 0.02
`0.019 1 0.001
`0.96 1 0.06
`0.83 1 0.04
`
`0.19 1 0.02
`0.16 1 0.01
`
`0.012 1 0.001
`0.025 1 0.005
`0.01 1 0
`0.40 1 0.06
`0.46 1 0.02
`
`Values in the table are mean 1 SD for three separate determinations. fm, unbound fraction in brain; fup, unbound fraction in plasma.
`
`Table 3
`In vivo CNS penetration of various antimuscarinic agents in rats following subcutaneous administration
`
`:
`
`Brain : plasma concentration
`ratio
`
`CSF : free plasma
`
`concentration ratio
`
`Compound
`
`‘lrospiutn
`5-HMT
`Darifenadn
`solifenacin
`Tolterodine
`Oxybutynin
`N-Inethylscopolamine
`Scopolamine
`
`Definition of terms: fm, unbound fraction in brain; fup, unbound fraction in plasma; Kp_free_ ratio of unbound concentrations in brain : plasma.
`
`comparator compounds that display significant (positive
`control) or marginal (negative control) CNS effects, respec-
`tively, at similar doses in mice [29] and in humans [30].
`Scopolamine, oxybutynin and solifenacin displayed B:P
`and CSF : free plasma concentration ratios greater than 1,
`consistent with significant CNS penetration and equilib-
`rium between CSF and free p|asma.Tolterodine had a B : P
`>1 (2.95) and CSF :free plasma of 0.1 6, consistent with sig-
`nificant CNS penetration but with CSF not in equilibrium
`with free plasma under these experimental conditions.
`5-HMT, darifenacin and trospium showed results that were
`similar to N-methy|scop|amine,with B : P in the range 0.03-
`0.16 and CSF:free plasma of 0.004—0.06, consistent with
`no significant CNS penetration.
`
`In vitro plasma and brain binding data and
`estimation of Kp, free
`The binding of compounds in rat brain tissue homogenate
`and plasma were determined in vitro and data expressed
`as unbound fraction in brain (f...,) and plasma (f.,,,).The ratio
`of f.,,, : f.,., was calculated in order to estimate the distribu-
`tion of compounds between brain and plasma assuming
`CNS penetration is due to passive diffusion [31]. 5—HMT,
`darifenacin, oxybutynin and trospium displayed slightly
`
`240 / 72:2 / Br]C|in Phannacol
`
`higher unbound fractions in plasma than in brain (f.,,,:f.,.,
`ratios
`1.9-4.5), whereas
`tolterodine and solifenacin
`
`showed even higher f.,,,:f,,;, ratios of 13 and 11, respec-
`tively. Scopolamine and N-methylscopolamine were found
`to have similar f.,,,: fur, ratios of 1.8 and 2.4, respectively.
`Assuming CNS distribution is due to passive permeability,
`the in vitro binding results would predict that all the anti-
`muscarinic agents have significant CNS penetration (f.,,, : f...,
`>1).
`
`The in vitro brain binding data and observed B : P ratio
`were used to calculate the unbound brain : unbound
`
`plasma ratio (Kp,free). Kp,free ratios were compared with
`the observed CSF :free plasma ratios to assess whether
`free brain and CSF concentrations were in equilibrium with
`free peripheral concentrations in the plasma (Table 3).
`5-HMT, trospium and darifenacin could be categorized as
`having low Kp,free with values ranging from 0.01 to 0.04
`and low CSF :free plasma with values between 0.004 and
`0.06. The low ratios were similar to Kp,free and CSF :free
`plasma of N-methylscopolamine, 0.02 and 0.01, respec-
`tively. Oxybutynin and scopolamine afforded a high
`Kp,free and CSF :free plasma approaching unity. To|terod-
`ine provided intermediate Kp,free and CSF :free plasma
`(0.23 and 0.16) while solifenacin had a similar Kp,free (0.28)
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2005 - 0006
`
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`Patent Owner, UCB Pharma GmbH – Exhibit 2005 - 0006
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`

`
`CNS penetration potential of OAB agents
`
`
`
`Tissueconcentration(ngml"orngg")
`
`f,,
`
`f“'
`
`
`
`Trospium 5-HMT
`
`0.72
`
`0. I 9
`
`0.12
`
`0. I 6
`
`Darlfen-
`acln
`0.074
`
`SoIifen-
`acln
`0. I 3
`
`ToItero- Oxybut- N-MS
`dine
`ynln
`0.32
`0.0 I 9
`
`0.96
`
`ScopoIa-
`mine
`0.83
`
`0.0 I 9
`
`0.0 I 2
`
`0.025
`
`0.0 I
`
`0.40
`
`0.46
`
`Figure 3
`Tissue concentrations of antimuscarinic agents following subcutaneous administration in rats. Mean concentrations of compounds in plasma, brain and CSF
`determined following subcutaneous closing to three animals are shown,where error bars represent SDs. Unbound fractions in plasma (f.,,,) and brain (f...) are
`shown underneath each compound. plasma (ng ml") E); brain (ng g“) (-);CSF (ng ml") (2)
`
`but higher CSF:free plasma of approximately 1.41. The
`results are consistent with low CNS penetration of S-HMT.
`darifenacin, N-methylscopolamine and trospium, and
`significant CNS penetration of oxybutynin, scopolamine.
`solifenacin and tolterodine. However, Kp,free values
`for solifenacin and tolterodine were significantly lower
`than unity, suggestive of some possible restriction in CNS
`penetration.
`
`Discussion
`
`The main objective of the present study was to detennine
`the CNS penetration of antimuscarinic agents used for the
`treatment of OAB. CNS penetration of drugs may be limited
`by the passive penneability properties and efflux trans-
`porters, such as P—gp, in the endothelial cells of the cere-
`brovascular capillaries that comprise the BBB. Hence, a
`secondary objective was to provide a mechanistic expla-
`nation of any apparent differences in the ability of OAB
`drugs to cross the BBB. The drugs were assessed in a rat
`model that determined their CNS penetration in vivo by
`measuring tissue concentration and binding of com-
`pounds.The in vivo results were then put into context with
`physical property assessment and results from permeabil-
`ity and P-gp substrate assays to provide a mechanistic
`interpretation of the CNS penetration results.
`
`CNS penetration of OAB agents in vivo
`In the present study, rats were administered subcutaneous
`doses of compounds and brain, plasma and CSF were
`
`sampled 1 h after dosing. As a result of theoretical pharma-
`cokinetic considerations combined with results of in vivo
`
`microdialysis experiments, it has been suggested that
`equilibration of drugs between brain and blood occurs
`very rapidly [32]. Other work that examined the time to
`reach brain equilibrium in rats dosed subcutaneously with
`a range of compounds found that compounds with a com-
`bination of low brain binding and high blood brain barrier
`permeability reached equilibrium (B: P at plateau) within
`10 min to 2 h [33]. However, tissue sampling at a single
`time point remains a potential limitation in experimental
`design because it is possible that compounds may not
`have attained steady-state distribution between brain and
`plasma within that time.The most rigorous protocol would
`employ tissue sampling over a time course,a||owing tissue
`concentration ratios to be detennined at a time point
`shown to represent steady state, and potentially over a
`range of doses. However, such a protocol is extremely time
`consuming and considerably increases the number of
`animals used to generate the data. The single dose and
`time point design employed in our study aimed to mini-
`mize animal use and provide plasma and tissue concentra-
`tions high enough to pennit their accurate measurement
`by specific LC/MS-MS assay, while avoiding saturation of
`transporter proteins that could affect tissue distribution.
`The ratio of total concentration in brain and plasma
`(B: P) is most commonly used to assess CNS penetration.
`However,
`if CNS penetration is solely detennined by
`passive penneability, steady-state B : P is dependent on the
`relative non—specific binding to plasma proteins (f..,,) and
`brain tissue (f.,.,) suggesting that the binding ratio can be a
`
`Br] Clin Pharmacol
`
`/ 72:2 / 241
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`Patent Owner, UCB Pharma GmbH — Exhibit 2005 - 0007
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`Patent Owner, UCB Pharma GmbH – Exhibit 2005 - 0007
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`

`
`E. Callegari et al.
`
`good surrogate for in vivo steady state B : P [20, 31]. The
`f..,, : f..;, values obtained for the OAB agents were found to
`be in the range of 1.9 to 13.Therefore, if these compounds
`have high passive permeability and are not substrates for
`efflux transporters, these results taken alone would predict
`that all OAB agents will have significant brain penetration,
`as suggested by their free fractions in plasma relative to
`brain. However, based on tissue concentration data in rats,
`
`the OAB agents may be broadly categorized into two
`groups: one consisting of trospium, 5—HMT and darifenacin
`with no significant CNS penetration and another consist-
`ing of oxybutynin, solifenacin and tolterodine with signifi-
`cant CNS penetration. Within each group, the positive
`and the negative
`controls,
`scopolamine
`and N-
`methylscopolamine helped confirm the categorization
`as CNS—penetrant and non—penetrant, respectively. The
`observed B : P ratios for solifenacin,to|terodine and oxybu-
`tynin were >1 and similar to scopolamine, consistent with
`significant CNS penetration. For these compounds the in
`vitro f.,,, : f.., was consistent with the in vivo results since
`predictions were within ~fourfold of observed. Tissue
`binding results were also used to calculate unbound tissue
`concentrations and enable comparison of free concentra-
`tions in plasma, brain and CSF, to indicate whether equilib-
`rium between the compartments was likely to exist. As the
`concentration of protein in CSF is very low. it was assumed
`that free concentrations of drug are measured in CSF. The
`Kp,free and CSF :free plasma ratios for oxybutynin and
`scopolamine afforded ratios of approximately 1, indicating
`that equilibrium between free concentrations in the brain,
`CSF and plasma had been achieved. However, for solifena-
`cin the CSF :free plasma was approximately 1, consistent
`with equilibrium between the CSF and free plasma com-
`partment, but Kp,free was 0.28 indicating that free brain
`and free plasma concentrations were not at equilibrium.
`Tolterodine also demonstrated significant CNS penetra-
`tion, yet Kp,free (0.23) and CSF :free plasma (0.16) did not
`reach unity indicating that equilibrium between CNS
`compartments had not been achieved. These results with
`solifenacin and tolterodine indicate some limitation in CNS
`
`penetration or may reflect slow attainment of equilibrium
`across the BBB relative to the other OAB agents. However, it
`cannot be ruled out that they act as substrates for a trans-
`porter protein other than P-gp that limits free brain and
`CSF concentrations. It is also possible that differences in
`the physiology of the CSF/plasma barrier and BBB play a
`role in the difference in Kp,free and CSF :free plasma ratio
`for solifenacin. CSF is produced at the choroid plexus, a
`fenestrated endothelial cell layer lacking the tight junc-
`tions of the BBB. Additionally, there have been reports of
`different expression and localization of transporter pro-
`teins in the choroid plexus relative to the BBB.These differ-
`ences in permeability and transporter expression may lead
`to different brain tissue and CSF free concentrations and
`
`different rates in reaching equilibrium across the OAB
`class [34].
`
`242 I 72:2 / Br] Clin Pharmacol
`
`The B: P, Kp,free and CSF :free plasma ratios for tro-
`spium, 5—HMT and darifenacin were similar to those of
`N—methy|scopo|amine, which is consistent with no signifi-
`cant CNS penetration. For these compounds, the P : B ratios
`of unbound fractions (f..,,: f...,) significantly over—predicted
`thei