British Journal of Pharmacology (2001) 133, 875 – 885
`
`ª 2001 Nature Publishing Group All rights reserved 0007 – 1188/01 $15.00
`www.nature.com/bjp
`
`Sirolimus, but not the structurally related RAD (everolimus),
`enhances the negative e€ects of cyclosporine on mitochondrial
`metabolism in the rat brain
`
`1,2Natalie Serkova, 1Wolfgang Jacobsen, 3Claus U. Niemann, 3Lawrence Litt, 1Leslie Z. Benet,
`2Dieter Leibfritz & *,1,4Uwe Christians
`
`1Department of Biopharmaceutical Sciences, University of California, San Francisco, California, CA 94143, U.S.A.; 2Institut fu¨ r
`Biologie/Chemie, Universita¨ t Bremen, 28359 Bremen, Germany; 3Department of Anesthesia and Perioperative Care, University
`of California, San Francisco, California, CA 94143, U.S.A and 4Department of Anesthesiology, University of Colorado Health
`Sciences Center, Denver, Colorado, CO 80262, U.S.A.
`
`1 Clinical studies have shown enhancement of cyclosporine toxicity when co-administered with the
`immunosuppressant sirolimus. We evaluated the biochemical mechanisms underlying the sirolimus/
`cyclosporine interaction on rat brain metabolism using magnetic resonance spectroscopy (MRS) and
`compared the e€ects of sirolimus with those of the structurally related RAD.
`2 Two-week-old rats (25 g) were allocated to the following treatment groups (all n=6): I. control,
`II. cyclosporine (10 mg kg71 d71), III. sirolimus (3 mg kg71 d71), IV. RAD (3 mg kg71 d71),
`V. cyclosporine+sirolimus and VI. cyclosporine+RAD. Drugs were administered by oral gavage
`for 6 days. Twelve hours after the last dose, metabolic changes were assessed in brain tissue extracts
`using multinuclear MRS.
`3 Cyclosporine significantly inhibited mitochondrial glucose metabolism (glutamate: 78+6% of
`control; GABA: 67+12%; NAD+: 76+3%; P50.05), but increased lactate production. Sirolimus
`and RAD inhibited cytosolic glucose metabolism via lactate production (sirolimus: 81+3% of
`control, RAD: 69+2%; P50.02). Sirolimus
`enhanced cyclosporine-induced inhibition of
`mitochondrial glucose metabolism (glutamate: 60+4%; GABA: 59+8%; NAD+: 45+5%;
`P50.02 versus cyclosporine alone). Lactate production was significantly reduced. In contrast,
`RAD antagonized the e€ects of cyclosporine (glutamate, GABA, and NAD+, not significantly
`di€erent from controls).
`4 The results can partially be explained by pharmacokinetic interactions: co-administration
`increased the distribution of cyclosporine and sirolimus into brain tissue, while co-administration
`with RAD decreased cyclosporine brain tissue concentrations. In addition RAD, but not sirolimus,
`distributed into brain mitochondria.
`5 The combination of cyclosporine/RAD compares favourably to cyclosporine/sirolimus in regards
`to their e€ects on brain high-energy metabolism and tissue distribution in the rat.
`British Journal of Pharmacology (2001) 133, 875 – 885
`tissue
`Keywords: Cyclosporine
`toxicity;
`sirolimus;
`everolimus; SDZ-RAD; pharmacodynamic drug interactions;
`distribution; brain metabolism; mitochondrial glucose metabolism; oxidative phosphorylation; magnetic
`resonance spectroscopy (MRS)
`Abbreviations: AMU, atomic mass units; CNS, central nervous system; GABA, g-amino butyric acid; MRS, magnetic
`resonance spectroscopy; MS, mass spectrometry; NAA, N-acetyl aspartate; NAD+, nicotinamide adenine
`dinucleotide; NDP, nucleoside diphosphate; NMP, nucleoside monophosphate; NTP, nucleoside triphosphate;
`OAA, oxaloacetate; TSP, trimethylsilyl propionic-2,2,3,3,-d4 acid
`
`Introduction
`
`The calcineurin inhibitor cyclosporine (INN: ciclosporin,
`Figure
`1)
`is
`used
`as
`an
`immunosuppressant
`after
`transplantation and for the treatment of immune diseases
`(Kahan, 1989; Faulds et al., 1993). The clinical use of
`cyclosporine is limited by its toxicity in combination with
`its narrow therapeutic index (Kahan, 1989). Neurotoxicity
`is one of
`the most
`serious
`side e€ects and incidences
`
`*Author for correspondence at: Department of Anesthesiology,
`University of Colorado Health Sciences Center, 4200 East Ninth
`Avenue, Room 2122, Campus Box B113, Denver, Colorado 80262,
`U.S.A. E-mail: uwe.christians@uchsc.edu
`
`between 25 and 55% have been reported in transplant
`patients (Hauben, 1996; Gijtenbeek et al., 1999). Since first
`reported by Calne
`al.
`(1979),
`a wide
`range of
`et
`cyclosporine-associated neurological symptoms have been
`described, ranging from mild symptoms such as tremor,
`agitation,
`insomnia,
`anxiety,
`amnesia, headache,
`and
`paraesthesia to severe neurotoxicity resulting in seizures,
`cortical blindness, aphasia, ataxia, and stroke-like episodes
`(Hauben, 1996; Gijtenbeek et al., 1999). The incidence of
`cyclosporine neurotoxicity is increased in patients with high
`cyclosporine blood levels, but neurotoxicity also occurs
`during long-term treatment with cyclosporine blood con-
`
`NOVARTIS EXHIBIT 2082
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`

`876
`
`N. Serkova et al
`
`Sirolimus, not RAD, enhances cyclosporine toxicity
`
`Cyclosporine and sirolimus/RAD have di€erent toxicity
`spectra, with the only exception being hyperlipidemia
`(Brattstro¨ m et al., 1998). Although sirolimus by itself is not
`nephrotoxic, animal (Andoh et al., 1996) and clinical studies
`(Kahan & The Rapamune US Study Group, 2000) have
`shown that sirolimus enhances cyclosporine nephrotoxicity.
`We showed in isolated, perfused rat brain slices that sirolimus
`and cyclosporine synergistically reduce high-energy phos-
`phate concentrations (Serkova et al., 1999). In the same in
`vitro model, RAD showed the opposite e€ect and antag-
`onized the cyclosporine-mediated inhibition of mitochondrial
`oxidative phosphorylation (Serkova et al., 2000a). However,
`these in vitro studies exclusively focused on high-energy
`phosphate metabolism. It
`is still unclear how sirolimus
`enhances the inhibition of oxidative phosphorylation by
`cyclosporine and why the structurally related RAD and
`sirolimus have opposite e€ects on high-energy metabolism in
`the presence of cyclosporine. In vitro (Serkova et al., 1999;
`2000a) potential pharmacokinetic and distribution interac-
`tions are ignored (Serkova et al., 2000b). In addition, in these
`in vitro studies brain tissue was exposed to the study drugs
`for a maximum period of 2 h, a time period most likely too
`short to detect potential e€ects of the study drugs on protein
`expression.
`Here, we treated rats with cyclosporine, sirolimus, and
`RAD as well as sirolimus or RAD in combination with
`cyclosporine for 6 days. The e€ects of the study drugs on
`brain metabolism were evaluated using multinuclear magnetic
`resonance spectroscopy (MRS). To assess pharmacokinetic
`interactions, concentrations of the study drugs in blood,
`brain tissue and brain mitochondria were quantified using
`h.p.l.c./ mass spectrometry. It was our goal to evaluate and
`compare the e€ects of sirolimus and RAD on brain cell
`metabolism in the presence of cyclosporine.
`
`Methods
`
`Materials
`
`Cyclosporine and RAD were kindly provided by Novartis
`Pharma AG (Basel, Switzerland). Sirolimus was purchased
`from Sigma (St. Louis, MO, U.S.A.). Stock solutions
`(10 g l71) of each drug were prepared in absolute ethanol
`(Aldrich Chemicals, Milwaukee, WI, U.S.A.). Perchloric acid
`(PCA, 60%) as well as potassium hydroxide (KOH) for the
`extraction of brain tissue homogenates were from Aldrich
`Chemicals (Milwaukee, WI, U.S.A.). Deuterated chemicals
`(D2O, NaOD, and DCl) for MRS were purchased from
`Cambridge
`Isotope Laboratories,
`Inc.
`(Andover, MA,
`U.S.A.) and NMR tubes of 5 mm diameter from Wilmad
`(Buena, NJ, U.S.A.). Cell metabolites in brain extracts were
`measured using a 500 MHz Bruker NMR spectrometer
`(Bruker Instruments Inc., Fremont, CA, U.S.A.). The system
`was controlled and data were processed using WINNMR
`software (Bruker Instruments Inc. Fremont, CA, U.S.A.).
`Methanol and zinc sulphate were purchased from Fisher
`Scientific (Fair Lawn, NJ, U.S.A.). Percoll and other
`chemicals for mitochondria isolation were from Aldrich
`Chemicals (Milwaukee, WI, U.S.A.). Concentrations of the
`study drug were measured using an h.p.l.c./h.p.l.c.-mass
`spectrometry (MS)
`system consisting of
`the
`following
`
`Figure 1 Structures of cyclosporine (A), sirolimus (B) and RAD
`(C). Numbering of the macrolide immunosuppressants sirolimus and
`RAD follows the IUPAC guidelines (IUPAC, 1993). (C) shows the
`region of the RAD molecule di€erent from sirolimus.
`
`centrations within the therapeutic target range (Hauben,
`1996; Gijtenbeek et al., 1999).
`In in vitro studies, we showed that cyclosporine primarily
`inhibits mitochondrial
`energy production in the brain
`(Serkova et al., 1996; 1999). Reduction of high-energy
`phosphate concentrations by cyclosporine was also found in
`kidney (Henke et al., 1992; Massicot et al., 1997),
`liver
`(Salducci et al., 1996), and intestine (Madsen et al., 1995;
`Gabe et al., 1998).
`sirolimus
`Recently,
`the macrolide immunosuppressants
`(rapamycin, Figure 1) and its 40-O-(2-hydroxyethyl) deriva-
`tive RAD (INN: everolimus, Figure 1) have emerged as
`promising combination partners for cyclosporine. In the
`lymphocyte, sirolimus and RAD bind to FK-binding proteins
`and the immunophilin/sirolimus complex and probably also
`the
`immunophilin/RAD complex bind to mTOR,
`the
`mammalian target of rapamycin (Gummert et al., 1999).
`Inhibition of mTOR arrests the interleukin-2 driven T-cell
`proliferation at the G1- S-interface (Sehgal, 1998). Since the
`calcineurin inhibitor cyclosporine and sirolimus/RAD inhibit
`subsequent steps in the T-cell-mediated immune response,
`combination of cyclosporine and sirolimus or RAD results in
`synergistic immunosuppressive activity (Kahan, 1997; Step-
`kowski et al., 1996; Schuurman et al., 1997; Hausen et al.,
`2000).
`
`British Journal of Pharmacology vol 133 (6)
`
`NOVARTIS EXHIBIT 2082
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`

`N. Serkova et al
`
`Sirolimus, not RAD, enhances cyclosporine toxicity
`
`877
`
`components (all series 1100 h.p.l.c. components, Hewlett-
`Packard, Palo Alto, CA, U.S.A.): h.p.l.c.
`I: G1311A
`quaternary pump, G1322A degasser and G1329A autosam-
`pler equipped with a G1330A thermostat; h.p.l.c. II: G1312A
`binary pump, G1322A degasser, G1316A column thermostat
`and G1946A mass selective detector. The two h.p.l.c. systems
`were connected via a 7240 Rheodyne 6-port switching valve
`mounted on a step motor (Rheodyne, Cotati, CA, U.S.A.).
`The system was controlled and data were processed using
`ChemStation Software Revision A.06.01 (Hewlett-Packard,
`Palo Alto, CA, U.S.A.). A 10·2 mm extraction column
`(Keystone Scientific, Bellefonte, PA, U.S.A.) filled with
`Hypersil ODS-1 of 10 mm particle size (Shandon, Chadwick,
`U.K.) and a 50·4.6 mm analytical column filled with C8
`material of 3.5 mm particle size (Zorbax XDB C8, Hewlett-
`Packard, Palo Alto, CA, U.S.A.) were used. The internal
`standard cyclosporin D was a kind gift from Novartis (Basel,
`Switzerland) and 28,40-O-diacetyl rapamycin was synthesized
`and purified as described by Streit et al. (1996).
`
`Animal studies
`
`All animal protocols were reviewed and approved by the
`University of California, San Francisco, Committee on
`Animal Research.
`The animals received humane care in compliance with the
`‘Principles of Laboratory Animal Care’ formulated by the
`National Society for Medical Research and the ‘Guide for the
`Care and Use of Laboratory Animals’, published by the
`National Institutes of Health (NIH publication No. 80-123,
`revised 1985).
`Seventy-two Wistar rats (two weeks old, 28+4 g body
`weight) were purchased from Charles River, Inc. (Wilmington,
`MA, U.S.A.) and were allocated to six treatment groups: (I)
`vehicle (control, n=12); (II) 10 mg kg71 cyclosporine (group
`Cs, n=12); (III) 3 mg kg71 sirolimus (group SRL, n=12); (IV)
`(V) 10 mg kg71
`3 mg kg71 RAD (group RAD, n=12);
`cyclosporine+3 mg kg71 sirolimus (group Cs+SRL, n=12),
`(VI) 10 mg kg71
`cyclosporine+3 mg kg71 RAD (group
`Cs+RAD, n=12). Stock solutions of the study drugs were
`dissolved in whole milk (1 : 10, final concentration 1 g l71). The
`study drugs were administered by oral gavage once daily. The
`final ethanol concentration was 10%. Animals in the control
`group were treated with 13 ml 10% ethanol/milk vehicle kg71
`body weight. This corresponded to the highest alcohol doses
`administered during the
`study (groups Cs+SRL and
`Cs+RAD). The doses were chosen on basis of a previous dose
`finding study (results not shown) with the goal to find oral
`doses of the study drugs resulting in brain tissue concentrations
`in the range of those that have previously been shown to have
`significant e€ects on the cell metabolism of isolated brain slices
`(Serkova et al., 1999; 2000a).
`During our study, the animals were closely monitored,
`including daily evaluations of appearance, grooming, appe-
`tite, behaviour, activity and body weight. At the end of the 6-
`day study period and 12 h after the last study drug dose, the
`animals were decapitated and whole blood and brain tissues
`were collected. Blood samples were drawn into tubes with
`EDTA as anticoagulant. Tissues were immediately frozen in
`liquid nitrogen and stored at 7808C until PCA extraction
`(n=6 per group) or h.p.l.c./h.p.l.c.-MS analysis (n=6 per
`group). Samples were analysed within 20 days.
`
`Measurement of brain cell metabolites using MRS
`
`Frozen brain tissues were weighed; 2 g (wet weight) of
`each tissue sample was homogenized in a mortar grinder
`in the presence of liquid nitrogen and extracted with 6 ml
`ice-cold PCA (12%). The extracts were centrifuged,
`the
`aqueous phase was removed and neutralized using KOH.
`The samples were centrifuged once again and the aqueous
`phase was
`lyophilized overnight. The lyophilized brain
`PCA extracts were reconstituted in 400 ml deuterium oxide
`(D2O) and adjusted to pH 7 using DCl and NaOD. The
`experiments with 1H-, 13C-, and 31P-MRS were carried out
`as described previously (Serkova et al., 1996). Trimethylsi-
`lyl propionic-2,2,3,3,-d4 acid (TSP) was used as an external
`standard for
`quantification
`in
`1H-MRS
`spectra.
`1H
`chemical shifts were referenced to TSP at 0 p.p.m. The
`use
`of
`an
`internal
`standard
`for
`1H-MRS
`allowed
`quantification based on areas under
`the 1H-signals of
`single metabolites. The C3 peak of
`lactate at 21 p.p.m.
`was used as the chemical shift reference in 13C-MRS. For
`31P-MRS, either phosphocreatine (PCr) at 72.33 p.p.m. or,
`if present, a-ATP at 79.90 p.p.m. were used as
`shift
`references. High-energy phosphate metabolism in the brain
`is
`a
`closed
`system, which means
`that
`a
`reduced
`concentration of one metabolite automatically leads to an
`increase of one or more other metabolites. Therefore, we
`calculated
`the
`relative
`concentrations
`of
`high-energy
`phosphates as the ratio of the 31P-MRS peak area of a
`metabolite to the total area of all high-energy phosphate
`metabolites quantified.
`
`Isolation of brain mitochondria
`
`After treatment with the study drugs or their combinations
`for 6 days, the concentrations of cyclosporine, sirolimus,
`and RAD in rat brain mitochondria were determined
`using
`h.p.l.c./h.p.l.c.-MS
`analysis
`(vide
`infra). Brain
`mitochondria from rats treated with the vehicle or the
`study drugs were isolated by a modified procedure as
`described previously (Kristal & Dubinsky, 1997). Briefly:
`after decapitation, brains were rapidly dissected and placed
`in 10 ml
`ice-cold isolation bu€er
`containing 350 mM
`sucrose, 2 mM HEPES and 1 mM EGTA (pH 7.4). After
`homogenization using a manual homogenizer,
`the brain
`suspension was centrifuged at 900 6g to remove cellular
`debris
`and nuclei
`for
`10 min. The
`supernatant was
`decanted into a centrifuge tube and re-centrifuged under
`the same conditions. The supernatant was transferred into
`another centrifuge tube and was centrifuged at 10,000 6g
`for
`10 min resulting
`in an olive-green mitochondrial
`precipitate. The mitochondrial
`fraction was homogenized
`in 1 part Percoll/7 parts bu€er. The homogenate was
`layered over preformed discontinuous Percoll gradients
`consisting of a bottom layer of 40% Percoll
`in bu€er
`and a top layer of 26% Percoll
`in bu€er. The gradients
`were centrifuged at 31,0006g for 45 min. The mitochon-
`drial fraction, which was concentrated at the interface of
`the two layers, was collected, re-constituted in bu€er, and
`centrifuged again. The supernatant was removed, and the
`pellet was reconstituted in 0.5 ml of 1 M KH2PO4 bu€er
`pH 7.4. Mitochondria were extracted and analysed using
`h.p.l.c./h.p.l.c.-MS as described below.
`
`British Journal of Pharmacology vol 133 (6)
`
`NOVARTIS EXHIBIT 2082
`Par v. Novartis, IPR 2016-01479
`Page 3 of 11
`
`

`

`878
`
`N. Serkova et al
`
`Sirolimus, not RAD, enhances cyclosporine toxicity
`
`Measurement of tissue distribution of
`immunosuppressants by h.p.l.c./h.p.l.c.-MS
`
`We used an h.p.l.c.-MS assay with automated online sample
`extraction (h.p.l.c./h.p.l.c.-MS) for quantification of the study
`drugs in blood,
`tissue and mitochondria, which was a
`modification of the assay we described previously (Christians
`et al., 2000). Brain tissues (1.5 g wet weight) were thawed and
`weighed. Brain samples were homogenized with 2 ml
`KH2PO4 bu€er pH 7.4 (1 M) using an electric homogenizer.
`For protein precipitation, 200 ml methanol/1 M ZnSO4 (80/
`20 v v71) was added to each 100 ml sample (homogenized
`brain, blood or isolated mitochondria). Cyclosporin D and
`28,40-O-diacetyl rapamycin were added as internal standards
`for cyclosporine and sirolimus/RAD, respectively, resulting in
`concentrations of 100 mg l71. After
`final
`centrifugation,
`100 ml of the supernatant was injected onto the extraction
`column. Samples were washed with a mobile phase of 40%
`methanol and 60% 0.1% formic acid supplemented with
`1 mmol l71 sodium formate. The flow was 5 ml min71 and the
`temperature for the extraction column was set to 658C. After
`0.75 min, the switching valve was activated and the analytes
`were eluted in the backflush mode from the extraction
`column onto analytical column (flow 0.5 ml min71). The
`mobile phase consisted of 90% methanol and 10% 0.1%
`formic acid supplemented with 1 mmol l71 sodium formate.
`The mass spectrometer was run in the selected ion mode and
`positive ions [M+Na]+ were recorded. For all matrices, the
`analytical recovery was 490%. In blood, brain and isolated
`mitochondria, the assay was linear from 1 mg l71 (lower limit
`to 500 mg l71
`of quantification)
`for
`cyclosporine and
`0.25 mg l71 to 100 mg l71 for the macrolides (r240.99).
`
`Data analysis
`
`All results are reported as means+standard deviation (s.d.).
`The results of the control and immunosuppressant-treated
`groups were compared using unpaired Student’s t-test or
`analysis of variance in combination with Duncan grouping as
`post hoc test (SPSS, version 9.0, SPSS Inc., Chicago, IL,
`U.S.A.).
`
`Results
`
`Clinical monitoring
`
`Figure 2 E€ect of the study drugs and their combinations on weight
`gain during the observation period. Data (mean+standard deviation,
`n=12) shows the per cent change at the beginning of each study day
`in comparison with the weight at day 1 prior to dosing (=100%).
`The study drugs were administered by oral gavage for 6 days. The
`rats (2-weeks old, mean weight 28+4 g) received the following doses:
`control, 13 ml vehicle (10% ethanol in milk); Cs, 10 mg kg71 d71
`cyclosporine; SRL, 3 mg kg71 d71 sirolimus; RAD, 3 mg kg71 d71
`10 mg kg71 d71
`cyclosporine+3 mg kg71 d71
`RAD; Cs+SRL,
`sirolimus; Cs+RAD, 10 mg kg71 d71 cyclosporine+3 mg kg71 d71
`RAD.
`(A) Comparison of groups Cs, SRL and Cs+SRL;
`(B)
`comparison of groups Cs, RAD and Cs+RAD. The results of the
`control group and Cs are shown in both (A) and (B) to facilitate
`comparison.
`
`On study day 6, the average weight (+s.d.) of vehicle treated
`animals in the control group was 166+12% of the initial
`body weight
`(Figure 2). In contrast, cyclosporine-treated
`animals (group Cs) showed no weight gain (91+10% of the
`initial body weight). Animals treated with sirolimus or RAD
`(groups SRL and RAD) were in better condition than the
`cyclosporine-treated animals and gained weight, however,
`than the control group (122+8 and 122+9%,
`slower
`respectively). Co-administration of cyclosporine and sirolimus
`had the most negative e€ect on the parameters recorded
`including body weight (85+8% of the initial body weight). In
`comparison to cyclosporine alone, animals treated with a
`combination of cyclosporine and RAD lost slightly but
`significantly less weight than those treated with cyclosporine
`alone (97+13%). On day 6, body weights di€ered signifi-
`
`cantly (P50.001) among groups as evaluated by analysis of
`variance in combination with Duncan grouping: control
`4RAD=SRL4Cs+RAD4Cs4Cs+SRL.
`
`Effect of the study drugs on brain cell metabolism
`
`1H-MRS In control rats,
`the following cell metabolites
`could be quantified: the neuronal marker N-acetyl aspartate
`(NAA), the excitatory amino acid neurotransmitters gluta-
`mate and aspartate, the inhibitory neurotransmitter g-amino
`butyric acid (GABA), the cellular osmolytes taurine and
`myo-inositol as well as lactate (Table 1). In comparison to
`the controls, cyclosporine (group Cs) decreased the brain
`tissue concentrations of NAA (59% of control, P50.002,
`Table 1) and those of the neurotransmitters glutamate (78%;
`
`British Journal of Pharmacology vol 133 (6)
`
`NOVARTIS EXHIBIT 2082
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`

`N. Serkova et al
`
`Sirolimus, not RAD, enhances cyclosporine toxicity
`
`879
`
`Table 1 The e€ects of the study drugs and their combinations on concentrations of neurotransmitters and cell metabolites in rat brain
`extracts analysed by 1H-mrs
`
`Control
`(I) n=7
`
`28.2+4.1
`6.8+2.0
`6.7+1.1
`15.0+2.6
`8.3+1.0
`7.5+0.8
`16.9+4.2
`10.9+0.8
`
`Cs
`(II) n=5
`
`16.5+1.8***
`4.9+1.8
`4.5+0.9*
`11.7+1.5**
`7.4+1.9
`10.4+1.0***
`16.8+4.4
`8.6+2.2*
`
`SRL
`(III) n=5
`
`21.6+2.8*
`5.7+1.3
`5.7+1.6
`13.2+2.5
`10.8+2.1*
`6.1+0.5**
`14.9+1.1
`7.4+1.1**
`
`RAD
`(IV) n=5
`
`20.6+5.1*
`5.5+1.5
`5.8+1.6
`13.4+0.9
`11.1+1.3**
`5.2+0.4***
`15.3+7.4
`7.3+0.9**
`
`Cs+SRL
`(V) n=5
`
`14.2+2.0***
`3.6+1.4*
`4.0+0.6**
`9.1+1.3***
`9.7+1.6
`4.0+0.8***
`15.1+4.2
`4.2+0.7***
`
`Cs+RAD
`(VI) n=5
`
`22.2+3.8
`5.1+0.6
`5.1+1.6
`13.7+0.6
`7.4+2.0
`6.5+0.3*
`15.4+3.0
`7.8+1.0*
`
`NAA
`Aspartate
`GABA
`Glutamate
`Glutamine
`Lactate
`Taurine
`Myo-Inositol
`
`All concentrations (mmol g71 wet weight) are means+s.d. Data was analysed using analysis of variance in combination with Duncan
`grouping: *(P50.05), **(P50.02) and ***(P50.002) in comparison to the control group. Abbreviations: Cs, cyclosporine; GABA, g-
`aminobutyric acid; NAA, N-acetyl aspartate; SRL, sirolimus.
`
`P50.02), and GABA (67%, P50.05). Lactate concentrations
`increased to 140% (P50.002). The e€ects of sirolimus (group
`SRL) and RAD (group RAD) were similar. In contrast to
`cyclosporine, the macrolide immunosuppressants decreased
`lactate concentrations (sirolimus: 81%, P50.02; RAD: 69%,
`P50.002). Sirolimus and RAD also slightly decreased the
`brain concentration of NAA and increased the concentra-
`tions of glutamine (Table 1).
`Co-administration of cyclosporine and sirolimus (group
`Cs+SRL) had the most
`significant e€ect. Among the
`treatment groups, brain tissue extracts from animals in the
`Cs+SRL group contained the lowest concentrations of
`NAA (50% of control, P50.002), of glutamate (60%,
`P50.002), of GABA (59%, P50.02), and of lactate (53%,
`P50.002). In comparison to the controls, co-administration
`of cyclosporine and RAD, however, did not significantly
`a€ect most of the parameters measured with the exception
`of a slight decrease in lactate and myo-inositol concentra-
`tions (Table 1).
`
`13C-MRS Although the natural abundance of 13C-isotopes is
`only 1.1%,
`intensities of
`13C-MRS signals in our brain
`for reliable quantification of 13C-
`extracts were su(cid:129)cient
`groups of glucose and the following intermediates of the
`glucose pathways: aspartate,
`lactate, GABA, glutamate,
`glutamine, and oxaloacetate. In addition, the 13C-signals of
`creatine, myo-inositol, NAA and taurine were detected.
`Representative 13C-MRS spectra of rat brain PCA extracts
`from rats of the control, Cs, SRL and RAD groups are
`shown in Figure 3.
`In comparison to the controls, brain extracts of cyclospor-
`ine treated animals showed reduced signal intensities of Krebs
`cycle-dependent metabolites such as glutamate, glutamine,
`GABA, and NAA (Figure 3). This reduction was accom-
`panied by glucose (at 92.9 and 96.8 p.p.m.) and oxaloacetate
`(at 50 p.p.m.) signals above the detection limit, which were
`not present in the spectra of the controls. Additionally, the
`13C-signal of
`lactate, an important anaerobic glucose
`metabolite, was slightly increased. Glucose and oxaloacetate
`signals were undetectable in the 31C-spectra of brain extracts
`from rats in the SRL and RAD groups. Lactate signals in
`these spectra were consistently smaller than in spectra of the
`control group. Characteristic changes after treatment with
`sirolimus or RAD were the significantly increased intensities
`of glutamine signals in comparison to the controls. Due to
`
`the lack of an internal standard, absolute concentrations of
`metabolites based on the 13C-MRS spectra could not be
`determined. We calculated the ratios of 13C-signal intensities
`of Krebs cycle metabolites (aerobic glucose metabolism)/
`glycolysis intermediates (anaerobic glucose metabolism):
`Krebs cycle/glycolysis (aerobic/anaerobic glucose metabo-
`lism) ratio=
`glutamate (cid:135) glutamine (cid:135) GABA (cid:135) aspartate (cid:135) NAA (cid:135) oxaloacetate
`lactate
`
`P
`
`P
`
`The ratios were 9.5+1.1 for the control group (n=7,
`mean+s.d.), 5.6+0.9 for the Cs group (n=5), 14.1+1.6 for
`the SRL group (n=5) and 14.7+2.9 for the RAD group
`(n=5).
`Co-administration of cyclosporine and sirolimus resulted in
`a reduction of all intermediates of glucose metabolism, from
`both Krebs cycle as well as from anaerobic glycolysis (Figure
`4B) resulting in an aerobic/anaerobic glucose metabolite ratio
`of 13.0+1.3. In comparison, glucose metabolism was less
`a€ected when cyclosporine and RAD were co-administered
`(ratio: 9.8+1.4, Figure 4C). The ratios were significantly
`di€erent with P50.002 (Duncan grouping, SRL=RAD=
`Cs+SRL4control=Cs+RAD).
`
`31P-MRS The results of the 31P-MRS analyses are summar-
`ized in Table 2. Treatment with cyclosporine reduced the
`relative concentrations of nucleoside triphosphates (NTP) to
`34%,
`those of phosphocreatine to 30% and those of
`nicotinamide adenine dinucleotide (NAD+) to 76% of the
`controls. In parallel, relative nucleoside diphosphate (NDP)
`concentrations were 2.4 fold and nucleoside monophosphate
`(NMP) concentrations 20.8 fold higher than in the control
`group. Sirolimus and RAD inhibited high-energy phosphate
`metabolism to a significantly smaller extent than cyclosporine
`(Table 2). Compared to the controls, sirolimus decreased
`NTP to 61% of the controls and increased relative NDP
`concentrations to 161% and NMP concentrations 5 fold.
`RAD decreased NTP concentration to 77% of controls,
`increased NDP concentrations
`to 171% but had no
`statistically significant e€ect on NMP concentrations. Both
`sirolimus and RAD did not significantly a€ect phosphocrea-
`tine or NAD+ concentrations.
`Again, co-administration of sirolimus and cyclosporine
`resulted in the most significant changes. At the end of the
`
`British Journal of Pharmacology vol 133 (6)
`
`NOVARTIS EXHIBIT 2082
`Par v. Novartis, IPR 2016-01479
`Page 5 of 11
`
`

`

`880
`
`N. Serkova et al
`
`Sirolimus, not RAD, enhances cyclosporine toxicity
`
`Figure 3 Representative 13C-MRS spectra of brain PCA extracts from rats in the control (A) and mono-treatment groups Cs (B),
`SRL (C), and RAD (D). Rats were treated with the following doses by oral gavage for 6 days: (A) control, 13 ml vehicle (10%
`ethanol in milk, total n=7), (B) Cs, 10 mg kg71 d71 cyclosporine (n=5), (C) SRL, 3 mg kg71 d71 sirolimus (n=5) and (D) RAD,
`3 mg kg71 d71 RAD (n=5). Abbreviations: Asp, aspartate; Cr, creatine; Cs, cyclosporine; GABA, g-aminobutyric acid; Glc,
`glucose; Gln, glutamine; Glu, glutamate; Ins, myo-inositol; Lac,
`lactate; NAA, N-acetyl aspartate; OAA, oxaloacetate; SRL,
`sirolimus; Tau, taurine.
`
`British Journal of Pharmacology vol 133 (6)
`
`NOVARTIS EXHIBIT 2082
`Par v. Novartis, IPR 2016-01479
`Page 6 of 11
`
`

`

`N. Serkova et al
`
`Sirolimus, not RAD, enhances cyclosporine toxicity
`
`881
`
`Figure 4 Representative 13C-MRS spectra of brain PCA extracts from rats in the control group (A) and after co-administration of
`cyclosporine and sirolimus (group Cs+SRL, B) and cyclosporine and RAD (group Cs+RAD, C). Rats were treated with the
`following doses by oral gavage for 6 days: (A) control, 13 ml vehicle (10% ethanol
`in milk, total n=7), (B) Cs+SRL,
`10 mg kg71 d71, cyclosporine+3 mg kg71 d71 sirolimus (n=5) and (C) Cs+RAD, 10 mg kg71 d71 cyclosporine+3 mg kg71 d71
`RAD. Abbreviations: Cr, creatine; Cs, cyclosporine; GABA, g-aminobutyric acid; Gln, glutamine; Glu, glutamate; Ins, myo-inositol;
`Lac, lactate; NAA, N-acetyl aspartate; SRL, sirolimus; Tau, taurine.
`
`the average relative NTP concentration in
`study period,
`brains of animals in group Cs+SRL was 22%, that of
`phosphocreatine 22% and that of NAD+ 45% of
`the
`
`controls. Consequently, relative NDP and NMP concentra-
`tions were significantly higher than in the controls. Again, co-
`administration of cyclosporine and RAD compared favorably
`
`British Journal of Pharmacology vol 133 (6)
`
`NOVARTIS EXHIBIT 2082
`Par v. Novartis, IPR 2016-01479
`Page 7 of 11
`
`

`

`882
`
`N. Serkova et al
`
`Sirolimus, not RAD, enhances cyclosporine toxicity
`
`(group
`combination
`cyclosporine/sirolimus
`the
`with
`Cs+SRL) and also with cyclosporine mono-treatment (group
`Cs). In the Cs+RAD group relative brain concentrations of
`NTP were reduced to 55% of the controls. However, there
`was no statistically significant di€erence between relative
`phosphocreatine and NAD+ concentrations between group
`Cs+RAD and the controls (Table 2). Comparison of the six
`study groups using analysis of variance indicated significant
`di€erences in the relative concentrations of the key para-
`meters NTP, phosphocreatine and NAD+ (all P50.0002).
`Duncan grouping gave the following results: NTP, control4
`RAD4SRL4Cs+RAD4Cs4Cs+SRL; phosphocreatine:
`control=SRL=RAD=Cs+RAD4Cs4Cs+SRL; NAD+:
`control=SRL=RAD=Cs+RAD4Cs4Cs+SRL.
`
`Blood and brain tissue concentrations of
`immunosuppressants
`
`The concentrations of the study drugs in blood and brain
`tissue 12 h after the last dose (C12h) are shown in Table 3.
`Comparison of
`the brain-to-blood partition coe(cid:129)cients
`showed that co-administration of cyclosporine and sirolimus
`increased brain penetration for both cyclosporine (0.68+0.03
`0.56+0.08
`in group Cs+SRL versus
`in group Cs,
`mean+s.d., P50.05) and sirolimus (10.9+2.0 in group
`Cs+SRL versus 6.0+1.6 in group SRL, P50.01).
`In
`contrast,
`co-administration of
`cyclosporine
`and RAD
`decreased the cyclosporine brain-to-blood partition coe(cid:129)cient
`(0.44+0.06 in group Cs+RAD versus 0.56+0.08 in group
`Cs, P50.05). The brain-to-blood partition coe(cid:129)cient
`for
`RAD was not a€ected by the presence of cyclosporine
`(3.4+0.5 in group Cs+RAD versus 3.4+1.2 in group RAD,
`statistically not significant).
`
`Distribution into brain mitochondria
`
`At the end of the study period, RAD and cyclosporine, but
`not sirolimus, were found in rat brain mitochondria (limit of
`the h.p.l.c/h.p.l.c.-MS assay: 0.25 mg l71). In
`detection of
`group Cs, the cyclosporine concentrations in mitochondria
`were 28%, and in the RAD group, RAD concentrations in
`the mitochondria were 82% of those in the brain tissue
`(Tables 3 and 4). Addition of RAD reduced cyclosporine
`concentrations in brain mitochondria by 65% (P50.005
`group Cs+RAD versus group Cs), while cyclosporine
`increased RAD penetration into mitochondria 2.5 fold
`(P50.0001 group Cs+RAD versus group RAD).
`Even though sirolimus itself could obviously not pass
`mitochondrial membranes (Table 4), it increased cyclosporine
`
`Table 4 Cyclosporine, sirolimus and RAD concentrations
`in isolated brain mitochondria of rats after oral treatment
`with the study drugs or their combinations for 6 days
`
`Cyclosporine
`[ng g71]
`
`68.3+14.8
`216.2+13.2**
`24.6+6.3*
`
`Sirolimus
`[ng g71]
`
`5LLOQ
`5LLOQ
`–
`
`RAD
`[ng g71]
`
`66.5+10.2
`–
`171.4+2.0***
`
`Mono
`Cs+SRL
`Cs+RAD
`
`Concentrations are presented as means+s.d. (n=3 for each
`group). Data was analysed using analysis of variance in
`combination with Duncan grouping: *(P50.05), **(P50.02)
`in comparison with the corresponding mono-treatment
`group. Abbreviations: 5LLOQ, concentration below lower
`limit of quantification; mono, mono-treatment (group Cs,
`SRL or RAD).
`
`Table 2 The e€ects of cyclosporine, sirolimus and RAD, as well as co-administration of cyclosporine/sirolimus and cyclosporine/RAD
`on brain phosphate metabolism
`
`Control
`(I) n=7
`
`40.5+1.5
`5.9+1.5
`0.5+0.4
`2.3+1.7
`5.1+0.7
`
`Cs
`(II) n=5
`
`13.6+2.7***
`14.2+0.9***
`10.4+0.7***
`0.7+0.5*
`3.9+0.3**
`
`SRL
`(III) n=5
`
`26.0+2.7**
`9.5+3.1*
`2.5+1.9*
`1.4+0.8
`4.5+1.1
`
`RAD
`(IV) n=5
`
`31.4+2.5*
`10.1+3.5*
`1.2+1.7
`2.2+1.3
`5.0+0.3
`
`Cs+SRL
`(V) n=5
`
`9.5+7.9c**
`11.2+4.5**
`7.5+5.2***
`0.5+0.5***
`2.3+0.9***
`
`Cs+RAD
`(VI) n=5
`
`22.3+8.0**
`12.0+2.4**
`5.0+2.5*
`1.7