APOTEX ET AL. - EXHIBIT 1053
`Apotex Inc. et al. v. Novartis AG
`IPR2017-00854
`
`

`

`sphingophospholipid (SPL)
`its
`antiapoptotic effects of
`component, sphingosine—i-phosphate [7]. Like SIP. sphin-
`gosylphosphorylcholine (SPC)
`represents a major SPL
`species circulating with HDL. Several groups have shown
`that SPC, similar to Si P, has an inhibitory effect on TNF-o-
`induced expression of cell adhesion molecules in endothelial
`cells [8, 9]. SPL traveling with HDL have been shown
`to induce vasodilatation in contracted vessels [2, 10, 11].
`There is, however, evidence for differential and even opposite
`effects when comparing SIP and SPC with respect
`to
`their effects in the cardiovascular system: SlP is a high-
`affinity ligand for the SIP—receptor family while SPC requires
`much higher concentrations to activate these G—protein—
`coupled receptors, which will activate NOS through Alst-
`phoSphorylation in both cases. In addition, there is evidence
`for additional intracellular receptors or direct effectors of
`SPC and SIP. Engagement of these different receptors could
`indeed be a source for adverse effects of SPC compared to
`81F.
`
`We, therefore, tested the hypotheses that (i) SPC—-like
`SIP—exerts cardioprotective effects in an in vivo mouse
`model of myocardial
`ischemia with reperfusion and (ii)
`that such cardioprotective effects of SPC—if detectable——
`are also mediated via SIP; receptors to ultimately result in
`reduced neutrophil recruitment and cardiomyocyte apop—
`tosis to afford protection from postischemic myocardial
`necrosis.
`
`2. Material and Methods
`
`2.}. Materials. SPC and S] P (Sigma, Tau fkirchen, Germany)
`from methanol stock solutions were air dried and dissolved
`
`in phosphate—buffered salineiWo bovine serum albumin and
`administered intravenously in 100 all I 0 g body weight doses.
`
`2.2. Myocordini isehemia/Reperfiision. To assure strain inde—
`pendent effects of SPC treatment we used Sle-deficient
`mice on a C57BLi6—background as well as an outbred Swiss
`strain [12]. Animals were strain matched, age matched, and
`sex matched and therefore used in a nonrandomized study
`design. Myocardial ischemia was indtlced with the approval
`ofthe institutional Review Board and in accordance with the
`
`Guidefor the Core and Use ofteborotory Animals published
`by the US National
`institutes of Health as previously
`published [7]. Briefly, thoracotomy and ligation of the. left
`anterior descending coronary artery (LAD) at the level of the
`left atrium were performed with silk-Y-U suture over a PEIO—
`tubing in barbiturate—anesthetized mice for 30 minutes. The
`chest was closed before the animals were weaned from the
`
`ventilator and extubated. After 24 hours of reperfusion,
`animals were reanesthetized and perfused with 0.9% saline
`through the abdominal aorta. The coronary ligation was
`retied. 2% coomassie blue solution was injected to delineate
`the area at risk. The heart was sectioned into 5 equal slices
`from the apex to the base and immersed in 2-, 3-, 5-
`triphenyltetrazolium chloride (TTC) solution at 37°C. TTC
`development
`lasted 10 minutes before the sections were
`scanned, processed. and morphonietrically analyzed for left
`
`Mediators of inflammation
`
`ventricular area, area at risk, and area of infarction using
`image I (NIH, Bethesda}. Data are presented as file average
`percent infarct size per area at risk. SPC (0.625, 1.25, and
`2.5 ,ugig body weight) was administered either 30 minutes
`before transient coronary ligation or therapeutically after
`myocardial ischemia with reinstitution of reperfusion (SPC;
`1,25 ygfg body weight).
`
`immunohisrochemisrry. Perfusion-fixed (4% paraform-
`2.3.
`aldehyde), paraffin-embedded sections of SPC-pretreated
`animals were stained for polymorphonuclear
`leucocytes
`(PMN) using the monoclonal antibody MCA771G (Serotec,
`Oxford, England), developed with antirat peroxidase—
`coupled secondary antibodies and DAB as a substrate {Vec—
`torstain, DAKO, Germany). TUNEL assays were performed
`using the ApopTag kit {Chemicon, Temecula, USA]. The
`number of stained cells was semiautomatically determined
`on three sections per heart using morphometrical analy-
`sis software {AnalySIS, Monster, Germany). Apoptosis of
`rat neonatal cardiomyocytes was induced by exposure to
`hypoxic conditions (0.8% 03 in the medium) for 210mm
`followed by 150 min of reoxygenation. SPC {mull/I; Sigma,
`Taufkirchen, Germany) was administered directly before
`onset of hypoxia. Apoptosis was assessed by TUN EL using
`the MEBSTAIN Apoptosis Kit
`11
`(MBL, Woburn, USA].
`TUNEL-positive nuclei were counted and expressed as
`TUN EL‘PUSitiVef total nuclei.
`
`in vitro effects of SFC on
`2.4. Flow Chamber Studies.
`endothelial adhesiveness for mouse PMNs was determined
`
`using a parallel-plate flow chamber model as described
`in detail previously [7]. PMNs were isolated from bone
`marrow of tnice [13] and labeled using cell tracker green
`(Molecular Probes, Leiden, Netherlands) before being per-
`fused at 100 s‘t across TN Fit-activated immortalized murine
`endothelioma cells [fEnd.5). The number of cells with firm
`adhesion was determined on pictures taken from 15 high-
`power fields after 5 minutes of cell perfusion followed by
`5 minutes of buffer wash and captured on an UltraView
`(Perkin Elmer,
`lugesheim, Germany) confocal scanning
`microscope. Quantification was performed using image I
`software.
`
`2.5. Statistical Analysis. Data are presented as mean i SEM.
`Nonparametric Kruskal-Wallis testing followed by Dunnet‘t's
`test was employed to identify significant differences between
`groups. Significant difierences were assumed at P < .05
`(inStat, GraphPad Inc., San Diego, USA).
`
`3. Results
`
`3.1. Sphingosyiphosphoryicholine {SPC} Reduces Infarct Size
`after Myocardial ischemia and Reperfiision in Vivo.
`[n wild-
`type mice, left ventricular cross-sectional area was 13.9 i
`0.7 mmz. Ligation of the LAB resulted in an ischemic area
`of7.6 : 0.5 mm2 (n = 11) constituting the area at risk. The
`infarcted area measured 3.4 x 0.4mm2 (n = 11). Neither
`
`

`

`Mediators oflnflamniation
`
`lnfarctiolti'areaatriskWis)
`
`SPC W liigi'g'}
`
`ischemiai’reperfiision
`FIGURE 1: SPC protects against myocardial
`injury in viva. SPC (0.625, 1.25 and 2.5 ngg body weight} and 1%
`bovine serum albumin in PBS were injected intravenously 30
`minutes before and after myocardial isehemia with reinstitution of
`reperfusion. Infarct size, after MUR as a function of area at risk is
`reduced in SPC treated mice.
`
`left ventricular area nor area at risk were statistically different
`between treatment groups. The mortality after myocardial
`ischemia with reperfusion was about 15% while adminis-
`tration of vehicle control or 513C showed no influence on
`
`the rate of mortality in the different treatment groups. In
`previous work, we showed that SIP l9 and 38 ngig body
`weight does-dependently reduced infarct size [7]. Since the
`lid ofSlP receptors for SPC is up to 40—fold higher than that
`for SIP [14, 15], we administered SPC in an equipotent dose
`range (0625, 1.25, and 2.5 ygfg body weight) 30 min before
`transient coronary artery ligation. Intravenous injection of
`SPC resulted in a dose-dependent reduction of infarct size
`by 23%, 36%, and 50%, respectively (n = 8,10,5, P «c
`.05;
`Figure 1). Interestingly, when administered therapeutically
`with reinstitution of reperfusion after myocardial ischemia
`we also observed a reduction ofinfarct size by 40% for SPC
`{1.25 |ugjg bw} compared to BSA treated controls (n = 6, P c
`.05; Figure l).
`
`in Vitro and in
`3.2. SPC Reduces Leukocyte Recruitment
`Vivo. Leukocyte recruitment plays a crucial role in ischemia/
`reperfusion damage. To test the effect ofSPC on leukocyte-
`endothelia] interactions in vitro. we used a parallel-plate flow
`chamber model where mouse macrophages or PMN were
`perfused over a confluent monolayer of activated murine
`endothelial cells (fEnd.5), and their adhesion was quantified.
`Stimulation with TNFa increased firm adhesion of PMNs to
`
`fEnd.5 by 296 t 19% (61 t 19 PMNsimm2 in unstimulated
`versus 180 i- 35 in TNFa-stimulated cells, n = 8, P c
`.001). Addition of lfllnM SPC reduced adhesion to 133
`:r 28 PMstmm3 in TNFu-stimulated cells (n = 6, P c
`.05 versus TNFa—stimulated cells in absence of SPC; Fig-
`ure 2(a)). in viva. during myocardial ischemiafreperfusion,
`PMN recruitment was decreased from 629 t 45 PMmem2
`
`in vehicle-treated hearts to 332 : 431’l\rll\lilnm2 in SPC—
`
`pretreated hearts (n = 6/7, P < .01; Figure 2(b)]. The
`observed antiadhesive effect of SPC in vitro does not
`
`size reduction
`role of SPC on infarct
`prove a causal
`but suggests that anti-inflammatory effects on endothelial
`cells may add up to the composite cardioprotective effect
`”I VII’D.
`
`3.3. SPC Protects Cardiomyocytesfl‘orn Apoptosis In Vitro and
`In Vivo. As SIP receptors are present and functional
`in
`cardiomyocytes [16] and both HDL and lysophospholipids
`are potent antiapoptotic signaling mediators in a number
`of experimental systems
`[7, 17, 18], we tested if SPC
`directly protects cardiomyocytes against apoptosis in vino.
`SPC had an antiapoptotic effect as it significantly reduced
`the amount of TUNEL~positive nuclei after simulated
`ischemialreperfusion {3.7 i- 0.6% versus 6.5 1 0.9% TUNEL—
`positive nuclei in control versus SPC treated cardiomyocytes.
`n = 3, P < .05; Figure 3(a)). in viva, apoptotic cell death
`was assessed in mice treated with SPC prior to ischemia.
`’I‘UNEL-staining in the area at risk {outside the TTC—positive
`area) was substantially reduced in lysophOspholipid—treated
`mice (920 i 225 versus 643 i 66 'FUNEL-positive cellslmm2
`in BSA versus SPC,—treated mice, resp.; n = S, P < .01;
`Figure 3(b}). By calculating the amount of apoptotic nuclei
`per total nuclei in the area at risk (outside the TTC-pOsitive
`area) we estimated the amount of viable cardiac muscle
`tissue lost due to apoptosis to be about 17% of the area at
`risk.
`
`is Mediated by the $in
`3.4. Cnrdioprotective SPC Eflect
`Lysophosphoiipid Receptor.
`In order to investigate which
`SIP receptor mediates the cardioprotection of SPC, we
`analyzed the effects ofSPC in knockout-SIP; receptor mice
`(Slim—"J that were available only on the C57BLf6 back-
`ground. Whereas the studies reported above were carried
`out in an outbted Swiss strain, the cardioprotection of SPC
`[1.25pgfgbw) is present to the same extent
`in wild-type
`C57BU6 mice (29 a 3,391: versus 34 i 2% infarctionlarea
`at risk, C57BIJ6 versus Swiss, n = 6, P < .05; Figure 4).
`However, in Sng’fimice no protection by SPC on infarct
`size was detectable (105 i 9% of vehicle—treated control.
`n = 5, P = ns; Figure 4).
`
`4. Discussion
`
`The salient findings of this study are that HDL-associated
`SPC,
`like SIP. exerts cardioprotective antiapoptotic and
`anti-inflammatory effects when administered preventively
`prior to ischemia or therapeutically to ischemic myocardium
`during reperfusion. This effect is mediated through the 5ng
`receptor and according to our previously published results
`likely to be nitric oxide dependent.
`Long-term beneficial atheroprotective effects oft-[D]. are
`generally accepted. Increasing EVidence points to additional
`effects of HDL in connection with acute tissue ischemia
`
`independent ofits role as cholesterol acceptor. A recent study
`demonstrated improved functional postischemic recovery of
`
`

`

`Mediators of Inflammation
`
`P<.01
`
`P<.05
`
`P<.Oi
`
`P<.t]5
`
`10
`
`200
`
`_ UIO
`
`VI0
`
`PMNadhering(mmz) E
`
`TUNEL-pos
`
`(at)
`
`Control
`
`+ SPC
`
`—
`
`—
`
`+
`
`+
`
`TNF-rx
`
`SPC
`
`1200
`
`800
`
`400
`
`TUNEL-poshnmz)
`
`Hypoxia reoxygenation
`
`(a)
`
`P < .01
`
`
`
`(bl
`
`FIGURE 3: SPC inhibits apoptosis of cardiomyocytes in vim: and
`in the infarction in viva. (a) Quantification of TUNELrpositive
`nuclei in rat neonatal cardiomyoeytes after simulated ischemiai’
`reperfusion in the presence or absence of 10 MM SPC. (b) Represen-
`tative terminal dUTP nick end-labeling (TUNELJ staining in the
`area at risk (outside the TTC-positive area} of control and SPC-
`treated mice 24 hours after ischemiaireperfusion. Morphometrie
`quantification is presented above.
`
`isolated rat hearts by HDL that was attributed to scavenging
`of myocardially released TNFa by HDL [ 19]. Former studies '
`demonstrated a reduced leukocyte-endothelial interaction in
`connection with atheroprotection in virm [20] and in vivo
`[21}-
`'
`
`—u-u-__——..---—-.—-n—.-_--
`
`—
`
`_
`
`+
`
`—
`
`+ P
`
`cfli
`
`
`
`Control
`
`SPC
`
`(b)
`
`.
`
`FIGURE 2: SPC inhibits PMN adhesion to activated endothelium
`under flow in vitro and PMN recruitment in the infarction area
`in viva. (a) PMN adhesion to TNF-a-activated endotheiial cells
`in vitro in the presence and absence of 1031M SPC as quanti-
`fied in a parallel—plate flow-chamber system. (b) Representative
`immunohistochemistry and morphometric quantification of PMN
`in infarcts of vehicle- and SPC-treated mice, respectively, 24 hours
`after ischerniaireperfiasion.
`
`

`

`Mediators oflnflammation
`
`60
`
`P<.05
`
`[[1ch[ioni'areaatrisk(‘iial
`
`csraIs—wr
`
`Step“-
`
`Fiouae 4: The SIP; lysophospholipid receptor is required for car-
`dioprotection by SPC.
`infarct sizer‘area at risk was determined
`in SlPa-deficient mice and their matching wild—type controls
`{C57BLi'6l after treatment with SPC (1.25pgt‘g body weight}.
`
`Rapid reperfusion is an established priority for treatment
`of myocardial
`ischemia. The underlying intention is
`to
`minimize tissue destruction and thereby infarct size with
`subsequent
`improved outcome of the patient. We have
`shown that HDL, in addition to its effects on reverse choles~
`teroi transport, stimulates NO release in human endothelial
`cells and induces vasodilatation [2, 101. According to the aim
`ofrapid reperfusion of an occluded vessel, this may account
`for direct beneficial effects ofHDL on ischemic myocardium.
`Scar size is, however, not only dependent on tissue loss
`during ischemia, but
`increases due to the inflammatory
`response during reperfusion [22]. Studies with isolated
`perfused hearts argue against a significant neutrophil-de-
`pendent component in cardioprotection, since postcondi-
`tioning reduced infarct size and necrosis in such leukocyte-
`free models [23]. However. studies from our group showed
`that three hours after ischemia, only a small part of the tissue
`defect is due to leukocytes whereas 24 hours after repert‘usion
`tissue loss is almost twice as big in controls compared to
`leukocyte depleted animals [7]. indicating that neutrophils
`contribute importantly to a second wave ofmyocardial tissue
`loss during later phases. We can not, however, exclude some
`degree of interdependence of inflammation and apoptosis.
`That is,
`inflammatory cell recruitment may contribute to
`cardiOmyocyte apoptosis. One argument supporting this
`notion is the earlier observation that antileukocyte strategies
`can entirely prevent tissue damage occurring during later
`phases of reperfusion [7].
`We have recently shown that HDL reduces cardiomyocyte
`apoptosis and leukocyte recruitment
`to the postischemic
`myocardium resulting in a cardioprotective elfect. This
`effect was mediated by HDL’s constituent sphingosine-I-
`phosphate that acts through its receptor SIPg. The SIP;
`effect, in turn, depends on nitric oxide synthase activity
`[7]. In addition to SIP, sphingosylphosphorylcholine (SPC)
`is another sphingophospholipid traveling with HDL, and
`because of their diverse affinities to different receptor subsets,
`there is an ongoing debate whether SPC and 51 P would exert
`
`similar or antagonistic effects in the cardiovascular system
`[2, 24—26]. Furthermore. the distinct role ofSPC in different
`cell types might be diverse. Nixon et al.
`[27I showed that
`SPC administered to vascular smooth muscle cells acts as
`a proinflammatory mediator. in contrast, we here show an
`anti—inflammatory role ofSPC in endothelial cells, suggesting
`that the balance between SPC effects in different cell types
`might be an important factor deciding ifbeneficial or adverse
`effects are realized in the cardiovascular system.
`Multiple protein kinase andfor phosphatase-signaling
`pathways are activated during ischemia with reperfusion
`[23]. Effects of SFC and SI? on downstream kinase
`phosphorylation have been reported to be divergent
`in
`vascular smooth muscle cells from rat cerebral arteries [28].
`Therefore, we analyzed the phosphorylation of ERKle and
`p3BMAPK in postischemic tissue and remote myocardium
`in SPC— versus BSA—treated mice, but we did not observe
`any significant differences (data not shown). This finding
`suggests that other signaling pathways might be involved in
`the in viva function ofSPC.
`
`lysophospholipid
`the SIP;
`We here demonstrate that
`receptor is required for cardioprotection by SPC, which is
`somewhat surprising because SPC is known to have only
`a low affinity for SIP-receptors [14, 15]. intracellular and
`extracellular sphingosine kinases i and 2 convert SPC 'to
`SIP, which could explain biological similarities of SPC and
`SlP. While ischemia induces the formation of ceramide and
`sphingosine by activation of sphingomyelinase, which have
`been shown to reveal negative effects on cardiac function.
`it is likely that a rapid and effective conversion of sphingo—
`sylphosphorylcholine to SIP catalyzed by sphingosine kinase
`[24] might be the underlying effect of cardioprotection by
`SPC.
`
`Activation ofsphingosine kinase {Sth) has been shown
`to play a crucial role in protection against apoptosis in
`oligodendrocyte survival by neutrophin-3 [29]. Further-
`more, [in and Karliner [30] reported cardioprotection via a
`PKCepsilon~Sth-Sl P-Altt pathway.
`We can not exclude that SPC pretreatments sets of
`cascades are also involved in preconditioning phenomena.
`especially since nitric oxide seems to be one of the active
`motifs. The postischemic treatment effects that we observed
`do not
`likewise exclude that postconditioning effects are
`aCcountable. Nevertheless, there is an antiapoptotic and anti—
`inflammatory efiect involved in SPCvfostered cardioprotec-
`tion.
`
`5. Conclusion
`
`like 31?, exerts
`that SPC,
`In aggregate, our data suggest
`cardioprotective effects during reperfusion injury regardless
`of the timing of its administration. Even if HDL rising
`strategies would,
`in parallel,
`increase circulating bioaclive
`SIP along with SPC, no adverse effects ofSPC will antagonize
`SlPs beneficial effects. The perspectives of interventions
`designed to acutely raise HDL levels in patients at high risk,
`for example. such with acute coronary syndromes to improve
`prognosis may be very attractive both for patients and
`clinicians.
`
`

`

`Acknowledgments
`
`This paper was supported in part by the Deutsche For-
`schungsgemeinschafi {Th667l6-l. LE 940-3. MU 1376-10).
`the Interdisciplinary Center for Clinical Research Munster
`llZKF. TheilfiBiOd). the National Institutes ofl'iealth (Grants
`P45048478 and DA019674 to I. Chum}. and the H.-H. Deich-
`mann Foundation for Atherosclerosis Research. The authors
`
`thankfully acknowledge the technical assistance of Marleen
`Lox. Gisela Gaecle. Stephan Liitke linking. Dirk Burger.
`K. Abouhamed, S. Mersrnann, V. Brinkrnann, Anelie Ahle,
`
`and Daniela AschhoFfi I. Mersrnann is currently located at
`the Department
`for Anesthesiology and Intensive Care
`Medicine, Iohannn Wolfgang Goethe University, Frankfurt.
`M. Winterhalter is currently located at the Department For
`Anesthesiology and Intensive Care Medicine ofthe Heinrich-
`Heine University Diisseldorf. C. Herring and M. Schmitz
`contributed equally to this work.
`
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