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`0022-3565/00/2941-0323$03.00/0
`THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
`Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
`JPET 294:323–332, 2000 /2243/834819
`
`Vol. 294, No. 1
`Printed in U.S.A.
`
`Tissue Distribution and Clinical Monitoring of the Novel
`Macrolide Immunosuppressant SDZ-RAD and Its Metabolites
`in Monkey Lung Transplant Recipients: Interaction with
`Cyclosporine1
`
`NATALIE SERKOVA, BERNARD HAUSEN, GERALD J. BERRY, WOLFGANG JACOBSEN, LESLIE Z. BENET,
`RANDALL E. MORRIS, and UWE CHRISTIANS
`
`Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, San Francisco, California (N.S., W.J., L.Z.B., U.C.);
`and Transplantation Immunology, Department of Cardiothoracic Surgery (B.H., R.E.M., U.C.), and Department of Pathology (G.J.B.), Stanford
`University Medical School, Stanford, California
`Accepted for publication March 27, 2000
`
`This paper is available online at http://www.jpet.org
`
`ABSTRACT
`We report the tissue distribution and clinical monitoring of the
`novel macrolide immunosuppressant SDZ-RAD [40-O-(2-hy-
`droxyethyl)-rapamycin] and its metabolites in monkey lung trans-
`plant recipients as well as its interaction with cyclosporine as the
`Neoral formulation. After left unilateral lung transplantation, cyno-
`molgus monkeys received by oral administration either 1) 1.5
`mg/kg/day SDZ-RAD (n 5 4); 2) 100 mg/kg/day cyclosporine (n 5
`4); 3) 0.3 mg/kg/day SDZ-RAD 1 100 mg/kg/day cyclosporine
`(n 5 6); 4) 1.5 mg/kg/day SDZ-RAD 1 50 mg/kg/day cyclosporine
`(n 5 5); or 5) SDZ-RAD and cyclosporine doses adjusted accord-
`ing to trough blood concentration measurements (n 5 6). At the
`end of the observation period (usually 29 days after transplanta-
`tion), and 24 h after the last doses, tissue samples were collected
`and analyzed with HPLC/mass spectrometry. Gall bladder, pan-
`creas, the transplant lung, cerebellum, kidneys, and spleen had
`the highest SDZ-RAD concentrations. Coadministration of cyclo-
`
`sporine increased SDZ-RAD concentrations in most tissues as
`well as tissue-to-blood distribution coefficients. In contrast, SDZ-
`RAD had only a small effect on cyclosporine blood and tissue
`concentrations. Rejection in lung grafts in monkeys treated with
`either of the cyclosporine/SDZ-RAD combinations was signifi-
`cantly less than in the monotherapy groups (P , .002). Histolog-
`ical rejection scores were inversely correlated with SDZ-RAD con-
`centrations in blood (r 5 20.68; P , .001; n 5 24), lymph nodes
`(P 5 20.58; P , .003; n 5 24), thymus (r 5 20.63; P , .001; n 5
`23) and transplant lung tissue (r 5 20.58; P , .003; n 5 24). We
`conclude that, in addition to the synergistic pharmacodynamic
`interaction, a pharmacokinetic interaction resulting in higher SDZ-
`RAD tissue concentrations contributed to the significantly better
`immunosuppressive efficacy when both drugs were combined
`compared with monotherapy.
`
`The novel macrolide SDZ-RAD [40-O-(2-hydroxyethyl)-
`rapamycin] is currently in phase II/III clinical trials as an
`immunosuppressant coadministered with microemulsion cy-
`closporine (Neoral;
`international nonproprietary name:
`ciclosporin) after organ transplantation. SDZ-RAD is a semi-
`synthetic derivative of rapamycin (international nonpropri-
`etary name: sirolimus). Although not yet proven, it is as-
`sumed that SDZ-RAD has the same molecular actions as
`rapamycin. The rapamycin/FK-binding protein (FKBP) com-
`plex, and probably also the SDZ-RAD/FKBP complex, binds
`
`Received for publication November 1, 1999.
`1 This study was supported in part by the Alexander von Humboldt-Foun-
`dation, Grant V-3-FLF-1052812 (to N.S.); the Deutsche Forschungsgemein-
`schaft Grants Ch 95/6-2 (to U.C.) and Ha 1967/2-1 (to B.H.); National Insti-
`tutes of Health Grants CA72006 and GM26691 (to L.Z.B.); the Ralph and
`Marian Falk Medical Research Fund; the Hedco Foundation; and a grant from
`Novartis Pharma AG, Basel, Switzerland (to R.E.M.).
`
`in T lymphocytes to mTOR, the mammalian target of rapa-
`mycin. The results are inhibition of the interleukin-2-stimu-
`lated phosphorylation activation of p70-kd S6 protein kinase
`and blockade of cell cycle progression at the G1-S-interface
`(Schuler et al., 1997; Bo¨hler et al., 1998). While the cal-
`cineurin inhibitor cyclosporine inhibits interleukin-2 synthe-
`sis, SDZ-RAD inhibits interleukin-2-mediated T-lymphocyte
`proliferation. It is generally thought that inhibition of the
`subsequent steps of T-lymphocyte proliferation is the main
`reason for the synergistic immunosuppressive interaction of
`cyclosporine and SDZ-RAD observed in in vitro and in vivo
`studies (Schuler et al., 1997; Schuurman et al., 1997, 1998).
`Major side effects of cyclosporine include nephrotoxicity, hep-
`atotoxicity, neurotoxicity, and hypertension (Kahan, 1989).
`The side effect pattern of SDZ-RAD in patients can be ex-
`pected to be similar to that of rapamycin (Murgia et al.,
`
`ABBREVIATIONS: FKBP, FK-binding protein; CYP, cytochrome P450; MS, mass spectrometry; amu, atomic mass units.
`
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`Serkova et al.
`
`1996): thrombocytopenia, hyperlipidemia, and gastrointesti-
`nal disorders.
`Access of cyclosporine to the various body compartments is
`governed by cytochrome P450 (CYP)3A, ATP-binding cas-
`sette protein transporters, and binding to immunophilins
`(Ryffel et al., 1991; Wacher et al., 1998). Like cyclosporine,
`SDZ-RAD is a substrate of both CYP3A and the ATP-binding
`cassette protein transporter p170-glycoprotein (Crowe and
`Lemaire, 1998; Crowe et al., 1999). Because many of the
`factors that determine cyclosporine pharmacokinetics also
`are involved in SDZ-RAD pharmacokinetics, we hypothe-
`sized that cyclosporine might have a significant effect on
`SDZ-RAD tissue distribution, thus influencing its immuno-
`suppressive efficacy and its tolerability.
`Due to the poor oral bioavailability of microemulsion cyclo-
`sporine in cynomolgus monkeys (Schuurman et al., 1996),
`significantly higher doses than in humans are required to
`maintain cyclosporine trough blood concentrations in the
`target range proposed for clinical lung transplantation. The
`cyclosporine (100 mg/kg) and SDZ-RAD (1.5 mg/kg) doses
`used in our study were based on those used in the same
`animal species by Schuurman et al. (1998). These doses re-
`sulted in trough blood concentrations in the therapeutic
`range for patients and were found to be effective in prevent-
`ing rejection of life-supporting kidney transplants for .50
`days. Because coadministration of 100 mg/kg cyclosporine
`and 1.5 mg/kg SDZ-RAD was tolerated poorly (Hausen et al.,
`2000), doses of 50 mg/kg cyclosporine and 1.5 mg/kg SDZ-
`RAD (for comparison with the SDZ-RAD monotherapy group)
`and of 100 mg/kg cyclosporine and 0.3 mg/kg (for comparison
`with the cyclosporine monotherapy group) were combined.
`These doses were based on the results of tolerability studies
`(Hausen et al., 2000). In addition to these fixed-dose drug
`regimens, we also included a study group in which, as in the
`clinical practice, doses were adjusted according to blood
`trough concentrations (20–40 mg/l for SDZ-RAD and 100–
`200 mg/l for cyclosporine). The cyclosporine target concentra-
`tions were based on those used in patients (Oellerich et al.,
`1995).
`
`Experimental Procedures
`Materials. SDZ-RAD, cyclosporine, and cyclosporin D were pro-
`vided by Novartis Pharma AG (Basel, Switzerland). Acetonitrile
`(HPLC grade), sulfuric acid (American Chemical Society grade),
`methanol, and methylene chloride were obtained from Fisher Scien-
`tific (Fairlawn, NJ). Zinc sulfate, formic acid, and sodium formate
`(all American Chemical Society grade) were purchased from Sigma
`Chemical Co. (St. Louis, MO). Extraction columns (bonded phase
`C18; 1 ml) were from Varian Sample Preparation Products (Harbor
`City, CA). The internal standard used for quantification of SDZ-
`RAD, 28-,40-O-diacetyl rapamycin, was synthesized as described by
`Streit et al. (1996). Analytical columns (250 3 4 mm) filled with
`Hypersil (Shandon, Chadwick, UK) C8, 3-mm material were from
`Keystone Scientific (Bellefonte, PA). HPLC micro vials, 100-ml in-
`serts, and Teflon screw caps were purchased from Hewlett Packard
`(Palo Alto, CA).
`Samples were analyzed on an HPLC/electrospray-mass spectrom-
`etry (MS) system consisting of a series 1100 HPLC system (G1322A
`degasser, G1312A binary pump, G1313A autosampler, and G1316A
`column thermostate), a 59887A electrospray interface equipped with
`an Iris hexapole ion guide (Analytica of Brandford, Brandford, CT),
`and a 5989B MS (Hewlett-Packard, Palo Alto, CA).
`
`Vol. 294
`
`Lung Transplantation in Cynomolgus Monkeys. The animals
`received humane care in compliance with the Principles of Labora-
`tory Animal Care (National Society for Medical Research) and the
`Guide for Care and Use of Laboratory Animals (National Academy of
`Sciences, published by the National Institutes of Health). The Insti-
`tutional Animal Care and Use Committee of Stanford University
`granted approval.
`imported by the
`Cynomolgus monkeys, Macaca fascicularis,
`Charles River Biomedical Research Foundation (Houston, TX) from
`Mauritius, were free of herpes B virus and Sendai virus. All animals
`were quarantined for a minimum of 2 months at Charles River and
`for 7 weeks at Stanford. While in quarantine, blood was drawn on
`each donor/recipient pair of animals for a mixed lymphocyte reaction
`assay after the protocol kindly provided by Dr. B. A. Cosimi (Harvard
`Medical School, Boston, MA). Donor and recipient monkeys were
`blood-group matched and mixed lymphocyte reaction mismatched to
`obtain a stimulation index of at least 2.5. After quarantine, left
`unilateral lung transplantation was carried out as described by
`Cooper (1989). For the first 24 postoperative hours, the monkeys
`were placed into intensive care cages supplied with oxygen and
`warm, humid air. They had free access to food and water 4 h after
`transplantation and were transferred to the regular animal room 1
`day later. The animals were assigned to one of five treatment groups
`after transplantation: 1) 1.5 mg/kg/day SDZ-RAD (group 1.5RAD;
`n 5 4); 2) 100 mg/kg/day cyclosporine (group 100Cs; n 5 4); 3) 0.3
`mg/kg/day SDZ-RAD 1 100 mg/kg/day cyclosporine (group 0.3RAD 1
`100Cs; n 5 6); 4) 1.5 mg/kg/day SDZ-RAD 1 50 mg/kg/day cyclospor-
`ine (group 1.5RAD 1 50Cs; n 5 5); and 5) concentration-controlled
`dose adjustments based on SDZ-RAD and cyclosporine trough blood
`concentrations (C24 h, group CCRAD 1 Cs; n 5 6). In the groups with
`100 mg/kg/day cyclosporine, the monkeys initially received 150 mg/
`kg/day. On postoperative day 7, the dose was lowered to 100 mg/kg/
`day and remained unchanged for the remainder of the study period.
`In the concentration-controlled dosing group (CCRAD 1 Cs), doses
`were adjusted to maintain SDZ-RAD and cyclosporine trough blood
`concentrations in target concentration ranges of 20 to 40 mg/l and 100
`to 200 mg/l, respectively. During the study period, the mean 6 S.D.
`daily SDZ-RAD dose in this group was 0.6 6 0.1 and 38 6 1 mg/kg for
`cyclosporine. Immunosuppressive therapy was started immediately
`after surgery (day 0). Cyclosporine (Neoral) and SDZ-RAD (in meth-
`ylcellulose vehicle) were given daily as a single oral dose. In addition,
`animals received a single dose of methylprednisolone (4 mg/kg i.v.) at
`the day of surgery, and the antibiotics cefazolin (25 mg/kg i.m.) and
`gentamycin (3 mg/kg s.c.) for the first 4 days after surgery. At either
`the end of the observation period (29 days after surgery) or when the
`animals’ health status seriously deteriorated, and 24 h after the last
`dose, the animals were sacrificed and the following samples were
`collected for measurement of drug tissue distribution: blood, brain
`stem, cerebellum, cerebrum, colon, duodenum, fat, gall bladder,
`heart, ileum, jejunum, kidney, liver, transplant lung, native lung,
`lymph nodes, pancreas, spleen, stomach, testis, and thymus. The
`tissues were frozen in liquid nitrogen immediately after collection
`and stored at 280°C until HPLC/MS analysis. Samples were ana-
`lyzed within 20 days.
`Extraction of Tissue Samples. Tissue samples were thawed,
`weighed, and homogenized with 2 ml of KH2PO4 buffer [pH 5 7.4
`(1 M)] with a Teflon-glass manual homogenizer. One milliliter of
`homogenate was taken for analysis. Cyclosporin D and 28-,40-di-
`acetyl sirolimus (in acetonitrile/sulfuric acid; pH 5 3; 90:10 v/v) were
`added as internal standards, resulting in final concentrations of 100
`mg/l of each. After addition of 2 ml of methanol/0.4 M ZnSO4 (80:20
`v/v) for protein precipitation, the samples were vortexed for 30 s and
`centrifuged at 1500g for 3 min. The organic supernatant was loaded
`on C18 extraction cartridges by drawing the samples through the
`columns with a 210 mm Hg vacuum. The extraction columns had
`previously been primed with 3 ml of water and 3 ml of acetonitrile.
`Immunosuppressants, metabolites, and internal standards were
`washed on the columns with 3 ml of water. The extraction columns
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`were dried by drawing air for 5 min. Samples were eluted with 1.5 ml
`of methylene chloride. The samples were evaporated to dryness
`under a stream of nitrogen at 50°C. The residues were reconstituted
`in 120 ml of acetonitrile/0.1% formic acid (75:25 v/v) and were trans-
`ferred into micro HPLC vials with conical 100-ml inserts and sealed
`with Teflon screw caps.
`HPLC/Electrospray-MS Analysis. Extracts (100 ml) were in-
`jected onto a 250- 3 4-mm analytical column filled with Hypersil
`C8-material of 3-mm particle size. SDZ-RAD, cyclosporine, their me-
`tabolites, and the internal standards were eluted from the analytical
`column with methanol and 0.1% formic acid supplemented with 1
`mmol/l sodium formate. The following linear gradient was used: 0
`min, 70% methanol; and 30 min, 90% methanol. The column was
`reequilibrated to starting conditions for 5 min before injection of the
`next sample. The flow rate was 0.4 ml/min and the column temper-
`ature was 65°C. The drying gas in the electrospray source was
`adjusted to a value of 42 (arbitrary units) and heated to 350°C. The
`pressure of the needle spray gas was 80 psi. The quadrupole was
`heated to 120°C. The mass spectrometer was run in the positive
`mode and the multiplier voltage was set to 1750 V and the X-ray
`voltage to 210 kV. For single ion detection, the mass spectrometer
`was focused on the [M 1 Na]1 of SDZ-RAD [980 atomic mass units
`(amu)], hydroxy SDZ-RAD (996 amu), desmethyl SDZ-RAD (966
`amu), the internal standard 28-,40-diacetyl sirolimus (1020 amu),
`cyclosporine (1224 amu), hydroxy cyclosporine (1240 amu), dihy-
`droxy cyclosporine (1256 amu), desmethyl cyclosporine (1210 amu),
`and the internal standard cyclosporin D (1238 amu). The dwell time
`per ion was 100 ms.
`Assay Validation and Quantification. The assay was validated
`for SDZ-RAD and cyclosporine in blood with the procedures de-
`scribed in detail by Segarra et al. (1998). The HPLC/MS assay had
`the following specifications for SDZ-RAD determined in blood: linear
`range 0.1 to 100 mg/l (y 5 0.96x 1 0.05; r2 5 0.99), mean intra-assay
`variability 6.9% (n 5 10), intraday accuracy 16.8% (n 5 10), inter-
`assay variability 8.0% (n 5 6; 3 days), and mean analytical recovery
`83%. In-process stability, freeze-thaw stability, dilution integrity,
`and partial volume verification were established and have been
`reported by Segarra et al. (1998). The specifications for cyclosporine
`were as follows: linear range 1 to 1000 mg/l (y 5 0.93x 1 15.1; r2 5
`
`SDZ-RAD Tissue Distribution
`
`325
`
`0.98), intra-assay variability 7.7% (n 5 10), intraday precision
`23.7% (n 5 10), interassay variability 9.8% (n 5 5; 3 days), and mean
`analytical recovery 86%. Abbreviated assay validations were carried
`out for each of the tissues, including analytical recovery, lower and
`upper limit of quantitation, linearity, interassay variability, and
`accuracy. Samples from tissues of untreated donor monkeys were
`collected. Tissue samples were homogenized and cyclosporine and
`SDZ-RAD were added and incubated at 37°C for 30 min to allow for
`distribution and protein binding. The following samples were pre-
`pared: blanks (n 5 3/tissue), calibration controls (four concentra-
`tions; n 5 3/concentration), precision controls (three concentrations;
`n 5 3/concentration), samples for determination of the lower (SDZ-
`RAD, 0.5 mg/l and cyclosporine, 10 mg/l; n 5 5) and upper limit of
`quantitation (SDZ-RAD, 100 mg/l and cyclosporine, 1000 mg/l; n 5 5).
`The samples were extracted and analyzed as described above. Re-
`coveries were calculated from the quality control samples (n 5 3 for
`each concentration). The mass spectrometer responses of the ex-
`tracted samples were compared with the response after injection of
`corresponding amounts of internal standard or with standard solu-
`tions of the immunosuppressants (in methanol/0.1% formic acid; 9:1
`v/v) directly on the analytical column. The lowest concentration that
`met the following criteria was accepted as the lower limit of quanti-
`tation: 80% of the samples analyzed had to be within 620% of the
`nominal value, and precision and accuracy variation had to be less
`than 20%. The upper limit of quantitation was determined similarly.
`No interferences with the assay were detected when blank tissues
`were analyzed. Recoveries of SDZ-RAD and cyclosporine from all
`tissues were .60%. Validation results are summarized in Table 1.
`The validation results in all tissues were similar and did not signif-
`icantly differ from those in blood. Therefore, tissue and blood con-
`centrations of the immunosuppressants in the study samples were
`calculated with external calibration curves prepared in blood and
`were corrected with the internal standards. When tissue sample
`concentrations exceeded the upper limit of quantification, samples
`were diluted with acetonitrile/0.1% formic acid (75:25 v/v) and rean-
`alyzed.
`Cyclosporine and SDZ-RAD metabolites were identified by com-
`
`TABLE 1
`Validation of the HPLC/MS assay for cyclosporine and SDZ-RAD in monkey organ tissues
`Tissue samples were collected from untreated monkeys. Tissues were homogenized and cyclosporine or SDZ-RAD was added and incubated at 37°C for 30 min to allow for
`distribution and protein binding. The samples were extracted as described in Experimental Procedures. A concentration was accepted as the lower or upper limit of
`quantitation when four of five samples (80%) were within 620% of the nominal concentration and the coefficient of variation of all samples was #20%. The calibration curve
`consisted of four different concentrations (n 5 3 for each concentration). Interassay coefficient of variation (CV) and accuracy (Acc) were measured at three different
`concentrations (n 5 3 per concentration). Means (n 5 9) are presented.
`
`Tissue
`
`Brainstem
`Cerebellum
`Cerebrum
`Kidney
`Liver
`Stomach
`Colon
`Ileum
`Duodenum
`Jejunum
`Pancreas
`Heart
`Lung (right)
`Lung (left)
`Spleen
`Testes
`Thymus
`Fat
`Lymph node
`Gall bladder
`
`Linear
`Range
`
`mg/l
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`50–1000
`
`Cyclosporine
`
`SDZ-RAD
`
`Regression Analysis
`
`CV
`
`Acc
`
`y 5 1.02x 2 7.5, r 5 0.996
`y 5 1.04x 2 9.5, r 5 0.997
`y 5 0.98x 2 4.6, r 5 0.998
`y 5 1.01x 2 4.1, r 5 0.995
`y 5 0.95x 2 4.6, r 5 0.992
`y 5 0.99x 2 1.5, r 5 0.993
`y 5 1.01x 1 3.9, r 5 0.993
`y 5 1.01x 2 5.7, r 5 0.988
`y 5 1.02x 1 0.8, r 5 0.990
`y 5 0.91x 1 0.8, r 5 0.995
`y 5 0.96x 1 5.1, r 5 0.998
`y 5 1.07x 2 5.9, r 5 0.996
`y 5 1.02x 2 2.6, r 5 0.999
`y 5 1.06x 1 2.2, r 5 0.998
`y 5 1.07x 2 2.1, r 5 0.997
`y 5 1.04x 2 2.7, r 5 0.995
`y 5 1.06x 2 5.3, r 5 0.996
`y 5 1.02x 2 1.4, r 5 0.996
`y 5 1.04x 2 1.5, r 5 0.995
`y 5 1.08x 2 7.8, r 5 0.999
`
`3.0
`5.7
`5.9
`9.8
`13.9
`4.1
`6.9
`6.5
`11.8
`6.0
`9.2
`6.8
`7.4
`6.0
`8.0
`4.4
`6.3
`3.6
`5.5
`10.9
`
`%
`
`20.8
`21.3
`23.9
`10.2
`210.3
`11.1
`16.6
`20.9
`110.2
`26.6
`26.6
`15.5
`10.4
`18.6
`17.2
`14.7
`13.5
`13.0
`12.7
`12.1
`
`Linear
`Range
`
`mg/l
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`0.5–100
`
`Regression Analysis
`
`CV
`
`Acc
`
`y 5 0.91x 1 0.22, r 5 0.999
`y 5 0.91x 2 0.31, r 5 0.999
`y 5 1.05x 2 0.25, r 5 0.995
`y 5 1.05x 2 0.03, r 5 0.997
`y 5 1.04x 2 0.37, r 5 0.999
`y 5 1.05x 2 1.38, r 5 0.997
`y 5 0.93x 1 1.02, r 5 0.997
`y 5 0.96x 1 0.28, r 5 0.997
`y 5 1.01x 1 0.42, r 5 0.999
`y 5 0.95x 2 0.91, r 5 0.997
`y 5 1.06x 2 1.67, r 5 0.996
`y 5 1.00x 2 0.69, r 5 0.999
`y 5 1.03x 2 0.02, r 5 0.997
`y 5 0.90x 1 1.75, r 5 0.997
`y 5 1.03x 2 0.46, r 5 0.999
`y 5 0.99x 2 1.25, r 5 0.999
`y 5 0.96x 2 0.5, r 5 0.998
`y 5 0.95x 2 0.43, r 5 0.996
`y 5 0.98x 6
`0, r 5 0.999
`y 5 1.11x 2 0.47, r 5 0.999
`
`8.3
`4.7
`7.1
`12.3
`9.8
`7.9
`11.5
`7.9
`11.5
`7.7
`15.4
`9.0
`7.6
`4.5
`4.3
`3.6
`8.0
`5.1
`3.8
`9.3
`
`%
`
`25.5
`22.4
`11.4
`16.0
`110.8
`21.1
`22.8
`12.8
`22.8
`22.7
`26.1
`25.6
`22.7
`23.7
`20.8
`22.3
`13.0
`23.4
`24.3
`28.1
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`Fig. 1. Distribution of cyclosporine (A), SDZ-RAD (B), and their main
`metabolites in tissues of lung transplant monkeys. Data are presented as
`mean 6 S.E. (n 5 4) and are given in nanograms per gram of wet tissue
`weight except for blood, which is given in micrograms per liter. The
`animals in A received 100 mg/kg/day (150 mg/kg the first week) cyclo-
`sporine (Neoral) and the animals in B received 1.5 mg/kg/day SDZ-RAD
`as a single daily oral dose for 4 weeks. TX, transplant.
`
`the median blood concentration of cyclosporine was 221 mg/l
`(range 131–255 mg/l; n 5 4). Cyclosporine concentrations in
`the native and transplant lung were similar (Fig. 1). The
`cyclosporine metabolites AM1 and AM9 were present in all
`tissues, except AM9 was not detectable in brain tissues.
`Median cyclosporine metabolite concentrations, except in
`gall bladder and cerebrum, were lower than cyclosporine
`concentrations. The concentrations of cyclosporine and its
`metabolites exceeded those in blood in most tissues (Table 2).
`Again, mainly due to the high variability, only cyclosporine
`concentrations in fat tissue, kidney, spleen, lymph nodes, gall
`bladder, and colon were significantly higher than in blood
`(P , .05).
`Effect of Cyclosporine on Tissue Distribution of
`SDZ-RAD (Group 1.5RAD 1 50Cs). Compared with the
`administration of each drug alone, combination of SDZ-RAD
`and cyclosporine affected the tissue concentrations of both
`drugs. As shown in Fig. 1B, SDZ-RAD concentrations had the
`tendency to increase in most tissues when coadministered
`with 50 mg/kg/day cyclosporine (group 1.5RAD 1 50Cs) com-
`pared with tissues from monkeys in the corresponding SDZ-
`RAD monotherapy group (group 1.5RAD). In contrast to most
`other organs, the average SDZ-RAD tissue concentrations in
`
`326
`
`Serkova et al.
`
`parison of the mass spectra and HPLC retention times with those of
`authentic standard material. Cyclosporine metabolites were isolated
`and their structures identified as previously described (Christians et
`al., 1991). SDZ-RAD was incubated with human liver microsomes
`and an NADPH-generating system. The metabolites were isolated by
`HPLC and their structures identified by MS and analysis of the
`fragment pattern after induction of nozzle-skimmer fragmentation
`as described by Vidal et al. (1998). Validation results for the quan-
`tification of metabolites were similar to those of the parent com-
`pounds.
`Clinical Monitoring of Transplant Animals. Laboratory
`screening was performed three times a week and included differen-
`tial blood counts, blood chemistry (serum lipid patterns, serum pro-
`tein, liver and kidney function parameters), and serum electrolytes.
`Lung transplant function and rejection was monitored by 1) chest
`radiographs (two times per week), 2) lung function tests with a
`Bicore CP-100 pulmonary monitor (Bicore Monitoring Systems, Ir-
`vine, CA), 3) bronchoscopy (day 4 and day 14 after surgery), and 4)
`open lung biopsies 2 and 4 weeks after transplantation. The histo-
`logical grade of rejection was classified according to the International
`Society for Heart and Lung Transplantation (A0, no rejection; A1,
`minimal rejection; A2, mild rejection; A3, moderate rejection; and
`A4, severe rejection; Yousem et al., 1996).
`Data Analysis. Data were processed with ChemStation software
`revision A04.02 for the HPLC system and C.03.00 for the electros-
`pray interface and MS (all from Hewlett-Packard). Concentrations of
`the immunosuppressants were calculated from an external standard
`curve and corrected on the basis of the internal standards. SPSS
`software, version 9.0, was used for statistical analysis (SPSS Inc.,
`Chicago, IL). Because data were not normally distributed, tissue
`concentrations in the combination therapy and respective control
`groups were compared with the nonparametric Mann-Whitney U
`test and results of distribution statistics are reported as median and
`range (minimum–maximum). Clinical chemical and biochemical
`data among groups were compared by multivariate ANOVA. Corre-
`lation (Pearson correlation coefficients, two-tailed test of signifi-
`cance) and stepwise regression analysis (probability-of-F-to-enter,
`.05) were based on the data of all study groups (n 5 25).
`
`Results
`Tissue Distribution of SDZ-RAD, Cyclosporine, and Their
`Metabolites
`Tissue Distribution of SDZ-RAD (Group 1.5RAD). Af-
`ter single daily oral SDZ-RAD doses (1.5 mg/kg) for 4 weeks
`and 24 h after the last dose, the blood concentrations of
`SDZ-RAD at the time of sacrifice ranged from 3.7 to 44 mg/l
`(median 5 9.2 mg/l) and those of its metabolites from 0 to 7.1
`mg/l (median 5 2.3 mg/l; n 5 4; Fig. 1). SDZ-RAD and its
`metabolites extensively distributed into tissues (Fig. 1; Table
`2). Median concentrations of SDZ-RAD and median total
`concentrations of its metabolites in most tissues exceeded
`those in blood. However, concentrations varied widely and,
`thus, the only organs that reached significantly higher tissue
`concentrations than in blood were pancreas and gallbladder
`(P , .05). Although there was a tendency to a higher median
`SDZ-RAD concentration in the transplant versus the native
`lung (Fig. 1), this difference was not statistically significant.
`It must be taken into account that due to the relatively small
`number of animals in the study groups, the statistical power
`for this analysis as well as all comparisons of the tissue
`concentrations reported below was less than 35%.
`Tissue Distribution of Cyclosporine (Group 100Cs).
`Cyclosporine was detected in all organ tissues examined in
`this study (Fig. 1). Twenty-four hours after the last oral dose,
`
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`SDZ-RAD Tissue Distribution
`
`327
`
`TABLE 2
`Comparison of tissue-to-blood distribution coefficients of SDZ-RAD and its metabolites with (50 mg/kg, group 1.5RAD 1 50Cs) and without
`cyclosporine (group 1.5RAD) and of cyclosporine and its metabolites with (0.3 mg/kg/day, group 0.3RAD 1 l00Cs) and without SDZ-RAD (group
`100Cs)
`The tissue-to-blood distribution coefficients were calculated by dividing the median concentration in tissue (nanograms per gram) by the median concentration in blood
`micrograms per liter.
`
`SDZ-RAD
`
`Metabolites
`
`Cyclosporine
`
`Metabolites
`
`Brainstem
`Cerebellum
`Cerebrum
`Colon
`Duodenum
`Fat
`Gall bladder
`Heart
`Ileum
`Jejunum
`Kidney
`Liver
`Lung (native)
`Lung (tx)
`Lymph node
`Pancreas
`Spleen
`Stomach
`Testes
`Thymus
`
`2Cs
`
`0.1
`4.3
`0.4
`1.7
`1.4
`0.2
`19.4
`0.8
`1.5
`1.2
`3.2
`0.6
`1.2
`12.6
`2.2
`82.6
`7.7
`0.7
`4.0
`2.3
`
`1Cs
`
`0.5
`1.1
`1.1
`5.8
`1.4
`1.1
`27.8
`1.1
`6.5
`3.2
`9.7
`2.2
`2.1
`1.7
`2.4
`8.8
`10.6
`1.8
`1.7
`1.7
`
`2Cs
`
`0.0
`3.3
`0.0
`5.1
`0.1
`1.7
`140.6
`2.1
`1.8
`1.8
`36.6
`8.6
`1.9
`21.4
`3.2
`551.4
`12.5
`1.3
`10.0
`30.9
`
`1Cs
`
`0.2
`0.2
`0.6
`2.9
`1.4
`0.5
`23.3
`1.0
`4.9
`3.8
`12.2
`2.8
`1.6
`3.5
`0.6
`3.7
`9.1
`0.7
`0.5
`1.2
`
`2RAD
`
`1RAD
`
`2RAD
`
`1RAD
`
`0.3
`0.2
`0.3
`13.5
`0.9
`4.1
`8.6
`1.6
`1.1
`1.6
`4.3
`2.9
`1.4
`2.1
`7.9
`2.8
`4.9
`1.3
`0.5
`2.9
`
`0.5
`0.4
`0.8
`3.5
`2.2
`1.5
`9.9
`1.4
`3.0
`3.5
`3.9
`2.1
`1.0
`2.0
`15.6
`1.9
`2.6
`1.4
`0.4
`2.3
`
`0.3
`0.3
`0.3
`6.1
`2.5
`1.0
`67.6
`3.9
`2.2
`2.1
`2.7
`3.1
`2.1
`3.3
`2.0
`5.5
`6.8
`2.1
`0.8
`2.4
`
`0.2
`0.3
`0.1
`0.8
`0.8
`0.3
`35.1
`0.8
`1.3
`2.1
`3.0
`3.4
`1.0
`2.2
`0.8
`1.5
`4.2
`0.6
`0.4
`0.7
`
`Cs; cyclosporine, RAD; SDZ RAD, tx, transplant.
`
`pancreas and cerebellum were lower in the presence of cyclo-
`sporine than in the SDZ-RAD monotherapy group (Fig. 2).
`This was because, compared with the other animals in this
`group, one monkey in the SDZ-RAD monotherapy group
`(1.5RAD) had extremely high concentrations in these organs
`(Fig. 1). Compared with the monotherapy group, with con-
`comitant cyclosporine the changes of SDZ-RAD concentra-
`tions in blood, ileum, duodenum, native lung, kidney, and
`brainstem (Fig. 1B) reached statistical significance (all P ,
`.04). As indicated by the tissue-to-blood partition coefficients
`[Ctissue (micrograms per gram)/Cblood (micrograms per liter)],
`the interaction with cyclosporine led to an increase of SDZ-
`RAD concentrations in most tissues, the extent of which was
`not predicted by blood concentrations (Table 2). In most tis-
`sues (Table 2; Fig. 2), cyclosporine increased the SDZ-RAD/
`metabolite ratio. Statistically significant correlations of SDZ-
`RAD trough blood concentrations with tissue concentrations
`were only observed in 10 of 20 tissues examined, and significant
`correlations for blood and tissue concentrations of SDZ-RAD
`metabolites were only observed in 2 of 20 tissues (Table 3).
`Effect of SDZ-RAD on Tissue Distribution of Cyclo-
`sporine (Group 0.3RAD 1 100Cs). SDZ-RAD had a
`smaller impact on cyclosporine tissue distribution than the
`inverse (Fig. 1, A and B, respectively). The Mann-Whitney U
`test did not show any statistically significant changes, in-
`cluding concentrations in blood and the transplant lung.
`Comparing the changes in all tissues, the effect of SDZ-RAD
`on median cyclosporine tissue/blood ratios lacked a consis-
`tent tendency (Table 2). In general, trough blood concentra-
`tions of cyclosporine and its metabolites showed a much
`better correlation with their tissue concentrations than SDZ-
`RAD and its metabolites (Table 3).
`
`Lung Transplant Rejection, Toxicity, and Clinical
`Monitoring (Table 4)
`Group 1.5RAD (1.5 mg/kg/day SDZ-RAD). At the end of
`the observation period, three of four animals exhibited severe
`
`rejection (A4) of the transplant lung. The median weight loss
`at the end of the study was 22% (Table 4). There were no
`signs of anemia or thrombocytopenia. Serum cholesterol con-
`centrations increased during the observation period (Table 4)
`but were not significantly different from the cyclosporine
`control group (group 100Cs).
`Group 100Cs (100 mg/kg/day Cyclosporine). At the
`end of the study, the transplant lungs in two animals showed
`moderate (A3) and in the other two animals severe rejection
`(A4). The median weight loss during the study was 15%.
`Serum cholesterol concentrations increased during the obser-
`vation period but the change was not statistically significant.
`No other changes in clinical chemical and biochemical pa-
`rameters were detected.
`Group 0.3RAD 1 100Cs (0.3 mg/kg/day SDZ-RAD 1
`100 mg/kg/day Cyclosporine). The combination of SDZ-
`RAD and cyclosporine was more effective in preventing re-
`jection of the lung allografts than monotherapy of either
`drug. At the end of the study, the biopsies of five animals
`showed a rejection score of A2 (mild acute rejection) and the
`biopsy of one animal had a rejection score of A3 (moderate
`acute rejection). However, in contrast to the monotherapy
`groups 1.5RAD and 100Cs, the animals developed significant
`anemia. Three animals required erythropoetin treatment
`and two animals required blood transfusions. Platelet counts
`were as low as 37,000/ml. These changes are not reflected in
`Table 4 because the table only includes the last values before
`sacrifice. From postoperative day 15 onward, serum choles-
`terol concentrations were higher in this group than in the
`control groups. However, the differences did not reach sta-
`tistical significance. The median weight loss in this group
`during the observation period was 22% (Table 4). Three of six
`animals had to be sacrificed early due to anemia and renal
`failure (n 5 2; days 20 and 23) or seizures (n 5 1; day 22).
`Group 1.5RAD 1 50Cs (1.5 mg/kg/day SDZ-RAD 1 50
`mg/kg/day Cyclosporine). At the end of the study, the lung
`
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`Vol. 294
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