MYLAN - EXHIBIT 1058
`Mylan Pharmaceuticals Inc. et al. v. Allergan, Inc.
`IPR2016-01127, -01128, -01129, -01130, -01131, & -01132
`
`

`

`CONSENSUS REPORT ON CYCLOSPORIN
`
`643
`
`7. In the immediate posttransplant period, the
`recommended frequency of monitoring is
`once-every 24 to 48 h.
`8. The laboratory should be able to provide
`same-day turnaround during the early post-
`transplant period.
`9. CsA concentrations need to be interpreted in
`conjunction with other laboratory data, clin-
`ical considerations, and concomitant immu—
`nosuppressive therapy.
`10. In the majority of clinical situations, the mon-
`itoring of CsA metabolites is not warranted.
`11. There is a need to develop assay systems ca-
`pable of measuring the individual patient
`state of immunosuppression.
`
`SAMPLE-COLLECTION TIME AND
`PHARMACOKINETIC MONITORING
`
`Whole blood with EDTA as anticoagulant is the
`preferred matrix for CsA measurement. Whenever
`possible, peripheral venopuncture sampling should
`be preferred over sampling from a resident central
`venous line or capillary testing. It is also important
`to note that CsA displays a circadian variation, eve-
`ning trough levels being significantly lower than
`morning trough levels (3).
`The marked intra— and interindividual variation in
`
`cyclosporin pharmacokinetics (4,5) complicates
`effective immunosuppressive therapy. There is a
`poor correlation between CsA dose and whole
`blood trough concentration. Early investigations of
`the therapeutic window of CsA documented that
`CsA trough levels far outside a therapeutic range
`tended to predict adverse events (4). The relation
`between cyclosporin trough concentration and im-
`munosuppressive efficacy or toxicity after organ
`transplantation has been widely studied (6), and
`currently most centers use trough CsA blood con-
`centrations as a guide for the dosage of this drug.
`However, patients displaying trough concentra-
`tions within a putative CsA therapeutic range are
`not always spared from either rejection or nephro—
`toxicity (7). In an attempt to understand the phar-
`macokinetic factors critical to outcome, a concen-
`tration-controlled strategy (8) based on serial con—
`centration—time profiles was applied to determine
`appropriate drug doses. Because of variations in-
`trinsic to the conventional oral liquid and gel cap
`formulations of CsA,
`this strategy was based on
`establishing a range of drug concentrations during
`continuous intravenous infusion and after oral ad-
`
`ministration. This range of concentrations allowed
`identification of optimal targets during therapy:
`steady-state concentrations of 400 ug/L during iv
`and initial l-month average concentrations of 550
`ug/L, tapering to a target of 400 pug/L from 6 to 12
`months (9). Strategies using 3-point samplings seem
`to yield reasonable estimates of actual AUC (10)
`and in practice have proven equally efficient as full
`7-point profiles (0, 2, 4, 6, 10, 14, and 24 h for q.d.
`and 0, 2, 4, 6, 8, 10, and 12 h for b.i.d.). The avail-
`ability of the new microemulsion formulation of
`CsA (Neoral) may streamline the endeavor, be-
`cause with this formulation, trough levels show an
`improved correlation with AUC (r2, 0.81 versus
`0.50 with a standard nonmicroemulsion formula-
`
`tion) and achieve good estimates of full AUC with
`only a 2— and 6-h sampling during a 12-h dosing
`interval (11). The use of the new microemulsion for-
`
`mulation is associated with reduced variability in
`CsA kinetics, implying that single trough levels will
`be more consistent. With this formulation, CsA ab-
`sorption is less dependent on bile acids and not af-
`fected by food intake (12). It
`is hoped that the
`greater reproducibility of CsA pharmacokinetic
`profiles when using the new microemulsion formu-
`lation (11,13) will facilitate dosage adjustment to
`compensate for inter— and intraindividual differ-
`ences and will facilitate the incorporation of phar-
`macokinetic approaches into transplant care. The
`major practical limitation, however, for AUC mon-
`itoring is the necessity for blood collections to be
`made at precisely timed intervals after oral dosing.
`So far, comparative studies on AUC versus
`trough-level monitoring have not consistently
`shown overall superiority of either method (9).
`Well—designed, preferably randomized prospective
`clinical trials, including the new microemulsion for-
`mulation and employing specific, well-validated as-
`says to measure CsA, will help to resolve this issue.
`In such studies, assay specificity and precision
`must be documented. There is widespread consen-
`sus that at present, trough-concentration monitor-
`ing is the most appropriate and practical (Table 1).
`
`FREQUENCY OF MONITORING
`
`The frequency of blood CsA concentration mon—
`itoring should depend on the time elapsed since
`transplantation, intercurrent illnesses, and concom-
`itant therapy with drugs affecting CsA metabolism.
`The immediate posttransplant period is character—
`ized by unstable graft function, extreme inter- and
`
`Ther Drug Monit, Vol. 17, No. 6, 1995
`
`

`

`644
`
`M. OELLERICH ET AL.
`
`TABLE 1. Therapeutic ranges for CsA obtained from a survey of transplant centers during 1994/1995
`Immuno-
`
`Analytic
`Dosing
`suppression
`Therapeutic
`Transplant
`
`Center [No.]
`method
`interval
`protocol
`ranges (pg/L)
`type
`
`HPLC
`
`b.i.d.
`
`University of
`Pennsylvania
`Medical Center,
`Philadelphia, PA,
`U.S.A. [1]
`
`mFPIA
`
`Oklahoma
`Transplantation
`Institute, Oklahoma
`City, OK, U.S.A.
`[2]
`Georg—August-Universitat, EMIT
`Gottingen, FRG [3]
`
`b.i.d.
`
`b.i.d.
`
`b.i.d.
`
`b.i.d.
`
`m 125I-RIA
`
`b.i.d., t.i.d.
`
`mFPIA
`
`b.i.d., t.i.d. in
`children <24
`mo
`
`St. Christophers
`Hospital for
`Children,
`Philadelphia, PA,
`U.S.A. [4]
`
`University of
`Cincinnati Medical
`Center, Cincinnati,
`OH, U.S.A. [5]
`
`Universitats
`Krankenhaus
`Eppendorf,
`Hamburg, FRG [6]
`Hospital for Sick
`Children, Toronto,
`ON, Canada [7]
`
`111
`
`111
`111
`111
`III
`III
`
`II
`
`111
`
`II + ALA
`
`IV
`
`111
`
`111; I, 12—18 mo
`
`III
`
`IV
`
`IV
`
`IV
`
`100—250 <3 mo
`80—125 >3 mo
`200—300
`200—300 <12 mo
`100-150 >12 mo
`250—350 <12 mo
`200—300 >12 mo
`
`400—500 <6 mo
`200—400 >6 mo
`
`150—200 <3 mo
`100—150 >3 mo
`150—200 <3 mo
`100—150 >3 mo
`250—350 <3 mo
`150—250 >3 mo
`
`100—200 <3 mo
`75—150 >3 mo
`250—350 <3 mo
`150—250 >3 mo
`250—350 <3 m0
`100—200 >3 mo
`
`250-375 <6 mo
`100—250 >6 mo
`350—450 <1 mo
`250—350 2—6 mo
`170—240 >6 mo
`300—420 <6 wks
`180—300 6—12 wks
`120—180 >12 wks
`
`mFPIA
`
`b.i.d.
`
`II, III in some
`patients
`
`200—250 <3 mo
`150—250 >3 mo
`
`HPLC
`
`b.i.d.
`
`III
`
`111
`
`III
`
`175—225 <3 mo
`150—175 3—12 mo
`100-125 >12 mo
`300—400 <3 mo
`250—300 3-12 mo
`200—250 12—18 mo
`80-200 >18 mo
`250—325 <6 mo
`200—250 6—12 mo
`150—200 >12 mo
`
`K
`
`L
`H
`Lu
`
`L
`
`K
`
`H
`
`K
`
`H
`
`K & K-Panc
`
`L
`
`H
`
`L
`
`K
`
`L
`
`H
`
`Ped K
`
`b.i.d.
`
`111
`
`University of
`California Los
`Angeles (UCLA)
`Medical Center, Los
`Angeles, CA,
`U.S.A. [8]
`
`HPLC and
`pFPIA
`Pediatric K
`only. The
`higher
`concentrations
`pertain to
`pFPIA
`
`250—375
`800—1,000 <1 mo
`225—300
`700—900 1—2 mo
`200-250
`500—750 2—3 mo
`125—200
`400—600 3—4 mo
`100—175
`350—500 4—6 mo
`100—150
`325—425 >1 year
`
`Ther Drug Monit, Vol. 17, No. 6, 1995
`
`

`

`CONSENSUS REPORT ON CYCLOSPORIN
`
`645
`
`TABLE 1. Continued
`
`Immuno-
`Transplant
`Therapeutic
`Dosing
`Analytic
`suppression
`method
`interval
`type
`Center [No.]
`protocol
`ranges (ug/L)
`350—450
`mFPIA and
`III
`b.i.d.
`BOO—1,000 <3 mo
`pFPIA. The
`100—150
`higher
`200—350 >3 mo
`concentrations
`280—300
`pertain to
`500—800 <3 mo
`pFPIA
`200—400
`600—800 <3 mo
`100—200
`400—600 >3 mo
`
`Adult K
`
`Adult L
`
`Adult H
`
`III
`
`III
`
`St George’s Hospital,
`The Medical School,
`London, UK. [9]
`
`Mayo Clinic,
`Rochester, MN,
`U.S.A. [10]
`
`m 125I-RIA
`
`b.i.d.
`
`HPLC
`
`b.i.d.
`
`III
`Steroids stopped
`at 3 mo
`
`III
`
`III
`
`IV
`
`III
`
`III <3 mo
`11 >3 mo
`
`300—350 <6 wk
`200—250 6 wk—6 mo
`150—200 6 mo—l year
`80—120 >1 year
`150—250 <2 wk
`150—200 <2 mo
`100—150 >4 mo
`350
`<2 wk
`250—350 <2 mo
`150—250 <4 mo
`100—150 >4 mo
`250—350 2 wks—2 mo
`75—125 >2 mo
`
`120—200 <3 mo
`100—160 >3 mo
`250—300 <3 mo
`200—250 3—12 mo
`100—150 >12 mo
`
`<1 mo
`550
`2—3 mo
`500
`3—6 mo
`450
`6—12 mo
`400
`12—24 mo
`350
`>24 mo
`300
`800—1,200 <1 mo
`700—1,200 >1 mo
`150—250
`200—300
`150—250
`200—350 <2 mo
`150—250 >3 mo
`
`200—250 <1 mo
`150—200 1—2 mo
`100—150 2—3 mo
`75—100 >3 mo
`200
`
`K, Panc
`
`Panc
`
`For H only,
`pFPIA and
`plasma/37°C
`HPLC
`
`b.i.d.
`
`mFPIA
`
`pFPIA
`
`mFPIA
`
`Dosing based on
`pharmacokinetic
`studies to
`achieve
`steady-state
`conc.
`b.i.d.
`
`b.i.d.
`
`mFPIA
`
`b.i.d.
`
`II
`
`II
`
`III
`III
`III
`III
`
`IV
`
`IV
`
`Princess Alexandra
`Hospital, Brisbane,
`Australia [1 1]
`
`University of Texas
`Health Science
`Center, Medical
`Center, Houston,
`TX, U.S.A. [12]
`
`University of Virginia
`Medical Center,
`Charlottesville, VA,
`U.S.A. [13]
`
`St. Johns Hospital &
`Medical Center,
`Detroit, Michigan,
`U.S.A. [14]
`
`University of Alberta
`Hospitals,
`Edmonton, AL,
`Canada [15]
`
`mFPIA
`
`b.i.d.
`
`III
`
`III
`
`300—400 <2 wks
`250—300 2—4 wks
`200—250 1—3 mo
`150—200 3—6 mo
`100—150 6—12 mo
`100-125 >12 mo
`300—350 <30 days
`250—350 30—60 days
`250—300 60—90 days
`200—250 90—180 days
`175—225 180 days—l year
`150—175 >1 year
`III
`350—500 <90 days
`
`300—350 >90 days
`
`

`

`646
`
`M. OELLERICH ET AL.
`
`TABLE 1. Continued
`Immuno-
`Analytic
`Dosing
`suppression
`Therapeutic
`Transplant
`
`Center [No.]
`method
`interval
`protocol
`ranges (pug/L)
`type
`111
`400—500 <90 days
`H-Lu
`400—500 >90 days
`
`Clin-Tox Associates,
`Germantowu, TN,
`U.S.A. [16]
`
`St. Vincent‘s Hospital,
`Darlinghurst, NSW,
`Australia [17]
`
`pFPIA
`Serum
`
`mFPIA
`
`b.i.d.
`
`b.i.d.
`
`West Virginia
`University
`Hospitals,
`Morgantown, WV,
`U.S.A. [18]
`
`University of North
`Carolina Hospitals,
`Chapel Hill, NC,
`U.S.A. [19]
`
`mFPIA
`
`b.i.d.
`
`mFPIA
`
`b.i.d.
`
`Queen Elizabeth
`Hospital,
`Birmingham, U.K.
`[20]
`
`Huddinge Hospital,
`Stockholm, Sweden
`[21]
`
`mFPIA
`
`b.i.d.
`
`m 125I—RIA
`
`b.i.d.
`
`Neues Allgemeines
`Krankenhaus,
`Vienna, Austria [22]
`Northwestern
`University Medical
`School, Chicago, IL,
`U.S.A. [23]
`
`mFPIA
`
`mFPIA
`
`HPLC
`
`b.i.d.
`
`b.i.d.
`
`b.i.d.
`
`III
`
`III
`
`111
`
`111
`
`III
`
`111
`
`IV
`
`IV in children.
`Prednisone
`stopped after
`3 mo
`IV
`
`111
`
`111
`
`III
`
`IV
`
`II
`
`II + ALA
`111
`
`IV
`
`175~225 <30 days
`125—175 30—90 days
`75—125 >90 days
`
`250-375 <6 mo
`100—250 >6 mo
`350—450 >2 mo
`300—400 2—3 mo
`250—300 3—6 mo
`200-300 6—12 mo
`150—200 >12 mo
`
`250—375 <6 mo
`100—250 >6 mo
`
`~200 initial post Tx
`150—200 >3 mo
`350
`>6 mo
`300
`6—12 mo
`200450 <12 the
`400—500 <1 wk
`250—350 2~3 wks
`200—300 3—4 wks
`180—280 >4 wks
`350
`<6 mo
`300
`6—12
`250
`>12
`
`400—500 <1 wk
`250—350 >2 wks
`
`100—300
`
`250—350 <1 mo
`200—300 1—2 mo
`150—250 2—3 mo
`70—150 >3 mo
`350—450 <9 days
`250—300 9 days—3 mo
`200—250 3—4 mo
`150—200 >4 mo
`300—400 <1 mo
`200-300 1—2 mo
`100—200 2—3 mo
`~100 >3 mo
`
`1254250 <3 mo
`100—200 >3 mo
`125—250
`250—400 <6 mo
`250—300 >6 mo
`200
`< 6 mo
`200
`<6 mo
`
`H
`
`K
`
`H, H-Lu
`
`K
`
`L
`
`H-Lu
`
`Panc
`
`L
`
`K
`
`L
`
`K-Panc, Pane
`
`K
`
`L
`K
`
`L
`Panc
`
`Univ.-Klinikum Rudolf
`Virchow, Berlin,
`PRO [24]
`Academisch
`Ziekenhuis,
`Groningen, Holland
`[25]WW—
`
`mFPIA
`
`HPLC
`
`b.i.d.
`
`b.i.d.
`
`IV
`
`III
`
`2004300 <4 wks
`100—200 >4 wks
`
`200—250 <4 wks
`100—150 >4 wks
`
`L
`
`L
`
`

`

`CONSENSUS REPORT ON CYCLOSPORIN
`
`647
`
`TABLE 1. Continued
`
`Immuno-
`Analytic
`Dosing
`suppression
`Therapeutic
`Transplant
`
`Center [No.]
`method
`interval
`protocol
`ranges (ptg/L)
`type
`
`Hopital Paul Brousse,
`Villejuif, France [26]
`
`mFPIA
`
`b.i.d.
`
`The Liver Unit, The
`Children’s Hospital,
`Birmingham, UK.
`[27]
`Addenbrooke’s
`Hospital,
`Cambridge, UK.
`[28]
`
`Papworth Hospital,
`Cambridge, UK.
`[29]
`
`University of
`Michigan, Ann
`Arbor, MI, U.S.A.
`[30]
`
`HPLC
`
`m 12SI-RIA
`
`EMIT
`
`HPLC
`
`King’s College,
`London, UK. [31]
`
`mFPIA
`
`Virginia
`Commonwealth
`University,
`Richmond, VA,
`U.S.A. [32]
`Hospital das Clinicas,
`Universidade de Sao
`Paulo, Brasil [33]
`University of
`Nebraska Medical
`Center, Omaha, NE,
`U.S.A. [34]
`
`’
`
`mFPIA
`
`m 125I-RIA
`
`pFPIA
`
`b.i.d.
`
`b.i.d.
`
`b.i.d.
`
`b.i.d.
`
`b.i.d.
`
`b.i.d.
`
`q.d.
`b.i.d.
`
`t.i.d.
`
`t.i.d.
`b.i.d.
`
`b.i.d.
`
`b.i.d.
`
`b.i.d.
`
`b.i.d.
`
`b.i.d.
`
`111
`
`III
`
`III
`
`III
`
`III
`
`III
`
`111
`
`IV
`IV
`
`IV
`
`III
`IV
`
`111
`
`111
`
`IV
`
`111
`
`II
`
`L
`
`L
`
`K
`
`Adult L
`
`Ped L
`
`H
`
`H-Lu
`
`K
`L
`
`H
`
`Lu
`Panc
`
`Adult L
`
`Ped L
`
`L
`
`K
`
`L
`
`400—500 1—15 days
`300—450 15—30 days
`250—300 30—90 days
`150—200 >12 mo
`
`<41/2 mo
`>250
`150—200 41/2—12 mo
`80—120 >12 mo
`
`200—250
`175—250
`200-250
`175—250
`150—250
`
`i.v.
`oral
`i.v.
`oral
`
`300—400 <3 mo
`200—300 3—12 mo
`100—250 >12 mo
`300—500 <3 mo
`200—300 3—12 mo
`100—250 > 12 mo
`
`100—150
`<3 mo
`150
`100—150 >3 mo
`200—250 <6 mo
`150—200 6—12 mo
`100—150 12—24 m0
`75—100 224 mo
`100—300
`225—275 <1 mo
`175—225 1—2 mo
`125—175 >2 m0
`
`150—250 <3 wks
`100—200 3—8 wks
`<125
`>12 wks
`150—250 <6 mo
`100—150 6—12 mo
`50—100 > 12 mo
`
`400
`300
`200
`
`<6 mo
`6—12 mo
`>12 mo
`
`160—200 <6 mo
`100—150 >6 mo
`
`900—1,100 >2 mo
`600—800 2—4 mo
`500—600 4—6 mo
`400—500 6—12 mo
`300—400 >12 mo
`
`Medizinische
`Hochschule,
`Hannover, FRG [35]
`
`EMIT
`
`b.i.d.
`
`100—200 <3 mo
`50—150 >3 mo
`250—300 <12 mo
`200—250 >12 mo
`L
`100—200 <3 mo
`11 + ALA
`50—150 >3 m0
`
`IV or
`II + ALA
`IV
`
`K
`
`H, H-Lu
`
`Except where listed otherwise, all centers use whole blood specimens, obtained just before the next dose, for CsA determinations.
`HPLC, high—performance liquid chromatography; mFPIA, monoclonal antibody Abbott TDx fluorescence polarization immunoassay;
`pFPIA, polyclonal antibody Abbott TDX fluorescence polarization immunoassay; m 125I-RIA, monoclonal antibody INCSTAR radio-
`immunoassay; EMIT, monoclonal antibody Syva enzyme-multiplied immunoassay; b.i.d., twice-daily dosing; t.i.d., three times daily
`dosing; I, CsA monotherapy; 11, “double therapy“: CsA + prednisone; III, “triple therapy”: CsA + prednisone + azathioprine; IV,
`“quadruple therapy,” induction therapy in which anti-lymphocyte antibody (ALA) is used as part of initial immunosuppression until
`good kidney function is achieved <14 days after surgery. Many centers use quadruple therapy in transplant patients with poor initial
`renal function only. This use of IV is not specifically defined for each center. Only when IV is used in all patients for a particular
`transplant type is this protocol noted; K, kidney; H, heart; L, liver; Lu, lung; Pane, pancreas; Ped, pediatric.
`
`

`

`648
`
`M. OELLERICH ET AL.
`
`intraindividual variability in the pharmacokinetics
`of the drug, and an enhanced risk of acute infection.
`During this early critical period, a monitoring
`schedule of four to seven samples per week would
`appear to be appropriate for kidney, liver, and heart
`transplant recipients (6). In liver transplantation,
`regular monitoring is essential after the start of oral
`CsA to compensate for inter— and intraindividual
`variation in pharmacokinetics at this stage of clini-
`cal and pharmacologic instability. Because the new
`microemulsion formulation of CsA appears to be
`less dependent on bile acids for its absorption, it
`may help to reduce such pharmacokinetic variabil-
`ity and thereby reduce requirements for such fre-
`quent CSA monitoring. After the intensive early
`monitoring, the measurement of cyclosporin blood
`levels can be gradually reduced; for example,
`in
`renal transplant recipients with an uncomplicated
`clinical course, CsA should be monitored once a
`month during the first year and at 1 to 3-month in-
`tervals thereafter. However, there are no hard and
`fast rules, and measurements should be performed
`if the clinical signs of symptoms suggest that dosage
`adjustment might be necessary. Furthermore, addi-
`tional measurements should be performed when the
`patient has to be treated with drugs known to inter-
`act with the metabolism or excretion (or both) of
`CsA. If adjustment is required, this should be fol—
`lowed—up with a second blood-concentration mea-
`surement within the following week. It is important
`that CsA measurements be made available on the
`
`same day if they are to influence therapy. This is
`feasible with the latest generation of semiautomated
`immunoassays, which offer a same-day service
`even with a large workload.
`
`METHODS OF MEASUREMENT
`
`Carefully validated high-performance liquid chro—
`matography (HPLC) methods that measure the par—
`ent drug specifically can be used as reference
`procedures for CsA measurement. A definitive
`method, however, or a reference method according
`to the IFCC criteria (14) is not yet available. Recent
`reports (6,15,16) have noted a trend toward the use
`of semiautomated immunoassay techniques involv—
`ing selective monoclonal antibodies rather than
`HPLC for routine measurement of CsA. Data from
`
`the UK Cyclosporine Quality Assessment Scheme
`(UKCQAS), which is predominantly European
`based, show that most laboratories are using one of
`the following three immunoassays: the INCSTAR
`
`Ther Drug Monit, Vol. 17, No. 6, 1995
`
`CYCLO-Trac SP radioimmunoassay (m 125I-RIA),
`the Abbott TDx monoclonal antibody fluorescence
`polarization immunoassay (mFPIA), and the Syva
`enzyme multiplied immunoassay technique
`(EMIT). For nonspecific measurements, both the
`Abbott TDx polyclonal antibody fluorescence po—
`larization assay (pFPIA) and the Incstar nonspecific
`monoclonal RIA are available. The nonspecific RIA
`detects a broader range of metabolites than the pF-
`PIA. Data from other schemes and a recent survey
`summarized in Table 1 are in broad agreement with
`these findings. A receptor assay approach for the
`quantification of CsA that utilizes the specific bind~
`ing of the latter to certain immunophilins (17) is
`currently being evaluated (18).
`The choice of analytic technique depends on a
`number of factors, including available instrumenta—
`tion,
`the technical expertise available, sample
`throughput, the requirement for after—hours work,
`the clinical indications for the use of cyclosporin,
`and local regulations governing the use of radio-
`chemicals.
`
`In the previous consensus conference (19), the
`following performance characteristics were recom—
`mended for a method to be acceptable for selective
`determination of the parent drug CsA:
`
`1. Imprecision: Coefficient of variation of S10%
`at a CsA concentration of 50 ug/L and of <5%
`at a concentration of 300 ug/L.
`2. Accuracy (method comparison with a vali—
`dated HPLC reference method): Slope of the
`line: s10% from the line of identity, intercept
`<15 ug/L; sy/XzsIS pug/L.
`
`These precision requirements should refer to be-
`tween-days imprecision. For the statistical evalua—
`tion of method comparison, a bivariate procedure
`[e.g., principal component analysis (20) or weighted
`Deming (21)], or a nonparametric rank procedure
`[e.g., Passing/Bablok method (21,22)] should be
`used.
`
`PERFORMANCE CHARACTERISTICS
`
`The performance characteristics of the specific
`semiautomated cyclosporine immunoassays have
`recently been reviewed (6). The coefficients of vari—
`ation at CsA concentrations between 50 ug/L and
`~350 ug/L ranged from 7 to 15% with EMIT, 3 to
`10% with mFPIA, and 5 to 11% with m IZSI-RIA.
`With an improved modification of the EMIT, be—
`tween-days coefficients of variation were 5 and 8%
`
`

`

`CONSENSUS REPORT ON CYCLOSPORIN
`
`649
`
`at CsA concentrations of 95 Mg/L and 225 ug/L,
`respectively (23). Precision requirements as pro-
`posed by the previous consensus conference were
`best fulfilled by mFPIA compared to the remaining
`immunoassays (6,24).
`The mean overestimation of CsA concentrations
`
`compared with HPLC due to the presence of cross-
`reactivity with CsA metabolites ranged from 8 to
`30% with EMIT, 24 to 48% with mFPIA, and 22 to
`30% with m 125I-RIA. As the EMIT showed the
`highest specificity and exact calibration, this test
`best fulfilled the proposed performance criteria for
`accuracy. The reasons for the discrepancies be-
`tween the methods include specificity of the anti-
`bodies used in the immunoassays and between-
`method differences in the calibration. Particularly
`in patients who may show an accumulation of CsA
`metabolites (e.g.,
`liver transplant recipients), re-
`sults for the same sample can differ by as much as
`100%, depending on which of the specific assays is
`used. Because CsA metabolites do not significantly
`contribute to overall immunosuppression, the use
`of less specific assays in such patients may result in
`the physician underdosing the patient. Assay-
`related differences contribute to the disparity in tar-
`get concentration ranges quoted by individual cen-
`ters (Table 1). The impact of such discrepancies on
`clinical decision making requires further clarifica—
`tion. One area in which analytic accuracy is of par—
`ticular relevance is in the conduct of randomized
`concentration-control clinical trials (25). Because
`such trials are likely to involve a multicenter design,
`constancy of results, in terms of both accuracy and
`precision, is of importance, particularly if multiple
`laboratories rather than a central facility are used to
`monitor concentrations.
`
`QUALITY ASSESSMENT
`
`Previous consensus documents on CsA monitor—
`
`ing have recommended that laboratories should par-
`ticipate in external quality assessment programs
`(16,19). It is also mandatory that laboratories offer—
`ing a service for measurement of cyclosporin have a
`system in place to verify the day-to—day constancy
`of their results using at least one measure of internal
`quality control. National external quality control
`schemes have been set up in various countries. The
`U.K. CQAS Scheme (6), for example, currently
`covers >280 centers in 38 countries. The most re-
`cent findings from this scheme show that >90% of
`the results reported relate to whole blood samples,
`
`and 89% of these are produced by methods with a
`high specificity for the parent compound (6). Re-
`sults from the Canadian Quality Assurance Program
`confirm that HPLC, RIA, and EMIT methods are
`more specific than FPIA (26). To verify correct cal-
`ibration, whole blood samples that have been
`spiked with standard reference material (Cyclospor-
`ine USP-RS; Cat. No. 15850) should be used. For
`the assessment of the specificity of the applied
`method, samples containing various amounts of the
`major cyclosporin metabolites,
`in addition to the
`parent drug, should be a part of the external quality
`control scheme.
`
`CYCLOSPORIN METABOLITES
`
`The clinical relevance of CsA metabolites is still a
`
`controversial issue (6,27). Some immunosuppres-
`sive activity has been found in vitro for metabolites
`AMI, AM9, and AM4N (28—30). Although overim-
`munosuppression induced by high concentrations
`of cyclosporin metabolites could increase the risks
`of serious infection, the relative toxicities of cyclo-
`sporin metabolites are probably more important
`clinically. There is some evidence that high blood
`concentrations of metabolites AMlc9 and AM19
`
`are associated with nephrotoxicity in the early post-
`operative period after liver transplantation (31).
`From the study design, however, it was not possible
`to ascertain whether high metabolite concentrations
`were the cause or the consequence of renal dys-
`function. In vivo studies in rats receiving subcuta-
`neous doses of AMI, AMIA, AMlc, and AM4N
`could not demonstrate renal or hepatic dysfunction
`after exposure to these metabolites (32). Because of
`the known differences in the metabolization of CsA
`in rats and humans, and interindividual susceptibil-
`ities to CsA toxicity, definite conclusions cannot be
`made as to the contribution of CsA metabolites to
`toxicity in humans.
`Some centers have advocated the parallel use of
`both nonspecific and specific methods to gain an
`insight into the ratio between CsA and its metabo-
`lites in transplant recipients with severely disturbed
`liver function (33). They suggested that concentra—
`tions of parent drug and metabolites >1,200 pug/L,
`as determined with the Incstar nonspecific mono-
`clonal RIA (33), should be avoided. For the large
`majority of clinical situations, however, monitoring
`of CsA metabolites seems not to be warranted. The
`failure of pFPIA to distinguish CsA nephrotoxicity
`from rejection (34,35) would argue against a signif-
`
`Ther Drug Monit, Vol. 17, No. 6, I995
`
`

`

`650
`
`M. OELLERICH ET AL.
`
`icant role of metabolites. From the available data, it
`cannot be decided whether the specific measure—
`ment of individual cyclosporin metabolites will be
`of clinical significance.
`
`THERAPEUTIC RANGES AND
`IMMUNOSUPPRESSION PROTOCOLS
`
`In contrast to the concept of reference intervals
`in clinical chemistry, there is no generally accepted
`concept or protocol on how to establish the thera-
`peutic range of a drug. There is consensus that it
`represents the range of drug concentrations within
`which the probability of the desired clinical re-
`sponse is relatively high and the risk of unaccept-
`able toxicity is relatively low. This implies that a
`given concentration within the therapeutic range is
`not necessarily safe and effective for every patient.
`It is difficult to establish a therapeutic range for
`CsA, because there are no simple parameters for
`the assessment of the immunosuppressive effect.
`On the other hand, the lack of such criteria and the
`marked intra— and interindividual variation in CsA
`
`pharmacokinetics are a strong argument for immu-
`nosuppressive drug monitoring to prevent over- or
`underimmunosuppression. There is general agree—
`ment that CsA monitoring has helped to avoid toxic
`concentrations. On the basis of their own and pub—
`lished experience, transplant centers have derived
`therapeutic ranges empirically for the different
`transplant types. These ranges show substantial
`variability between centers (Table 1). Differences
`relate mainly to the specificity of the analytic meth—
`ods used for measurement of CsA and the various
`immunosuppression protocols employed, including
`center-specific preference in the degree of initial ex-
`posure of patients to CsA.
`To facilitate the establishment of consensus ther-
`
`apeutic ranges for the different transplant types, the
`following points have to be considered: use of se—
`lective assays with standards and samples in whole
`blood matrix, sampling time, and time interval from
`transplantation, biochemical and morphologic crite-
`ria for rejection or toxicity, and influence of immu-
`nosuppression protocol.
`The data obtained by us in a survey conducted in
`1994 through 1995 on the acceptable therapeutic
`ranges for CsA concentration are summarized in
`Table 1. The use of time—dependent target ranges of
`CsA is widespread. Most centers recommend a
`higher range of target concentrations during the
`
`Ther Drug Monit, Vol. 17, No. 6, 1995
`
`early postoperative period. Doses are then tapered
`to a lower maintenance concentration range, usu-
`ally 3 to 6 months after transplantation, although
`some centers extend this tapering period over 12
`months or more.
`The median values for the minimum and maxi-
`
`mum concentrations in pre-dose trough samples ob-
`tained with mFPIA, m 125I-RIA, and HPLC are
`shown in Table 2. As can be expected, with similar
`immunosuppression protocols, centers using mF-
`PIA for monitoring report median upper and lower
`limits that are on average ~28% (range, 0—67%)
`higher than those given by centers using specific
`HPLC methods. Surprisingly, the median m 125I-
`RIA/HPLC ratio of the therapeutic window limits
`was 1.00 (range, 0.72—1.59). The similarity in the
`therapeutic ranges from centers using either HPLC
`or m 125I—RIA is even more astonishing in View of
`the fact that the m 125I—RIA overestimates the CsA
`concentration by 22—30%. The target ranges for
`CsA should preferably be defined on the basis of
`specific measurement of the parent drug. Manufac-
`turers should be encouraged to improve their im-
`munoassays accordingly. However, it is also clear
`(Table 1) that organ- and time-specific therapeutic
`ranges vary widely, even between centers using the
`same analytic procedure to determine CsA and
`comparable immunosuppression protocols. As an
`example, Fig. 1 shows therapeutic CsA ranges used
`in kidney transplant recipients. This possibly re-
`flects the different assessment of CsA toxicity risk
`on the one hand and the consequences of underdos-
`ing (acute or chronic rejection) on the other hand.
`Thus there has been increasing emphasis in some
`centers over the past few years on using higher dos-
`ages and maintaining higher CsA blood concentra-
`tions (36). In a recent publication, Soin et a1. (37)
`found a significantly lower incidence of chronic re-
`jection in those patients who were maintained on
`median CsA whole blood trough levels of 2175
`ug/L in the first 28 days after transplantation.
`A review of the information provided in Table 1
`shows that the concentration ranges are somewhat
`higher for heart transplant than for kidney and liver
`transplant patients. Higher CsA doses are used in
`heart or heart—lung transplant recipients as an extra
`measure of immunosuppression because these or-
`gans are more difficult to replace. The consequence
`of intractable acute rejection in heart or heart—lung
`recipients is usually death. In heart—lung recipients,
`the CsA target whole blood concentrations range
`from 350 to 600 tug/L during induction therapy (ap-
`
`

`

`CONSENSUS REPORT ON CYCLOSPORIN
`
`651
`
`TABLE 2. Therapeutic ranges for cyclosporin stratified according to
`transplanted organ, immunosuppressive regimen, induction/maintenance therapy
`and immunoassay technique
`
`
`Kidney
`Heart
`L‘Ver
`
`Method
`Triple therapy
`Triple therapy
`Triple therapy
`Double therapy
`Induction“
`HPLC
`mFPIA
`m 12sI-RIA
`EMIT
`Maintenance
`HPLC
`mFPIA
`m 125I-RIA
`EMIT
`
`150—225 (5)
`250-375 (6)
`160—200 (5)
`125—200 (2)
`
`100—150 (5)
`100—250 (8)
`75—150 (5)
`75—150 (2)
`
`250—325 (1)
`300—400 (3)
`250—325 (2)
`275—375 (2)
`
`125—175 (2)
`150—250 (5)
`90—160 (2)
`150—250 (3)
`
`225—300 (4)
`250—313 (8)
`250—300 (3)
`
`100—150 (6)
`135—200 (8)
`150—238 (4)
`
`300—375 (2)
`
`125—200 (2)
`
`100—150 (1)
`150—250 (3)
`
`75—150 (2)
`
`The ranges are the median values (pg/L) for the minimum and maximum trough cyclosporin
`concentrations (whole blood) calculated from the data of those centers listed in Table 1 that
`fitted the particular category. The number of contributing centers is given in parentheses.
`HPLC, high—performance liquid chromatography; mFPIA, monoclonal antibody fluorescence
`polarization immunoassay; m ‘ZSI-RIA, monoclonal antibody INCSTAR radioimmunoassay;
`EMIT, enzyme-multiplied immunoassay technique.
`a In some centers, anti-lymphocyte antibodies were also included as part of induction ther-
`apy.
`
`prox. S3 months after transplantation) and from
`200 to 300 pug/L during maintenance therapy.
`A high fluctuation in blood CsA levels was found
`to increase the short—term risk of acute lung or kid-
`ney rejection (38,39). Thus efforts should be di-
`rected to maintaining stable blood CsA concentra-
`tions. The tendency of reducing maintenance immu-
`nosuppression over time to reduce the incidence of
`infection and chronic nephrotoxicity should be
`weighed against the emerging evidence of the ben-
`efit of higher CsA doses and blood levels for long-
`term graft survival (36). CsA dosage individualiza-
`tion is particularly important in the cystic fibrosis
`subgroup of heart—lung transplant recipients. Be-
`cause of poor CsA absorption, these patients re—
`quire 2—3 times the daily dose, usually administered
`in three to four divided doses, to achieve equivalent
`blood CsA conccntrations compared to noncystic
`heart—lung recipients (40).
`For kidney transplant recipients, double (CsA
`and steroids), triple, and quadruple induction treat—
`ment protocols are being used (Table 1). Most are
`designed to introduce CsA slowly, particularly in
`patients with delayed kidney function, while pro-
`viding effective immunosuppressive cover with al-
`ternative agents. Some patients can be managed on
`CsA monotherapy or can be switched to monother—
`apy from other regimens (41). At various centers,
`there is an apparent increase in attempts to elimi—
`nate steroids in selected populations (Table 1). Rou—
`tine CsA monitoring is particularly valuable in kid-
`
`ney transplant recipients, because an increasing se-
`rum creatinine can be the result of either rejection
`or nephrotoxicity. A recent study (7) investigated
`the diagnostic utility of whole blood CsA levels
`measured by the m 125I-RIA within the first 100
`days after renal transplantation in patients receiving
`triple therapy. It was concluded that when using
`low-dose triple—therapy regimens, CsA levels be-
`tween 150 and 400 lug/L were of little diagnostic
`value in acute allograft dysfunction, whereas levels
`outside this range were useful in the clinical diag-
`nosis of CsA nephrotoxicity and acute allograft re-
`jection. A review of the existing data on the use of
`CsA in kidney transplantation, particularly with re-
`gard to monitoring of CsA concentrations, has re—
`cently been published (42).
`In pediatric kidney transplant recipients with a
`CsA—steroid regimen, CsA target whole blood con—
`centrations of 150—250 pug/L and 100—150 ug/L have
`been recommended for induction and maintenance
`
`therapy, respectively (43). In general, therapeutic
`CsA ranges used in pediatric patients are similar to
`those employed in adults (Table 1). However, dos-
`age requirements (mg/kg) to achieve these ranges
`are greater in children.
`Recent investigations in patients with simulta—
`neous pancreas—kidney (SPK) transplants point to
`an increased incidence of acute rejection at CsA
`levels S200 pig/L as determined by HPLC (unpub-
`lished data). From the results of a further study in
`SPK recipients, it