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New developments in immunosuppressive drug monitoring

Michael Oellerich Abteilung Klinische Chemie Georg-August-Universität GÜttingen Germany www.clinchem.med.uni-goettingen.de


Immunosuppressive drug monitoring - key issues -

• Optimal monitoring strategies • PK/PD relationships and target concentrations • Identification of clinical settings where TDM is recommendable

• Biomarkers for immunosuppressive drug effects


Criteria for a pharmacokinetic parameter to be useful for routine TDM • easy to obtain • short turn-around time (same day) • cost effective • sufficient correlation with AUC (drug exposure)

• related to outcome (e.g. ADR`s, acute rejection)


TDM of tacrolimus Is bioequivalence of TDM given for different formulations?


Once daily modified release tacrolimus (Advagraf)

Mean Concentration (ng/ml)

Advagraf versus Prograf

20

AUC0-24 vs C24

Day 14 Prograf

Advagraf r2: 0.85 – 0.89

Day 28 Advagraf

Prograf

r2: 0.79 – 0.85

Day 42 Prograf Day 56 Advagraf

15

10

5

0 0

4

8

12 Time (hr)

16

20

24

Undre, 2008 unpublished

Adapted from van Hooff J et al, Am J Transplant 2005;5(suppl 11):190


TacroBell® vs Prograf® in healthy Korean adults

Tacrolimus (µg/L)

n=29

AUC0-24 vs C0 TacroBell®

: r2 = 0.89

Prograf®

: r2 = 0.92

Park K et al, Clin Ther 2007;29:154-162


PK-PD relationship for MMF in renal transplantation Triple therapy: CsA, MMF, steroids Target MPA-AUC (mg*h/L)

16.1

32.2

60.6

Percentage of patients

40 35 30 25 20 15 10 5 0

rejection

adverse event

MPA measured by HPLC Adapted from Hale et al. Clin Pharmac Ther. 1998;64:672-83


Rationale for TDM of MPA

• MPA-AUC shows a substantial between patient pharmacokinetic variability (44%) with a fixed – dose MMF regimen • One dose does not fit all patients

Adapted from van Hest et al, Ther Drug Monit 2005;27:163-167


Variables associated with MPA exposure Variable Hypalbuminemia Hyperbilirubinemia Cyclosporine

Change in MPA Presumed Mechanism Exposure Clearance Free Decreased protein binding MPA enterohepatic recirculation

Corticosteroid withdrawal Decreased creatinine clearance (moderate) Decreased creatinine clearance (< 25 ml/min) Low UGT1A9 expression UGT1A9 promoter region 275 T > A, 2152 C > T SNPs

MRP-2-mediated transport inhibition Loss of enzyme induction

variable

Decreased MPAG clearance Increased free MPA fraction Decreased MPA conjugation to MPAG Increased MPA conjugation to MPAG

Adapted from Weimert et al, Ther Drug Monit 2007; 29: 141-149


Mycophenolate mechanism of action

Mycophenolate mofetil

IMPDH

Weimert et al, Ther Drug Monit 2007; 29: 141-149


MPA induces cleavage of Rho GDI -2 • Rho GDI -2: regulatory protein of Rho-GTPases • Caspase 1 cleaves Rho GDI-2 at position Asp55 Cleavage product is unable to bind and regulate GTPases (might influence cell signalling)1

• Caspase 3 cleaves Rho GDI-2 at position Asp19 Cleavage product translocates from the cytosol to the nucleus and mitochondria (influences cell signalling2, translocation of proteins from the cytosol to mitochondria upon caspase cleavage is an important mechanism for pro-apoptotic signalling to the mitochondria3) 1. Danley DE J Immunol 1996 2. Krieser RJ Cell Death Differ 1999 3. Thiede B Proteomics 2002 Heller T., Asif A.R. et al Ther Drug Monit 2009;31:211-217


Cleavage of Rho GDI -2* by caspase 3 in T-lymphoblast cells treated with MPA (100 µmol/L)

•Rho GDP dissociation inhibitor 2

Heller T., Asif A.R. et al Ther Drug Monit 2009;31:211-217


Pharmacokinetics of mycophenolic acid and main metabolizing enzymes

van Agteren et al, Ther Drug Monit 2008;30:439-444


Methods for MPA determination in plasma

Immunoassays - EMIT - CEDIA COBAS INTEGRA MPA Chromatographic assays - HPLC - LC-MS/MS * under development

Bias from AcMPAG

Total MPA

Free MPA

+ +

-

~ 20 â&#x20AC;&#x201C; 30 %

+

+*

~2- 6%

+ +

+ +

-


MPA concentration by Roche total MPA assay and LC-MS/MS

Brandhorst et al Ther Drug Monit 2008;30:428-433


TDM strategies – estimation of MPA AUC as surrogate marker of drug exposure • Pre-dose level (Co) (r2 = 0.003 – 0.7)

• Sparse sampling algorithms - sampling time points : 0 , 0.5 , 2 h (r2 = 0.75 – 0.86)

• Bayesian forecasting - sampling time points : 20 min, 1 h , 3 h www.chu.limoges.fr (Æ activité médicale) email: stp@chu-limoges.fr


Concentration-time profiles of MPA in kidney transplant recipients

EC-MPS Conc/Dose (mg/L/MPA eq)

Conc/Dose (mg/L/MPA eq)

MMF

Time (min)

Time (min)

Cattaneo et al, Clin J Am Soc Nephrol 2007;2:1147-55


Relationship between MPA predose level (C0) and acute rejection (AR) Significant results (p<0.05)

Author

Renal transplantation ↑ AR risk with lower C0

Krumme, 1998; Van Gelder, 1999; Weber, 2002; Lu, 2004; Borrows, 2006; Pawinski, 2006; van Gelder, 2008; Gaston, 2009

No significant or only weak relationship

Smak Gregoor, 2000; Pillans, 2001; Kuypers, 2003; Kuypers, 2003; Kiberd, 2004; Barbari 2005; Hazzan, 2005; Okamoto, 2005

Cardiac transplantation ↑ AR risk with lower C0

Meiser, 1999; Yamani, 2000; Gajarski 2004

No significant relationship

DeNofrio, 2000; Cantin, 2002

Hepatic transplantation ↑ AR risk with trough <1 µg/ml

Tredger 2004

Adapted from Kaplan B, Current Medical Research and Opinion 2006;22: 2355-2364


Relationship between MPA - AUC concentration and clinical outcome in kidney transplantation Study

N

IS

Takahashi et al 1995 Hale et al 1998

32

PK

Outcome

CsA HPLC

AUC

AR, (Inf)

150 CsA HPLC

AUC

AR

Mourad et al 2001 31

method

Pillans et al 2001

27

ATG EMIT CsA CsA HPLC

Weber et al 2002

54

CsA HPLC

AUC (C0) Hem (AR) AUC AR AUC (C0) AR free MPA Hem, Inf AUC AR

Le Meur et al 2007 130 CsA HPLC LC-MS/MS van Gelder et al 901 CsA HPLC AUC (C0) AR 2008 TAC EMIT


Early MPA exposure: Area under ROC curve in prediction of rejection and toxicity Rejection

Toxicity

Day 3

Area

p

Area

p

MPA C0

0.65

0.08

0.49

0.90

MPA C2

0.68

0.025

0.51

0.77

MPA AUC0-12

0.72

0.007

0.53

0.29

C2 (CyA)

0.63

0.11

0.46

0.98

Adult Ktx patients, MMF, CsA (basiliximab) Kiberd et al, Am J Transplant 2004; 4:1079-1083


Relationship between MPA predose level und adverse events (AEs) Significant results (p<0.05)

Author

Renal transplantation ↑ incidence of AEs with higher C0

Hübner, 2000; Mourad, 2001; Lu, 2004; Kuypers 2004; Borrows 2006; Kuypers 2008

No significant relationship

van Gelder, 1999; Pillans, 2001; Mourad, 2001; Cattaneo, 2001; Weber, 2002; Kuypers, 2003; Kiberd 2004; Okamoto, 2005; Pawinski, 2006

Cardiac transplantation ↑ incidence of AEs with higher C0

Zakliczynski, 2005

Hepatic transplantation ↑ incidence of AEs with higher C0

Tredger 2004

Adapted from Kaplan B, Current Medical Research and Opinion 2006;22: 2355-2364


MPA AUC in the presence and absence of drug-related toxicity (n=464) 5y follow-up study in renal transplant recipients

Kuypers et al, Clin Ther 2008;30:673-683


Therapeutic ranges for MPA early after transplantation (≤ 3 month) Renal transplant recipients on triple therapy (CsA, corticosteroids, MMF) MPA - AUC0-12

: 30-60 mg * h / L 1)

MPA - C0

: 1.0-3.5 mg/L 1)

MPA – AUC > 30 mg*h/L [82% of pts]: - MPA – C0 ≥ 1.3mg/L (MMF,CsA) - MPA – C0 ≥ 1.9 mg/L (MMF, TAC) No established therapeutic range for maintenance therapy 1)

Measured by HPLC or LC/MS-MS

Based on RCCT data from van Gelder et al, Transplantation 1999;68:261-6; Shaw et al; unpublished


Long – term pharmacokinetics of MMF in pediatric renal transplant recipients 140 120

Dose-adjusted MPA-AUC 0-12(µg x h/mL)

Dose-adjusted MPA-AUC 0-12(µg x h/mL)

CsA, MMF, steroids A

100 80 60 40 20 0 0 3 6 9 12 15 18 21 24 27 30 33 36 39 Time after Rtx (months)

MPA measured by HPLC

140 B 120 100 80

*

#

*

#

*

*

3 6

9 12 15 18 21 24 27 30 33 36 39

60 40 20 0 0

Time after Rtx (months) *p<0.05 vs. 1 week

#p<0.05

vs. 3 months

Weber et al, Ther Drug Monit 2008;30:570-575


Banff score

Anti-fibrotic effect of MMF in renal transplant recipients

Nankivell et al, Am J. Transplant 2007; 7: 366-376


Influence of MPA on COS-7 fibroblast proliferation test

120

100

Proliferation [% of control]

80

MPA

*

MPA+G/8AG

60

AcMPAG AcMPAG+G/8AG

* P < 0.05

40

20

0 0

*32 mg/L

0.1

1

10

100*

Conc. [Âľmol/L]

Petrova et al Clin Chem Lab Med 2008;46:A195


MMF improves kidney function in a mouse model for progressive renal fibrosis â&#x20AC;&#x153;Alport syndromeâ&#x20AC;?, COL4A3- deficient mice

2.5

Creatinine [mg/dL]

2.0

P < 0.01, r= - 0.655

1.5

1.0

0.5

0.0 0

5

10 15 20 MPA concentration [mg/L]

25

30

Brandhorst et al. 2009, submitted


Fixed (2g) vs concentration – controlled dosing of MMF in renal transplant recipients APOMYGRE

• Adults (n = 137) • CsA, MMF • Induction • Target MPA AUC: 40 mg.h/l • Bayesian estimator Benefit of TDM: demonstrated BPAR CC vs FD - 7.7% vs 24.6% (p < 0.02)

FDCC • Adults + pediatrics (n = 901) • CsA or Tacro, MMF • Induction or no induction

Opticept • Adults (n = 720) • Standard or reduced dose CsA or Tacro, MMF • Induction or no induction

• Target MPA AUC:

• Target MPA Co:

45 mg.h/l • 3-point methods

not demonstrated BPAR CC vs FD - 14.9% vs 15.5% (p = 0.956)

≥ 1.3 mg/l for CsA ≥ 1.9 mg/l for Tacro potential utility in CNI-sparing regimens Co > 1.6 mg/l lower BPAR (p < 0.001) in pts on Tacro (n = 590)

Le Meur et al Am J Transplant 2007;7:2496-2503 Van Gelder et al Transplantation 2008;86:1043-1051 Gaston et al Am J Translant 2009;9:1-13


Opticept Trial design

Gaston et al, Am J Transplant 2009;9:1-13


MPA exposure and time to first BPAR

Gaston et al, Am J Transplant 2009;9:1-13


FDCC-Trial: MPA-AUC in FD vs CC patients Concentration controlled (CC) Fixed dose (FD)

Concentration controlled (CC) Fixed dose (FD)

Tacrolimus Subpopulation

MPA AUC (mgâ&#x20AC;˘h/l)

Cyclosporin Subpopulation 105

105

90

90

75

75

60

60

45

45

30

30

15

15

0

0 D3

D 10 Wk 4

M3

M6

M 12

D3

D 10 Wk 4

M3

M6

M 12

van Gelder et al, Transplantation 2008;86:1043-1051


Logistic Regression Analysis of Biopsy Proven Acute Rejection (BPAR) during the 1st year

MPA AUC / Day 3

p-value

Regression Estimate*

p-value Likelihood Ratio Test

0.0060

-0.0169

0.0032

â&#x20AC;˘ Regression estimate > 0 indicates a positive relationship between MPA AUC on day 3 and BPAR van Gelder et al, Transplantation 2008;86:1043-1051


APOMYGRE study: MPA exposure 80

Fixed dose

70

Concentration controlled

**

AUC (h.mg/L)

60

**

50 40 30 20 10 D7

FD (mg/day) 2000 CC (mg/day) 2000

D14

M1

M3

M6

M12

2000 2698

1960 2969

1852 2279

1778 1924

1958 1827

Adapted from Le Meur Y et al, Am J Transplant 2007; 7:2496 - 2503


Conclusions – MMF monitoring • TDM of MPA is still not fully established • Target population for MPA TDM

- Dual immunosuppressive therapy - Reduced dose CNI therapy (incl. delayed introduction of CNI) - CNI switch or withdrawal - High immunological risk patients - Delayed graft function - Altered gastrointestinal /hepatic /renal function - Compliance problems (e.g. adolescents) - Drug interactions Adapted from van Gelder et al, Ther Drug Monit. 2006; 28: 145-154 Kuypers et al, Transplantation 2009, in prep


Suggested schedule for MMF monitoring in renal transplant patients on standard CNI regimens • Initial evaluation (preferably abbreviated AUC) once during the first week (days 3 - 5) • Once during days 10-14 (C0 or abbr. AUC) • At weeks 3 or 4 (C0 or abbr. AUC) MPA-AUC MPA-AUC

30 – 60 mg*h/L -> no dosage change < 30 mg*h/L -> dosage change > 60 mg*h/L -> dosage change in case of AEs -> confirmation of AUC after a new steady state is reached (3 – 5 days) Adapted from Shaw et al, Am J. Transplant 2003; 3:534-542 Oellerich et al, Ther Drug Monit 2006; 28:720-725


Pharmacodynamic monitoring - Proposed biomarkers - Drug target enzymes IMPDH (Inosine monophosphate dehydrogenase) CN (Calcineurin phosphatase) p70 S6 k phosphorylation

- Cytokines IL-2,IFN-Îł

- Marker of immune cell response PHA-stimulated ATP-production (ImmuknowR Cylex)

- Marker of lymphocyte proliferation PCNA (proliferating cell nuclear antigen)

- Markers of lymphocyte activation CD25 (IL â&#x20AC;&#x201C; 2 receptor), CD71

- Predictor of tolerance CD4+ CD25high Foxp3+ (Treg)


Association between proposed MMF exposure group and acute rejection IMPDH > cut-off */ Dose reduction

O%

-/-

37 %

-/+

36 %

+/-

82 %

+/+

no rejection rejection

4

0

12

7 9

5 2 0

2

9 4

6

8

10

12

14

Number of patients *8.53 nmol/mg protein/h Adapted from Glander et al, Am J Transplant 2004; 2045-2051


Logistic regression results on biopsy-proven acute rejection for IMPDH2 T3757C polymorphism at 12 months

Effect

OR [95% Wald CI]

P-value

IMPDH2 T3757C T3757C (CC,CT/TT)

3.39 [1.42 â&#x20AC;&#x201C; 8.09]

0.0061

Adapted from Grinyo et al, Transpl Int 2008;21:879-891


Global CD4+ cellular response measured by iATP synthesis Sodium heparinate whole blood

Kowalski et al, Clin Transplant. 2003 Apr; 17(2):77-88


Immune response of solid organ transplant recipients during periods of rejection, infection and stability

Immune Response (ATP ng/mL)

1000

Zone of minimal risk

P < 0.001

800 P < 0.001 600 468

400 249

200 111 0

Rejection (n=39)

Stable (n=504)

Infection (n=66)

Adapted from Kowalski et al, Transplantation 2006; 82:663-8


Association between pretransplant iATP levels (ImmuKnow, Cylex) with kidney graft outcome p = 0.04

600

iATP ng/ml

500 400

300 200 100 n = 57

No clinical reason

n= 8

n = 18

Positive AR

Negative

n= 4

Ab-mediated AR

Biopsies

Adapted from Reinsmoen et al, Transplantation 2008; 85:462 - 470


ImmuKnow (iATP) values in lung transplant recipients with infections CMV: cytomegalovirus; FC: fungal colonization; FD: fungal disease; PNEU: bacterial pneumomia TB: tracheobronchitis; VIRAL: viral infection STABLE: control group

Husain et al, Transplantation 2009;87:1852-1857


Individualized response of a stable small bowel transplant recipient to tacrolimus weaning 1 : 5 mg tac 2x/w

immune activation â&#x2020;&#x201C;

2 : 5 mg tac 3x/w 3 : 8 mg tac 2x/w

1

2

3

Zeevi et al Transplant Immunology 2005;15:17-24


Individualized response of a stable small bowel transplant recipient to tacrolimus weaning acute rejection â&#x2020;&#x201C;

Zeevi et al Transplant Immunology 2005;15:17-24


Correlation of immune response with therapy changes based on clinical indications

Zeevi et al Transplant Immunology 2005;15:17-24


Trough level (ng/ml)

Immune cell response (iATP ng/ml)

Immune cell response and CNI trough levels in stable pediatric liver transplant recipients (< 12 y)

Healthy controls

CsAbased

Tacrolimusbased

(n=11)

(n=11)

(n=15)

CsA

Tacrolimus

(n=11)

(n=15)

Schulz-J端rgensen et al (submitted)


Correlation between (PHA) â&#x20AC;&#x201C; stimulated (ATP) immune response and CNI trough levels 1200

Immune cell response (iATP ng/ml)

600

Immune cell response (iATP ng/ml)

1000

800

600

400

r = 0.152

200

p = 0.575 0

5

10

15

20

Tacrolimus (ng/ml)

25

30

500

400

300

200

100

r = 0.037 p = 0.903

0

35

0

50

100

150

200

250

CsA (ng/ml)

Schulz-JĂźrgensen et al (submitted)

300


Immune cell response in a liver transplant recipient with EBV infection

Immune cell response (iATP ng/ml)

2 y, boy 500

Tacrolimus 19.8 Âľg/ l

Tacrolimus removal strong

400

395

Recovery from infection < 103 copies

EBV infection 1.4 x 105 copies

300

Aciclovir

moderate 279

200

175

low

181

100

0

13.03.09 01.01.09

27.05.09 01.06.09

Schulz-JĂźrgensen et al (unpublished)


Immune cell response in a liver transplant recipient on MMF monotherapy with acute rejection MPA: 2.7 mg/l

15 y, girl

acute rejection

MPA: 0.4 mg/l

1000

> 1000

Immune cell response (iATP ng/ml)

900 800

MPA: 3.3 mg/l

AST: 97 U/l ALT: 143 U/l γ-GT: 449 U/l

> 1000

AST: 257 U/l ALT: 379 U/l γ-GT: 690 U/l

700 600 500

AST: 49 U/l ALT: 67 U/l γ-GT: 328 U/l

strong

400 300

395

moderate

325

200

175

low

100 0 27.02.09 Jan 09

Feb17.06.09 09

20.07.09

Schulz-Jürgensen et al (unpublished)


Immune response distributions in adult liver transplant recipients 1: acute rejection (ACR) 2: active recurrent hepatitis C (RHC) [HAI>5] 3: ACR and mild RHC [HAI<5]

n=12

n=15

n=15 Cabrera R. et al Liver Transpl 2009;15:216-222


Relative increase in FoxP3 mRNA expression during IS withdrawal in liver transplant recipients Gr-T: BT S, T Â&#x201E; Gr-R: |

Pons et al. Transplantation 2008;86:1370-1378


T cell subsets in peripheral blood from liver transplant patients with renal dysfunction CD3+CD8+ cells/nL

CD3+CD4+ cells/nL

CD4+CD25highFoxp3+ cells/nL

Baseline

0.613 ± 0.092

1.068 ± 0.099

0.062 ± 0.016

Month 12

0.424 ± 0.055+ 0.756 ± 0.085+

0.056 ± 0.010

Baseline

0.474 ± 0.085

0.645 ± 0.096

0.051 ± 0.037

Month 12

0.530 ± 0.091

0.728 ± 0.089

0.034 ± 0.008†

MMF group (n=22)*

Control group (n=14)**

+

P < 0.001 vs. baseline; † P < 0.05 vs. baseline; mean ± S.E.M.

* MMF 2x1 g; CsA 25-50 µg/l or TAC 2-4 µg/l ** CsA 80-120 µg/l or TAC 5-7 µg/l Adapted from Cicinnati et al, Aliment Pharmacol Ther 2007:26,1195-1208


Bio-markers for immunosuppressive drug effects Potential complementary tools in addition to TDM - may identify candidates for minimization of immunosuppressive therapy

- may identify patients at risk for acute rejection or infection

- may be useful to manage the timing and rate of immunosuppressant weaning -

sequential monitoring may allow maintenance of an individualized immunosuppressive regimen


Bio-markers for immunosuppressive drug effects PD-monitoring using bio-markers is still in its early stages - no prospectively validated target ranges for adult and pediatric patients - independent prospective clinical outcome studies are in progress â&#x20AC;˘ Development of "tolerance permissive" immunosuppressive regimens would be desirable


Department of Clinical Chemistry - George-August University Gรถttingen -

Thank you

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