THE CHANGING FACE OF SIROLIMUS MONITORING

1

• THE CHANGING FACE OFSIROLIMUS MONITORING

David W HoltProfessor of Bioanalytics St
Georges Hospital Medical SchoolLondon, UK
2
Learning Objectives

• Provide background on sirolimus and use in
  immunosuppression.
• Review current monitoring methods and changes.
• Present future directions in sirolimus monitoring.

3
Introduction

• Immunosuppressive drug monitoring
• Immunoassays basis of most services
• cyclosporine / tacrolimus
• convenience / simplicity
• differences in absolute values
• calibration
• antibody specificity

4
Sirolimus

• Clinical indication
• prophylaxis of organ rejection in patients
  receiving kidney transplants
• use initially in a regimen with cyclosporine and
  corticosteroids
• in patients at low to moderate immunologic risk
  cyclosporine should be withdrawn 2 to 4 months
  after transplantation
• sirolimus dose should be increased to reach
  recommended blood concentrations

5
Sirolimus

• Dosing
• oral solution and tablet formulations
• sirolimus and cyclosporine
• 2mg per day fixed dose
• sirolimus following cyclosporine withdrawal
• monitor blood concentrations
• 12-24ng/mL (chromatographic assay)

6
Sirolimus

• Rationale
• reduce calcineurin inhibitor toxicity
• improve long-term renal function
• historical data suggest better long-term outcome

Study 310
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Sirolimus

• Pharmacokinetics
• tmax 1 to 2 hours post dose
• systemic availability low
• 14 (95 CI, 10 to 17)
• metabolised by CYP3A4
• substrate for p-glycoprotein
• half-life - 6216h
• clearance ? to liver blood flow
• high bloodplasma ratio

8
Sirolimus

• Measured in whole blood
• Preferred sample EDTA anticoagulated
• Low concentrations
• pre-dose measurements
• 3 20ng/mL

9
Sirolimus

• No platform assay for sirolimus
• May have influenced adoption of drug
• Currently, HPLC used
• Has lead to centralised measurement

10
Sirolimus Clinical Trials

• Began against a background of TDM
• concentration-controlled dosing
• validated LC assays
• central laboratories
• external proficiency testing
• emphasis on
• accuracy
• specificity
• sensitivity

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Sirolimus Measurement

• Definitive studies
• HPLC with MS and UV detection
• Phase III/IIIa - immunoassay
• later withdrawn
• for reasons unrelated to performance
• results 1.25 - 1.35 ? HPLC
• Most centres still using HPLC
• immunoassay launched in Europe 8/04

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Sirolimus Measurement
Spiked sample
Pooled patientsample
International Sirolimus PT Scheme
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Requirements for an Immunoassay

• On-site or local laboratory
• Widely available analytical platform
• Technically simple
• Reliable
• Sensitive
• Reproducible
• Turn-around time within dosage interval

14
Immunoassay for Sirolimus

• Abbott Diagnostics
• IMx platform
• MEIA technology
• Similar format to tacrolimus assay

Submitted to FDA for review. Assay not
available in the US
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IMx Immunoassay Design Goals

• Sample (150µL)
• Add precipitating reagent (300µL)
• Mix and centrifuge
• Decant supernatant
• Press the button!
• Calibration stable at least 5 days
• 20 samples in one run
• Analysis time 1.5 hours

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IMx Immunoassay

• Abbott sponsored studies
• International participants
• FAM1 and FAM2
• Precision
• Analytical Sensitivity
• Functional Sensitivity
• Proficiency Testing Sample Panels
• Correlation with HPLC

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Study Participants
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IMx Sirolimus Assay Precision

• 3 control samples
• nominal values
• 5, 11, 22ng/mL
• 2 runs on each of 5 days
• 4 international sites
• NCCLS protocol

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IMx Sirolimus Assay Precision
Submitted to FDA for review. Assay not
available in the US
20
IMx Sirolimus Assay Sensitivity

• 10 replicates of A-calibrator, 2 of B-calibrator
• Mean value plus 2 SD calculated

Submitted to FDA for review. Assay not
available in the US
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IMx Sirolimus AssayFunctional Sensitivity

• Sensitivity Panel
• EDTA human blood spiked with 5 low concentrations
  of sirolimus
• Four sites tested each sample
• 10 replicates per run
• 1 run per day for 2 days
• Mean and CV for each run calculated
• Functional sensitivity determined by the
  concentration corresponding to 20 CV on the
  curve of best-fit

Submitted to FDA for review. Assay not
available in the US
22
IMx Sirolimus AssayFunctional Sensitivity
Study site - UK
Submitted to FDA for review. Assay not
available in the US
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IMx Sirolimus AssayFunctional Sensitivity
Submitted to FDA for review. Assay not
available in the US
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Proficiency Testing Panel

• 22 spiked samples
• range 5-28ng/mL
• 11 pooled samples
• kidney Tx
• 2 sirolimus-free
• 1 recovery
• pooled sample
• 10ng/mL added
• Assayed in batches
• IMx vs in-house

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Proficiency Testing Panel

• Recovery
• HPLC/MS mean 103.4
• Analytical Unit IMx 102.1
• Blank samples

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Proficiency Testing Panel
Spiked Samples
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Proficiency Testing Panel
30
25
20
IMx Sirolimus (ng/mL)
15
10
y 1.0491x - 0.2301
r 0.94
n 36
5
0
0
5
10
15
20
25
30
UK - IMx vs LCMS mean
LC/MS (ng/mL)
Submitted to FDA for review. Assay not
available in the US
28
Proficiency Testing Panel
35
20
30
15
25
20
IMx
IMx
10
15
10
5
5
0
0
0
5
10
15
20
25
30
35
0
5
10
15
20
Mean HPLC/MS
Mean HPLC/MS
Spiked Samples
Pooled Patient Samples
Study site - UK
Submitted to FDA for review. Assay not
available in the US
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Correlation Study

• 100 clinical samples tested
• kidney Tx
• IMx and in-house method
• Statistics
• slope using Passing-Bablok Conversion
• correlation coefficient

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Correlation Study
70
60
50
40
IMx Sirolimus (ng/mL)
30
20
y 1.2477x - 0.5055
r 0.93
n 655
10
0
0
10
20
30
40
50
60
70
LC/MS Sirolimus (ng/mL)
5 study sites
Submitted to FDA for review. Assay not
available in the US
31
Correlation Study
30
25
20
IMx Sirolimus (ng/mL)
15
10
y 1.276x - 0.360
r 0.93
5
n 858
Samples 0
0
5
10
15
20
25
30
LC/MS Sirolimus (ng/mL)
Submitted to FDA for review. Assay not
available in the US
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Correlation Study
50
40
30
IMx
IMx 1.12HPLC 0.97 r² 0.92 mean difference
19.5
20
10
0
0
10
20
30
40
50
HPLC/MS
Kidney Tx Samples (n129)
Analytical Unit
Submitted to FDA for review. Assay not
available in the US
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Correlation Study
Mean
Kidney Tx Samples (n129)
Analytical Unit
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Correlation Study
Study site - Austria
Includes 6 lung transplant patients
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Implementing the IMx Assay

• Centres currently using HPLC
• In-house comparison
• 50-100 samples run in parallel
• discuss results with clinical colleagues
• agree new reference range
• Centres starting with IMx assay
• alert clinical colleagues to assay differences
• agree reference range

Submitted to FDA for review. Assay not
available in the US
36
Implementing the IMx Assay

• Current ranges
• without cyclosporine 12-24ng/mL
• New reference ranges
• HPLC range 25-30
• without cyclosporine 15-30ng/mL

Submitted to FDA for review. Assay not
available in the US
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Sirolimus TDM

• Advised
• for pediatric patients
• for patients with hepatic insufficiency
• when strong inducers (e.g. rifampin, rifabutin)
  or strong inhibitors (e.g. ketoconazole) of
  CYP3A4 are introduced or discontinued
• when cyclosporine dosing is markedly changed or
  if discontinued

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Information on Monitoring

• Web site
• URL www.bioanalytics.co.uk
• International PT Schemes
• summaries of results
• Publications
• bibliography updated monthly
• Information on upcoming meetings
• summaries from presentations

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Conclusions

• Sirolimus monitoring well-established
• pre-dose sample useful
• related to pharmacodynamic parameters
• TDM not the sole basis for dosing
• interpret in the light of
• clinical signs
• other laboratory measurements
• tissue biopsy findings

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Conclusions

• New immunoassay already implemented
• tested in samples from
• kidney Tx patients
• some other Tx indications
• bias vs HPLC
• 25-30
• previous IMx immunoassay used in clinical studies
• suitable for routine monitoring
• will allow local monitoring

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Immunoassay Testing

• Cross-reactivity of original IMx assay
• hydroxy-metabolites (50)
• 41-O-demethyl (127)
• Test differences between patient groups
• liver dysfunction
• Test stability of metabolite pattern
• between transplant groups
• with time after transplantation
• with concomitant therapy

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Study UK02 Kidney Tx
Expressed as percentage of total metabolite area
Days Post Tx
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Kidney Transplant Patients
100
10
1
Expressed as percentage of total metabolite area
n 7
0
0
20
40
60
80
100
120
140
160
180
Days Post Tx