20130617-ADavison-transplantation and the Laboratory

8/12/2019 20130617-ADavison-transplantation and the Laboratory

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Andrew Davison FRCPath

Department of Clinical Biochemistry and Metabolic Medicine

Royal Liverpool and Broadgreen University Hospitals Trust

ACB Training Course 2, Warwick June 2013

Overview

Types of Organ Donation

Therapeutic Drug Monitoring & Immunosuppression

Measurement of Immunosuppressants

Renal and Liver Transplantation


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Types of Organ Donation Living Donor

Heart Beating Donor/Donors after Brain Death

Organ procured from individuals having suffered an intracranial catastropheresulting in brain stem death, whilst on life support.

Withdrawal of treatment is a carefully orchestrated event, which is inevitablyfollowed by respiratory, and cardiac arrest, with death certified usingneurological/brain stem death criteria

Non-Heart Beating Donor/Donors after Circulatory Death

Organ procured from individuals post cardio-respiratory arrest, and aretherefore certified dead based on cardiac criteria.

Death is uncontrolled and often occurs without pre warning.

It is expected therefore that organs procured from these often unexpectedfatalities will have the added disadvantage of an increased warm ischemia time

Modified Maastricht Classification

of Donor Organs

Category I Dead on arrival

Category II Unsuccessful resuscitation

Category III Awaiting cardiac arrest

Category IV Cardiac arrest in a brainstem dead donor.

Category V Unexpected cardiac arrest in a critically illpatient

Kootstra at al. Transpl Proc 1995;27:28934


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Organ DonationHow Big is the Problem?

www.organdonation.nhs.uk/statistics/transplant_activity_report/

Liver Donors



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Cardiac Donors


Kidney Donors



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Suitability for Transplantation

Not just about good match it is about best use of organ

Any condition with an expected prognosis of less than 2 yearsfollowing transplantation absolute contra-indication.

Any condition with an expected prognosis of 2-5 years survival post-transplantation relative contra-indication.

Anatomical abnormalities which make surgery technically difficultmay be regarded as relative / absolute contraindications asdetermined by the transplant surgeon.

Transplantation vs. dialysis

Newcastle scoreAge Score


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Therapeutic Drug Monitoring

There should be a clear relationship between drug concentrationand effect

The drug should have a narrow therapeutic index i.e. the difference in the concentrations exerting therapeutic benefit

and those causing adverse events should be small

There should be considerable between-subject pharmacokineticvariability and, therefore, a poor relationship between dose and

drug concentration/response

The pharmacological response of the drug should be difficult toassess or to distinguish from adverse events.

Purpose of Immunosuppression

To try and prevent acute cellular rejection (ACR) andpromote graft survival

4-stages to ACR:

Allograft recognition T-cell activation

Clonal expansion

Inflammation


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Immunosuppressant agents

Non

biological agents

Calcineurin inhibitors Ciclosporin Tacrolimus

mTOR inhibitors Sirolimus Everolimus

Anti-metabolites

Mycophenolic acid Azathioprine

Corticosteroids

Immunosuppressant agentsBiological agents

Polyclonal Antibodies Anti-thymocyte globulin

Anti-lymphocyte globulin

Monoclonal Antibodies Anti-IL-2 receptorAbs

Basiliximab (Simulect) (T1/2~20 days)

Daclizumab (Zenapax) (T1/2~6 days)

Anti-CD-52 Ab Alemtuzumab (Campath-1)

Muromonab-CD3 (OKT3)


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Polyclonal Antibodies Rabbit/horse

Role is to promote depletion of lymphocytes

Act on: T-cells CD2-4, CD8, CD-28 B-cells CD20, CD40 HLA-1/2 and NK cells

Cell death via receptor mediated opsonisation and apoptosis

Examples Anti-thymocyte globulin Anti-lymphocyte globulin

Monoclonal Antibodies Anti-IL-2 receptorAbs

Inhibit T-cell proliferation e.g. Basiliximab (Simulect), Daclizumab (Zenapax)

Anti-CD-52 Ab Depletion of thymocytes, T/B-cells

Receptor modulation of T-cells e.g. Alemtuzumab (Campath-1)

Muromonab-CD3 (OKT3) Depletion and receptor modulation of T/B-cells and Thymocytes

Fever, hypotension, diarrhoea and vomiting


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Ciclosporin A (CsA)

Highly protein bound (lipoproteins mainly) and excreted via biliary route mainly

In the cytoplasm CsA binds to its immunophilin, cyclophylin (CpN), forming acomplex

CsACpN complex binds and blocks the function of the enzyme calcineurin (CaN,serine/threonine phosphatase )

CaN fails to dephosphorylate the cytoplasmic component of the nuclear factor ofactivated T cells (NF-ATc), and thereby the transport of NF-ATc to the nucleus andthe binding of NF-ATc to the nuclear component of the nuclear factor of activatedT cells (NF-ATn).

Normally NF-ATcNF-ATn complex binds to the promoter of the IL-2 gene andinitiates IL-2 production.

Consequently, T cells do not produce IL-2, which is necessary for full T-cellactivation.

How Does Ciclosporin A Work?


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15 metabolites: AM1, AM9 & Am4n estimated to have 10-15% of the biologicalactivity of CsA important when metabolites accumulate

Ciclosporin Adrug interactions Inducers of CYP450 including phenytoin, phenobarbital and

rifampicin are reported to decrease CsA blood concentrations,presumably through increased metabolism

Ketoconazole and cimetidine significantly increase the bloodconcentration by inhibiting metabolism

Erythromycin increases blood concentrations by inhibitingclearance

An alternate form of the drug Ciclosporin G has been found tobe much less nephrotoxic than CsA


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Tacrolimus Tacrolimus (FK506) binds to FK506-binding protein

(FKBP), forming a FK506FKBP complex, which binds toand blocks CaN.

FK506FKBPCaN complex inhibits the activation of NF-ATc, thus preventing its entrance into the nucleus.

Suppresses IL-2, IL-3, IL-4, IL-5, TNF, GMCSF and IL-2R

and IL-7R

Major metabolite is 13-demethyl-FK506

How Does Tacrolimus Work?


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Sirolimus Sirolimus (SRL) binds to FK506-binding protein (FKBP)

The complex that is formed between SRL and FKBP binds to the mammaliantarget of rapamycin (mTOR).

The SRLFKBPmTOR complex inhibits biochemical pathways that arerequired for cell progression through the late G1 phase or entry into the Sphase of the cell cycleSRL blocks cytokine signal transduction.

SRL is thought to target:(1) 70-kD S6 protein kinase p70S6K

(2) eukaryotic initiation factor eIF-4F(3) G1-controlling cyclin-dependent kinase proteins e.g. cdk2, cdk6(4) kinase inhibitory protein Kip1 (p27kip), which blocks cell progression tothe S phase

How Does Sirolimus Work?


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Everolimus

2ndmTOR inhibitor in transplantation

Differs in structure by just one hydroxyethylgroup from sirolimus

Shorter life (28 vs. 62 hours)

Approved for use in Europe, not FDA

approved

Drug name Metabolism route

Tacrolimus Sirolimus

Chloramphenicol Unknown (possibly UGT)

Clotrimazole CYP3A4, possibly P-gp CYP3A4

Mibefradil (withdrawn) CYP3A4, CYP2D6

Danazol, Cimetidine,

Metoclopramide,Bromocriptine, Proteaseinhibitors, Nicardipine,Cisapride

CYP3A4 CYP3A4 and/or

P-gp

Felodipine, Lansoprazole,Levofloxacin, Nefazodone,Pomelo juice,Methylprednisolone

CYP3A4

Drugs that Increase the Level ofTacrolimus/Sirolimus (1)

Samaniego M et al. Nat Clin Pract Neprol 2006;2:688699


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Drug name Metabolism routeTacrolimus Sirolimus

Quinidine P-gp

Metronidazole, Nifedipine CYP3A4, CYP3A5

Omeprazole Conflicting data withCYP3A4 ()

Ergotamine, Ethinylestradiol, Josamycin,Miconazole, Midazolam,Tamoxifen

CYP3A (in-vitro, in-vivo data not available)

Diltiazem, Verapamil,Grapefruit juice,Cyclosporin, Azole-antifungals, Macrolideantibiotics

CYP3A4, P-gp CYP3A4, P-gp

Drugs that Increase the Level of

Tacrolimus/Sirolimus (2)


Drugs that Decrease the Level ofTacrolimus/Sirolimus

Drug nameMetabolism route


Corticosteroids, Rifampin, St. JohnsWort

CYP3A4, P-gp CYP3A4, P-gp

Phenytoin, Phenobarbital,

Carbamazepine,

CYP3A4 CYP3A4

Rifapentin CYP3A4



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Drugs that are Increased by

Tacrolimus/Sirolimus

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Drug nameMetabolism route


Irinotecan UGT1A1

Rifampin CYP3A4, P-gp

Simvastatin CYP3A4

Cerivastatin(withdrawn)

Probably through CYP3A4

Erythromycin, S-(-)Verapamil

CYP3A4, CYP3A5, P-gp


Selective inhibition of T-lymphocyte proliferation at S phase of cell cycle

Inhibits inosine monophosphate dehydrogenase, the enzyme that controls the rate ofsynthesis of guanine monophosphate in the de novopathway of purine synthesis usedin the proliferation of B and T lymphocytes.

Mycophenolate


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Mycophenolic Acid (MPA) is animmunosuppressive agent availableeither as an ester pro-drug or as a

sodium salt.

Mycophenolate mofetil (MMF) isthe 2-morpholinoethyl ester pro-drug of MPA formulated toimprove its bioavailability

Mycophenolate Sodium is adelayed release formulation thatdelivers MPA in the small intestinewithout being first released intothe stomach.

[MPA-Glu] ~40 fold higher thanMPA

Debate over whether MPA TDM isnecessary

CES carboxylesterases, UGT UDP-glucuronosyl transferse

Ciclosporin Tacrolimus Sirolimus Mycophenolate

Oral bioavailability(%)

10-45 5-90 10-15 80-94

Half life (h) 11 11 60 9-17

Time to peakconcentration (h)

20.5 3.52.5 21.5 1.0

Mechanism of actionInhibition ofcalcineurin

Inhibition ofcalcineurin

Inhibition ofprotein kinase

mTORInhibits IMPDH

MetabolismHepaticCYP3A4

Intestinal andhepaticCYP3A4

Intestinal andhepaticCYP3A4

Intestinal andHepaticCES 1 and CES2

CYP3A4/5CYP2C8

Sample type Whole blood Whole blood Whole blood Whole blood

Suggested therapeuticrange (g/L)

100-300 5-20 5-10 1.0-3.5**

**=mg/L. MPA glucuronide=35-100mg/L


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Azathioprine (Aza)

Azathioprine (AZA) and mercaptopurine (MP) are thefirst-line immunomodulators for inflammatory boweldisease and have proven efficacy in induction of remissionin Crohnsdisease (CD) and maintenance of diseaseremission in both ulcerative colitis and CD.

Role in fistula healing, as steroid-sparing agents and asconcomitant immunosuppression to prevent loss ofresponse to biologic therapy

~60% of those diagnosed with CD receive AZA at somepoint in their disease course

Smith et al. Int J Clin Pract 2013, 67, 2, 161169

Azathiopurine Metabolism

Karran P & Natalie Attard N. Nature Cancer 2008:8;24-36


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Thioguanine Nucleotides

TGN level monitoring has been proposed as a way of optimisingand individualising thiopurine treatment.

A consensus is emerging that TGN levels: Correlate with response to thiopurine treatment

Inversely correlate with HarveyBradshaw Index

Are lower in those with active disease

Useful tool to detect non-adherence to treatment, suboptimal

dosing and biochemical resistance (predominant methylation) inpatients who are non-responders to thiopurine treatment

TGN levels may also predict dose-dependent toxicity and detectnon-adherence in those who are in remission.


Use of TGN & MeMP in Clinical Practice



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Corticosteroids

Inhibit production of T-cell cytokines IL-2, IL-6, IF-(requiredto enhance lymphocyte/macrophage response to allograftantigens)

Suppress Ab and C binding

Stimulate migration of T-cells from intravascular space tolymphoid tissue

Selective lysis of immature cortical lymphocytes

Avoid use in hepatitis as virus contain steroid response element

Corticosteroids Side Effects

Mukherjee S et al. J Trans 2009


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What is the Optimal Therapeutic Target?

It depends!!

Must consider:

Transplant type

Time post-transplant

Concomitant immunosuppressive therapy

Trough vs. C2 vs. C6 level

Risk factors

Proposed that Bayesian approach employed

Emphasis should be on therapeutic effect and not numbers

Press RR et al. Cur Pharm Des 2010;16:176-186


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Immunosuppression policy - RLUHPre-operativelySimulect/Zenapax should be considered for all transplants

Intra-operatively500-1000mg of Methylprednisolone is given by slow intravenous injection prior to re-vascularisation of the graft

Post-operativelyTreat all patients with CNI (either Tacrolimus or Neoral) monotherapy except where there are clinical indicationsto use other types of immunosuppression

The choice of CNI depends on individual patients. Steroid avoidance is the mainstay of the units policy.

CNI Monotherapy

Initial DoseWhen the patient can swallow, Neoral is commenced at 8 mg/kg/d or Tacrolimus 0.15mg/kg/d in two doses,12h. This dose is altered according to blood Ciclosporin, or Tacrolimus levels.

Maintenance Dose

Dose of Neoral is altered to achieve 12h trough level of 200 250g/L.These levels are maintained for the first three months after transplantation and reduced after that to achievelevels after 6 months after transplantation of between 150200g/L.

Tacrolimus level target is 1012g/L in the first 6 months then 810g/L.

Indications for dual therapy and triple therapy

Delayed graft function Immunosuppression is altered in case of delayed graft function to

protect the graft from CNI nephrotoxicity.

Ciclosporin should be reduced to 5mg/kg/d, Tacrolimus to0.1mg/kg/d. With Neoral, Mycophenolate Mofetil 1g bd is initiated,and with Tacrolimus 750mg bd.

Ciclosporin trough should not be greater than 150g/L. Tacrolimuslevels are maintained at 8-10g/L. (Dual therapy levels)

High immunological risk Patients who have history of severe rejection of previous graft,

leading to graft loss, or high cytotoxic antibodies MMF is added toTacrolimus/Neoral


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HLA Mismatch Patients with poorly matched kidneys MMF is added to

Tacrolimus/NeoralCNI Nephrotoxicity Patients with acute CNI toxicity, MMF is added to allow

reduction of Tacrolimus/Neoral.

Original kidney disease Patients with autoimmune disease with high rate of

recurrence.

Acute rejection When monotherapy fails to control acute rejection,

immunosuppression should be changed after the secondacute or after first severe acute rejection from monotherapyto dual therapy with addition of Mycophenolate Mofetil.

If the patient is already on Neoral, Tacrolimus should beconsidered as a replacement.

Initial Immunosuppression

Managing Kidney Transplant Recipients. KDIGO, 2010


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Risk Factors for Acute rejection


Treatment of Acute Rejection



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Treatment of Chronic Allograft Injury


Screening Post Transplantation



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Screening for Recurrent Disease


Cardiovascular Disease



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Cancer


Infection



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Other




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Biochemical Consequences of

Immunosuppression

Adverse effect PrednisoloneCsA FK506 mTORiMMF AZAHyperglycaemia x x xx x xHypercholestrolaemia x x xHypertriglyceridaemia x x xHypertension xx xx xOsteopenia xx x (x)Anaemia & Leucopenia x x x x x xDelayed wound healing xNausea/diarrhoea x xxProteinuria xxReduced eGFR (> creatinine)* x xHypomagnesaemia x

Hypokalaemia

x

x

x

Hyperkalaemia x xHypophosphataemia x xHyperuricaemia x xHypercalcaemia xAcid base disturbance xHyperbilirubinaemia x x x xHypocalcaemia xAdrenal suppression x

*=Inhibition of peptidyl-propyl-cis-trans-isomerase activity


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Measurement of Immunosuppressants

Immunoassay Limited specificity due to cross reactivity of metabolites

Haematocrit can influence concentrations e.g. FK506increases with decreasing haematocrit

Cost of reagents is high compared to LC-MS/MS

LC-MS/MS Simple sample preparation protein precipitation and

injection Potential to measure CsA, FK506 and Sirolimus in a

single run

Labelled isotope IS available

External Quality Assurance


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Renal Transplantation First successful renal transplant between identical

twins in 1954

Preferred treatment for patients with end-stage renaldisease. It offers better quality of life and confersgreater longevity than long-term dialysis

Hypothermic machine perfusion

Newcastle Clinical ViabilityProtocol:

8-10C, 8h

Perfusion f low indices>0.4mL/min/100g/mmHg

Perfusate GST


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Benefits of hypothermic machine

perfusion Maintains patency of the vascular bed

Provides nutrients and low demand oxygen to support reduced energy demands

Removes metabolic by-products and toxins

Provides access for administration of cytoprotective agents and/or

immunomodulatory drugs

Increased availability of assays for organ viability assessment and tissue matching

Facilitates a change from emergency to elective surgery (cheaper/better outcome)

Improves stabilization or rescue of DCD kidneys or organs from DBD that increasethe size of the donor pool

Significant economic benefit for the transplant centers and reduced costs

Provides technology for ex-vivo use of non-transplanted human organs forpharmaceutical development research.

Taylor and Baicu. Cryobiology 2009;33:472-482

The Value of Machine Perfusion Perfusate Biomarkersfor Predicting Kidney Transplant Outcome

Moers et al. Transplantation 2010;90: 966973

Hypothermic machine perfusion (MP) versus staticcold storage in deceased-donor kidneytransplantationMP reduced the risk of delayedgraft function.

Retrospective evidence suggests that LDH, AST, totalglutathione-S-transferase (GST), ALT,N-acetyl--D-glucosaminidase, and heart Type fatty acid bindingprotein measured during kidney machine perfusion(MP) could have predictive value for post transplant

outcome

GST, NAG, and H-FABP were independent predictorsof delayed graft function

Increased GST, NAG, or H-FABP concentrationsduring MP are an indication to adjust post transplantrecipient management.


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Liver Transplantation First successful transplantation 1967 in an 18 month

old with hepatoblastoma

Elective Liver Transplantation

Chronic liver disease or failure

Hepatocellular carcinoma

A variant syndrome

Have been accepted through the National AppealsPanel

NHS Blood and Transplant Liver Advisory Group, Sept 2009. Version 1


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Liver Transplantation


Super Urgent Liver TransplantSelection Criteria

Adult (aged 17 years and older) liver transplantation

An individual can be considered either for a super-urgent liver transplant when theyhave the most severe form of liver disease or an elective liver transplant.

To be registered on the super-urgent liver scheme, at least one of the followingcriteria must be met:

Paracetamol poisoning (1 of the risk factors below):

pH 100 seconds or INR >6.5, and serum creatinine >300mol/L or anuria, and grade 3-4 encephalopathy.

Serum lactate more than 24h after overdose >3.5 mmol/L on admission or >3.0mmol/L after fluid resuscitation.

Two of the three criteria from category 2 with clinical evidence of deterioration (e.g.increased ICP, FiO2 >50%, increasing inotrope requirements) in the absence ofclinical sepsis.



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Seronegative hepatitis, hepatitis A or hepatitis B, or an idiosyncratic drug reaction.Prothrombin time >100 seconds or INR >6.5, and any grade of encephalopathy.

Seronegative hepatitis, hepatitis A or hepatitis B or an idiosyncratic drug reaction.Any grade of encephalopathy, and any three from the following: unfavourableaetiology (idiosyncratic drug reaction, seronegative hepatitis), age >40 years,jaundice to encephalopathy time >7 days, serum bilirubin >300mol/L, prothrombintime >50 seconds or INR >3.5.

Acute presentation of Wilsons disease, or Budd-Chiari syndrome. A combination ofcoagulopathy, and any grade of encephalopathy.

Hepatic artery thrombosis on days 0 to 21 after liver transplantation.

Early graft dysfunction on days 0 to 7 after liver transplantation with at least two ofthe following: AST >10,000IU/L, INR >3.0, serum lactate >3 mmol/L, absence of bileproduction.


Questions?

20130617-ADavison-transplantation and the Laboratory

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