Immunopharmacology:Immunosuppression in Organ

Transplanation

Prof D. A. Joyce Pharm 3320/3321

http://www.aihw.gov.au/WorkArea/DownloadAsset.aspx?id=10737422288

Organ Transplants, Australia

Organ Donors (2013)

Kidney 630

Liver 248

Heart 77

Lung 167

Pancreas 33

Bone Marrow 1,812 (2012)

http://www.anzdata.org.au/anzod/updates/anzod1989toCurrent.pdf

http://www.abmtrr.org/index.php/resources/data‐management/

Kidney

Inflammation and Immunity

Inflammation is what you see, clinically, in the pathological specimen or under the microscope. There are diagnostic “signs” at each level. EG, clinically: heat, redness, pain, swelling, disordered function

Immune response describes the events that take place during inflammation. Immunity results from an immune response.

Tuberculosis

Destroys & removes

foreign &damaged tissue, initiates repair

Chemicalmediators

Dilate blood vesselsFluid & protein extravasationRecruit more leukocytes

PainSwelling

Redness, Heat

A very rough summary of how an immune response to infection

causes inflammation

LeukocytesInfectiousagent

LymphoidMyelomonocytic

Erythroid

Megakaryocytic

Lymphoid lineage has left bone marrow by early postnatal life. Other lineages proliferate and differentiate in bone marrow through life

Haemopoetic Differentiation in Bone Marrow

Bone Marrow

Gut-associatedlymphatic tissue

Lymph Nodes

LymphaticVessels

Lymph NodesThymus

Spleen

Adenoids& Tonsils

Lymph Nodes

TissueCirculation

Thymus

Bone Marrow

Lymphoidtissue

Lymphaticvessels

Lymphaticvessels

Lymphocytes: Origins and Circulation

Lymphoid tissue includes nodes, thymus, spleen, adenoids, tonsils & gut-associated lymphoid tissue

Lymph Node Architecture

Various collections of LymphocytesChannels for lymph and lymphocytes in (from tissue) and out (to other nodes & blood)

Lymphocytes

Artery & vein

Some macropha

Lymph nodes and related organs T

Lymphocytes lineages:B lymphocytes make antibodiesT lymphocytes make “cell-mediated immunity”

B

T Lymphocyte

T

Cell Death

T

T

“Cytotoxic” T Cell

Virus-infected,foreign orcancer cell

MacrophageActivation, etc

“Helper” T Cell

BAntibody

EliminatedSpecifically BindsForeign Protein orof Cell (eg bacteria)

B Lymphocyte

Antibody Mediated Immunity Cell Mediated Immunity

The lymphocyte is the central player in acquired immunity

Somalia, 1979

1796

Acquired Immunity• Specific: to a foreign protein (antigen)

• Learned: takes days – weeks to learn.

• Remembered: can be recalled years later

1157 b.c.

An antigen-specific lymphocyte learns and remembers

Is it only infection that causes immunity and inflammation?

• Infections, acute and chronic: – viral - bacterial, – protozoal - parasitic

• Foreign material– Appropriate– Excessive (allergies, eg, allergic asthma)– Transplanted organ (“Allogeneic” transplant)

• Tissue injury: – trauma - burns, – surgery - radiation (eg, sunburn)

• Autoimmunity: abnormal inflammatory reaction to normal tissue

1. Prevention of rejection in organ transplantation

- suppression of an “appropriate” immune reaction to a foreign tissue (eg heart, kidney, lung)

2. Treatment of autoimmune diseases

- Suppression of an inappropriate immune reaction to a “self” tissue (eg lupus nephritis)

• Treatment of severe allergy

- Suppression of an inappropriate immune reaction to a foreign antigen (eg glucocorticoids for allergic asthma)

Why Suppress Acquired Immunity?

Suppressing the Immune Response to a Transplanted Organ:

Control of T-lymphocyte Clonal Expansion

T-Lymphocyte infiltration in chronic allograft rejection

Kidney

Artery in heart

Liver

Lung

Allograft antigen carried by antigen presenting cells via lymphatics to lymphoid tissue

Chronic Allograft Rejection: Role of T‐Lymphocytes

Allograft‐specific T‐lymphocytes return to attack allograft

Allograft‐derived Antigen

Specific T‐lymphocyte clonal expansion

Allograft Recognition and T‐Lymphocyte Clonal Expansion in Lymphoid Tissue

TT

T

T

T

TT

Allograft cell destruction  Rejection

Antigen Processing Cell

T-Cell Receptor recognises processed antigen

Specific for the antigen, i.e. highly variable

Variability generated by gene rearrangement during clonal selection in the thymus

Antigen-presenting cell (APC)

T-lymphocyte

Processed antigen is presented, bound by a specific protein complex (HLA complex) on the APC surface

1

2

3

APC

Receptor Binding& Signaling

Cell - cell signaling(surface & secreted proteins)

Autocrinesignaling

(IL-2)

4 Proliferation

4 Steps to T-Lymphocyte Clonal Expansion

IL-2

IL-2R

IL-2

Mitogenesis

Signaling for T-Cell Clonal Expansion: Role of IL-2

TCR

IL‐2

IL‐2R

IL‐2

Mitogenesis

Anti‐CD3

mTOR inhibitorsSirolimusEverolimus

Mycophenolate

Drugs that Prevent IL‐2‐Driven T‐Cell Clonal Expansion

Calcineurin InhibitorsCyclosporinTacrolimus Azathioprine

Glucocorticoids

IL‐2

IL‐2R

IL‐2

Mitogenesis

Anti‐CD3

Calcineurin InhibitorsCyclosporinTacrolimus

Glucocorticoids

Prevent TCR signalingfor IL-2 gene transcription

IL-2

TCR

Receptor-associated tyrosinekinases (ZAP-70, lck & fyn)

phosphatidylinositol

IP3

Ca2+

CalcineurinNF-AT

P

Oct

PNF-AT

Oct

IL-2

Proximal IL-2 Promoter Transcription Factor Binding

NF-AT

P Oct

P

NF

-AT

Oct

NF-B

NF

-AT

Oct

AP

-1

c-Fos

c-Jun

Calcineurin

Rel-A

NF-B I-B

Cyclosporin

NCH3

OH

Tacrolimus (FK506)

Fungal-derived macrolide

Fungal peptide

IL-2

Action of Cyclosporin or Tacrolimus

NF-AT

P Oct

P

NF

-AT

Oct

NF-B

NF

-AT

Oct

AP

-1

Calcineurin

Cyclo

Cyclophilin

Cyclo

Tac

FKBP

Tac

Cyclophilin & FKBP (Immunophilin)

• Intracellular peptidyl-prolyl topoisomerases

• Normal role is in post-translational folding of proteins

• Targets of cyclosporin (cyclophilin) and tacrolimus (FKBP = FK506-binding protein)

• Ligand-bound, cyclophilin & FKBP acquire affinity for calcineurin inhibit enzyme

IL-2

Action of Cyclosporin or Tacrolimus

NF-AT

P Oct

P

NF

-AT

Oct

NF-B

NF

-AT

Oct

AP

-1

Calcineurin

Cyclo/Tacro

Cyclophilinor FKBPCyclo/Tacro

Cyclo/Tacro

IL-2

Action of Cyclosporin or Tacrolimus

NF-AT

P Oct

P

NF-B

AP

-1

Calcineurin

Cyclo/Tacro

Cyclophilinor FKBPCyclo/Tacro

Cyclo/Tacro

Glucocorticoids

• Suppress IL-2 gene transcription

• Reduce IL-2 mRNA stability

Glucocorticoids

=O in cortisone

Hydrocortisone (cortisol)

**

F

PrednisolonePotent synthetic glucocorticoid

DexamethasoneVery potent glucocorticoid(Methylated & fluorinated)

Also many other potent synthetic glucocorticoids

*

Steroid bound to circulating corticosteroid binding globulin

Steroid binds cytosolic glucocorticoid receptor, which translocates to nucleus

Steroid-receptor complex binds recognition sequences on promoter of responsive genes, stimulating transcription.

The Glucocorticoid Receptor (GR)

Ligand-binding domain

Regulatory domain: binds chaperones & other proteins

Zn fingers in DNA-binding domain

Transactivation domain: binds proteins that

activate transcription

Rang et al, Pharmacology, Elsevier

Glucocorticoid Receptor Activation

GRNLS

GCGC

GRNLS

GC

GRNLS

GC

GRNLS

GC

GRE

Cytoplasmic glucocorticoid

receptor & chaperone proteins

XXXXXXXXXXXXXGGTACAnnnTGTTCTXXXXXXXXXXXXX

Ligand binding releases chaperones & reveals nuclear localisation signal &

dimerisation site

Nuclear localisation signal allows recognition by nuclear pore complex

and access to nucleus

Receptor interacts with glucocorticoid response element (GRE) through Zinc

FingersTranscriptional

activation

2

3

1

4

5

IL-2

Glucocorticoids & AP-1

NF

-AT

Oct

NF-B

NF

-AT

Oct

AP

-1

GRNLS

GCGC

GRNLS

GCc-

Jun

P

c-F

os

AP-1

P

IL-2

Glucocorticoids & AP-1

NF

-AT

Oct

NF-B

NF

-AT

Oct

AP

-1

GRNLS

GCGC

GRNLS

GC

c-Ju

nP

c-F

os

AP-1

Cytoplasm

Nucleus

Leucine-zipped dimers

Rel/NF-B Family Transcription Factors

NF-B family includes– NF-B1 (p50)

– Rel-A (p65)

– NF-B2 (p52)

– c-Rel (p75)

– Rel-B (p68)

IB family includes– IB ,,,,

Rel-A

NF-B1 I-BComplexed to IB protein

Cytoplasm-resident, until activation

NF-B Signalling

Rel-A

NF-B I-B

Rel

-A

NF

-B

I-B Degradation

Cytoplasm

Nucleus

A C C GC T T C C T C CA GA T GA GC T C A T GGGT T T C T C CA C C A A GGA A GT T T T C C GC T GG

NF-B Binding Sequence

Gene Promoter

Cell Surface Receptor Activation

Phosphorylation of Serine 32 & 36 of I-B

Nuclear Localisation Signal on NF-B revealed

Binds NF-B-response element

2

3

1

4

5

IL-2

Glucocorticoids & NFB

NF

-AT

Oct

NF-B

NF

-AT

Oct

AP

-1

Rel-A

NF-B I-B

Rel

-A

NF

-B

GRNLS

GCGC

Glucocorticoids Induce IB Gene Transcription

Rel-A

NF-B I-B

GRNLS

GC

GRNLS

GC

I-B

I-B

Rel-A

NF-B

GGTACAnnnTGTTCT

c-Fos

c-Jun

IL-2

NF

-AT

Oct

NF-B

NF

-AT

Oct

AP

-1

Glucocorticoids & IL-2 Gene Transcription: Summary

GRNLS

GC

GRNLS

GC

Rel-A

NF-B I-B

Coding Region 3’ UTRPromoter

mRNA Synthesis

Coding

3’UTR

AAAAAAAAA

Poly-A

AU-rich region

IL-2

AcceleratedDegradation

IL-2 mRNA Degradationglucocorticoids

• Fungal-derived macrolides

• Structurally related to cyclosporine & tacrolimus

• Bind immunophilin FKBP and the complex targets a serine – threonine kinase “Mammalian Target of Rapamycin” (mTOR)

• Do not inhibit calcineurin

• Inhibit mitogenic response to IL-2 and other growth factors

For detailed pathways, see: http://www.biocarta.com/pathfiles/m_mTORPathway.asp

Sirolimus (Rapamycin) and Everolimus:targeting growth factor (incl IL-2) response

Sirolimus Everolimus

Sirolimus (Rapamycin) and Everolimus:targeting growth factor (incl IL-2) response

Prevent mitogenic response to IL-2 & other growth factors

Sirolimus & Everolimus

IL-2 & other growth factors

IL-2R & other growth factor receptors

Mitogenesis

TCR PI3Kinase/AKT pathway

mTORSir/Ever

Serine/threonine kinase

Translation of mRNA for cell-cycle progression proteins (eg. cyclin D1)

Degradation of p27 cell-cycle inhibitor

FKBP

Sir/Ever

CDK: cyclin-dependent kinase

mTOR

P27 degradation in late G1

Cyclin E

CDK2 p27

Cyclin E

CDK2

G0

G1

G2

S

M

CDK2/cyclin E complex inhibited by p27-CDK-inhibitor during G0 & G1

G1 S Transition

FKBP

Sir/Ever

Sir/Ever

For detailed pathways, see: http://www.biocarta.com/pathfiles/m_mTORPathway.asp

mTOR regulation of translation

mRNA’s for many labile cell-cycle regulating proteins have characteristic sequences or structures

mTOR has two targets that are particularly important in regulating translation of these mRNA’s

They are:

EIF-4E-binding protein-1, which releases eukaryotic initiation factor-4E when it is phosphorylated. EIF-4E contributes to initiation of translation

40-S ribosome subunit

Sirolimus – FKBP and everolimus – FKBP complexes inhibits these mTOR actions also

mTOR

mTOR

p70s6 KinasemTOR

40

FKBP

EIF-4E

40-S ribosome subunit

mTOR regulation of translation

mRNA

Cell-cycle proteins

Sir/Ever

Sir/Ever

Mycophenolate

• Fungal derived

• Inhibitor of de-novo guanosine monophosphate synthesis

• B and T cells depend on de-novo synthesis:-lack salvage pathway for guanine recovery

• Specific inhibition of DNA synthesis, RNA synthesis & other GTP or cGMP requiring pathways in lymphocytes

Ribose-5P + ATP

5-phosphoribosyl-1-pyrophosphate (PRPP)

Guanine Guanosine MP Inosine MP Adenosine MP

Salvage Pathway(deficient in T & B cells)

IMP dehydrogenase

Mycophenolate

CD3- protein of the TCR- contributes to signaling

APC

T

IL-2

Prevent activation of T-cell via TCRAnti-CD3 Antibody

T

CD3

IL-2

Anti-CD3 Antibody

IL-2

Anti-CD3

Mitogenesis

Sirolimus

Mycophenolate

IL-2

Summary:Preventing IL-2-Driven T-Cell Clonal Expansion

CyclosporinTacrolimusGlucocorticoids

Glucocorticoids

Clinical Pharmacology: Clinical Use of Immunosuppressive Drugs in

Preventing Transplant Rejection

The Clinical Problem• Inadequate immunosupression allows transplant rejection• Excessive immunosupression brings infection risk• Calcineurin and mTOR have widespread physiological

functions, so inhibition brings toxicity in various tissues• Drug effects relate to tissue exposure• The drugs have variable pharmacokinetics, obscuring the

relationship between doses and tissue exposure

Clinical Pharmacology: Main reasons of variable pharmacokinetics

• Cyclosporin, tacrolimus, sirolimus and everolimus are all eliminated largely by cytochrome P450 3A4 (CYP 3A4), which is subject to inhibition and induction by other drugs

• Cyclosporin, tacrolimus, sirolimus and everolimus are all also substrates for the ABC-family transporter, p-glycoprotein* in the gut, so are subject to absorption interactions. Especially relevant for cyclosporin.

• Mycophenolate is glururonidated in the liver. Glucuronides are excreted into the bile, reabsorbed and renally excreted. They may be retained in renal failure. De-conjugation by gut bacteria, which is normally inconsequential, may then allow accumulation of the parent mycophenolate.

* aka multi-drug resistance transporter-1; PGP/MDR-1

Improving the Risk-Benefit Ratio: Multiple Drug Targets

• Typically, two or more drug are used together, at doses less than would be needed individually to prevent rejection– Additive efficacy

– Different toxicity

EG: sirolimus or mycophenolate + low dose calcineurin inhibitor + low dose prednisolone during maintenance

Optimising Tissue Exposure: Therapeutic Drug Monitoring

• Tacrolimus, sirolimus, everolimus have long t½ . Pre-dose

blood concentration (ie, trough, also called “C0”) provide

good guide to tissue exposure

• Cyclosporin AUC predicts response & toxicity, but very

variable absorption, so C0 is a poor predictor of AUC. C2 is

a better predictor

0

500

1000

1500

2000

0 4 8 12 16 20 24

Time – hours

C2 C0

Cyclosporin µg/L

AUC0‐4

Mycophenolate TDMMPA

MPA glucuronides

Biliary excretionGut hydrolysis

MPA

MPA reabsorbed

More variable relationship between C0 (C12) and AUC

Figurski et al. Ther Drug Monit 2009;31:717–726