immunosuppressive medications mechanisms usage side effects

Immunosuppressive medications

MechanismsUsage

Side effects

Allograft rejection

T cell (CD4+) dependentrecognize intact MHC class II antigens of

the surface of transplanted cellsrecognize processed antigens on self APCs

T cell receptors (TCRs)major TCR heterodimerminor TCR heterodimerconstant and variable portions

Allograft rejection

TCR engages presented alloantigenT cell is activatedTCR-CD3 complexes transduce signal 1TCR-CD3 complexes and CD4 co-

receptors are phosphorylatedTranscription factors induce expression

of new proteins involved in cell division and proliferation

Allograft rejectionActivated CD4+ cells differentiate into two

distinct populationsType 1 T helper cells (Th1 cells)

IL-2, IFN-mediate activation of macrophages, and delayed

hypersensitivity

Type 2 T helper cells (Th2 cells)IL-4, IL-5, IL-10, IL-13involved in B cells function

Allograft rejection

Activated CD4+ cells secrete cytokines which activate CD8+ cytotoxic cells

CD8+ cells recognize HLA class I molecules, and, if T helper cytokines (IL-2, IL-4, IL-5) are present differentiate, divide, and destroy cells with the recognized markers.

Allograft rejection

CD8+ secretory mechanismgranzymes (serine esterases)perforins kill by DNA degradations and osmotic lysis

CD8+ contact mechanismFas (CD95) on target meets the Tcell Fas

ligand engagement of Fas ligand triggers

apoptosis

Allograft rejectionIL-2 from macrophages induces Th1 cellsIL-4 from other cells induces Th2 cellsTh2 cells down regulate Th1 cells (yin-yang)All T cell responses require two initiating

factors: a TCR-MHC interaction and a “co-stimulatory” signal

Tolerance is related to 1/2 stimulation: TCR-MHC but no second “hit” as in absent or blocked CD40, B7 interactions

Allograft rejection

CD8 knockouts still reject transplantsCD4 knockouts do not rejectB cell responses are important in the

hyperacute rejection and IgG plays a role in chronic rejection

Natural killer (NK) cells recognize alloMHC directly and kill MHC negative and alloMHC cells

Allograft rejection

APCs take up foreign HLA proteins; HLA on a donor APC is recognized directly

Ag specific T cells are activated, proliferate, and enter circulation

T cells move to tissue in response to selectins or chemoattractantsselectins, ICAMs, VCAMs, etc

Transplantation: therapy options

Avoid need - disease prevention / modificatioin

Designer organsImmune toleranceImmunosuppression

Immunosuppresion: historyPrednisoneAzathioprineTotal body (lymph node) irradiationSplenectomyAnti lymphocyte globulin ---- OKT3Cyclosporine, FK506, rapamycinMycophenolate mofetil Daclizumab, Basiliximab

Immunosuppression timeline

Prednisone

Azathioprine

Cyclosporin

Mycophenolate

Tacrolimus

Sirolimus

60’s 80’s 90’s 00’s

Immunosuppression: induction

SteroidsAzathioprineCyA, FK 506 (tacrolimus), and rapamycinOKT3, ALG, ATGMycophenolateDaclizumabBasiliximab

Immunosuppression: rescue

SteroidsOKT3, ATG, thymoglobulinMycophenolate? role of receptor-blockers ?

Steroids: phamacokinetics

Agent specificPrednisone half life 18 - 36 hours70% protein boundActions do not correlate with plasma

concentrationsVolume of distribution variableElderly have decreased clearance, as do

women. Obesity increases clearance

Prednisone: drug interactions

P450 inducers increase the rate of steroid metabolism

Nephrotic patients have more toxicity at the same dosage and level

CyA and FK-506 may interfere

Prednisone: mechanisms

Glucocorticoids act upon steroid receptors and induce genes which shut off cytokine production

AP-1 complexes formed of Jun and Fos proteins are bound by steroid/GR complex

Similar reactions occur with Il-1, Il-3, Il-4, Il-6, IGF-1, TNF, IFN-, Il-8, RANTES, and MCAF

Prednisone: mechanisms

Downregulates: collagenases, elastases, plasminogen activator complex

Decreases: WBC numbersUpregulates: neural endopeptidase

which degrades neuropeptides such as substance P and bradykinin

Alters: adhesion molecules (ICAMs)

Prednisone: mechanisms

Decreases: release of Il-1, TNFLocally inhibits: arachadonic acid

metabolism (blocks PLA-A2)Inhibits: cytokine dependent COXInhibits: platelet activation factorPrevents: synthesis of inducible NO

synthase

Prednisone: side effects

ImmunosuppressionBone and cartilage metabolismCushing’s syndromeGastric symptomsWeight gainDiabetesCataracts

AzathioprineImidazole purine analogue which is the

prodrug for 6-mercaptopurine (6MP)Imidazole side group preserves purine

structure from degradationConverted by HGPRT to a precursor of

thionuclides which inhibits PRPP synthaseInhibits AMP and GMP formationA methylnitroimidazole product may have

separate effects on Ag recognition etc.

Azathioprine

Absorbed in 2 hrsParent drug bioavailability is about 20%Metabolite availability is >40%Total availablity is > 60%Oxidized by GSSHXanthine oxidase inhibitors increase toxicity,

as do genetic factors in AsiansClonal mutation can lead to resistance

Azathioprine

Half lives of drug and active metabolite are short (50 min, 75 min)

Renal eliminationHepatic disease interferes with

metabolism and leads to accumulation of drug

Azathioprine

Excessive marrow suppression5 - 10 days to marrow recoveryAsians and patients taking allopurinol

may become very cytopenicLiver disease: cholestatic disease and

VODInterstitial pneumonitisIncreased malignancies

Cyclosporines

Norwegian soil fungus productMonstrous macrolide antibiotic? Calcineurin inhibitor ?

(E)-14,17,26,32-tetrabutyl-5-ethyl-8-(1-hydroxy-2-methylhex-4-enyl) -1,3,9,12,15,18,20,23,27-nonamethyl-11,29-dipropyl-1,3,6,9,12,15,18,21,24,27,30- undecaazacyclodotriacontan-2,4,7,10,13,16,19,22,25,28,31-undecaone

Cyclosporine A: mechanisms

Disrupts the calcium dependent cascade of events which follows binding of antigen to the T cell receptors and leads to activation and proliferation.

Interferes with secondary messengers in cell

Suppresses genes for c-myc, Il-2, Il-3, Il-4, Il-5, IFN-, and Il-2 receptors

Cyclosporine

CyA/Cyp inhibits calcineurinThis prevents the phosphorylation of nuclear

factor (NF-Atc) of activated T cells The NF-ATc cannot enter nucleus to activate

its nuclear (DNA) subnunitPrevents promotion of: Il-2 gene and thus Il-

2, Il-3, Il-4, IFN-, TNF, also: AP-1, AP-3, OAP, Oct-1 and NF-

Cyclosporine

Prodrug: only active after it binds to intracellular ligands

Immunophilins are small proteins which comprise 0.2 - 0.4% of cellular proteins

Cyclophilins A, B, C, and DComplexes bind to calcineurin catalytic

subunit and render complex inactiveInihibition stops activation of T cells

Cyclosporine

May upregulate TGF-TGF- increases endothelin 1, and

extracellular matrix production

CyclosporineInhibits B cellsCyA inhibits CD40 ligand expression by

inhibiting NF-ATC phosphorylationThe CD40 ligand is involved in T cell, B

cell interactionsAlters TNF production by macrophagesComplex effects on bone marrow

Cyclosporine: Side effects

Nephrotoxicityrelated to trough levelspatchy, striped, interstitial fibrosis

HypertensionCholesterol metabolism Neurotoxicity

peripheral neuropathy

Lethality> 1500

Cyclosporine: drug interactions

Far too many to recallInterference by drugs which are

metabolized by the P450 Interactions at the P-glycoprotein pump

Cyclosporine-drugs : useful things

Erythromycin - itraconazole - ketoconazoleinhibit p450 and reduce CyA dosage (save

money)reduce infections

Diltiazem - verapamil (p-glycoprotein)Interfere with metabolism, other effects

Nifedipine (p-glycoprotein)effect on transplant independent of CyA

FK-450, Tacrolimus (Prograf)

Japanese fungus product Giant macrolide moleculeNot structurally related to CyAWorks much like CyA

3S-[3R*[E(1S*,3S*,4S*)],4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*,18S*,19S*,26aR*]]-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a -hexadecahydro-5, 19-dihydroxy-3-[2-(4-hydroxy-3-methoxycyclohexyl) -1-methylethenyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(2-propenyl) -15,19-epoxy-3H-pyrido[2,1-c] [1,4] oxaazacyclotricosine-1,7,20,21(4H,23H) -tetrone, monohydrate

Tacrolimus

Well absorbedFood slows absorptionPeak 0.5 to 8 hours post doseProtein bound, variable volume of

distributionHepatic metabolism by P-450-3A4Monitoring less critical than with CyA

Tacrolimus: mechanismsProdrug which binds to an immunophilinFK-BP (high affinity receptor)FK-BP not related to cyclophilinsInhibits rotamase activity of FK-BP onlyDoes bind to rapamycin BPComplex (FK-506/FK-BP) binds to and

inhibits a calcium/calmodulin-dependent serine-threonine phosphatase

Tacrolimus: mechanism

Inhibition of the phosphatase prevents the enzyme from dephosphorylating the transcription factors for NF-AT, AP-3, Oct-1 and others which inhibits gene turn on

The result is less Il-2, Il-3, Il-4, TNF-, IFN-Also affects a rapamycin inhibitable system

which is not calcium dependent

Tacrolimus

Clinically, tacrolimus is very close to CyA in terms of effects, though the binding proteins and the precise mechanisms differ

Pharmacologically, the drug is 50 times more active wt for wt

Blood levels are said to be much more stable

Tacrolimus: toxicity

NephrotoxicitySimilar to CyAMechanism of toxicity is unknown

Inhibits the P-glycoprotein multidrug transporter

Tacrolimus: drug interactions

Pharmacological antagonist for CyASimilar profile of drug interactions

Rapamycin

Yet another macrolide Not nephrotoxic ?Acts at a point beyond tacrolimus and

CyAActs on the same binding protein as

tacrolimus, but the mechanisms differSome protocols use in combination with

CyA

(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S, 26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26, 27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]- 1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26- hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]-oxaazacyclohentriacontine-1,5,11,28,29 (4H,6H,31H)-pentone

Rapamycin

Binds to TOR (target of rapamycin)Inhibits signal transduction downstream

from growth factor receptors (IL-2)Blocks progression from G1 to S phase

of cell cycleAntagonist of tacrolimusCooperative with cyclosporine A

Mycophenolate mofetil:

Inhibts inosine monophosphate dehydrogenase (IMPDH)

Mycophenolate is the morpholino-ethyl ester of mycophenolic acid (a penicillium product)

Non-competitive inhibitor of IMPDH

Mycophenolate: mechanism

Inhibits IMPDHIMPDH is a key enzyme in purine nucleoside

synthesisIMPDH inhibition lowers GMP levels which

occupy a key step in T cell responseTCR /CD3 complex activity may need GMPThus cell activation is inhibited

Mycophenolate: mechanism

Lymphocytes do not have purine salvage pathway enzymes

Lymphocytes depend on IMPDH for purines

Lymphocyte proliferation is blocked by MPA effect on purines

Most other cells have purine salvage and are not affected

Mycophenolate: mechanisms

Effects can be reversed by administration of guanosine nucleotide

Guanosine does not reverse AZA effectsIMDPH acts if given up to 24 hours after

T cell stimulationDoes not act on early response genes

and IlsArrests lymphocytes in G1 phase

Mycophenolate: toxicity

LeukopeniaAnemiaGI toxicityNote: despite the “targeting” of the

drug to cells without a salvage pathway, the toxicity tends to indicate all high turnover cells are at risk…….. ? a rate issue ?

Antibodies

ALG goingATG goingMM AB goneOKT3 only option

OKT3

Murine IgG2a directed against chain of CD3 molecule

Cells may be activated and then release cytokines which cause acute toxic responses

Block recognition site by internalizationCells are then opsonized and

sequestered by macrophages

OKT3

Given IV only, 5mg a day for up to 14 days

First 2 to 3 doses may trigger severe reactions which result in flu-like symptoms, acute pulmonary edema, and chemical meningitis

Wt must be within 3% of dryProfound lymphopenia results

OKT3

Very high risk for viral infectionsUse immune globulin for CMV

If dose excessive, high risk for lymphoproliferative malignancyDon’t exceed 70 mg

Antibodies may developTest for Ab titer before second dose

IL-2 receptor antibodies

IL-2 is a major factor in the proliferation of activated Tcells in response to grafts

CyA, tacrolimus inhibit IL-2 production by interfering with secondary messages which lead to stimulation of the IL-2 promoter

Rapamycin inhibits secondary messenger responses (by Tyr Kinase pathways) to IL-2 stimulation

Activation of T cells

APC T cellPresentation of Ag to Tcell Ag receptor

Proliferation/differentiation signal

Activation of T cells

APC T cell

IL-2

rejection

IL-2 and tolerance

Since IL-2 stimulates Tcells in recursive manner, it would seem that IL-2 inhibition should block rejection and memory

But, IL-2 knockout mice reject heart transplants

Anti IL-2 treatment slows but does not prevent later rejection

IL-2 neutralizing antibodies reduce toleranceIL-2 causes apoptosis of activated T cells

(clonal deletion role)

IL-2 effectsActivation of T cellsInduction of “memory cells”

Induction of T cell apoptosis

T cells and tolerance

T cell activation is required for tolerance to develop

Complete suppression of T cell response (total blockade of IL-2, for example) leads to ignorance, and the host remains primed to recognize and reject

Tolerance is preferable to ignoranceIL-2 inhibits the maintenance of memory

cells

Memory cellsNaïve cells Memory cells

Survival Long (years) Very long (life)

Homing Circulate to secondary lymph sites

Circulate to lymphatic and non lymph tissue *

Response Slow (high threshold)

Fast (low threshold)

*Memory cells do not require secondary lymphatic structures to react

T cell responses

T cellDelete (die – apoptosis)

Suppress ( T regulatory)

Deviate (Th2)

Anergy

Ignore -- tolerance

Memory

time

die offexpandclone

memory

tolerance

exposure toforeign antigen

IL-2 receptor antibodies

Rather than inhibit the secondary messages which lead to IL-2 production or the response to IL-2, directly attack the major element -- IL-2

IL-2 blockers are clinically available

IL-2 R antibodies

Daclimuzab (Zenapax) is a “humanized” anti-T cell chain IL-2 receptor antibody

Basilixamb (Simulect) is a chimeric mouse anti-CD 25 monoclonal antibody with affinity for the alpha chain of the IL-2 R

IL-2 receptor antibodies

Vicente et al (NEJM 98): Induction protocol which included CyA and daclimizab (IL-2 receptor antibodies)

Results of the study indicated there was an increase in transplant survival (95 vs 90%), and decreased rejection episodes in cadaver kidney recipients

IL-2 receptor antibody

Nashan et al used basiliximab as induction protocol (Lancet 350:1193-1198, 1999)

They reported a reduction in biopsy proven rejection (30% vs 44%) European trial