immunosuppressive medications mechanisms usage side effects
TRANSCRIPT
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