TRANSPLANT 1

Question 1

PATHOPHYSIOLOGY OF ORGAN REJECTION
Categories of graft rejection:
Two types of immunity:

Answer 1

Categories of graft rejection:
 Acute cellular: infiltration of T cells into the allograft → inflammatory and cytotoxic
effects
 Humoral/chronic: cellular cytokines, CD4+ and CD8+ T cells, B-cells, antibodies
Two types of immunity:
 Innate: complements, granulocytes, monocytes, macrophages, natural killer cells, mast
cells, basophils (fast acting, no memory, does not require priming)
 Adaptive: B and T cells (relatively slower in response, has memory, requires priming)
 Antibodies generated from plasma B cells
 Helper, cytolytic, and regulatory T cells:
 T-cells sense/recognize foreign antigens (i.e., protein, peptide fragments) as major
histocompatibility complex (or human leukocyte antigens) presented by antigen
presenting cells (APC)
 APCs: macrophages, dendritic cells
 Human leukocyte antigens / major histocompatibility complex: genetic coding of
proteins that allow the individual to differentiate self from non-self
 B and T cell activation → cell division, release of chemokines/cytokines/antibodies

Question 2

T-CELL ACTIVATION
PROCESS

Answer 2

1) T-cell receptor identifies antigen bound
to MHC
2) Co-stimulatory signal is needed for T-cell
activation (CD80/86 – CD28 interaction)
3) Increased interleukin-2 (IL-2) generation.
Feedback amplification

Question 3

TRANSPLANT PHARMACOTHERAPY

Answer 3

1) Optimize ABO blood type and HLA match (organ donation
team, Canadian Blood Services)
2) Combinatorial pharmacotherapy:
Induction: short duration, maximum immunosuppression, peritransplant
Maintenance: to be discussed in detail in these lectures
Target different pharmacological targets; use of lower doses→
maximize efficacy and minimize toxicity
3) Maintain a fine balance between drug efficacy and toxicity,
in the setting of multiple co-morbidities (cardiovascular
disease, endocrinological disease, bone-mineral disease,
infectious disease)

Question 4

MAINTENANCE THERAPY

Answer 4

Minimize / prevent acute and chronic rejection
Minimize immunosuppressant-associated toxicities
Combinatorial therapy required in transplantation (multiple targets)
Antimetabolites: azathioprine or mycophenolic acid – reduces purine
synthesis and decreases T-cell proliferation
Calcineurin inhibitors: cyclosporine or tacrolimus – reduces interleukin
2 (IL-2) production and decreases T-cell activation
Corticosteroids: prednisone vs. steroid-free – multiple mechanisms
 Mammalian target of rapamycin (mTOR) receptor inhibitors: sirolimus
reduces IL-2 production (not routinely used)

Question 5

MYCOPHENOLIC ACID

Answer 5

*Indication:
*Solid organ transplantation (kidney, liver, heart, lung)
*Off-label for autoimmune hepatitis, lupus, psoriasis, autoimmune disorder,
graft-vs-host disease
*Largely replaced azathioprine as the “anchor” antimetabolite due to
improved efficacy, increased patient/graft survival (evidence: Wagner et al.
Cochrane Database Syst Rev. 2015; 12:CD007746. doi(12):CD007746)
*Mechanism:
*Non-competitive binding to inosine monophosphate dehydrogenase
(IMPDH- type II [lymphocyte specific] – reduced off-target toxicity)
*Blocks guanosine nucleotide synthesis, reduces DNA polymerase
activities
*Reduces T and B cell proliferations

Question 6

MYCOPHENOLIC ACID
*Dosing and formulations:

Answer 6

q

Question 7

MYCOPHENOLIC ACID
Pharmacokinetics

Answer 7

q

Question 8

MYCOPHENOLIC ACID
*Absorption
distribution

Answer 8

MYCOPHENOLIC ACID
*Absorption:
*Different absorption behaviours between mycophenolate mofetil (MMF,
Cellcept) and enteric-coated mycophenolate sodium (EC-MPS, Myfortic) – not
interchangeable
*Cmax with MMF formulation (1-1.5hr) vs. EC-MPS (1.5-3hr)
*bioavailability (F) with MMF (>90%) vs. EC-MPS (~70%)
Food  Cmax of both formulations by 33-40% but does not change areaunder the curve (AUC). **consistency with food intake is key.
*Distribution:
*Primarily into the plasma (drug monitoring in plasma)
*~3.6 L/kg for the MMF formulation
*Extensively bound (<97%) to albumin
*May distribute into fetus and milk. Contraindicated during
pregnancy and lactation

Question 9

MYCOPHENOLIC ACID *Metabolism and Excretion

Answer 9

*MMF is a pro-drug→
bioactivation
→ mycophenolic acid
(MPA)
*MPA undergoes extensive intestinal
and hepatic metabolism
(conjugation) by UDP
- glucuronosyltransferase 1A9 to
form MPA
-glucuronide (inactive)
* Potential for drug
-drug interactions
*Minor pathway: UGT2B7
-mediated
conjugation to form the acyl
- glucuronide (bioactive?)

Mycophenolic acid glucuronide
(MPAG, the primary metabolite)
Mycophenolic acid
(MPA)
Mycophenolic acid acyl glucuronide
(AcMPAG, the minor metabolite)

Question 10

MYCOPHENOLIC ACID
*Metabolism and Excretion

Answer 10

*MPAG excreted into the bile by multidrug resistant protein 2 (MRP2)
*MPAG gets deconjugated in the intestines by bacteria and gets recycled
back into systemic circulation (entero-hepatic recirculation)
*Potential for drug-drug interactions
*MPAG excreted into the urine (up to 87% of dose) by organic anion
transporter 3 (OAT3) and MRP2
*Potential for drug-drug interactions
*Half-life: ~ 18±7 hr (MMF) vs. 8-16 hr (EC-MPS)
*How much time to reach steady-state after dosage adjustment?

Question 11

MYCOPHENOLIC ACID
*Adverse effects:

Answer 11

Gastrointestinal (e.g., abdominal pain, nausea, vomiting,
diarrhea)
*Mitigation: switch MMF to EC-MPS to minimize stomach upset. Mechanism:
potentially due to  local irritation at different site of absorption
*Infections (e.g., bacterial, viral, fungal)
*Hematological (e.g., neutropenia, leukopenia, anemia).

Question 12

MYCOPHENOLIC ACID
*Drug-Drug interactions:

Answer 12

*Clinically relevant pharmacokinetic interactions:
*Aluminum, magnesium-containing antacids:  AUC (absorption)
*Cholestyramine:  AUC (absorption)
*Proton pump inhibitors:  AUC (absorption) However, the majority of patients will be given the PPI on discharge hey? So the key there is to ask them to be consistent with the PPI
- decrease absorption because of change changing the gastric acid.

*Acyclovir:  AUC (renal excretion) Inhibit OATs, renal secretion is blocked by it

*Cyclosporine:  AUC (entero-hepatic recirculation). No interaction with
tacrolimus. Dose adjustment should be considered when MPA given with
cyclosporine.
*Antibiotics:  AUC (entero-hepatic recirculation).
*Clinically relevant Pharmacodynamic (PD) interactions:
*Drugs that can cause immunosuppression / leukopenia / neutropenia

Question 13

MYCOPHENOLIC ACID
*Therapeutic drug monitoring

Answer 13

*Not consistently practiced, except for
*Active rejection
* Evidence of side effects
*Abnormal kinetics (erratic clinical response)
*Limited sampling strategy (use of a
limited number of blood samples to
estimate the full exposure)
* Specific to the population in which the
equation was developed
Therapeutic target: AUC of 30 – 60
mgh/L

So this is based on the total concentration. It’s 30 to 60 milligram hour per year. Total concentration.
Okay. So this is based on public concentration, and we we discuss why this may not be the optimal
way to monitor the drug, because it’s a total concentration. There’s scenarios where the free concentration may not change where the total concentration can change. So this has its limitations.

Question 14

MYCOPHENOLIC ACID
*Pharmacogenomics

Answer 14

*No clinically relevant single nucleotide polymorphisms or linkage
dis-equilibriums as of the year 2023
*No genomic-based dosing recommended to date

Question 15

MYCOPHENOLIC ACID – CLINICAL CASES

CJ (50 yrs old, 70 kg) just received a cadaveric kidney
transplant (panel reactive antibody 5%) and the transplant
team wants to start her on mycophenolate, tacrolimus, and
valganciclovir (cytomegalovirus mismatch). She was not
prescribed a regular maintenance steroid regimen because of
the low immunologic risk. She has a history of hypertension
and takes amlodipine 5mg PO daily. All of her blood work is
within normal limits. Her current GFR is 15mL/min. The site
protocol also starts the patient on cotrimoxazole 3 times
weekly as antibacterial prophylaxis and pantoprazole 40mg
po daily as GI prophylaxis. On what dose of mycophenolate
would you start CJ? Is therapeutic drug monitoring warranted
in this setting?

Answer 15

Loading dose typically not administered
Conventional starting dose (not weight-based): 1g PO BID
MMF
Insufficient evidence to support therapeutic drug monitoring
unless suspecting altered PK or evidence of rejection/side
effects
Limited sampling approach for AUC estimation
Do not use trough concentration – not reliable

Question 16

It has been 1-month post transplant and CJ’s renal function has
deteriorated to ~ 10mL/min. Her serum creatinine has been
trending up slightly over 2 weeks. Her current
immunosuppressants are mycophenolate mofetil 1g PO BID
and tacrolimus 2.5mg PO BID (trough concentration within
target). Her blood pressure is adequately controlled on
amlodipine. She is seronegative with respect to
cytomegalovirus and BK virus. Her clinical blood work is within
normal limits. She has not experienced any adverse effects
from drugs. The team suspects acute graft rejection and has
ordered a renal ultrasound to be followed by renal biopsy. Is
it appropriate to monitor mycophenolate exposure in this
setting? How would you adjust the dose?

Answer 16

Assuming no other causes, it may be appropriate to monitor
MPA exposure
Best approach is to estimate AUC using limited sampling
equations (only used in a similar patient population for which
the equation was originally developed/validated)
Steroid-free:
MPA exposure = 9.328 + 1.311 (C1hr) + 1.455 (C2hr) +
2.901 (C4hr)

. It just needs plasma concentration, Mpa: 1 h after dosing
2 h after dosing and 4 h after dosing. So the timing for these
concentrations is critical.

Question 17

You asked for and the laboratory reported the following MPA
concentrations (dose given at 9am):
10:05am: 4 mg/L
11:03am: 1.5 mg/L
12:58pm: 1 mg/L
Are you able to calculate CJ’s mycophenolate exposure based
on this information?

Answer 17

MPA exposure = 9.328 + 1.311 (C1hr) + 1.455 (C2hr) +
2.901 (C4hr)
MPA exposure = 9.328 + 1.311 (4mg/L) + 1.455 (1.5 mg/L)
+ 2.901 (1mg/L)
= ~ 20 mghr/L
Therapeutic target is 30 – 60 mghr/L, assuming a
proportional dose response:
Desired dose = (desired exposure / current exposure) *
current dose
Desired dose = 30mghr/L / 20 mghr/L * 2 g/day
Desired dose = 3 g/day or 1.5g PO Q12hr MMF

Proportional:
So if you increase the dose
by one fold. You would also expect an increase in AUC by one fold approximately.

Question 18

CJ’s kidney rejection episode was reversed with steroid pulse, and
her renal function has gradually normalized (GFR ~ 50 mL/min) at
2 months post transplant. Her current immunosuppressants are
mycophenolate mofetil 1.5g PO BID and tacrolimus 3 mg PO BID
(trough concentrations within target). Her blood pressure is
adequately controlled (130/80 mm HG) on amlodipine 10mg PO
daily. She is still on valgancyclovir 900mg PO daily and
cotrimoxazole DS tablet 3 times weekly. Her clinic blood work is
within normal limits, but she complains of stomach cramps and
occasional diarrhea for the past 1 month, which became more
intensified/intolerable in the past week. The symptoms are more
intense after the ingestion of her BID medication regimens. How
would you manage this complaint?

Answer 18

Thorough history of stomach ailments should be taken
If no other causes (e.g. peptic ulcer disease, infection), then
consider drug adverse effects
Diarrhea/cramp more likely associated with MMF (timing
associated with dose increase and with MMF dosing times)
Option 1: split the dose into QID dosing (i.e. 0.75g QID) with
food and snack
It’s gonna affect the C. Max, but it’s not going to affect the Auc.

Option 2: switch to enteric-coated mycophenolic acid sodium
(Myfortic). Closest “equivalent” dosing ~ 1.08g PO BID).

Question 19

A year after CJ’s transplant, her renal function has remained
stable (GFR ~ 60 mL/min). Her current immunosuppression regimen
includes enteric-coated mycophenolate sodium 1.08g PO BID and
tacrolimus 3.5 mg PO BID (trough concentration within range). Her
cotrimoxazole had been taken off 2 weeks ago because of a
significant drop in white blood cell count. Her valganciclovir was
stopped 3 months ago because of several, consecutive seronegative cytomegalovirus readings in the blood. Her blood
pressure is well controlled with amlodipine. Her current clinic blood
work still reveals a depressed white blood cell count and
neutrophil count. She is afebrile and otherwise asymptomatic.
Virology indicates no detectable amounts of BK or
cytomegalovirus in the blood. How would you advise the team?

Answer 19

Neutropenia is common within 1-2 years of transplant due to many
reasons
If no other causes (e.g. virus-associated), must consider drugs.
Valganciclovir and cotrimoxazole had already been stopped.
MPA can cause neutropenia; exposure can increase over time after
transplant
Can you estimate AUC of MPA in this case (limited sampling
equation available for EC-MPA)
Empirically drop the dose by 25 – 50% and monitor the patient
closely (CBC, renal function, signs of infection, signs of rejection)

Question 20

AZATHIOPRINE

Answer 20

Indication:
*Kidney transplantation and rheumatoid arthritis
*Off-label Crohn’s disease, lupus, ulcerative colitis, and psoriasis
*2
nd line therapy in transplantation
*May be considered in severe MPA allergy/toxicity, pregnancy
*Mechanism:
*A prodrug
*Azathioprine→ 6-mercaptopurine→6-thioguanine-nucleotide
(halts lymphocyte DNA replication)
*Deactivation by xanthine oxidase, thiopurine Smethyltransferase (TPMT), and nudix hydrolase 15 (NUDT15)
*Drug/gene-interaction implications

Xanthine oxidase - specifically problematic w respect to drug-drug intx
Allopurinol contraindication w full dose azathioprine

Question 21

AZATHIOPRINE
*Dosing and formulations:

Answer 21

Loading dose not usually done

Usually we just start with the maintenance those because the patient would have been
adequately immune, suppressed either with induction therapy or with something else.

typically once daily

w/ allopurinol -> reduce azathiprine to 1/4 of orig dose

Question 22

AZATHIOPRINE
*Absorption:
distribution

Answer 22

*Absorption:
*F ~ 40%
*Tmax ~ 1-2 hrs
*Effects of food on absorption not well characterized (consistency with
food is key)
*Distribution:
*6-mercaptopurine distributes to total body water
(approximates its Vd)
*Not extensively protein bound (~70% free)
*Distributes into the placenta, but considered “acceptable” for
use in kidney transplant patients planning pregnancy

Question 23

AZATHIOPRINE
*Metabolism and Excretion:

Answer 23

*Hepatic glutathione S-transferase: converts azathioprine to 6-
mercaptopurine
*Hypoxanthine guanine phosphoribosyltransferase: converts 6-
mercaptopurine → 6-thioguanine-nucleotide (TGN, active, multi-step
process)
*Deactivation of 6-mercaptopurine by xanthine oxidase and TPMT
*Allopurinol and febuxostat (xanthine oxidase inhibition):   6-mercaptopurine
plasma concentrations
*Deactivation of TGN by nudix hydrolase 15 (NUDT15) (triphosphate→
monophosphate)
*Urinary excretion of formed metabolites
*Half-life: ~ 3 hr (6-mercaptopurine)

Question 24

AZATHIOPRINE metabolism

Answer 24

further bioactivated into 6-TGNs, and these are the metabolites which intercalate with DNA and lead to T cell suppression and bone marrow suppression

So if you block the deactivation pathways, you will increase the active metabolites.

Question 25

AZATHIOPRINE
*Adverse effects:

Answer 25

*Gastrointestinal toxicity (nausea, vomiting, diarrhea)
*Food may help
*Hematological toxicities (thrombocytopenia, leukopenia, anemia)
*Can occur within weeks of starting azathioprine (typically
managed with dose reduction)
*Correlated with reduced TPMT / NUDT15 activities (loss of
function polymorphism) or with concurrent xanthine oxidase
inhibitors
*Dose-dependent liver toxicity (cholestatic and hepatocellular)
*Alopecia, pancreatitis (very rare)

Question 26

AZATHIOPRINE
*Drug-Drug interactions:

Answer 26

*Xanthine oxidase inhibitors:  6-mercaptopurine concentration by
up to 4x (reduce the dose of azathioprine by up to 75%)
*Shunts the metabolism to  production of 6-thioguanine-nucleotide
→ bone marrow suppression
Drug-Gene interactions (pharmacogenomics):
TPMT
NUDT15
 minor allele frequencies: https://cpicpgx.org/guidelines/guideline-for-thiopurines-and-tpmt/

Question 27

AZATHIOPRINE
pharmacogenomics

Answer 27

You can determine whether they are a normal metabolizer of Tpmt.
the intermediate metabolizer. or a poor metabolizer. So it a normal metabolizer will be a patient carrying 2 copies, the wild type allele
Intermediate: 1 wild type

if their intermediate metabolizer. They also recommended starting with a a
reduce those 30 to 80% reduction.
Okay. And if they’re a poor metabolizer
in the setting of transplantation, they tell you to avoid azathioprine

Same recommendations for NUDT15

Question 28

AZATHIOPRINE
*Therapeutic drug monitoring

Answer 28

*Concentration monitoring not commonly done
*Monitor for clinical signs/symptoms/blood biochemistry

Question 29

SAMPLE FINAL EXAM QUESTION
*Which of the following is correct about the therapeutic drug
monitoring of mycophenolic acid?
1) Trough concentration monitoring is routinely done in kidney transplant
patients.
2) Limited sampling strategy equations may be used in the clinic in select
clinical scenarios, but should be tailored to the patient population they were
originally derived from.
3) Therapeutic drug monitoring of mycophenolic acid is never conducted in
kidney transplant patients.
4) All are correct.
5) None are correct

Answer 29

2) Limited sampling strategy equations may be used in the clinic in select
clinical scenarios, but should be tailored to the patient population they were
originally derived from.