2. A patient with congestive heart failure
has been successfully treated with
digoxin for several years, but recently
developed renal failure. Laboratory
records indicate semi-annual peak
digoxin concentrations that have all
been within the therapeutic range. A
serum specimen was collected upon
admission. Although the digoxin
concentration is elevated, the
physician indicates the patient is not
exhibiting signs or symptoms of
toxicity
Case study 2
5. Definition
Drug monitoring
Process of studying effects
of a chemical substance
administered to an
individual
Therapeutic Drug monitoring:
Process used to measure
blood drug levels so that
the most effective dosage
is maintained and toxicity
prevented
5
6. Introduction
• Measurement of plasma concentration involving
analysis, assessment, and evaluation of
circulating concentrations of drugs in serum,
plasma, or whole blood
• Clinical laboratory measurement with
appropriate medical interpretation
• Individualize therapeutic regimens for optimal
patient benefit
6
8. Pharmacokinetics
What body does
to a drug?
Mathematica description of
physiological disposition of
xenobiotics or endogenous
chemicals
Indicated by
Absorption
Distribution
Metabolism
Excretion
8
9. Pharmacokinetics
• All of these are dependent on:
• Disease state
• Distribution
• Co-medication
• Age
• Sex
• Amount of drug absorbed relative to quantity given is referred
to as its bioavailability
9
10. Pharmacokinetics-
Absorption
• Most direct route of
administering a drug is
intravenous delivery
• Frequently delivered via
oral administration
• Pass from gastrointestinal
tract into vascular system
• Compound must
dissociate from its dosing
formulation into digestive
fluids
10
11. Pharmacokinetics-Drug Distribution 11
• Spreads throughout the systemic circulation
and into various tissues
• Distribution of a drug to a particular site in
body depends on numerous factor
• Molecular size,
• Degree of ionization,
• Lipid solubility,
• Extent of protein binding
• Body composition
13. Therapeutic Range/Window
Therapeutic range/ therapeutic
window is concentration range of
drug in plasma where drug has been
shown to be efficacious without
causing toxic effects in most people
13
14. Drug Metabolism
• Biotransformation of a compound, whether endogenous or exogenous
• Many drug metabolites are active (must be considered)
• Metabolic enzymes are expressed body, with largest concentrations in
oLiver
oGastrointestinal tract
oKidneys
• Most drugs undergo first pass metabolism
• Biochemical pathway responsible for metabolism: Hepatic mixed
function oxidase (MFO) system
14
15. Drug Elimination
• Plasma free fraction of a parent
drug or its metabolites is subject
to glomerular filtration, renal
secretion, or both
• If there is no reabsorption,
elimination rate directly relates to
GFR
• Drugs are eliminated through
hepatic metabolism, renal
filtration, or a combination of both
• Most often occur as a first-order
process indicating an exponential
rate of loss
15
16. Protein Binding
• Most drugs bind to Proteins in plasma (e.g. bilirubin)
• Acidic drug bind to albumin while basic bind to glycoproteins
• Proportion: 0%-100%
• Effects of changes of protein binding esp. during interpretation
• Free fraction of a drug change within an individual over
time e.g. pregnancy or withdrawal of drugs
• Highly abnormal binding proteins concentration in plasma
• Pathological states
16
17. Main Pharmacokinetic Parameters
• Bioavailability
• Volume of distribution and distribution phases.
• Clearance
• Half-life
• Protein binding of drug
17
18. Pharmacodynamics
• Action of drug on body
• Optimum dosage can be arrived at by commencing
treatment with a standard dose
• Modifying this as necessary in light of the observer
response
• Effect also depends on drugs competing for same
receptor
• Genetic conditions/ Tolerance status for drug
18
19. Factors influencing
pharmacokinetics and Dynamics
• Correlation between
plasma concentration and
pharmacological effect
provides the rationale for
the use of concentration
measurement in
therapeutic drug
monitoring
19
20. Pharmacodynamic monitoring
• Study of biological effect of a drug at target site
• Mostly applied to area of immunosuppressive therapy and cancer
therapy
• Eg: Effects of cyclosporin and tacrolimus assessed by direct
measurement of calcineurin phosphatase activity
• Disadvantage: Assays involved are often complex and time
consuming
20
21. Role of Biomarkers
• Biochemical measurement used to determine efficacy,
extent of toxicity or individual pharmacodynamics
• Marker of toxicity more then pharmacodynamics
• Eg: Red cell 6-thioguanine nucleotide concentrations
• Integrate biomarker monitoring~ Define therapeutic
ranges
• Eg:
21
22. Pharmacogenetics
• Study of genetic influences on drug metabolism and application of drugs
to enhance safety and/or efficacy
• Pharmacogenetic polymorphism is defined as existence in a population
of 2 or more alleles at same locus resulting in phenotypes in respect to
effect of drug
• Administration of test dose and integrating information
• Eg: CYP3A4 isoforms
• Clinical application
• Anticoagulation (Warfarin-CYP2CA)
• Oncology/immunosuppression (DPD gene mutations)
• Psychiatry (COMT genotying)
• Epilepsy etc.
22
23. Why
therapeutic
Drug
monitoring?
Analysis, assessment, and
evaluation of circulating
concentrations of drugs
Certain drugs have a
narrow therapeutic range
Not all patients have the
same response at similar
doses
Effective and safe drug therapy
in individual patient using
serum drug concentration
23
24. Criteria for clinically useful TDM
• Absence of good clinical marker of drug
effect
• Poor correlation between dose and clinical
effect
• Good correlation between plasma drug
concentration and clinical effect
• Narrow concentration interval between
therapeutic and toxic effects
24
25. Criteria for effective drug monitoring 25
• Appropriate clinical question
• Accurate patient information
• Appropriate sample
• Accurate analysis
• Relevant clinical interpretation
• Effective action taken
28. Appropriate sample
• Most preferred: Blood sample
• Serum Vs. Plasma
• Depending on drug (Cyclosporin concentration in RBC)
• Avoid hemolysis
• Protein bound Vs. Free bound
• Urine, Saliva etc.
• Timing: Need a baseline and drug dependent
28
30. Saliva, Upcoming TDM Specimen
• Non-invasive
• Concentration of a drug in saliva is proportional to the
concentration of unbound drug in plasma
• Easier in children and neonates
• Limitations:
• Discrepancies in plasma/salivary ratios
• Salivary flow may be reduced in patients taking anti cholinergic drugs
• High interferences
30
32. Accurate Analysis
Purpose required
• On-site analysis in clinics
• Urgent analysis
• Batch analysis, single drug
• Batch analysis, Multi-drug
• Single analysis with metabolite
patterns
Most appropriate method
• Point of case immunoassay
• Optical immunoassays
• Optical immunoassays, HPLC,
Gas Chromatography, Mass
Spectrometry (MS)
• HPLC, Gas chromatography
(GS)
• HPLC, MS or LC/MS
32
33. Current techniques to assess TDM
• HPLC: High Pressure Liquid Chromatography is a common analytical method
used to measure therapeutic drug levels. Eg: Bupropion
• GC/MS and LC/MS: Gas-liquid chromatography is a separation method using
very high temperatures to cause sample vaporization. molecules separated on
the basis of molecular weight therefore establishes a “fingerprint” for
identification
• RIA: Radioimmunoassay not commonly used any longer due to waste disposal
issues
• EIA or enzyme immunoassay: Most of drug testing today is performed using
homogeneous EIA techniques in a single step. Eg: Digitoxin
• Chemiluminescence: This is a chemical reaction that emits energy in the form
of light. Most common is enzyme-amplified. Eg: Phenobarbital, Valproic acid
33
34. Newer techniques to assess TDM
• PETINIA: An immunoturbidimetric method that is used today for TDM testing is
PETINIA or Particle Enhanced Turbidimetric Inhibition Immunoassay. This
method uses the creation of light scattering particles to measure drug levels
• EMIT (Enzyme Multiplied Immunoassay Technique): competition for target
analyte antibody binding sites. Eg: Methotrexate
• FPIA (Fluorescence Polarization Immunoassay): fluorescent molecule as label
• ACMIA: Affinity Chrome-Mediated Immunoassay. ACMIA is a technique to
measure drug concentrations in which free and drug-bound antibody enzyme
conjugates are separated using magnetic (chrome) particles
• CEDIA: Cloned Enzyme Donor Immunoassay. CEDIA employs a recombinant DNA
technology
34
35. Relevant clinical interpretation
• Target ranges to be set
• Therapeutic ranges to be set to patient response and not
to be inflexible
• Therapeutic decisions not to solely rely on serum drug
concentrations
• Treat Patient, not drug concentration
35
36. Effective action taken
• Patient symptomology
• Drug concentration based on therapeutic window and
response
• Adverse effects
• Achievement of steady state concentration
• Drug taken time to be known
36
38. Lithium
This Photo by Unknown Author is licensed under CC BY-SA
• Treatment of Bi-polar disorder
• Mechanism of action: unknown
• Half life: 20-40 hours depending on treatment duration
• Narrow therapeutic window; Highly nephrotoxic
• Dosage: Slow release preparations~ 12hourly
• Elimination: Kidneys
• Effective concentrations: ~1.2mmol/L (acute mania)
• Method of estimation: Fluorimetry and
spectrophotometry, ISE
38
39. Lithium
This Photo by Unknown Author is licensed under CC BY-SA
Plasma concentration response relationship
• ˃ 0.4 mmol/L: Little therapeutic effect
• 0.4 to 1 mmol/L: Optimum range for prophylaxis of mania
• 0.8 to 1.2 mmol/L: Optimum range for acute mania
• 1.2 to 1.5 mmol/L: Causes possible renal impairment
• 1.5 to 3 mmol/L: Renal impairment, weakness, drowsiness, thirst
and diarrhoea
• 3 to 5 mmol/L: Confusion, spasticity, convulsions, coma and death
39
40. Lithium
This Photo by Unknown Author is licensed under CC BY-SA
• Problems faced with estimation being done here
• No history of patient
• Sample type unknown
• No monitoring being reported
• Lack of involvement and participation
40
41. Phenytoin
• Antiepileptic Drug
• Mechanism of action: voltage-dependent block of
voltage gated sodium channels
• Long half life
• Elimination: Kidneys
• Effective concentrations: 5-20mg/L (20-80 µmol/L)
• Timing: from time of administration to 6-7 days
• Method of estimation: HPLC
This Photo by Unknown Author is licensed under CC BY-SA
41
42. Phenytoin
• Narrow therapeutic window
• Highly protein-bound; drug-drug interactions, drug-
disease interactions
• Non-linear pharmacokinetics even within the
therapeutic range
Approximately 90% of phenytoin is bound to albumin
and must be corrected according to albumin levels:
This Photo by Unknown Author is licensed under CC BY-SA
42
43. Phenytoin
• When taken in doses >20mg/L
Neurotoxicity (Concentration dependent):Far gaze nystagmus
Gastrointestinal: Nausea, vomiting, Anorexia
• Doses >30mg/L: 45º lateral gaze nystagmus and ataxia
• Doses >40mg/L: Decreased mentation
• Doses >100mg/L: Death
This Photo by Unknown Author is licensed under CC BY-SA
43
44. Tacrolimus
• Immunosuppressants- Calcineurin phosphastase inhibitor
• Prevent graft rejection
• Metabolism: CYP3A in liver
• EDTA whole blood preferred
• Not nephrotoxic but causes vascular renal constriction
• Monitoring very important in hepatic dysfunction
• Preferred to used in combined therapy
• Target concentrations: 5-15μg/L
• Methods of estimation: Immunoassays or LC-MS preferred
44
This Photo by Unknown Author is licensed under CC BY-SA
45. Digoxin
• Cardiac Glycosides
• Mechanism of action: Inhibition of Na+, K+ ATPase pump
• Long term patients: production of additional pump
• Long half-life (20-60 hours)
• Dose: Once daily
• Elimination: Kidneys
• Effective plasma concentration : 0.5-2.0 μg/L (0.6-2.6
nmol/L)
• Heart failure: 0.5-1.0 μg/L (0.6-1.3 nmol/L)
• Timing of blood sampling: 6hour post dose
• Method of estimation: liquid chromatography-tandem mass
spectrometry (LC/MS)
This Photo by Unknown Author is licensed under CC BY-SA
45
46. Digoxin
• Plasma concentration –response relationship
• 0.5µg/L: No therapeutic effect
• 0.7 µg/L: some ↑ in force of contraction of heart
• 0.8- 2 µg/L: Optimum therapeutic range
• 2 -2.5 µg/L: ↑ risk of toxicity although tolerated in some
patients
• ˃ 2.5 µcg/L: Gastrointestinal, cardiovascular and CNS
toxicity including death
• Rule out Digoxin like interfering substances (DLS)
This Photo by Unknown Author is licensed under CC BY-SA
46
47. Aminoglycosides
• Amikacin, Gentamycin, Tobramycin
• Mechanism of action: bind to receptors on the 30S subunit of bacterial
ribosomes inducing misreading of genetic code
• Dose: once a day
• Elimination: Kidneys
• Method of Estimation: X-ray crystallography, nuclear magnetic resonance
(NMR) spectroscopy and mass spectrometry (MS)
• Exhibit significant systemic toxicity
• Nephrotoxicity (Reversible): cause a viscous cycle
• Ototoxicity: Mild-reversible, but if left untreated, Irreversible
This Photo by Unknown Author is licensed under CC BY-SA
47
48. Theophylline
• Bronchodilator
• Half-life: 3-13 hours; Sustained release-12-24 hour intervals
• Narrow therapeutic range and wide pharmacokinetic variability
• Elimination: Liver vis cytochrome p450
• Concentration: Adults-10-20mg/L (55-110 µmol/L)
Neonates 5-15mg/L (25-80 µmol/L)
• Caffeine replacing Theophylline in treatment of neonatal apnoea
• Method of estimation: Gas liquid chromatography, HPLC, radioimmunoassay and
enzyme immunoassay (EIA)
This Photo by Unknown Author is licensed under CC BY-SA
48
49. Theophylline
• PK problems - Bioavailability varies widely between preparations.
• 90% eliminated by the liver & 10% unchanged in the urine
• Dose to be calculated in presence of impaired hepatocellular function
• Toxicity - manifest as tachy-arrythmias, vomiting & convulsions
• Mild: Nausea, Headache, Jitteriness
• Serious: Tremor, agitation, insomnia, diarrhoea, palpitations, seizures,
cardiac arrythmias
• TDM of great assistance in patients receiving intravenous therapy of
theophylline
This Photo by Unknown Author is licensed under CC BY-SA
49
50. Methotrexate
• Chemotherapy/ Immunosuppressant
• Mechanism of action: Inhibitor of folic acid metabolism
• Half life: 6-12hours
• Used in high dose
• Rationale: Short exposure period, kill rapid dividing cell sparing slow normal
growing cells
• In case of longer exposure period, leucovorin given
• Method of Estimation: HPLC, fluorescence detection, radioimmunoassay,
dihydrofolate reductase inhibition assay
• Require monitoring of liver function (Hepatotoxic)
• N-terminal propeptide of collagen type III (Hepatic fibrosis)
This Photo by Unknown Author is licensed under CC BY-SA
50
51. Cyclosporin
• Immunosuppressant: Graft rejections
• Mechanism of action: Inhibit Calcineurin phosphatase and limit T-cell
action
• Nephrotoxic/ balance risk of undertreatment
• not a useful marker for prediction of acute rejection
• Half life: 5-18 hours
• High lipophilic so EDTA sample preferred
• Elimination: Liver via CYP3A (Multiple metabolites)
• Method of estimation: Immunoassays and LC/MS
This Photo by Unknown Author is licensed under CC BY-SA
51
52. Cyclosporin
• Cyclosporine blood levels determination : 2-3 days then monthly after 3
months
• Target of TDM: avoidance of nephrotoxicity and too high immunosuppression
This Photo by Unknown Author is licensed under CC BY-SA
52
57. Challenges in Therapeutic Drug Monitoring
• Quality drug assay should be performed within a time frame that is clinically useful
• Biological sample is collected to provide a clinically meaningful measurement
• Properly timed blood specimens
• Absorption is variable after oral administration
• Factors such as slow absorption can significantly delay peak concentrations
• Plasma samples should be drawn at trough
• Both the parent drug and the metabolites must be measured
• Not possible in routine monitoring
• Clinical features that may affect the relationship between concentration and
clinical effects
• Cost effectiveness and infrastructure
57
58. Summary
• TDM is monitoring of plasma concentration of drug for
individualization of dose in patients
• Mainly indicated for drugs having narrow therapeutic index, or to
check compliance and titration of dose
• Most common drugs to undergo TDM are anticonvulsants, lithium,
digoxin, gentamicin
• HPLC, LC/MS are most common techniques used for TDM
• There are several issues pertaining to TDM
58
59. References
• Blameris TL.Therapeutic Drug Monitoring. In: Clinical Chemistry. 8th
ed. Philedelphia: Wolters Kluwers; 2018. p. 1508–52
• Snozek CLH, Mcmillin GA. Therapeutic Drug Monitoring. In: Teitz's
textbook of clinical chemistry and molecular diagnosis. 7th ed.
Missouri: Elseiver's; 2015. p. 562–84
• Marshal WJ. Therapeutic drug monitoring and chemical aspects of
toxicology. In: Marshal's textbook of Clinical Chemistry. 7th ed.
British Library:Elseiver’s ; 2012
• Hallworth M. Therapeutic drug monitoring. In: Marshal’s textbook of
Medical Biochemistry. 3rd ed. British Library:Elseiver’s 2015. 767-86
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